Calvin Kuo

All Publications

• Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions. Nature communications Lo, Y. H., Horn, H. T., Huang, M. F., Yu, W. C., Young, C. M., Liu, Q., Tomaske, M., Towers, M., Dominguez, A., Bassik, M. C., Lee, D. F., Qi, L. S., Weissman, J. S., Chen, J., Kuo, C. J. 2025; 16 (1): 7566

  Abstract

  Understanding how genes influence drug responses is critical for advancing personalized cancer treatments. However, identifying these gene-drug interactions in a physiologically relevant human system remains a challenge, as it requires a model that reflects the complexity and heterogeneity among individuals. Here we show that large-scale CRISPR-based genetic screens, including knockout, interference (CRISPRi), activation (CRISPRa), and single-cell approaches, can be applied in primary human 3D gastric organoids to systematically identify genes that affect sensitivity to cisplatin. Our screens uncover genes that modulate cisplatin response. By combining CRISPR perturbations with single-cell transcriptomics, we resolve how genetic alterations interact with cisplatin at the level of individual cells and uncover an unexpected link between fucosylation and cisplatin sensitivity. We identify TAF6L as a regulator of cell recovery from cisplatin-induced cytotoxicity. These results highlight the utility of human organoid models for dissecting gene-drug interactions and offer insights into therapeutic vulnerabilities in gastric cancer.

  View details for DOI 10.1038/s41467-025-62818-3

  View details for PubMedID 40813572

  View details for PubMedCentralID PMC12354852

• FZD5 controls intestinal crypt homeostasis and colonic Wnt surrogate agonist response. Developmental cell Mu, Q., Ha, A., Santos, A. J., Lo, Y. H., van Unen, V., Miao, Y., Tomaske, M., Guzman, V. K., Alwahabi, S., Yuan, J. J., Deng, L., Li, L., Garcia, K. C., Kuo, C. J. 2024

  Abstract

  The rapidly regenerating intestinal epithelium requires crypt intestinal stem cells (ISCs). Wnt/β-catenin signaling maintains crypt homeostasis and Lgr5+ ISCs, and WNT ligands bind Frizzled receptors (FZD1-10). Identifying specific FZD(s) essential for intestinal homeostasis has been elusive; however, bioengineered antagonists blocking Wnt binding to FZD5 and FZD8 deplete the gut epithelium in vivo, highlighting potential roles. Here, an epithelial-specific Fzd5 knockout (KO) elicited lethal pan-intestinal crypt and villus loss, whereas an Lgr5+ ISC-specific Fzd5 KO depleted Lgr5+ ISCs via premature differentiation and repressed Wnt target genes. Fzd5-null phenotypes were rescued by constitutive β-catenin activation in vivo and in both mouse and human enteroids. KO of Fzd5, not Fzd8, in enteroids ablated responsiveness to dual-specificity FZD5/FZD8-selective Wnt surrogate agonists, which ameliorated DSS-induced colitis in wild-type and Fzd8 KO mice. Overall, FZD5 is a dominant and essential regulator of crypt homeostasis, Lgr5+ ISCs, and intestinal response to Wnt surrogate agonists, with implications for therapeutic mucosal repair.

  View details for DOI 10.1016/j.devcel.2024.10.022

  View details for PubMedID 39579768

• A human autoimmune organoid model reveals IL-7 function in coeliac disease. Nature Santos, A. J., van Unen, V., Lin, Z., Chirieleison, S. M., Ha, N., Batish, A., Chan, J. E., Cedano, J., Zhang, E. T., Mu, Q., Guh-Siesel, A., Tomaske, M., Colburg, D., Varma, S., Choi, S. S., Christophersen, A., Baghdasaryan, A., Yost, K. E., Karlsson, K., Ha, A., Li, J., Dai, H., Sellers, Z. M., Chang, H. Y., Dunn, J. C., Zhang, B. M., Mellins, E. D., Sollid, L. M., Fernandez-Becker, N. Q., Davis, M. M., Kuo, C. J. 2024

  Abstract

  In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.

  View details for DOI 10.1038/s41586-024-07716-2

  View details for PubMedID 39048815

• Cancer organoids 2.0: modelling the complexity of the tumour immune microenvironment. Nature reviews. Cancer Polak, R., Zhang, E. T., Kuo, C. J. 2024

  Abstract

  The development of neoplasia involves a complex and continuous interplay between malignantly transformed cells and the tumour microenvironment (TME). Cancer immunotherapies targeting the immune TME have been increasingly validated in clinical trials but response rates vary substantially between tumour histologies and are often transient, idiosyncratic and confounded by resistance. Faithful experimental models of the patient-specific tumour immune microenvironment, capable of recapitulating tumour biology and immunotherapy effects, would greatly improve patient selection, target identification and definition of resistance mechanisms for immuno-oncology therapeutics. In this Review, we discuss currently available and rapidly evolving 3D tumour organoid models that capture important immune features of the TME. We highlight diverse opportunities for organoid-based investigations of tumour immunity, drug development and precision medicine.

  View details for DOI 10.1038/s41568-024-00706-6

  View details for PubMedID 38977835

  View details for PubMedCentralID 7046529

• Functional screening of amplification outlier oncogenes in organoid models of early tumorigenesis. Cell reports Salahudeen, A. A., Seoane, J. A., Yuki, K., Mah, A. T., Smith, A. R., Kolahi, K., De la O, S. M., Hart, D. J., Ding, J., Ma, Z., Barkal, S. A., Shukla, N. D., Zhang, C. H., Cantrell, M. A., Batish, A., Usui, T., Root, D. E., Hahn, W. C., Curtis, C., Kuo, C. J. 2023; 42 (11): 113355

  Abstract

  Somatic copy number gains are pervasive across cancer types, yet their roles in oncogenesis are insufficiently evaluated. This inadequacy is partly due to copy gains spanning large chromosomal regions, obscuring causal loci. Here, we employed organoid modeling to evaluate candidate oncogenic loci identified via integrative computational analysis of extreme copy gains overlapping with extreme expression dysregulation in The Cancer Genome Atlas. Subsets of "outlier" candidates were contextually screened as tissue-specific cDNA lentiviral libraries within cognate esophagus, oral cavity, colon, stomach, pancreas, and lung organoids bearing initial oncogenic mutations. Iterative analysis nominated the kinase DYRK2 at 12q15 as an amplified head and neck squamous carcinoma oncogene in p53-/- oral mucosal organoids. Similarly, FGF3, amplified at 11q13 in 41% of esophageal squamous carcinomas, promoted p53-/- esophageal organoid growth reversible by small molecule and soluble receptor antagonism of FGFRs. Our studies establish organoid-based contextual screening of candidate genomic drivers, enabling functional evaluation during early tumorigenesis.

  View details for DOI 10.1016/j.celrep.2023.113355

  View details for PubMedID 37922313

• Therapeutic blood-brain barrier modulation and stroke treatment by a bioengineered FZD4-selective WNT surrogate in mice. Nature communications Ding, J., Lee, S., Vlahos, L., Yuki, K., Rada, C. C., van Unen, V., Vuppalapaty, M., Chen, H., Sura, A., McCormick, A. K., Tomaske, M., Alwahabi, S., Nguyen, H., Nowatzke, W., Kim, L., Kelly, L., Vollrath, D., Califano, A., Yeh, W., Li, Y., Kuo, C. J. 2023; 14 (1): 2947

  Abstract

  Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/beta-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

  View details for DOI 10.1038/s41467-023-37689-1

  View details for PubMedID 37268690

• Deterministic evolution and stringent selection during preneoplasia. Nature Karlsson, K., Przybilla, M. J., Kotler, E., Khan, A., Xu, H., Karagyozova, K., Sockell, A., Wong, W. H., Liu, K., Mah, A., Lo, Y. H., Lu, B., Houlahan, K. E., Ma, Z., Suarez, C. J., Barnes, C. P., Kuo, C. J., Curtis, C. 2023

  Abstract

  The earliest events during human tumour initiation, although poorly characterized, may hold clues to malignancy detection and prevention1. Here we model occult preneoplasia by biallelic inactivation of TP53, a common early event in gastric cancer, in human gastric organoids. Causal relationships between this initiating genetic lesion and resulting phenotypes were established using experimental evolution in multiple clonally derived cultures over 2 years. TP53 loss elicited progressive aneuploidy, including copy number alterations and structural variants prevalent in gastric cancers, with evident preferred orders. Longitudinal single-cell sequencing of TP53-deficient gastric organoids similarly indicates progression towards malignant transcriptional programmes. Moreover, high-throughput lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programmes repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a marked degree of phenotypic convergence in premalignant epithelial organoids. These data imply predictability in the earliest stages of tumorigenesis and show evolutionary constraints and barriers to malignant transformation, with implications for earlier detection and interception of aggressive, genome-instable tumours.

  View details for DOI 10.1038/s41586-023-06102-8

  View details for PubMedID 37258665

  View details for PubMedCentralID 5656752

• Modeling human adaptive immune responses with tonsil organoids. Nature medicine Wagar, L. E., Salahudeen, A. n., Constantz, C. M., Wendel, B. S., Lyons, M. M., Mallajosyula, V. n., Jatt, L. P., Adamska, J. Z., Blum, L. K., Gupta, N. n., Jackson, K. J., Yang, F. n., Röltgen, K. n., Roskin, K. M., Blaine, K. M., Meister, K. D., Ahmad, I. N., Cortese, M. n., Dora, E. G., Tucker, S. N., Sperling, A. I., Jain, A. n., Davies, D. H., Felgner, P. L., Hammer, G. B., Kim, P. S., Robinson, W. H., Boyd, S. D., Kuo, C. J., Davis, M. M. 2021

  Abstract

  Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.

  View details for DOI 10.1038/s41591-020-01145-0

  View details for PubMedID 33432170

• A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation. Cancer discovery Lo, Y. H., Kolahi, K. S., Du, Y. n., Chang, C. Y., Krokhotin, A. n., Nair, A. n., Sobba, W. D., Karlsson, K. n., Jones, S. J., Longacre, T. A., Mah, A. T., Tercan, B. n., Sockell, A. n., Xu, H. n., Seoane, J. A., Chen, J. n., Shmulevich, I. n., Weissman, J. S., Curtis, C. n., Califano, A. n., Fu, H. n., Crabtree, G. R., Kuo, C. J. 2021

  Abstract

  Mutations in ARID1A rank amongst the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity and mucinous differentiation. Genetic Wnt/B-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation. ARID1A mutation induced transcriptional regulatory modules characteristic of MSI and EBV subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wnt-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells.

  View details for DOI 10.1158/2159-8290.CD-20-1109

  View details for PubMedID 33451982

• Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature Salahudeen, A. A., Choi, S. S., Rustagi, A., Zhu, J., van Unen, V., de la O, S. M., Flynn, R. A., Margalef-Catala, M., Santos, A. J., Ju, J., Batish, A., Usui, T., Zheng, G. X., Edwards, C. E., Wagar, L. E., Luca, V., Anchang, B., Nagendran, M., Nguyen, K., Hart, D. J., Terry, J. M., Belgrader, P., Ziraldo, S. B., Mikkelsen, T. S., Harbury, P. B., Glenn, J. S., Garcia, K. C., Davis, M. M., Baric, R. S., Sabatti, C., Amieva, M. R., Blish, C. A., Desai, T. J., Kuo, C. J. 2020

  Abstract

  The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. We generated long-term feeder-free, chemically defined culture of distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential while basal cell organoids developed lumens lined by differentiated club and ciliated cells. Single cell analysis of basal organoid KRT5+ cells revealed a distinct ITGA6+ITGB4+ mitotic population whose proliferation further segregated to a TNFRSF12Ahi subfraction comprising ~10% of KRT5+ basal cells, residing in clusters within terminal bronchioles and exhibiting enriched clonogenic organoid growth activity. Distal lung organoids were created with apical-out polarity to display ACE2 on the exposed external surface, facilitating SARS-CoV-2 infection of AT2 and basal cultures and identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and establishes a facile in vitro organoid model for human distal lung infections including COVID-19-associated pneumonia.

  View details for DOI 10.1038/s41586-020-3014-1

  View details for PubMedID 33238290

• Organoid Modeling of the Tumor Immune Microenvironment. Cell Neal, J. T., Li, X., Zhu, J., Giangarra, V., Grzeskowiak, C. L., Ju, J., Liu, I. H., Chiou, S., Salahudeen, A. A., Smith, A. R., Deutsch, B. C., Liao, L., Zemek, A. J., Zhao, F., Karlsson, K., Schultz, L. M., Metzner, T. J., Nadauld, L. D., Tseng, Y., Alkhairy, S., Oh, C., Keskula, P., Mendoza-Villanueva, D., De La Vega, F. M., Kunz, P. L., Liao, J. C., Leppert, J. T., Sunwoo, J. B., Sabatti, C., Boehm, J. S., Hahn, W. C., Zheng, G. X., Davis, M. M., Kuo, C. J. 2018; 175 (7): 1972

  Abstract

  Invitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor Tcell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.

  View details for PubMedID 30550791

• Organoids reveal cancer dynamics NATURE Kuo, C. J., Curtis, C. 2018; 556 (7702): 441–42

  View details for Web of Science ID 000430793000032

  View details for PubMedID 29686366

• Non-equivalence of Wnt and R-spondin ligands during Lgr5(+) intestinal stem-cell self-renewal NATURE Yan, K. S., Janda, C. Y., Chang, J., Zheng, G. X., Larkin, K. A., Luca, V. C., Chia, L. A., Mah, A. T., Han, A., Terry, J. M., Ootani, A., Roelf, K., Lee, M., Yuan, J., Li, X., Bolen, C. R., Wilhelmy, J., Davies, P. S., Ueno, H., von Furstenberg, R. J., Belgrader, P., Ziraldo, S. B., Ordonez, H., Henning, S. J., Wong, M. H., Snyder, M. P., Weissman, I. L., Hsueh, A. J., Mikkelsen, T. S., Garcia, K. C., Kuo, C. J. 2017; 545 (7653): 238-?

  Abstract

  The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5(+) intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5(+) ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5(+) ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5(+) ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

  View details for DOI 10.1038/nature22313

  View details for Web of Science ID 000400963800037

• Surrogate Wnt agonists that phenocopy canonical Wnt and beta-catenin signalling NATURE Janda, C. Y., Dang, L. T., You, C., Chang, J., de Lau, W., Zhong, Z. A., Yan, K. S., Marecic, O., Siepe, D., Li, X., Moody, J. D., Williams, B. O., Clevers, H., Piehler, J., Baker, D., Kuo, C. J., Garcia, K. C. 2017; 545 (7653): 234-?

  Abstract

  Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.

  View details for DOI 10.1038/nature22306

  View details for PubMedID 28467818

• Gpr124 is essential for blood-brain barrier integrity in central nervous system disease NATURE MEDICINE Chang, J., Mancuso, M. R., Maier, C., Liang, X., Yuki, K., Yang, L., Kwong, J. W., Wang, J., Rao, V., Vallon, M., Kosinski, C., Zhang, J. J., Mah, A. T., Xu, L., Li, L., Gholamin, S., Reyes, T. F., Li, R., Kuhnert, F., Han, X., Yuan, J., Chiou, S., Brettman, A. D., Daly, L., Corney, D. C., Cheshier, S. H., Shortliffe, L. D., Wu, X., Snyder, M., Chan, P., Giffard, R. G., Chang, H. Y., Andreasson, K., Kuo, C. J. 2017; 23 (4): 450-?

  Abstract

  Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

  View details for DOI 10.1038/nm.4309

  View details for PubMedID 28288111

• Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states NATURE MEDICINE Furman, D., Chang, J., Lartigue, L., Bolen, C. R., Haddad, F., Gaudilliere, B., Ganio, E. A., Fragiadakis, G. K., Spitzer, M. H., Douchet, I., Daburon, S., Moreau, J., Nolan, G. P., Blanco, P., Dechanet-Merville, J., Dekker, C. L., Jojic, V., Kuo, C. J., Davis, M. M., Faustin, B. 2017; 23 (2): 174-184

  Abstract

  Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N(4)-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.

  View details for DOI 10.1038/nm.4267

  View details for Web of Science ID 000393729000009

  View details for PubMedID 28092664

• Toward recreating colon cancer in human organoids. Nature medicine Salahudeen, A. A., Kuo, C. J. 2015; 21 (3): 215-216

  View details for DOI 10.1038/nm.3818

  View details for PubMedID 25742455

• Ascl2 reinforces intestinal stem cell identity. Cell stem cell Yan, K. S., Kuo, C. J. 2015; 16 (2): 105-106

  Abstract

  Ascl2 is a Wnt-responsive master transcription factor that controls the Lgr5(+) intestinal stem cell gene expression program. Now in Cell Stem Cell, Schuijers et al. (2015) report an Ascl2 positive feedback loop, tuned by previous Wnt pathway activity, that perpetuates intestinal stem cell identity in response to Wnt/R-spondin stimulation.

  View details for DOI 10.1016/j.stem.2015.01.014

  View details for PubMedID 25658363

• Identification and specification of the mouse skeletal stem cell. Cell Chan, C. K., Seo, E. Y., Chen, J. Y., Lo, D., McArdle, A., Sinha, R., Tevlin, R., Seita, J., Vincent-Tompkins, J., Wearda, T., Lu, W., Senarath-Yapa, K., Chung, M. T., Marecic, O., Tran, M., Yan, K. S., Upton, R., Walmsley, G. G., Lee, A. S., Sahoo, D., Kuo, C. J., Weissman, I. L., Longaker, M. T. 2015; 160 (1-2): 285-298

  Abstract

  How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

  View details for DOI 10.1016/j.cell.2014.12.002

  View details for PubMedID 25594184

• Through-skull fluorescence imaging of the brain in a new near-infrared window NATURE PHOTONICS Hong, G., Diao, S., Chang, J., Antaris, A. L., Chen, C., Zhang, B., Zhao, S., Atochin, D. N., Huang, P. L., Andreasson, K. I., Kuo, C. J., Dai, H. 2014; 8 (9): 723-730

  Abstract

  To date, brain imaging has largely relied on X-ray computed tomography and magnetic resonance angiography with limited spatial resolution and long scanning times. Fluorescence-based brain imaging in the visible and traditional near-infrared regions (400-900 nm) is an alternative but currently requires craniotomy, cranial windows and skull thinning techniques, and the penetration depth is limited to 1-2 mm due to light scattering. Here, we report through-scalp and through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy utilizing the intrinsic photoluminescence of single-walled carbon nanotubes in the 1.3-1.4 micrometre near-infrared window. Reduced photon scattering in this spectral region allows fluorescence imaging reaching a depth of >2 mm in mouse brain with sub-10 micrometre resolution. An imaging rate of ~5.3 frames/s allows for dynamic recording of blood perfusion in the cerebral vessels with sufficient temporal resolution, providing real-time assessment of blood flow anomaly in a mouse middle cerebral artery occlusion stroke model.

  View details for DOI 10.1038/NPHOTON.2014.166

  View details for Web of Science ID 000342600100016

  View details for PubMedCentralID PMC5026222

• Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture NATURE MEDICINE Li, X., Nadauld, L., Ootani, A., Corney, D. C., Pai, R. K., Gevaert, O., Cantrell, M. A., Rack, P. G., Neal, J. T., Chan, C. W., Yeung, T., Gong, X., Yuan, J., Wilhelmy, J., Robine, S., Attardi, L. D., Plevritis, S. K., Hung, K. E., Chen, C., Ji, H. P., Kuo, C. J. 2014; 20 (7): 769-777

  Abstract

  The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

  View details for DOI 10.1038/nm.3585

  View details for Web of Science ID 000338689500021

• Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer. Genome biology Nadauld, L. D., Garcia, S., Natsoulis, G., Bell, J. M., Miotke, L., Hopmans, E. S., Xu, H., Pai, R. K., Palm, C., Regan, J. F., Chen, H., Flaherty, P., Ootani, A., Zhang, N. R., Ford, J. M., Kuo, C. J., Ji, H. P. 2014; 15 (8): 428-?

  Abstract

  Gastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis.Both the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity.We document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.

  View details for DOI 10.1186/s13059-014-0428-9

  View details for PubMedID 25315765

  View details for PubMedCentralID PMC4145231

• Interfollicular Epidermal Stem Cells Self-Renew via Autocrine Wnt Signaling SCIENCE Lim, X., Tan, S. H., Koh, W. L., Chau, R. M., Yan, K. S., Kuo, C. J., van Amerongen, R., Klein, A. M., Nusse, R. 2013; 342 (6163): 1226-1230

  Abstract

  The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using mouse lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/β-catenin signaling to proliferate. The same cells contribute robustly to wound healing, with no requirement for a quiescent stem cell subpopulation. By means of double-labeling RNA in situ hybridization in mice, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self-renewal.

  View details for DOI 10.1126/science.1239730

  View details for Web of Science ID 000327857900046

  View details for PubMedID 24311688

• A liver Hif-2a-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition. Nature medicine Wei, K., Piecewicz, S. M., McGinnis, L. M., Taniguchi, C. M., Wiegand, S. J., Anderson, K., Chan, C. W., Mulligan, K. X., Kuo, D., Yuan, J., Vallon, M., Morton, L. C., Lefai, E., Simon, M. C., Maher, J. J., Mithieux, G., Rajas, F., Annes, J. P., McGuinness, O. P., Thurston, G., Giaccia, A. J., Kuo, C. J. 2013; 19 (10): 1331-1337

  Abstract

  Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis. The liver possesses a rich sinusoidal capillary network with a higher degree of hypoxia and lower gluconeogenesis in the perivenous zone as compared to the rest of the organ. Here, we show that diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in nondiabetic C57BL/6 and diabetic db/db mice, potentiating hepatic insulin signaling with lower gluconeogenic gene expression, higher glycogen storage and suppressed hepatic glucose production. VEGF inhibition induced hepatic hypoxia through sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia-inducible factor-2α (Hif-2α, encoded by Epas1) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling through direct and indirect induction of insulin receptor substrate-2 (Irs2), an essential insulin receptor adaptor protein. Further, liver Irs2 was both necessary and sufficient to mediate Hif-2α and Vegf inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between Hif-2α-mediated hypoxic signaling and hepatic insulin action through Irs2 induction, which can be co-opted by Vegf inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for Hif-1α, which promotes glycolysis, and Hif-2α, which suppresses gluconeogenesis, and suggest new treatment approaches for type 2 diabetes mellitus.

  View details for DOI 10.1038/nm.3295

  View details for PubMedID 24037094

• Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes. Nature medicine Taniguchi, C. M., Finger, E. C., Krieg, A. J., Wu, C., Diep, A. N., Lagory, E. L., Wei, K., McGinnis, L. M., Yuan, J., Kuo, C. J., Giaccia, A. J. 2013; 19 (10): 1325-1330

  Abstract

  Signaling initiated by hypoxia and insulin powerfully alters cellular metabolism. The protein stability of hypoxia-inducible factor-1 alpha (Hif-1α) and Hif-2α is regulated by three prolyl hydroxylase domain-containing protein isoforms (Phd1, Phd2 and Phd3). Insulin receptor substrate-2 (Irs2) is a critical mediator of the anabolic effects of insulin, and its decreased expression contributes to the pathophysiology of insulin resistance and diabetes. Although Hif regulates many metabolic pathways, it is unknown whether the Phd proteins regulate glucose and lipid metabolism in the liver. Here, we show that acute deletion of hepatic Phd3, also known as Egln3, improves insulin sensitivity and ameliorates diabetes by specifically stabilizing Hif-2α, which then increases Irs2 transcription and insulin-stimulated Akt activation. Hif-2α and Irs2 are both necessary for the improved insulin sensitivity, as knockdown of either molecule abrogates the beneficial effects of Phd3 knockout on glucose tolerance and insulin-stimulated Akt phosphorylation. Augmenting levels of Hif-2α through various combinations of Phd gene knockouts did not further improve hepatic metabolism and only added toxicity. Thus, isoform-specific inhibition of Phd3 could be exploited to treat type 2 diabetes without the toxicity that could occur with chronic inhibition of multiple Phd isoforms.

  View details for DOI 10.1038/nm.3294

  View details for PubMedID 24037093

  View details for PubMedCentralID PMC4089950

• A liver Hif-2 alpha-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition NATURE MEDICINE Wei, K., Piecewicz, S. M., McGinnis, L. M., Taniguchi, C. M., Wiegand, S. J., Anderson, K., Chan, C. W., Mulligan, K. X., Kuo, D., Yuan, J., Vallon, M., Morton, L. C., Lefai, E., Simon, M. C., Maher, J. J., Mithieux, G., Rajas, F., Annes, J. P., McGuinness, O. P., Thurston, G., Giaccia, A. J., Kuo, C. J. 2013; 19 (10): 1331-?

  Abstract

  Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis. The liver possesses a rich sinusoidal capillary network with a higher degree of hypoxia and lower gluconeogenesis in the perivenous zone as compared to the rest of the organ. Here, we show that diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in nondiabetic C57BL/6 and diabetic db/db mice, potentiating hepatic insulin signaling with lower gluconeogenic gene expression, higher glycogen storage and suppressed hepatic glucose production. VEGF inhibition induced hepatic hypoxia through sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia-inducible factor-2α (Hif-2α, encoded by Epas1) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling through direct and indirect induction of insulin receptor substrate-2 (Irs2), an essential insulin receptor adaptor protein. Further, liver Irs2 was both necessary and sufficient to mediate Hif-2α and Vegf inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between Hif-2α-mediated hypoxic signaling and hepatic insulin action through Irs2 induction, which can be co-opted by Vegf inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for Hif-1α, which promotes glycolysis, and Hif-2α, which suppresses gluconeogenesis, and suggest new treatment approaches for type 2 diabetes mellitus.

  View details for DOI 10.1038/nm.3295

  View details for Web of Science ID 000325531700034

  View details for PubMedID 24037094

  View details for PubMedCentralID PMC3795838

• Restriction of intestinal stem cell expansion and the regenerative response by YAP NATURE Barry, E. R., Morikawa, T., Butler, B. L., Shrestha, K., de la Rosa, R., Yan, K. S., Fuchs, C. S., Magness, S. T., Smits, R., Ogino, S., Kuo, C. J., Camargo, F. D. 2013; 493 (7430): 106-?

  Abstract

  A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

  View details for DOI 10.1038/nature11693

  View details for Web of Science ID 000312933800040

  View details for PubMedID 23178811

  View details for PubMedCentralID PMC3536889

• beta-Catenin-Driven Cancers Require a YAP1 Transcriptional Complex for Survival and Tumorigenesis CELL Rosenbluh, J., Nijhawan, D., Cox, A. G., Li, X., Neal, J. T., Schafer, E. J., Zack, T. I., Wang, X., Tsherniak, A., Schinzel, A. C., Shao, D. D., Schumacher, S. E., Weir, B. A., Vazquez, F., Cowley, G. S., Root, D. E., Mesirov, J. P., Beroukhim, R., Kuo, C. J., Goessling, W., Hahn, W. C. 2012; 151 (7): 1457-1473

  Abstract

  Wnt/β-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic β-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of β-catenin in transformation, we classified β-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that β-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with β-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of β-catenin-dependent cancers in both cell lines and animal models. These observations define a β-catenin-YAP1-TBX5 complex essential to the transformation and survival of β-catenin-driven cancers.

  View details for DOI 10.1016/j.cell.2012.11.026

  View details for Web of Science ID 000312890300012

  View details for PubMedID 23245941

  View details for PubMedCentralID PMC3530160

• The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yan, K. S., Chia, L. A., Li, X., Ootani, A., Su, J., Lee, J. Y., Su, N., Luo, Y., Heilshorn, S. C., Amieva, M. R., Sangiorgi, E., Capecchi, M. R., Kuo, C. J. 2012; 109 (2): 466-471

  Abstract

  The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.

  View details for DOI 10.1073/pnas.1118857109

  View details for PubMedID 22190486

• Essential Regulation of CNS Angiogenesis by the Orphan G Protein-Coupled Receptor GPR124 SCIENCE Kuhnert, F., Mancuso, M. R., Shamloo, A., Wang, H., Choksi, V., Florek, M., Su, H., Fruttiger, M., Young, W. L., Heilshorn, S. C., Kuo, C. J. 2010; 330 (6006): 985-989

  Abstract

  The orphan G protein-coupled receptor (GPCR) GPR124/tumor endothelial marker 5 is highly expressed in central nervous system (CNS) endothelium. Here, we show that complete null or endothelial-specific GPR124 deletion resulted in embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, whereas in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, vascular endothelial growth factor-independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.

  View details for DOI 10.1126/science.1196554

  View details for PubMedID 21071672

• Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nature medicine Ootani, A., Li, X., Sangiorgi, E., Ho, Q. T., Ueno, H., Toda, S., Sugihara, H., Fujimoto, K., Weissman, I. L., Capecchi, M. R., Kuo, C. J. 2009; 15 (6): 701-706

  Abstract

  The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.

  View details for DOI 10.1038/nm.1951

  View details for PubMedID 19398967

  View details for PubMedCentralID PMC2919216

• WNT7A/B assemble a GPR124-RECK-LRP5/6 co-receptor complex to activate β-catenin signaling in brain endothelial cells. The Journal of biological chemistry Heiden, R., Hannig, L., Kuo, C. J., Ergün, S., Braunger, B. M., Vallon, M. 2025: 110682

  Abstract

  WNT7A and WNT7B, secreted by neural cells, are essential regulators of developmental brain angiogenesis and blood-brain barrier integrity. In brain endothelial cells, WNT7 proteins activate β-catenin signaling through the ligand-specific receptor complex GPR124-RECK and classical WNT receptors of the FZD and LRP families. Previous studies suggested that WNT7 isoforms assemble a GPR124-RECK-FZD-LRP5/6 multi-receptor complex for signaling. However, direct biochemical evidence for this complex and its signaling mechanisms remains elusive. Here, we investigated the formation and signaling mechanisms of WNT7 co-receptor complexes in brain endothelial cells using CRISPR/Cas9, biochemical analyses, and cell-based assays. Unexpectedly, cells with knockout of all FZD isoforms retained ∼25% of WNT7 responsiveness, whereas classical WNT3A signaling was completely abolished. Similarly, knockout of all Dvl paralogs, key mediators of FZD signaling, preserved ∼50% of WNT7 signaling activity but fully blocked WNT3A responses. In contrast, knockout of Gpr124, Reck, or Lrp5/6 completely abrogated WNT7 signaling. While both WNT7A and WNT3A triggered LRP6 phosphorylation, only WNT3A induced DVL2 phosphorylation. Biochemical analyses revealed WNT7-dependent recruitment of LRP5/6, but not FZD, to the GPR124-RECK heterodimer, forming a GPR124-RECK-WNT7-LRP5/6 complex. In GPR124-deficient cells, WNT7 proteins still assembled a RECK-WNT7-LRP5/6 core complex, yet this complex lacked signaling activity and LRP6 phosphorylation. Clustering of RECK-WNT7-LRP5/6 complexes with recombinant dimeric GPR124 ectodomain or a RECK antibody partially restored signaling, suggesting that GPR124 mediates formation of higher-order complexes. Our findings indicate that WNT7 signaling in brain endothelial cells is driven by distinct co-receptor complexes: a FZD-independent GPR124-RECK-LRP5/6 complex and FZD-dependent complexes that likely act in synergy.

  View details for DOI 10.1016/j.jbc.2025.110682

  View details for PubMedID 40914247

• Defining the antitumor mechanism of action of a clinical-stage compound as a selective degrader of the nuclear pore complex. Cancer discovery Yuan, L., Ji, W., Dwyer, B. G., Lu, J., Bian, J., Colombo, G. M., Martinez, M. J., Fernandez, D., Phillips, N. A., Tang, M. T., Zhou, C. W., Quispe Calla, N. E., Guzman Huancas, C., Eckart, M., Tran, J., Jones, H. M., Qiu, T., Doench, J. G., Rees, M. G., Roth, J. A., Cameron, M. D., Charville, G. W., Kuo, C. J., Dixon, S. J., Zhang, T., Hinshaw, S. M., Gray, N. S., Corsello, S. M. 2025

  Abstract

  Cancer cells are acutely dependent on nuclear transport due to elevated transcriptional activity, suggesting an unrealized opportunity for selective therapeutic inhibition of the nuclear pore complex. Through large-scale phenotypic profiling of cancer cell lines, genome-scale functional genomic modifier screens, and mass spectrometry-based proteomics, we discovered that the clinical drug PRLX-93936 is a molecular glue that binds and reprograms the TRIM21 ubiquitin ligase to degrade the nuclear pore complex. Upon compound-induced TRIM21 recruitment, the nuclear pore is ubiquitylated and degraded, resulting in the loss of short-lived cytoplasmic mRNA transcripts and induction of cancer cell apoptosis. Direct compound binding to TRIM21 was confirmed via surface plasmon resonance and x-ray crystallography, while compound-induced TRIM21-nucleoporin complex formation was demonstrated through multiple orthogonal approaches in cells and in vitro. Phenotype-guided optimization yielded compounds with 10-fold greater potency and drug-like properties with robust pharmacokinetics and efficacy against pancreatic cancer xenografts and patient-derived organoids.

  View details for DOI 10.1158/2159-8290.CD-25-0271

  View details for PubMedID 40891634

• A quantitative spatial cell-cell colocalizations framework enabling comparisons between in vitro assembloids and pathological specimens. Nature communications Bouchard, G., Zhang, W., Ilerten, I., Li, I., Bhattacharya, A., Li, Y., Trope, W., Shrager, J. B., Kuo, C., Ozawa, M. G., Giaccia, A. J., Tian, L., Plevritis, S. K. 2025; 16 (1): 1392

  Abstract

  Spatial omics is enabling unprecedented tissue characterization, but the ability to adequately compare spatial features across samples under different conditions is lacking. We propose a quantitative framework that catalogs significant, normalized, colocalizations between pairs of cell subpopulations, enabling comparisons among a variety of biological samples. We perform cell-pair colocalization analysis on multiplexed immunofluorescence images of assembloids constructed with lung adenocarcinoma (LUAD) organoids and cancer-associated fibroblasts derived from human tumors. Our data show that assembloids recapitulate human LUAD tumor-stroma spatial organization, justifying their use as a tool for investigating the spatial biology of human disease. Intriguingly, drug-perturbation studies identify drug-induced spatial rearrangements that also appear in treatment-naïve human tumor samples, suggesting potential directions for characterizing spatial (re)-organization related to drug resistance. Moreover, our work provides an opportunity to quantify spatial data across different samples, with the common goal of building catalogs of spatial features associated with disease processes and drug response.

  View details for DOI 10.1038/s41467-024-55129-6

  View details for PubMedID 39915493

  View details for PubMedCentralID 7479520

• Author Correction: Targeting colorectal cancer with small-molecule inhibitors of ALDH1B1. Nature chemical biology Feng, Z., Hom, M. E., Bearrood, T. E., Rosenthal, Z. C., Fernández, D., Ondrus, A. E., Gu, Y., McCormick, A. K., Tomaske, M. G., Marshall, C. R., Kline, T., Chen, C. H., Mochly-Rosen, D., Kuo, C. J., Chen, J. K. 2024

  View details for DOI 10.1038/s41589-024-01810-2

  View details for PubMedID 39653787

• Frizzled5 controls murine intestinal epithelial cell plasticity through organization of chromatin accessibility. Developmental cell Deng, L., He, X. C., Chen, S., Zhang, N., Deng, F., Scott, A., He, Y., Tsuchiya, D., Smith, S. E., Epp, M., Malloy, S., Liu, F., Hembree, M., Mu, Q., Haug, J. S., Malagola, E., Hassan, H., Petentler, K., Egidy, R., Maddera, L., Russell, J., Wang, Y., Li, H., Zhao, C., Perera, A., Wang, T. C., Kuo, C. J., Li, L. 2024

  Abstract

  The homeostasis of the intestinal epithelium relies on intricate yet insufficiently understood mechanisms of intestinal epithelial plasticity. Here, we elucidate the pivotal role of Frizzled5 (Fzd5), a Wnt pathway receptor, as a determinant of murine intestinal epithelial cell fate. Deletion of Fzd5 in Lgr5+ intestinal stem cells (ISCs) impairs their self-renewal, whereas its deletion in Krt19+ cells disrupts lineage generation, without affecting crypt integrity in either case. However, a broader deletion of Fzd5 across the epithelium leads to substantial crypt deterioration. Integrated analysis of single-cell RNA sequencing (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) identifies that Fzd5 governs chromatin accessibility, orchestrating the regulation of stem- and lineage-related gene expression mainly in ISCs and progenitor cells. In summary, our findings provide insights into the regulatory role of Fzd5 in governing intestinal epithelial plasticity.

  View details for DOI 10.1016/j.devcel.2024.10.021

  View details for PubMedID 39579769

• Microdissection tools to generate organoids for modeling the tumor immune microenvironment. Microsystems & nanoengineering Cordts, S. C., Yuki, K., Henao Echeverri, M. F., Narasimhan, B., Kuo, C. J., Tang, S. K. 2024; 10 (1): 126

  Abstract

  Patient-derived tumor organoids have emerged as promising models for predicting personalized drug responses in cancer therapy, but they typically lack immune components. Preserving the in vivo association between tumor cells and endogenous immune cells is critical for accurate testing of cancer immunotherapies. Mechanical dissection of tumor specimens into tumor fragments, as opposed to enzymatic digestion into single cells, is essential for maintaining these native tumor-immune cell spatial relationships. However, conventional mechanical dissection relying on manual mincing is time-consuming and irreproducible. This study describes two microdissection devices, the Dicer and Grater, to facilitate the generation of intact tumor fragments from mouse B16 melanoma, a common model of human melanoma. The Dicer- and Grater-cut tumor fragments were used to generate air‒liquid interface (ALI) organoids that copreserve tumor cells with infiltrating immune subsets without artificial reconstitution. The Dicer, consisting of a hexagonal array of silicon microblades, was employed to investigate the effect of organoid size. The viability of ALI organoid immune cells appeared insensitive to organoid sizes exceeding ~400m but diminished in organoids ~200m in size. The Grater, consisting of an array of submillimeter holes in stainless steel, was employed to accelerate dissection. For the samples studied, the Grater was 4.5 times faster than manual mincing. Compared with those generated by manual mincing, ALI organoids generated by the Grater demonstrated similar viability, immune cell composition, and responses to anti-PD-1 immunotherapy. With further optimization, the Grater holds potential for integration into clinical workflows to support the advancement of personalized cancer immunotherapy.

  View details for DOI 10.1038/s41378-024-00756-8

  View details for PubMedID 39251611

• Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair. Cancer discovery Rose, J. C., Belk, J. A., Wong, I. T., Luebeck, J., Horn, H. T., Daniel, B., Jones, M. G., Yost, K. E., Hung, K. L., Kolahi, K. S., Curtis, E. J., Kuo, C. J., Bafna, V., Mischel, P. S., Chang, H. Y. 2024

  Abstract

  Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for ecDNA induction with extensive characterization of newly formed ecDNA to examine their biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of 800kb, 1 Mb, and 1.8 Mb ecDNAs reaching or exceeding 15%. We show non-homologous end joining and microhomology-mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar can form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

  View details for DOI 10.1158/2159-8290.CD-23-1117

  View details for PubMedID 39109936

• Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids. Nature materials LeSavage, B. L., Zhang, D., Huerta-López, C., Gilchrist, A. E., Krajina, B. A., Karlsson, K., Smith, A. R., Karagyozova, K., Klett, K. C., Huang, M. S., Long, C., Kaber, G., Madl, C. M., Bollyky, P. L., Curtis, C., Kuo, C. J., Heilshorn, S. C. 2024

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix. However, how the altered cell/extracellular-matrix signalling contributes to the PDAC tumour phenotype has been difficult to dissect. Here we design and engineer matrices that recapitulate the key hallmarks of the PDAC tumour extracellular matrix to address this knowledge gap. We show that patient-derived PDAC organoids from three patients develop resistance to several clinically relevant chemotherapies when cultured within high-stiffness matrices mechanically matched to in vivo tumours. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, matrix-induced chemoresistance occurs within a stiff environment due to the increased expression of drug efflux transporters mediated by CD44 receptor interactions with hyaluronan. Moreover, PDAC chemoresistance is reversible following transfer from high- to low-stiffness matrices, suggesting that targeting the fibrotic extracellular matrix may sensitize chemoresistant tumours. Overall, our findings support the potential of engineered matrices and patient-derived organoids for elucidating extracellular matrix contributions to human disease pathophysiology.

  View details for DOI 10.1038/s41563-024-01908-x

  View details for PubMedID 38965405

  View details for PubMedCentralID 5704175

• DRA involvement in linaclotide stimulated bicarbonate secretion during loss of CFTR function. JCI insight Sarthi, J. B., Trumbull, A. M., Abazari, S. M., van Unen, V., Chan, J. E., Jiang, Y., Gammons, J., Anderson, M. O., Cil, O., Kuo, C. J., Sellers, Z. M. 2024

  Abstract

  Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) datasets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression (Cftr knockout mice) or function (CFTRinh-172). NHE3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

  View details for DOI 10.1172/jci.insight.172364

  View details for PubMedID 38869953

• Engineered CD47 protects T cells for enhanced antitumour immunity. Nature Yamada-Hunter, S. A., Theruvath, J., McIntosh, B. J., Freitas, K. A., Lin, F., Radosevich, M. T., Leruste, A., Dhingra, S., Martinez-Velez, N., Xu, P., Huang, J., Delaidelli, A., Desai, M. H., Good, Z., Polak, R., May, A., Labanieh, L., Bjelajac, J., Murty, T., Ehlinger, Z., Mount, C. W., Chen, Y., Heitzeneder, S., Marjon, K. D., Banuelos, A., Khan, O., Wasserman, S. L., Spiegel, J. Y., Fernandez-Pol, S., Kuo, C. J., Sorensen, P. H., Monje, M., Majzner, R. G., Weissman, I. L., Sahaf, B., Sotillo, E., Cochran, J. R., Mackall, C. L. 2024

  Abstract

  Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47E), which engages SIRPα and provides a 'don't eat me' signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3, the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.

  View details for DOI 10.1038/s41586-024-07443-8

  View details for PubMedID 38750365

  View details for PubMedCentralID 4182950

• Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer. Nature communications Haderk, F., Chou, Y. T., Cech, L., Fernández-Méndez, C., Yu, J., Olivas, V., Meraz, I. M., Barbosa Rabago, D., Kerr, D. L., Gomez, C., Allegakoen, D. V., Guan, J., Shah, K. N., Herrington, K. A., Gbenedio, O. M., Nanjo, S., Majidi, M., Tamaki, W., Pourmoghadam, Y. K., Rotow, J. K., McCoach, C. E., Riess, J. W., Gutkind, J. S., Tang, T. T., Post, L., Huang, B., Santisteban, P., Goodarzi, H., Bandyopadhyay, S., Kuo, C. J., Roose, J. P., Wu, W., Blakely, C. M., Roth, J. A., Bivona, T. G. 2024; 15 (1): 3741

  Abstract

  Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

  View details for DOI 10.1038/s41467-024-47423-0

  View details for PubMedID 38702301

  View details for PubMedCentralID 5384713

• Design and Evaluation of a Robust CRISPR Kinetic Assay for Hot-Spot Genotyping. Analytical chemistry Blanluet, C., Kuo, C. J., Bhattacharya, A., Santiago, J. G. 2024

  Abstract

  Next-generation sequencing offers highly multiplexed and accurate detection of nucleic acid sequences but at the expense of complex workflows and high input requirements. The ease of use of CRISPR-Cas12 assays is attractive and may enable highly accurate detection of sequences implicated in, for example, cancer pathogenic variants. CRISPR assays often employ end-point measurements of Cas12 trans-cleavage activity after Cas12 activation by the target; however, end point-based methods can be limited in accuracy and robustness by arbitrary experimental choices. To overcome such limitations, we develop and demonstrate here an accurate assay targeting a mutation of the epidermal growth factor gene implicated in lung cancer (exon 19 deletion). The assay is based on characterizing the kinetics of Cas12 trans-cleavage to discriminate the mutant from wild-type targets. We performed extensive experiments (780 reactions) to calibrate key assay design parameters, including the guide RNA sequence, reporter sequence, reporter concentration, enzyme concentration, and DNA target type. Interestingly, we observed a competitive reaction between the target and reporter molecules that has important consequences for the design of CRISPR assays, which use preamplification to improve sensitivity. Finally, we demonstrate the assay on 18 tumor-extracted amplicons and 100 training iterations with 99% accuracy and discuss discrimination parameters and models to improve wild type versus mutant classification.

  View details for DOI 10.1021/acs.analchem.3c05657

  View details for PubMedID 38684052

• GPR124 regulates murine brain embryonic angiogenesis and BBB formation by an intracellular domain-independent mechanism. Development (Cambridge, England) Yuki, K., Vallon, M., Ding, J., Rada, C. C., Tang, A. T., Vilches-Moure, J. G., McCormick, A. K., Echeverri, M. F., Alwahabi, S., Braunger, B. M., Ergün, S., Kahn, M. L., Kuo, C. J. 2024

  Abstract

  The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion 7-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7, which is transferred to Frizzled (FZD) to initiate canonical b-catenin signaling. GPR124 remains enigmatic; while its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124/FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function by mouse deletion (Gpr124ΔC). Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, versus Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Further, Gpr124ΔC and recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function is exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion 7-pass transmembrane receptors.

  View details for DOI 10.1242/dev.202794

  View details for PubMedID 38682276

• Integrative multi-omic profiling of adult mouse brain endothelial cells and potential implications in Alzheimer's disease. Cell reports Yu, M., Nie, Y., Yang, J., Yang, S., Li, R., Rao, V., Hu, X., Fang, C., Li, S., Song, D., Guo, F., Snyder, M. P., Chang, H. Y., Kuo, C. J., Xu, J., Chang, J. 2023; 42 (11): 113392

  Abstract

  The blood-brain barrier (BBB) is primarily manifested by a variety of physiological properties of brain endothelial cells (ECs), but the molecular foundation for these properties remains incompletely clear. Here, we generate a comprehensive molecular atlas of adult brain ECs using acutely purified mouse ECs and integrated multi-omics. Using RNA sequencing (RNA-seq) and proteomics, we identify the transcripts and proteins selectively enriched in brain ECs and demonstrate that they are partially correlated. Using single-cell RNA-seq, we dissect the molecular basis of functional heterogeneity of brain ECs. Using integrative epigenomics and transcriptomics, we determine that TCF/LEF, SOX, and ETS families are top-ranked transcription factors regulating the BBB. We then validate the identified brain-EC-enriched proteins and transcription factors in normal mouse and human brain tissue and assess their expression changes in mice with Alzheimer's disease. Overall, we present a valuable resource with broad implications for regulation of the BBB and treatment of neurological disorders.

  View details for DOI 10.1016/j.celrep.2023.113392

  View details for PubMedID 37925638

• A microwell platform for high-throughput longitudinal phenotyping and selective retrieval of organoids. Cell systems Sockell, A., Wong, W., Longwell, S., Vu, T., Karlsson, K., Mokhtari, D., Schaepe, J., Lo, Y., Cornelius, V., Kuo, C., Van Valen, D., Curtis, C., Fordyce, P. M. 2023; 14 (9): 764

  Abstract

  Organoids are powerful experimental models for studying the ontogeny and progression of various diseases including cancer. Organoids are conventionally cultured in bulk using an extracellular matrix mimic. However, bulk-cultured organoids physically overlap, making it impossible to track the growth of individual organoids over time in high throughput. Moreover, local spatial variations in bulk matrix properties make it difficult to assess whether observed phenotypic heterogeneity between organoids results from intrinsic cell differences or differences in the microenvironment. Here, we developed a microwell-based method that enables high-throughput quantification of image-based parameters for organoids grown from single cells, which can further be retrieved from their microwells for molecular profiling. Coupled with a deep learning image-processing pipeline, we characterized phenotypic traits including growth rates, cellular movement, and apical-basal polarity in two CRISPR-engineered human gastric organoid models, identifying genomic changes associated with increased growth rate and changes in accessibility and expression correlated with apical-basal polarity. A record of this paper's transparent peer review process is included in the supplemental information.

  View details for DOI 10.1016/j.cels.2023.08.002

  View details for PubMedID 37734323

• The colocatome as a spatial -omic reveals shared microenvironment features between tumour-stroma assembloids and human lung cancer. bioRxiv : the preprint server for biology Bouchard, G., Zhang, W., Li, I., Ilerten, I., Bhattacharya, A., Li, Y., Trope, W., Shrager, J. B., Kuo, C., Tian, L., Giaccia, A. J., Plevritis, S. K. 2023

  Abstract

  Computational frameworks to quantify and compare microenvironment spatial features of in-vitro patient-derived models and clinical specimens are needed. Here, we acquired and analysed multiplexed immunofluorescence images of human lung adenocarcinoma (LUAD) alongside tumour-stroma assembloids constructed with organoids and fibroblasts harvested from the leading edge (Tumour-Adjacent Fibroblasts;TAFs) or core (Tumour Core Fibroblasts;TCFs) of human LUAD. We introduce the concept of the "colocatome" as a spatial -omic dimension to catalogue all proximate and distant colocalisations between malignant and fibroblast subpopulations in both the assembloids and clinical specimens. The colocatome expands upon the colocalisation quotient (CLQ) through a nomalisation strategy that involves permutation analysis and thereby allows comparisons of CLQs under different conditions. Using colocatome analysis, we report that both TAFs and TCFs protected cancer cells from targeted oncogene treatment by uniquely reorganising the tumour-stroma cytoarchitecture, rather than by promoting cellular heterogeneity or selection. Moreover, we show that the assembloids' colocatome recapitulates the tumour-stroma cytoarchitecture defining the tumour microenvironment of LUAD clinical samples and thereby can serve as a functional spatial readout to guide translational discoveries.

  View details for DOI 10.1101/2023.09.11.557278

  View details for PubMedID 37745466

  View details for PubMedCentralID PMC10515823

• Report of the Assay Guidance Workshop on 3-Dimensional Tissue Models for Antiviral Drug Development. The Journal of infectious diseases Jordan, R., Ford-Scheimer, S. L., Alarcon, R. M., Atala, A., Borenstein, J. T., Brimacombe, K. R., Cherry, S., Clevers, H., Davis, M. I., Funnell, S. G., Gehrke, L., Griffith, L. G., Grossman, A. C., Hartung, T., Ingber, D. E., Kleinstreuer, N. C., Kuo, C. J., Lee, E. M., Mummery, C. L., Pickett, T. E., Ramani, S., Rosado-Olivieri, E. A., Struble, E. B., Wan, Z., Williams, M. S., Hall, M. D., Ferrer, M., Markossian, S. 2023

  Abstract

  The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.

  View details for DOI 10.1093/infdis/jiad334

  View details for PubMedID 37669225

• Critical role of down-regulated in adenoma bicarbonate transporter in linaclotide stimulated intestinal bicarbonate secretion. bioRxiv : the preprint server for biology Sarthi, J. B., Trumbull, A. M., Abazari, S. M., van Unen, V., Chan, J. E., Joo, N. S., Jiang, Y., Kuo, C. J., Sellers, Z. M. 2023

  Abstract

  Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also alter duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum. Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression or function. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA) inhibition, regardless of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Linaclotide increased apical membrane expression of DRA in non-CF and CF differentiated enteroids. These data provide insights into the action of linaclotide and suggest linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

  View details for DOI 10.1101/2023.05.05.539132

  View details for PubMedID 37205513

  View details for PubMedCentralID PMC10187319

• Organoid modeling of lung-resident immune responses to SARS-CoV-2 infection. Research square Choi, S. S., van Unen, V., Zhang, H., Rustagi, A., Alwahabi, S. A., Santos, A. J., Chan, J. E., Lam, B., Solis, D., Mah, J., Röltgen, K., Trope, W., Guh-Siesel, A., Lin, Z., Beck, A., Edwards, C., Mallajosyula, V., Martin, B. A., Dunn, J. C., Shrager, J., Baric, R. A., Pinsky, B., Boyd, S. D., Blish, C. A., Davis, M. M., Kuo, C. J. 2023

  Abstract

  Tissue-resident immunity underlies essential host defenses against pathogens, but analysis in humans has lacked in vitro model systems where epithelial infection and accompanying resident immune cell responses can be observed en bloc. Indeed, human primary epithelial organoid cultures typically omit immune cells, and human tissue resident-memory lymphocytes are conventionally assayed without an epithelial infection component, for instance from peripheral blood, or after extraction from organs. Further, the study of resident immunity in animals can be complicated by interchange between tissue and peripheral immune compartments. To study human tissue-resident infectious immune responses in isolation from secondary lymphoid organs, we generated adult human lung three-dimensional air-liquid interface (ALI) lung organoids from intact tissue fragments that co-preserve epithelial and stromal architecture alongside endogenous lung-resident immune subsets. These included T, B, NK and myeloid cells, with CD69+CD103+ tissue-resident and CCR7- and/or CD45RA- TRM and conservation of T cell receptor repertoires, all corresponding to matched fresh tissue. SARS-CoV-2 vigorously infected organoid lung epithelium, alongside secondary induction of innate cytokine production that was inhibited by antiviral agents. Notably, SARS-CoV-2-infected organoids manifested adaptive virus-specific T cell activation that was specific for seropositive and/or previously infected donor individuals. This holistic non-reconstitutive organoid system demonstrates the sufficiency of lung to autonomously mount adaptive T cell memory responses without a peripheral lymphoid component, and represents an enabling method for the study of human tissue-resident immunity.

  View details for DOI 10.21203/rs.3.rs-2870695/v1

  View details for PubMedID 37205380

  View details for PubMedCentralID PMC10187413

• Complete Remission of Widely Metastatic Human Epidermal Growth Factor Receptor 2-Amplified Pancreatic Adenocarcinoma After Precision Immune and Targeted Therapy With Description of Sequencing and Organoid Correlates. JCO precision oncology King, D. A., Smith, A. R., Pineda, G., Nakano, M., Michelini, F., Goedegebuure, S. P., Thyparambil, S., Liao, W. L., McCormick, A., Ju, J., Cioffi, M., Zhang, X., Hundal, J., Griffith, M., Grandori, C., Pollastro, M., Rosati, R., Margossian, A., Chatterjee, P., Ainge, T., Flory, M., Ocampo, P., Chen, L. M., Poultsides, G. A., Baron, A. D., Chang, D. T., Herman, J. M., Gillanders, W. E., Park, H., Hoos, W. A., Nichols, M., Fisher, G. A., Kuo, C. J. 2023; 7: e2100489

  View details for DOI 10.1200/PO.21.00489

  View details for PubMedID 37079860

• Regulation of the Blood-Brain Barrier in Health and Disease. Cold Spring Harbor perspectives in medicine Rada, C. C., Yuki, K., Ding, J., Kuo, C. J. 2023

  Abstract

  The neurovascular unit is a dynamic microenvironment with tightly controlled signaling and transport coordinated by the blood-brain barrier (BBB). A properly functioning BBB allows sufficient movement of ions and macromolecules to meet the high metabolic demand of the central nervous system (CNS), while protecting the brain from pathogenic and noxious insults. This review describes the main cell types comprising the BBB and unique molecular signatures of these cells. Additionally, major signaling pathways for BBB development and maintenance are highlighted. Finally, we describe the pathophysiology of BBB diseases, their relationship to barrier dysfunction, and identify avenues for therapeutic intervention.

  View details for DOI 10.1101/cshperspect.a041191

  View details for PubMedID 36987582

• RHAMM marks proliferative subpopulation of human colorectal cancer stem cells. Cancer science Nakano, M., Taguchi, R., Kikushige, Y., Isobe, T., Miyawaki, K., Mizuno, S., Tsuruta, N., Hanamura, F., Yamaguchi, K., Yamauchi, T., Ariyama, H., Kusaba, H., Nakamura, M., Maeda, T., Kuo, C. J., Baba, E., Akashi, K. 2023

  Abstract

  The cancer stem cell (CSC) theory features typically rare self-renewing subpopulation that reconstitute the heterogeneous tumor. Identification of molecules which characterize the feature of CSCs is a key imperative for further understanding of tumor heterogeneity and for the development of novel therapeutic strategies. However, the use of conventional markers of CSCs is still insufficient for the isolation of bona fide CSCs. We investigated organoids which are miniature forms of tumor tissues with reconstructing cellular diversity to identify specific marker to characterize CSCs in heterogeneous tumors. Here, we report that receptor for hyaluronan-mediated motility (RHAMM) expresses in a subpopulation of CD44+ conventional human colorectal CSC fraction. Single-cell transcriptomics of organoids highlighted RHAMM positive proliferative cells that revealed distinct characteristics among the various cell types. Prospectively isolated RHAMM+ CD44+ cells from the human colorectal cancer tissues showed highly proliferative character with self-renewal ability in comparison with the other cancer cells. Furthermore, inhibition of RHAMM strongly suppressed organoids formation in vitro and inhibited the tumor growth in vivo. Our findings suggest that RHAMM is a potential therapeutic target because it is a specific marker of the proliferative subpopulation within the conventional CSC fraction.

  View details for DOI 10.1111/cas.15795

  View details for PubMedID 36945114

• Pharmacological targeting of TFIIH suppresses KRAS mutant pancreatic ductal adenocarcinoma and synergizes with TRAIL. Cancer research Moser, R., Annis, J., Nikolova, O., Whatcott, C. J., Gurley, K. E., Mendez, E., Moran-Jones, K., Dorrell, C., Sears, R. C., Kuo, C. J., Han, H., Biankin, A. V., Grandori, C., Von Hoff, D. D., Kemp, C. J. 2022

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (Triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TNF-related apoptosis inducing ligand (TRAIL), effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy with direct translational implications for current clinical trials on metastatic pancreatic cancer patients.

  View details for DOI 10.1158/0008-5472.CAN-21-4222

  View details for PubMedID 35819261

• Immune organoids: from tumor modeling to precision oncology. Trends in cancer Dao, V., Yuki, K., Lo, Y., Nakano, M., Kuo, C. J. 2022

  Abstract

  Cancer immunotherapies, particularly immune checkpoint inhibitors, are rapidly becoming standard-of-care for many cancers. The ascendance of immune checkpoint inhibitor treatment and limitations in the accurate prediction of clinical response thereof have provided significant impetus to develop preclinical models that can guide therapeutic intervention. Traditional organoid culture methods that exclusively grow tumor epithelium as patient-derived organoids are under investigation as a personalized platform for drug discovery and for predicting clinical efficacy of chemotherapies and targeted agents. Recently, the patient-derived tumor organoid platform has evolved to contain more complex stromal and immune compartments needed to assess immunotherapeutic efficacy. We review the different methodologies for developing a more holistic patient-derived tumor organoid platform and for modeling the native immune tumor microenvironment.

  View details for DOI 10.1016/j.trecan.2022.06.001

  View details for PubMedID 35773148

• R-SPONDIN2+ mesenchymal cells form the bud tip progenitor niche during human lung development. Developmental cell Hein, R. F., Wu, J. H., Holloway, E. M., Frum, T., Conchola, A. S., Tsai, Y., Wu, A., Fine, A. S., Miller, A. J., Szenker-Ravi, E., Yan, K. S., Kuo, C. J., Glass, I., Reversade, B., Spence, J. R. 2022

  Abstract

  The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

  View details for DOI 10.1016/j.devcel.2022.05.010

  View details for PubMedID 35679862

• IRBIT as a Regulator of Bicarbonate Transport in the Small Intestine. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Sarthi, J. B., van Unen, V., Chan, J. E., Abazari, S., Trumbull, A., Guh-Siesel, A., Kuo, C. J., Sellers, Z. M. 2022; 36 Suppl 1

  Abstract

  BACKGROUND: Small intestinal bicarbonate transport, is critical for epithelial protection, digestion, and absorption. Membrane trafficking and/or recycling of transporters is a critical means to modulate their function, however, the cellular mechanisms that drive these processes in the duodenum remain unclear. The inositol 1,4,5-trisphosphate (IP3 ) receptor binding protein released with IP3 (IRBIT) is an intracellular protein that has been shown to regulate localization of bicarbonate transporters outside of the intestine, yet its role in regulating duodenal bicarbonate secretion is unknown.AIM: To examine duodenal expression and function of IRBIT in regulating bicarbonate transport.METHODS: Duodenal endoscopic biopsies from subjects without gross or histological evidence of disease were used directly or cultured using traditional apical-in or flipped apical-out enteroids. mRNA expression was evaluated using qPCR and re-analysis of a duodenal sc-RNAseq dataset. Protein expression and localization was determined by Western blot and confocal immunofluorescence, respectively. In vivo bicarbonate secretion was measured in anesthetized mice by duodenal perfusion and back-titration.RESULTS: IRBIT is highly expressed in human duodenal biopsies, with protein densitometric analysis showing ≥3.5-fold higher expression than Calu-3 airway submucosal gland cells or HEK293 kidney-derived cells (n=1 each). Analysis of duodenal sc-RNAseq data (1,625 cells) showed ACHYL1 in crypts and villi. Immunofluorescence staining of human duodenal biopsies (n=3) showed IRBIT staining along the crypt-villus axis, primarily localizing in E-cadherin positive epithelial cells, but also present in the lamina propria. In patient-derived duodenal enteroids, IRBIT was expressed in both crypt-like (undifferentiated) and villus-like (differentiated) enteroids (n=3 each), with greater IRBIT mRNA and protein expression in the latter, a pattern similar to SLC26A3 and SLC26A6 chloride/bicarbonate exchangers (n=3 each). In sc-RNAseq analysis, AHCYL1 and SLC26A3 were co-expressed in 38% of all villus cells, compared to only 5% co-expressing AHCYL1 and SLC26A6. CFTR-independent duodenal bicarbonate secretion stimulated by linaclotide (10-7 M) was significantly inhibited by phospholipase C inhibition (U7312, 10-6 M; 91±11%, n=3), which we showed disrupts IRBIT membrane localization, or SLC26A3 inhibition (DRAinh -A250, 10-5 M; 68±8%, n=10). Linaclotide stimulation of apical-out enteroids increased both IRBIT (n=3) and SLC26A3 (n=12) membrane trafficking.SUMMARY AND CONCLUSIONS: IRBIT is highly expressed in the duodenum and appears to functionally interact with SLC26A3. IRBIT may be an attractive target to modulate small intestinal pH in diseases like cystic fibrosis, where impaired bicarbonate secretion contributes to intestinal disease and malnutrition.

  View details for DOI 10.1096/fasebj.2022.36.S1.R5339

  View details for PubMedID 35553821

• Lumbar Motion Is Maintained with Paraspinous Tension Band for Degenerative Spondylolisthesis: Results from 24-month FDA Study Kim, K. D., Sasso, R., Hu, S., Villavicencio, A., Yoon, S., Lavelle, W., Sandhu, H., Bae, H., Deutsch, H., Sethi, K., Fischgrund, J., Stauff, M., Perez-Cruet, M., Yu, E., Berven, S., Mermer, M., Davis, R., Bains, R., Kuo, C., Ray, W., Alamin, T., Fielding, L., Welch, W. AMER ASSOC NEUROLOGICAL SURGEONS. 2022

  View details for Web of Science ID 001132701400225

• Targeting colorectal cancer with small-molecule inhibitors of ALDH1B1 Nature Chemical Biology Feng, Z., Hom, M. E., Bearrood, T. E., Rosenthal, Z. C., Fernández, D., Ondrus, A. E., Gu, Y., McCormick, A. K., Tomaske, M. G., Marshall, C. R., Chen, C., Mochly-Rosen, D., Kuo, C. J., Chen, J. K. 2022

  View details for DOI 10.1038/s41589-022-01048-w

• Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report. Annals of the New York Academy of Sciences Cable, J., Pei, D., Reid, L. M., Wang, X. W., Bhatia, S., Karras, P., Melenhorst, J. J., Grompe, M., Lathia, J. D., Song, E., Kuo, C. J., Zhang, N., White, R. M., Ma, S. K., Ma, L., Chin, Y. R., Shen, M. M., Ng, I. O., Kaestner, K. H., Zhou, L., Sikandar, S., Schmitt, C. A., Guo, W., Wong, C. C., Ji, J., Tang, D. G., Dubrovska, A., Yang, C., Wiedemeyer, W. R., Weissman, I. L. 2021

  Abstract

  The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability.On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.

  View details for DOI 10.1111/nyas.14719

  View details for PubMedID 34850398

• CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment. Cell reports Klomp, J. E., Lee, Y. S., Goodwin, C. M., Papke, B., Klomp, J. A., Waters, A. M., Stalnecker, C. A., DeLiberty, J. M., Drizyte-Miller, K., Yang, R., Diehl, J. N., Yin, H. H., Pierobon, M., Baldelli, E., Ryan, M. B., Li, S., Peterson, J., Smith, A. R., Neal, J. T., McCormick, A. K., Kuo, C. J., Counter, C. M., Petricoin, E. F., Cox, A. D., Bryant, K. L., Der, C. J. 2021; 37 (9): 110060

  Abstract

  We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC.

  View details for DOI 10.1016/j.celrep.2021.110060

  View details for PubMedID 34852220

• High-resolution positron emission microscopy of patient-derived tumor organoids. Nature communications Khan, S., Shin, J. H., Ferri, V., Cheng, N., Noel, J. E., Kuo, C., Sunwoo, J. B., Pratx, G. 2021; 12 (1): 5883

  Abstract

  Tumor organoids offer new opportunities for translational cancer research, but unlike animal models, their broader use is hindered by the lack of clinically relevant imaging endpoints. Here, we present a positron-emission microscopy method for imaging clinical radiotracers in patient-derived tumor organoids with spatial resolution 100-fold better than clinical positron emission tomography (PET). Using this method, we quantify 18F-fluorodeoxyglucose influx to show that patient-derived tumor organoids recapitulate the glycolytic activity of the tumor of origin, and thus, could be used to predict therapeutic response in vitro. Similarly, we measure sodium-iodine symporter activity using 99mTc- pertechnetate and find that the iodine uptake pathway is functionally conserved in organoids derived from thyroid carcinomas. In conclusion, organoids can be imaged using clinical radiotracers, which opens new possibilities for identifying promising drug candidates and radiotracers, personalizing treatment regimens, and incorporating clinical imaging biomarkers in organoid-based co-clinical trials.

  View details for DOI 10.1038/s41467-021-26081-6

  View details for PubMedID 34620852

• Treatment-induced arteriolar revascularization and miR-126 enhancement in bone marrow niche protect leukemic stem cells in AML. Journal of hematology & oncology Zhang, B., Nguyen, L. X., Zhao, D., Frankhouser, D. E., Wang, H., Hoang, D. H., Qiao, J., Abundis, C., Brehove, M., Su, Y., Feng, Y., Stein, A., Ghoda, L., Dorrance, A., Perrotti, D., Chen, Z., Han, A., Pichiorri, F., Jin, J., Jovanovic-Talisman, T., Caligiuri, M. A., Kuo, C. J., Yoshimura, A., Li, L., Rockne, R. C., Kortylewski, M., Zheng, Y., Carlesso, N., Kuo, Y., Marcucci, G. 2021; 14 (1): 122

  Abstract

  BACKGROUND: During acute myeloid leukemia (AML) growth, the bone marrow (BM) niche acquires significant vascular changes that can be offset by therapeutic blast cytoreduction. The molecular mechanisms of this vascular plasticity remain to be fully elucidated. Herein, we report on the changes that occur in the vascular compartment of the FLT3-ITD+ AML BM niche pre and post treatment and their impact on leukemic stem cells (LSCs).METHODS: BM vasculature was evaluated in FLT3-ITD+ AML models (MllPTD/WT/Flt3ITD/ITD mouse and patient-derived xenograft) by 3D confocal imaging of long bones, calvarium vascular permeability assays, and flow cytometry analysis. Cytokine levels were measured by Luminex assay and miR-126 levels evaluated by Q-RT-PCR and miRNA staining. Wild-type (wt) andMllPTD/WT/Flt3ITD/ITD mice with endothelial cell (EC) miR-126 knockout or overexpression served as controls. The impact of treatment-induced BM vascular changes on LSC activity was evaluated by secondary transplantation of BM cells after administration of tyrosine kinase inhibitors (TKIs) to MllPTD/WT/Flt3ITD/ITD mice with/without either EC miR-126 KO or co-treatment with tumor necrosis factor alpha (TNFalpha) or anti-miR-126 miRisten.RESULTS: In the normal BM niche, CD31+Sca-1high ECs lining arterioles have miR-126 levels higher than CD31+Sca-1low ECs lining sinusoids. We noted that during FLT3-ITD+ AML growth, the BM niche lost arterioles and gained sinusoids. These changes were mediated by TNFalpha, a cytokine produced by AML blasts, which induced EC miR-126 downregulation and caused depletion of CD31+Sca-1high ECs and gain in CD31+Sca-1low ECs. Loss of miR-126high ECs led to a decreased EC miR-126 supply to LSCs, which then entered the cell cycle and promoted leukemia growth. Accordingly, antileukemic treatment with TKI decreased the BM blast-produced TNFalpha and increased miR-126high ECs and the EC miR-126 supply to LSCs. High miR-126 levels safeguarded LSCs, as shown by more severe disease in secondary transplanted mice. Conversely, EC miR-126 deprivation via genetic or pharmacological EC miR-126 knock-down prevented treatment-induced BM miR-126high EC expansion and in turn LSC protection.CONCLUSIONS: Treatment-induced CD31+Sca-1high EC re-vascularization of the leukemic BM niche may represent a LSC extrinsic mechanism of treatment resistance that can be overcome with therapeutic EC miR-126 deprivation.

  View details for DOI 10.1186/s13045-021-01133-y

  View details for PubMedID 34372909

• A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation. Lo, Y., Kolahi, K. S., Du, Y., Chang, C., Krokhotin, A., Nair, A., Sobba, W. D., Karlsson, K., Jones, S. J., Longacre, T. A., Mah, A. T., Sockell, A., Seoane, J. A., Chen, J., Weissman, J. S., Curtis, C., Califano, A., Fu, H., Crabtree, G. R., Kuo, C. J. AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000680263502064

• Nanoparticle-enabled innate immune stimulation activates endogenous tumor-infiltrating T cells with broad antigen specificities PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yin, Q., Yu, W., Grzeskowiak, C. L., Li, J., Huang, H., Guo, J., Chen, L., Wang, F., Zhao, F., von Boehmer, L., Metzner, T. J., Leppert, J. T., Chien, Y., Kuo, C. J., Davis, M. M. 2021; 118 (21)

  View details for DOI 10.1073/pnas.2016168118|1of11

  View details for Web of Science ID 000659437300004

• Nanoparticle-enabled innate immune stimulation activates endogenous tumor-infiltrating T cells with broad antigen specificities. Proceedings of the National Academy of Sciences of the United States of America Yin, Q., Yu, W., Grzeskowiak, C. L., Li, J., Huang, H., Guo, J., Chen, L., Wang, F., Zhao, F., von Boehmer, L., Metzner, T. J., Leppert, J. T., Chien, Y., Kuo, C. J., Davis, M. M. 2021; 118 (21)

  Abstract

  Tumors are often infiltrated by T lymphocytes recognizing either self- or mutated antigens but are generally inactive, although they often show signs of prior clonal expansion. Hypothesizing that this may be due to peripheral tolerance, we formulated nanoparticles containing innate immune stimulants that we found were sufficient to activate self-specific CD8+ T cells and injected them into two different mouse tumor models, B16F10 and MC38. These nanoparticles robustly activated and/or expanded antigen-specific CD8+ tumor-infiltrating T cells, along with a decrease in regulatory CD4+ T cells and an increase in Interleukin-17 producers, resulting in significant tumor growth retardation or elimination and the establishment of immune memory in surviving mice. Furthermore, nanoparticles with modification of stimulating human T cells enabled the robust activation of endogenous T cells in patient-derived tumor organoids. These results indicate that breaking peripheral tolerance without regard to the antigen specificity creates a promising pathway for cancer immunotherapy.

  View details for DOI 10.1073/pnas.2016168118

  View details for PubMedID 34021082

• Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids. Advanced science (Weinheim, Baden-Wurttemberg, Germany) Hunt, D. R., Klett, K. C., Mascharak, S., Wang, H., Gong, D., Lou, J., Li, X., Cai, P. C., Suhar, R. A., Co, J. Y., LeSavage, B. L., Foster, A. A., Guan, Y., Amieva, M. R., Peltz, G., Xia, Y., Kuo, C. J., Heilshorn, S. C. 2021; 8 (10): 2004705

  Abstract

  Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin-like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue-derived, epithelial-only intestinal organoids. HELP enables the encapsulation of dissociated patient-derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal-derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin-ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid-matrix interactions and potential patient-specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G' ≈ 1 kPa), slower stress relaxation rate (t1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10-3-1 × 10-3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal-derived products or synthetic polyethylene glycol for potential clinical translation.

  View details for DOI 10.1002/advs.202004705

  View details for PubMedID 34026461

  View details for PubMedCentralID PMC8132048

• A new open-access platform for measuring and sharing mTBI data. Scientific reports Domel, A. G., Raymond, S. J., Giordano, C., Liu, Y., Yousefsani, S. A., Fanton, M., Cecchi, N. J., Vovk, O., Pirozzi, I., Kight, A., Avery, B., Boumis, A., Fetters, T., Jandu, S., Mehring, W. M., Monga, S., Mouchawar, N., Rangel, I., Rice, E., Roy, P., Sami, S., Singh, H., Wu, L., Kuo, C., Zeineh, M., Grant, G., Camarillo, D. B. 2021; 11 (1): 7501

  Abstract

  Despite numerous research efforts, the precise mechanisms of concussion have yet to be fully uncovered. Clinical studies on high-risk populations, such as contact sports athletes, have become more common and give insight on the link between impact severity and brain injury risk through the use of wearable sensors and neurological testing. However, as the number of institutions operating these studies grows, there is a growing need for a platform to share these data to facilitate our understanding of concussion mechanisms and aid in the development of suitable diagnostic tools. To that end, this paper puts forth two contributions: (1) a centralized, open-access platform for storing and sharing head impact data, in collaboration with the Federal Interagency Traumatic Brain Injury Research informatics system (FITBIR), and (2) a deep learning impact detection algorithm (MiGNet) to differentiate between true head impacts and false positives for the previously biomechanically validated instrumented mouthguard sensor (MiG2.0), all of which easily interfaces with FITBIR. We report 96% accuracy using MiGNet, based on a neural network model, improving on previous work based on Support Vector Machines achieving 91% accuracy, on an out of sample dataset of high school and collegiate football head impacts. The integrated MiG2.0 and FITBIR system serve as a collaborative research tool to be disseminated across multiple institutions towards creating a standardized dataset for furthering the knowledge of concussion biomechanics.

  View details for DOI 10.1038/s41598-021-87085-2

  View details for PubMedID 33820939

• Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids ADVANCED SCIENCE Hunt, D. R., Klett, K. C., Mascharak, S., Wang, H. Y., Gong, D., Lou, J., Li, X., Cai, P. C., Suhar, R. A., Co, J. Y., LeSavage, B. L., Foster, A. A., Guan, Y., Amieva, M. R., Peltz, G., Xia, Y., Kuo, C. J., Heilshorn, S. C. 2021

  View details for DOI 10.1002/advs.202004705

  View details for Web of Science ID 000627768200001

• An expanded universe of cancer targets. Cell Hahn, W. C., Bader, J. S., Braun, T. P., Califano, A., Clemons, P. A., Druker, B. J., Ewald, A. J., Fu, H., Jagu, S., Kemp, C. J., Kim, W., Kuo, C. J., McManus, M., B Mills, G., Mo, X., Sahni, N., Schreiber, S. L., Talamas, J. A., Tamayo, P., Tyner, J. W., Wagner, B. K., Weiss, W. A., Gerhard, D. S., Cancer Target Discovery and Development Network, Dancik, V., Gill, S., Hua, B., Sharifnia, T., Viswanathan, V., Zou, Y., Dela Cruz, F., Kung, A., Stockwell, B., Boehm, J., Dempster, J., Manguso, R., Vazquez, F., Cooper, L. A., Du, Y., Ivanov, A., Lonial, S., Moreno, C. S., Niu, Q., Owonikoko, T., Ramalingam, S., Reyna, M., Zhou, W., Grandori, C., Shmulevich, I., Swisher, E., Cai, J., Chan, I. S., Dunworth, M., Ge, Y., Georgess, D., Grasset, E. M., Henriet, E., Knutsdottir, H., Lerner, M. G., Padmanaban, V., Perrone, M. C., Suhail, Y., Tsehay, Y., Warrier, M., Morrow, Q., Nechiporuk, T., Long, N., Saultz, J., Kaempf, A., Minnier, J., Tognon, C. E., Kurtz, S. E., Agarwal, A., Brown, J., Watanabe-Smith, K., Vu, T. Q., Jacob, T., Yan, Y., Robinson, B., Lind, E. F., Kosaka, Y., Demir, E., Estabrook, J., Grzadkowski, M., Nikolova, O., Chen, K., Deneen, B., Liang, H., Bassik, M. C., Bhattacharya, A., Brennan, K., Curtis, C., Gevaert, O., Ji, H. P., Karlsson, K. A., Karagyozova, K., Lo, Y., Liu, K., Nakano, M., Sathe, A., Smith, A. R., Spees, K., Wong, W. H., Yuki, K., Hangauer, M., Kaufman, D. S., Balmain, A., Bollam, S. R., Chen, W., Fan, Q., Kersten, K., Krummel, M., Li, Y. R., Menard, M., Nasholm, N., Schmidt, C., Serwas, N. K., Yoda, H. 2021; 184 (5): 1142–55

  Abstract

  The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.

  View details for DOI 10.1016/j.cell.2021.02.020

  View details for PubMedID 33667368

• Targeted replacement of full-length CFTR in human airway stem cells by CRISPR/Cas9 for pan-mutation correction in the endogenous locus. Molecular therapy : the journal of the American Society of Gene Therapy Vaidyanathan, S. n., Baik, R. n., Chen, L. n., Bravo, D. T., Suarez, C. J., Abazari, S. M., Salahudeen, A. A., Dudek, A. M., Teran, C. A., Davis, T. H., Lee, C. M., Bao, G. n., Randell, S. H., Artandi, S. E., Wine, J. J., Kuo, C. J., Desai, T. J., Nayak, J. V., Sellers, Z. M., Porteus, M. H. 2021

  Abstract

  Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

  View details for DOI 10.1016/j.ymthe.2021.03.023

  View details for PubMedID 33794364

• Models for Immuno-oncology Research CANCER CELL Kuo, C. J., Voest, E., Parrini, M., Zou, W., Teng, M. W. L., Greten, T. F., Palucka, K., Gill, S., Joshi, N. S. 2020; 38 (2): 145–47

  Abstract

  The interactions between cancer cells and immune cells are complex and context dependent. Choosing the right model to study these interactions is a crucial step in the development of immunotherapies. From cell co-cultures to organoids, organs-on-chip, and a variety of mouse models, experts share their model of choice for immuno-oncology research and discuss their strengths and caveats.

  View details for Web of Science ID 000559591600015

  View details for PubMedID 32781038

• Applications of Organoids for Cancer Biology and Precision Medicine. Nature cancer Lo, Y. H., Karlsson, K., Kuo, C. J. 2020; 1 (8): 761-773

  Abstract

  Organoid technologies enable the creation of in vitro physiologic systems that model tissues of origin more accurately than classical culture approaches. Seminal characteristics, including three-dimensional structure and recapitulation of self-renewal, differentiation, and disease pathology, render organoids eminently suited as hybrids that combine the experimental tractability of traditional 2D cell lines with cellular attributes of in vivo model systems. Here, we describe recent advances in this rapidly evolving field and their applications in cancer biology, clinical translation and precision medicine.

  View details for DOI 10.1038/s43018-020-0102-y

  View details for PubMedID 34142093

  View details for PubMedCentralID PMC8208643

• Development of a miniaturized 3D organoid culture platform for ultra-high throughput screening. Journal of molecular cell biology Du, Y., Li, X., Niu, Q., Mo, X., Qui, M., Ma, T., Kuo, C. J., Fu, H. 2020

  Abstract

  The recent advent of robust methods to grow human tissues as 3-dimensional (3D) organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery. However, organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening (HTS)-based drug discovery due to technical difficulties. Using genetically engineered human colon organoids as a model system, here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS. We first established organoid culturing in a 384-well plate format and validated its application in a cell viability HTS assay by screening a 2036-compound library. We further miniaturized the 3D organoid culturing in a 1536-well ultra-high throughput screening format and demonstrated its robust performance for large-scale primary compound screening. Our miniaturized organoid culturing method may be adapted to other types of organoids. By leveraging the power of 3D organoid culture in a high-density plate format, we provide a physiologically relevant screening platform to model tumors to accelerate organoid-based research and drug discovery.

  View details for DOI 10.1093/jmcb/mjaa036

  View details for PubMedID 32678871

• Organoid Models of Tumor Immunology. Trends in immunology Yuki, K., Cheng, N., Nakano, M., Kuo, C. J. 2020

  Abstract

  Cellular interactions in the tumor microenvironment (TME) significantly govern cancer progression and drug response. The efficacy of clinical immunotherapies has fostered an exponential interest in the tumor immune microenvironment, which in turn has engendered a pressing need for robust experimental systems modeling patient-specific tumor-immune interactions. Traditional 2D in vitro tumor immunotherapy models have reconstituted immortalized cancer cell lines with immune components, often from peripheral blood. However, newly developed 3D in vitro organoid culture methods now allow the routine culture of primary human tumor biopsies and increasingly incorporate immune components. Here, we present a viewpoint on recent advances, and propose translational applications of tumor organoids for immuno-oncology research, immunotherapy modeling, and precision medicine.

  View details for DOI 10.1016/j.it.2020.06.010

  View details for PubMedID 32654925

• Organoid modeling of tumor and tissue microenvironments. Kuo, C. J. AMER ASSOC CANCER RESEARCH. 2020: 17

  View details for Web of Science ID 000537844900006

• Insertion of the CFTR cDNA in the Endogenous Locus in Airway Stem Cells Using CRISPR/Cas9 Restores CFTR Function to Wild-Type Levels in Differentiated Epithelia Vaidyanathan, S., Sellers, Z. M., Bravo, D. T., Le, W., Randell, S. H., Desai, T. J., Kuo, C. J., Nayak, J. V., Porteus, M. H. CELL PRESS. 2020: 569–70

  View details for Web of Science ID 000530089302407

• CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities. Nature Han, K., Pierce, S. E., Li, A., Spees, K., Anderson, G. R., Seoane, J. A., Lo, Y. H., Dubreuil, M., Olivas, M., Kamber, R. A., Wainberg, M., Kostyrko, K., Kelly, M. R., Yousefi, M., Simpkins, S. W., Yao, D., Lee, K., Kuo, C. J., Jackson, P. K., Sweet-Cordero, A., Kundaje, A., Gentles, A. J., Curtis, C., Winslow, M. M., Bassik, M. C. 2020; 580 (7801): 136-141

  Abstract

  Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

  View details for DOI 10.1038/s41586-020-2099-x

  View details for PubMedID 32238925

• Organoids as Oracles for Precision Medicine in Rectal Cancer. Cell stem cell Kolahi, K. S., Nakano, M., Kuo, C. J. 2020; 26 (1): 4-6

  Abstract

  Two recent papers in Cell Stem Cell and Nature Medicine (Yao et al. [2019] and Ganesh et al. [2019]) demonstrate the successful use of rectal cancer patient-derived organoids to predict patient responses to neoadjuvant chemoradiation therapy, paving the way toward a new paradigm for precision medicine.

  View details for DOI 10.1016/j.stem.2019.12.003

  View details for PubMedID 31951587

• Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo. Cell stem cell Miao, Y. n., Ha, A. n., de Lau, W. n., Yuki, K. n., Santos, A. J., You, C. n., Geurts, M. H., Puschhof, J. n., Pleguezuelos-Manzano, C. n., Peng, W. C., Senlice, R. n., Piani, C. n., Buikema, J. W., Gbenedio, O. M., Vallon, M. n., Yuan, J. n., de Haan, S. n., Hemrika, W. n., Rösch, K. n., Dang, L. T., Baker, D. n., Ott, M. n., Depeille, P. n., Wu, S. M., Drost, J. n., Nusse, R. n., Roose, J. P., Piehler, J. n., Boj, S. F., Janda, C. Y., Clevers, H. n., Kuo, C. J., Garcia, K. C. 2020

  Abstract

  Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.

  View details for DOI 10.1016/j.stem.2020.07.020

  View details for PubMedID 32818433

• Integrated genomic characterization of ERBB2/HER2 alterations in invasive breast carcinoma: a focus on unusual FISH groups. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc Yang, S. R., Bouhlal, Y. n., De La Vega, F. M., Ballard, M. n., Kuo, C. J., Vilborg, A. n., Jensen, G. n., Allison, K. n. 2020

  Abstract

  In patients with invasive breast cancer, fluorescence in situ hybridization (FISH) testing for HER2 typically demonstrates the clear presence or lack of ERBB2 (HER2) amplification (i.e., groups 1 or 5). However, a small subset of patients can present with unusual HER2 FISH patterns (groups 2-4), resulting in diagnostic confusion. To provide clarity, the 2018 CAP/ASCO HER2 testing guideline recommends additional testing using HER2 immunohistochemistry (IHC) for determining the final HER2 status. Despite this effort, the genomic correlates of unusual HER2 FISH groups remain poorly understood. Here, we used droplet digital PCR (ddPCR) and targeted next-generation sequencing (NGS) to characterize the genomic features of both usual and unusual HER2 FISH groups. In this study, 51 clinical samples were selected to represent FISH groups 1-5. Furthermore, group 1 was subdivided into two groups, with groups 1A and 1B corresponding to cases with HER2 signals/cell ≥6.0 and 4-6, respectively. Overall, our findings revealed a wide range of copy number alterations in HER2 across the different FISH groups. As expected, groups 1A and 5 showed the clear presence and lack of HER2 copy number gain, respectively, as measured by ddPCR and NGS. In contrast, group 1B and other uncommon FISH groups (groups 2-4) were characterized by a broader range of HER2 copy levels with only a few select cases showing high-level gain. Notably, these cases with increased HER2 copy levels also showed HER2 overexpression by IHC, thus highlighting the correlation between HER2 copy number and HER2 protein expression. Given the concordance between the genomic and protein results, our findings suggest that HER2 IHC may inform HER2 copy number status in patients with unusual FISH patterns. Hence, our results support the current recommendation for using IHC to resolve HER2 status in FISH groups 2-4.

  View details for DOI 10.1038/s41379-020-0504-5

  View details for PubMedID 32161378

• Organoids as Oracles for Precision Medicine in Rectal Cancer CELL STEM CELL Kolahi, K. S., Nakano, M., Kuo, C. J. 2020; 26 (1): 4–6

  View details for DOI 10.1016/j.stem.2019.12.003

  View details for Web of Science ID 000505213700003

• Retinoic Acid and Lymphotoxin Signaling Promote Differentiation of Human Intestinal M Cells. Gastroenterology Ding, S. n., Song, Y. n., Brulois, K. F., Pan, J. n., Co, J. Y., Ren, L. n., Feng, N. n., Yasukawa, L. L., Sánchez-Tacuba, L. n., Wosen, J. E., Mellins, E. D., Monack, D. M., Amieva, M. R., Kuo, C. J., Butcher, E. C., Greenberg, H. B. 2020

  Abstract

  Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids.We analyzed transcriptome data from mouse Peyer's Patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells.A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids.We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.

  View details for DOI 10.1053/j.gastro.2020.03.053

  View details for PubMedID 32247021

• Applications of organoids for cancer biology and precision medicine. Nature Cancer Lo, Y., Karlsson, K., Kuo, C. J. 2020; 1: 761–773

  View details for DOI 10.1038/s43018-020-0102-y

• Surrogate R-spondins for tissue-specific potentiation of Wnt Signaling. PloS one Luca, V. C., Miao, Y. n., Li, X. n., Hollander, M. J., Kuo, C. J., Garcia, K. C. 2020; 15 (1): e0226928

  Abstract

  Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/β-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.

  View details for DOI 10.1371/journal.pone.0226928

  View details for PubMedID 31914456

• Immune receptor inhibition through enforced phosphatase recruitment. Nature Fernandes, R. A., Su, L. n., Nishiga, Y. n., Ren, J. n., Bhuiyan, A. M., Cheng, N. n., Kuo, C. J., Picton, L. K., Ohtsuki, S. n., Majzner, R. G., Rietberg, S. P., Mackall, C. L., Yin, Q. n., Ali, L. R., Yang, X. n., Savvides, C. S., Sage, J. n., Dougan, M. n., Garcia, K. C. 2020

  Abstract

  Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

  View details for DOI 10.1038/s41586-020-2851-2

  View details for PubMedID 33087934

• Engineered materials for organoid systems NATURE REVIEWS MATERIALS Kratochvil, M. J., Seymour, A. J., Li, T. L., Pasca, S. P., Kuo, C. J., Heilshorn, S. C. 2019; 4 (9): 606–22

  View details for DOI 10.1038/s41578-019-0129-9

  View details for Web of Science ID 000484683300006

• Engineered materials for organoid systems. Nature reviews. Materials Kratochvil, M. J., Seymour, A. J., Li, T. L., Paşca, S. P., Kuo, C. J., Heilshorn, S. C. 2019; 4 (9): 606-622

  Abstract

  Organoids are 3D cell culture systems that mimic some of the structural and functional characteristics of an organ. Organoid cultures provide the opportunity to study organ-level biology in models that mimic human physiology more closely than 2D cell culture systems or non-primate animal models. Many organoid cultures rely on decellularized extracellular matrices as scaffolds, which are often poorly chemically defined and allow only limited tunability and reproducibility. By contrast, the biochemical and biophysical properties of engineered matrices can be tuned and optimized to support the development and maturation of organoid cultures. In this Review, we highlight how key cell-matrix interactions guiding stem-cell decisions can inform the design of biomaterials for the reproducible generation and control of organoid cultures. We survey natural, synthetic and protein-engineered hydrogels for their applicability to different organoid systems and discuss biochemical and mechanical material properties relevant for organoid formation. Finally, dynamic and cell-responsive material systems are investigated for their future use in organoid research.

  View details for DOI 10.1038/s41578-019-0129-9

  View details for PubMedID 33552558

  View details for PubMedCentralID PMC7864216

• Human Intestinal Enteroids Model MHC-II in the Gut Epithelium FRONTIERS IN IMMUNOLOGY Wosen, J. E., Ilstad-Minnihan, A., Co, J. Y., Jiang, W., Mukhopadhyay, D., Fernandez-Becker, N. Q., Kuo, C. J., Amieva, M. R., Mellins, E. D. 2019; 10

  View details for DOI 10.3389/fimmu.2019.01970

  View details for Web of Science ID 000481776300001

• Human Intestinal Enteroids Model MHC-II in the Gut Epithelium. Frontiers in immunology Wosen, J. E., Ilstad-Minnihan, A., Co, J. Y., Jiang, W., Mukhopadhyay, D., Fernandez-Becker, N. Q., Kuo, C. J., Amieva, M. R., Mellins, E. D. 2019; 10: 1970

  Abstract

  The role of intestinal epithelial cells (IECs) in mucosal tolerance and immunity remains poorly understood. We present a method for inducing MHC class II (MHC-II) in human enteroids, "mini-guts" derived from small intestinal crypt stem cells, and show that the intracellular MHC-II peptide-pathway is intact and functional in IECs. Our approach enables human enteroids to be used for novel in vitro studies into IEC MHC-II regulation and function during health and disease.

  View details for DOI 10.3389/fimmu.2019.01970

  View details for PubMedID 31481960

  View details for PubMedCentralID PMC6710476

• Inhibition of VEGF (Vascular Endothelial Growth Factor)-A or its Receptor Activity Suppresses Experimental Aneurysm Progression in the Aortic Elastase Infusion Model. Arteriosclerosis, thrombosis, and vascular biology Xu, B., Iida, Y., Glover, K. J., Ge, Y., Wang, Y., Xuan, H., Hu, X., Tanaka, H., Wang, W., Fujimura, N., Miyata, M., Shoji, T., Guo, J., Zheng, X., Gerritsen, M., Kuo, C., Michie, S. A., Dalman, R. L. 2019: ATVBAHA119312497

  Abstract

  OBJECTIVE: We examined the pathogenic significance of VEGF (vascular endothelial growth factor)-A in experimental abdominal aortic aneurysms (AAAs) and the translational value of pharmacological VEGF-A or its receptor inhibition in aneurysm suppression. Approaches and Results: AAAs were created in male C57BL/6J mice via intra-aortic elastase infusion. Soluble VEGFR (VEGF receptor)-2 extracellular ligand-binding domain (delivered in Ad-VEGFR-2), anti-VEGF-A mAb, and sunitinib were used to sequester VEGF-A, neutralize VEGF-A, and inhibit receptor tyrosine kinase activity, respectively. Influences on AAAs were assessed using ultrasonography and histopathology. In vitro transwell migration and quantitative reverse transcription polymerase chain reaction assays were used to assess myeloid cell chemotaxis and mRNA expression, respectively. Abundant VEGF-A mRNA and VEGF-A-positive cells were present in aneurysmal aortae. Sequestration of VEGF-A by Ad-VEGFR-2 prevented AAA formation, with attenuation of medial elastolysis and smooth muscle depletion, mural angiogenesis and monocyte/macrophage infiltration. Treatment with anti-VEGF-A mAb prevented AAA formation without affecting further progression of established AAAs. Sunitinib therapy substantially mitigated both AAA formation and further progression of established AAAs, attenuated aneurysmal aortic MMP2 and MMP9 protein expression, inhibited inflammatory monocyte and neutrophil chemotaxis to VEGF-A, and reduced MMP2, MMP9, and VEGF-A mRNA expression in macrophages and smooth muscle cells in vitro. Additionally, sunitinib treatment reduced circulating monocytes in aneurysmal mice.CONCLUSIONS: VEGF-A and its receptors contribute to experimental AAA formation by suppressing mural angiogenesis, MMP and VEGF-A production, myeloid cell chemotaxis, and circulating monocytes. Pharmacological inhibition of receptor tyrosine kinases by sunitinib or related compounds may provide novel opportunities for clinical aneurysm suppression.

  View details for DOI 10.1161/ATVBAHA.119.312497

  View details for PubMedID 31294623

• HAT1 Coordinates Histone Production and Acetylation via H4 Promoter Binding. Molecular cell Gruber, J. J., Geller, B., Lipchik, A. M., Chen, J., Salahudeen, A. A., Ram, A. N., Ford, J. M., Kuo, C. J., Snyder, M. P. 2019

  Abstract

  The energetic costs of duplicating chromatin are large and therefore likely depend on nutrient sensing checkpoints and metabolic inputs. By studying chromatin modifiers regulated by epithelial growth factor, we identified histone acetyltransferase 1 (HAT1) as an induced gene that enhances proliferation through coordinating histone production, acetylation, and glucose metabolism. In addition to its canonical role as a cytoplasmic histone H4 acetyltransferase, we isolated a HAT1-containing complex bound specifically at promoters of H4 genes. HAT1-dependent transcription of H4 genes required an acetate-sensitive promoter element. HAT1 expression was critical for S-phase progression and maintenance of H3 lysine 9 acetylation at proliferation-associated genes, including histone genes. Therefore, these data describe a feedforward circuit whereby HAT1 captures acetyl groups on nascent histones and drives H4 production by chromatin binding to support chromatin replication and acetylation. These findings have important implications for human disease, since high HAT1 levels associate with poor outcomes across multiple cancer types.

  View details for DOI 10.1016/j.molcel.2019.05.034

  View details for PubMedID 31278053

• Receptor subtype discrimination using extensive shape complementary designed interfaces NATURE STRUCTURAL & MOLECULAR BIOLOGY Dang, L. T., Miao, Y., Ha, A., Yuki, K., Park, K., Janda, C. Y., Jude, K. M., Mohan, K., Ha, N., Vallon, M., Yuan, J., Vilches-Moure, J. G., Kuo, C. J., Garcia, K., Baker, D. 2019; 26 (6): 407-+

  View details for DOI 10.1038/s41594-019-0224-z

  View details for Web of Science ID 000470110200005

• Receptor subtype discrimination using extensive shape complementary designed interfaces. Nature structural & molecular biology Dang, L. T., Miao, Y., Ha, A., Yuki, K., Park, K., Janda, C. Y., Jude, K. M., Mohan, K., Ha, N., Vallon, M., Yuan, J., Vilches-Moure, J. G., Kuo, C. J., Garcia, K. C., Baker, D. 2019

  Abstract

  To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. We describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.

  View details for PubMedID 31086346

• Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions. Cell reports Co, J. Y., Margalef-Catala, M., Li, X., Mah, A. T., Kuo, C. J., Monack, D. M., Amieva, M. R. 2019; 26 (9): 2509

  Abstract

  Human enteroids-epithelial spheroids derived from primary gastrointestinal tissue-are a promising model to study pathogen-epithelial interactions. However, accessing the apical enteroid surface ischallenging because it is enclosed within the spheroid. We developed a technique to reverse enteroid polarity such that the apical surface everts to face the media. Apical-out enteroids maintain proper polarity and barrier function, differentiate into the major intestinal epithelial cell (IEC) types, and exhibit polarized absorption of nutrients. We used this model to study host-pathogen interactions and identified distinct polarity-specific patterns of infection by invasive enteropathogens. Salmonella enterica serovar Typhimurium targets IEC apical surfaces for invasion via cytoskeletal rearrangements, and Listeria monocytogenes, which binds to basolateral receptors, invade apical surfaces at sites of cellextrusion. Despite different modes of entry, both pathogens exit the epithelium within apically extruding enteroid cells. This model will enable further examination of IECs in health and disease.

  View details for PubMedID 30811997

• Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions CELL REPORTS Co, J. Y., Margalef-Catala, M., Li, X., Mah, A. T., Kuo, C. J., Monack, D. M., Amieva, M. R. 2019; 26 (9): 2509-+

  View details for DOI 10.1016/j.celrep.2019.01.108

  View details for Web of Science ID 000460279100022

• Introduction to themed series on intestinal stem cells and the NIDDK Intestinal Stem Cell Consortium AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Wang, T. C., Martin, M. G., Kuo, C. J., Klein, O. D., Niland, J. 2019; 316 (2): G247–G250

  View details for DOI 10.1152/ajpgi.00146.2018

  View details for Web of Science ID 000457877300002

• High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia. Cell stem cell Vaidyanathan, S. n., Salahudeen, A. A., Sellers, Z. M., Bravo, D. T., Choi, S. S., Batish, A. n., Le, W. n., Baik, R. n., de la O, S. n., Kaushik, M. P., Galper, N. n., Lee, C. M., Teran, C. A., Yoo, J. H., Bao, G. n., Chang, E. H., Patel, Z. M., Hwang, P. H., Wine, J. J., Milla, C. E., Desai, T. J., Nayak, J. V., Kuo, C. J., Porteus, M. H. 2019

  Abstract

  Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

  View details for DOI 10.1016/j.stem.2019.11.002

  View details for PubMedID 31839569

• RECK in Neural Precursor Cells Plays a Critical Role in Mouse Forebrain Angiogenesis. iScience Li, H. n., Miki, T. n., Almeida, G. M., Hanashima, C. n., Matsuzaki, T. n., Kuo, C. J., Watanabe, N. n., Noda, M. n. 2019; 19: 559–71

  Abstract

  RECK in neural precursor cells (NPCs) was previously found to support Notch-dependent neurogenesis in mice. On the other hand, recent studies implicate RECK in endothelial cells (ECs) in WNT7-triggered canonical WNT signaling essential for brain angiogenesis. Here we report that RECK in NPCs is also critical for brain angiogenesis. When Reck is inactivated in Foxg1-positive NPCs, mice die shortly after birth with hemorrhage in the forebrain, with angiogenic sprouts stalling at the periphery and forming abnormal aggregates reminiscent of those in EC-selective Reck knockout mice and Wnt7a/b-deficient mice. The hemorrhage can be pharmacologically suppressed by lithium chloride. An effect of RECK in WNT7-producing cells to enhance canonical WNT-signaling in reporter cells is detectable in mixed culture but not with conditioned medium. Our findings suggest that NPC-expressed RECK has a non-cell-autonomous function to promote forebrain angiogenesis through contact-dependent enhancement of WNT signaling in ECs, implying possible involvement of RECK in neurovascular coupling.

  View details for DOI 10.1016/j.isci.2019.08.009

  View details for PubMedID 31445376

• Introduction to Themed Series on Intestinal Stem Cells and the NIDDK Intestinal Stem Cell Consortium. American journal of physiology. Gastrointestinal and liver physiology Wang, T. C., Martin, M. G., Kuo, C. J., Klein, O. D., Niland, J. C. 2018

  View details for PubMedID 30548077

• Organoid Modeling of the Tumor Immune Microenvironment CELL Neal, J. T., Li, X., Zhu, J., Giangarra, V., Grzeskowiak, C. L., Ju, J., Liu, I. H., Chiou, S., Salahudeen, A. A., Smith, A. R., Deutsch, B. C., Liao, L., Zemek, A. J., Zhao, F., Karlsson, K., Schultz, L. M., Metzner, T. J., Nadauld, L. D., Tseng, Y., Alkhairy, S., Oh, C., Keskula, P., Mendoza-Villanueva, D., De La Vega, F. M., Kunz, P. L., Liao, J. C., Leppert, J. T., Sunwoo, J. B., Sabatti, C., Boehm, J. S., Hahn, W. C., Zheng, G. X. Y., Davis, M. M., Kuo, C. J. 2018; 175 (7): 1972-+

  View details for DOI 10.1016/j.cell.2018.11.021

  View details for Web of Science ID 000453242200023

• The Intestinal Stem Cell Niche: Homeostasis and Adaptations TRENDS IN CELL BIOLOGY Santos, A. J. M., Lo, Y., Mah, A. T., Kuo, C. J. 2018; 28 (12): 1062–78

  View details for DOI 10.1016/j.tcb.2018.08.001

  View details for Web of Science ID 000450302500009

• Reserve Stem Cells in Intestinal Homeostasis and Injury GASTROENTEROLOGY Bankaitis, E. D., Ha, A., Kuo, C. J., Magness, S. T. 2018; 155 (5): 1348–61

  Abstract

  Renewal of the intestinal epithelium occurs approximately every week and requires a careful balance between cell proliferation and differentiation to maintain proper lineage ratios and support absorptive, secretory, and barrier functions. We review models used to study the mechanisms by which intestinal stem cells (ISCs) fuel the rapid turnover of the epithelium during homeostasis and might support epithelial regeneration after injury. In anatomically defined zones of the crypt stem cell niche, phenotypically distinct active and reserve ISC populations are believed to support homeostatic epithelial renewal and injury-induced regeneration, respectively. However, other cell types previously thought to be committed to differentiated states might also have ISC activity and participate in regeneration. Efforts are underway to reconcile the proposed relatively strict hierarchical relationships between reserve and active ISC pools and their differentiated progeny; findings from models provide evidence for phenotypic plasticity that is common among many if not all crypt-resident intestinal epithelial cells. We discuss the challenges to consensus on ISC nomenclature, technical considerations, and limitations inherent to methodologies used to define reserve ISCs, and the need for standardized metrics to quantify and compare the relative contributions of different epithelial cell types to homeostatic turnover and post-injury regeneration. Increasing our understanding of the high-resolution genetic and epigenetic mechanisms that regulate reserve ISC function and cell plasticity will help refine these models and could affect approaches to promote tissue regeneration after intestinal injury.

  View details for PubMedID 30118745

• A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability. Cell reports Vallon, M., Yuki, K., Nguyen, T. D., Chang, J., Yuan, J., Siepe, D., Miao, Y., Essler, M., Noda, M., Garcia, K. C., Kuo, C. J. 2018; 25 (2): 339

  Abstract

  WNT7A and WNT7B control CNS angiogenesis and blood-brain barrier formation by activating endothelial Wnt/beta-catenin signaling. The GPI-anchored protein RECK and adhesion G protein-coupled receptor GPR124 critically regulate WNT7-specific signaling in concert with FZD and LRP co-receptors. Here, we demonstrate that primarily the GPR124 ectodomain, but not its transmembrane and intracellular domains, mediates RECK/WNT7-induced canonical Wnt signaling. Moreover, RECK is the predominant binding partner of GPR124 in rat brain blood vessels in situ. WNT7A and WNT7B, but not WNT3A, directly bind to purified recombinant soluble RECK, full-length cell surface RECK, and the GPR124:RECK complex. Chemical cross-linking indicates that RECK and WNT7A associate with 1:1 stoichiometry, which stabilizes short-lived, active, monomeric, hydrophobic WNT7A. In contrast, free WNT7A rapidly converts into inactive, hydrophilic aggregates. Overall, RECK is a selective WNT7 receptor that mediates GPR124/FZD/LRP-dependent canonical Wnt/beta-catenin signaling by stabilizing active cell surface WNT7, suggesting isoform-specific regulation of Wnt bioavailability.

  View details for PubMedID 30304675

• A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability CELL REPORTS Vallon, M., Yuki, K., Nguyen, T. D., Chang, J., Yuan, J., Siepe, D., Miao, Y., Essler, M., Noda, M., Garcia, K., Kuo, C. J. 2018; 25 (2): 339-+

  View details for DOI 10.1016/j.celrep.2018.09.045

  View details for Web of Science ID 000446691400009

• The Intestinal Stem Cell Niche: Homeostasis and Adaptations. Trends in cell biology Santos, A. J., Lo, Y., Mah, A. T., Kuo, C. J. 2018

  Abstract

  The intestinal epithelium is a rapidly renewing cellular compartment. This constant regeneration is a hallmark of intestinal homeostasis and requires a tightly regulated balance between intestinal stem cell (ISC) proliferation and differentiation. Since intestinal epithelial cells directly contact pathogenic environmental factors that continuously challenge their integrity, ISCs must also actively divide to facilitate regeneration and repair. Understanding niche adaptations that maintain ISC activity during homeostatic renewal and injury-induced intestinal regeneration is therefore a major and ongoing focus for stem cell biology. Here, we review recent concepts and propose an active interconversion of the ISC niche between homeostasis and injury-adaptive states that is superimposed upon an equally dynamic equilibrium between active and reserve ISC populations.

  View details for PubMedID 30195922

• Three-dimensional organoid model for acquired drug resistance in non-small cell lung cancer Shukla, N. D., Salahudeen, A. A., Padda, S. K., Neal, J. W., Wakelee, H. A., Kuo, C. J. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1557-3265.AACRIASLC18-B30

  View details for Web of Science ID 000582244900061

• Spinal constraint modulates head instantaneous center of rotation and dictates head angular motion JOURNAL OF BIOMECHANICS Kuo, C., Fanton, M., Wu, L., Camarillo, D. 2018; 76: 220–28

  View details for DOI 10.1016/j.jbiomech.2018.05.024

  View details for Web of Science ID 000439674600028

• Facile generation of single-cell transcriptome and immune repertoire freshly isolated from clinical tumor specimens Zhu, J., Salahudeen, A. A., Giangarra, V., Montesclaros, L., Sapida, J., Sharifi, O., Lee, J., Zheng, G. X., Wagh, D., Coller, J., Sabatti, C., Kuo, C. J. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-5672

  View details for Web of Science ID 000468819505089

• Organoid-based characterization of patient tumors and microenvironments at single cell resolution Salahudeen, A. A., Zhu, J., Ju, J., Batish, A., Sutha, K., Neal, J. T., Giangarra, V., Montesclaros, L., Sapida, J., Sharifi, O., Lee, J., Zheng, G. X., Wagh, D. A., Coller, J. A., Neal, J. W., Padda, S. K., Sabatti, C., Kuo, C. J. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-987

  View details for Web of Science ID 000468818902486

• Facile single cell profiling and clonotype analysis of NSCLC immune microenvironments. Salahudeen, A., Zhu, J., Ju, J., Giangarra, V., Montesclaros, L., Sapida, J., Sharifi, O., Lee, J., Wagh, D., Coller, J., Neal, J. W., Padda, S., Wakelee, H. A., Sabatti, C., Kuo, C. AMER SOC CLINICAL ONCOLOGY. 2018

  View details for DOI 10.1200/JCO.2018.36.15_suppl.12014

  View details for Web of Science ID 000442916004029

• STAG2 deficiency induces interferon responses via cGAS-STING pathway and restricts virus infection. Nature communications Ding, S., Diep, J., Feng, N., Ren, L., Li, B., Ooi, Y. S., Wang, X., Brulois, K. F., Yasukawa, L. L., Li, X., Kuo, C. J., Solomon, D. A., Carette, J. E., Greenberg, H. B. 2018; 9 (1): 1485

  Abstract

  Cohesin is a multi-subunit nuclear protein complex that coordinates sister chromatid separation during cell division. Highly frequent somatic mutations in genes encoding core cohesin subunits have been reported in multiple cancer types. Here, using a genome-wide CRISPR-Cas9 screening approach to identify host dependency factors and novel innate immune regulators of rotavirus (RV) infection, we demonstrate that the loss of STAG2, an important component of the cohesin complex, confers resistance to RV replication in cell culture and human intestinal enteroids. Mechanistically, STAG2 deficiency results in spontaneous genomic DNA damage and robust interferon (IFN) expression via the cGAS-STING cytosolic DNA-sensing pathway. The resultant activation of JAK-STAT signaling and IFN-stimulated gene (ISG) expression broadly protects against virus infections, including RVs. Our work highlights a previously undocumented role of the cohesin complex in regulating IFN homeostasis and identifies new therapeutic avenues for manipulating the innate immunity.

  View details for PubMedID 29662124

• STAG2 deficiency induces interferon responses via cGAS-STING pathway and restricts virus infection NATURE COMMUNICATIONS Ding, S., Diep, J., Feng, N., Ren, L., Li, B., Ooi, Y., Wang, X., Brulois, K. F., Yasukawa, L. L., Li, X., Kuo, C. J., Solomon, D. A., Carette, J. E., Greenberg, H. B. 2018; 9

  View details for DOI 10.1038/s41467-018-03782-z

  View details for Web of Science ID 000430057800001

• Bone marrow niche trafficking of miR-126 controls the self-renewal of leukemia stem cells in chronic myelogenous leukemia NATURE MEDICINE Zhang, B., Le Xuan Truong Nguyen, Li, L., Zhao, D., Kumar, B., Wu, H., Lin, A., Pellicano, F., Hopcroft, L., Su, Y., Copland, M., Holyoake, T. L., Kuo, C. J., Bhatia, R., Snyder, D. S., Ali, H., Stein, A. S., Brewer, C., Wang, H., McDonald, T., Swiderski, P., Troadec, E., Chen, C., Dorrance, A., Pullarkat, V., Yuan, Y., Perrotti, D., Carlesso, N., Forman, S. J., Kortylewski, M., Kuo, Y., Marcucci, G. 2018; 24 (4): 450-+

  Abstract

  Leukemia stem cells (LSCs) in individuals with chronic myelogenous leukemia (CML) (hereafter referred to as CML LSCs) are responsible for initiating and maintaining clonal hematopoiesis. These cells persist in the bone marrow (BM) despite effective inhibition of BCR-ABL kinase activity by tyrosine kinase inhibitors (TKIs). Here we show that although the microRNA (miRNA) miR-126 supported the quiescence, self-renewal and engraftment capacity of CML LSCs, miR-126 levels were lower in CML LSCs than in long-term hematopoietic stem cells (LT-HSCs) from healthy individuals. Downregulation of miR-126 levels in CML LSCs was due to phosphorylation of Sprouty-related EVH1-domain-containing 1 (SPRED1) by BCR-ABL, which led to inhibition of the RAN-exportin-5-RCC1 complex that mediates miRNA maturation. Endothelial cells (ECs) in the BM supply miR-126 to CML LSCs to support quiescence and leukemia growth, as shown using mouse models of CML in which Mir126a (encoding miR-126) was conditionally knocked out in ECs and/or LSCs. Inhibition of BCR-ABL by TKI treatment caused an undesired increase in endogenous miR-126 levels, which enhanced LSC quiescence and persistence. Mir126a knockout in LSCs and/or ECs, or treatment with a miR-126 inhibitor that targets miR-126 expression in both LSCs and ECs, enhanced the in vivo anti-leukemic effects of TKI treatment and strongly diminished LSC leukemia-initiating capacity, providing a new strategy for the elimination of LSCs in individuals with CML.

  View details for PubMedID 29505034

• Identification and validation of a novel drug target in an organoid model of esophageal cancer. Shukla, N., Salahudeen, A., de la O, S., Hart, D., Taylor, G., Zhu, J., Yuki, K., Seoane, J., Ma, Z., Ding, J., Han, K., Morgens, D., Bassik, M., Curtis, C., Kuo, C. AMER SOC CLINICAL ONCOLOGY. 2018

  View details for DOI 10.1200/JCO.2018.36.4_suppl.46

  View details for Web of Science ID 000436174100045

• Detection of American Football Head Impacts Using Biomechanical Features and Support Vector Machine Classification SCIENTIFIC REPORTS Wu, L. C., Kuo, C., Loza, J., Kurt, M., Laksari, K., Yanez, L. Z., Senif, D., Anderson, S. C., Miller, L. E., Urban, J. E., Stitzel, J. D., Camarillo, D. B. 2017; 7

  View details for DOI 10.1038/s41598-017-17864-3

  View details for Web of Science ID 000425901700001

• Organoids lead the cancer attack NATURE MEDICINE Smith, A. R., Kuo, C. J. 2017; 23 (12): 1399–1400

  View details for PubMedID 29216041

• Expanding tumor chemical-genetic interaction map using next-generation cancer models Tseng, Y., Hong, A., Gill, S., Keskula, P., Raghavan, S., Cheah, J., Tsherniak, A., Vazquez, F., Alkhairy, S., Peng, A., Sayeed, A., Deasy, R., Ronning, P., Kantoff, P., Garraway, L., Rubin, M., Kuo, C., Puram, S., Gazdar, A., Wagle, N., Bass, A., Ligon, K., Janeway, K., Root, D., Schreiber, S., Clemons, P., Golub, T., Hahn, W., Boehm, J. AMER ASSOC CANCER RESEARCH. 2017

  View details for Web of Science ID 000412270800002

• Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity. Cell stem cell Yan, K. S., Gevaert, O., Zheng, G. X., Anchang, B., Probert, C. S., Larkin, K. A., Davies, P. S., Cheng, Z. F., Kaddis, J. S., Han, A., Roelf, K., Calderon, R. I., Cynn, E., Hu, X., Mandleywala, K., Wilhelmy, J., Grimes, S. M., Corney, D. C., Boutet, S. C., Terry, J. M., Belgrader, P., Ziraldo, S. B., Mikkelsen, T. S., Wang, F., von Furstenberg, R. J., Smith, N. R., Chandrakesan, P., May, R., Chrissy, M. A., Jain, R., Cartwright, C. A., Niland, J. C., Hong, Y. K., Carrington, J., Breault, D. T., Epstein, J., Houchen, C. W., Lynch, J. P., Martin, M. G., Plevritis, S. K., Curtis, C., Ji, H. P., Li, L., Henning, S. J., Wong, M. H., Kuo, C. J. 2017; 21 (1): 78-90.e6

  Abstract

  Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ISCs, the most well-defined ISC pool, but Bmi1-GFP+cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

  View details for DOI 10.1016/j.stem.2017.06.014

  View details for PubMedID 28686870

  View details for PubMedCentralID PMC5642297

• Rapid characterization of candidate loss of function genes in primary organoid culture. Hart, D., Salahudeen, A., de la O, S., Han, K., Morgens, D., Bassik, M., Kuo, C. AMER SOC CLINICAL ONCOLOGY. 2017

  View details for Web of Science ID 000443281700075

• Linked read sequencing resolves complex genomic rearrangements in gastric cancer metastases. Genome medicine Greer, S. U., Nadauld, L. D., Lau, B. T., Chen, J. n., Wood-Bouwens, C. n., Ford, J. M., Kuo, C. J., Ji, H. P. 2017; 9 (1): 57

  Abstract

  Genome rearrangements are critical oncogenic driver events in many malignancies. However, the identification and resolution of the structure of cancer genomic rearrangements remain challenging even with whole genome sequencing.To identify oncogenic genomic rearrangements and resolve their structure, we analyzed linked read sequencing. This approach relies on a microfluidic droplet technology to produce libraries derived from single, high molecular weight DNA molecules, 50 kb in size or greater. After sequencing, the barcoded sequence reads provide long range genomic information, identify individual high molecular weight DNA molecules, determine the haplotype context of genetic variants that occur across contiguous megabase-length segments of the genome and delineate the structure of complex rearrangements. We applied linked read sequencing of whole genomes to the analysis of a set of synchronous metastatic diffuse gastric cancers that occurred in the same individual.When comparing metastatic sites, our analysis implicated a complex somatic rearrangement that was present in the metastatic tumor. The oncogenic event associated with the identified complex rearrangement resulted in an amplification of the known cancer driver gene FGFR2. With further investigation using these linked read data, the FGFR2 copy number alteration was determined to be a deletion-inversion motif that underwent tandem duplication, with unique breakpoints in each metastasis. Using a three-dimensional organoid tissue model, we functionally validated the metastatic potential of an FGFR2 amplification in gastric cancer.Our study demonstrates that linked read sequencing is useful in characterizing oncogenic rearrangements in cancer metastasis.

  View details for PubMedID 28629429

• Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity Cell Stem Cell Yan, K., Gevaert, O., Zheng, G., Anchang, B., Probert, C., et al 2017; 21 (1): 78 - 90.e6

  Abstract

  Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ISCs, the most well-defined ISC pool, but Bmi1-GFP+cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

  View details for DOI 10.1016/j.stem.2017.06.014

  View details for PubMedCentralID PMC5642297

• Wnt pathway regulation of intestinal stem cells. journal of physiology Mah, A. T., Yan, K. S., Kuo, C. J. 2016; 594 (17): 4837-4847

  Abstract

  Wnt signalling is involved in multiple aspects of embryonic development and adult tissue homeostasis, notably via controlling cellular proliferation and differentiation. Wnt signalling is subject to stringent positive and negative regulation to promote proper development and homeostasis yet avoid aberrant growth. Such multi-layer regulation includes post-translational modification and processing of Wnt proteins themselves, R-spondin (Rspo) amplification of Wnt signalling, diverse receptor families, and intracellular and extracellular antagonists and destruction and transcription complexes. In the gastrointestinal tract, Wnt signalling is crucial for development and renewal of the intestinal epithelium. Intestinal stem cells (ISCs) undergo symmetric division and neutral drift dynamics to renew the intestinal epithelium. Sources of Wnts and Wnt amplifers such as R-spondins are beginning to be elucidated as well as their functional contribution to intestinal homeostasis. In this review we focus on regulation of ISCs and intestinal homeostasis by the Wnt/Rspo pathway, the potential cellular sources of Wnt signalling regulators and highlight potential future areas of study.

  View details for DOI 10.1113/JP271754

  View details for PubMedID 27581568

  View details for PubMedCentralID PMC5009769

• Transforming Big Data into Cancer-Relevant Insight: An Initial, Multi-Tier Approach to Assess Reproducibility and Relevance The Cancer Target Discovery and Development Network MOLECULAR CANCER RESEARCH Clemons, P. A., Shamji, A., Hon, C., Wagner, B. K., Schreiber, S. L., Krasnitz, A., Sordella, R., Sander, C., Lowe, S. W., Powers, S., Smith, K., Aburi, M., Lavarone, A., Lasorella, A., Silva, J., Stockwell, B., Califano, A., Boehm, J. S., Vazquez, F., Weir, B. A., Golub, T. R., Hahn, W. C., Khuri, F. R., Moreno, C. S., Du, Y., Cooper, L., Ivanov, A. A., Johns, M. A., Fu, H., Nikolova, O., Mendez, E., Gadi, V. K., Margolin, A. A., Grandori, C., Kemp, C. J., Warren, E. H., Riddell, S. R., McIntosh, M. W., Gevaert, O., Ji, H. P., Kuo, C. J., Dhruv, H., Finlay, D., Kiefer, J., Kim, S., Vuori, K., Berens, M. E., Weissman, J., Bivona, T., Bandyopadhyay, S., Hangauer, M., Boettcher, M., McManus, M., McCormick, F., Aksoy, O., Simonds, E. F., Zheng, T., Chen, J., An, Z., Balmain, A., Weiss, W. A., Chen, K., Liang, H., Scott, K. L., Mills, G. B., Posner, B. A., Macmillan, J., Minna, J., White, M. A., Roth, M. G., Jagu, S., Mazerik, J. N., Gerhard, D. S. 2016; 14 (8): 675-682

  View details for DOI 10.1158/1541-7786.MCR-16-0090

  View details for Web of Science ID 000382283900001

• Kruppel-like Factor 4 Modulates Development of BMI1(+) Intestinal Stem Cell-Derived Lineage Following gamma-Radiation-Induced Gut Injury in Mice STEM CELL REPORTS Kuruvilla, J. G., Kim, C., Ghaleb, A. M., Bialkowska, A. B., Kuo, C. J., Yang, V. W. 2016; 6 (6): 815-824

  Abstract

  In response to ionizing radiation-induced injury, the normally quiescent intestinal stem cells marked by BMI1 participate in the regenerative response. Previously, we established a protective role for Krüppel-like factor 4 (KLF4) in the intestinal epithelium where it reduces senescence, apoptosis, and crypt atrophy following γ-radiation-induced gut injury. We also described a pro-proliferative function for KLF4 during the regenerative phase post irradiation. In the current study, using a mouse model in which Klf4 is deleted from quiescent BMI1(+) intestinal stem cells, we observed increased proliferation from the BMI1(+) lineage during homeostasis. In contrast, following irradiation, Bmi1-specific Klf4 deletion leads to decreased expansion of the BMI1(+) lineage due to a combination of reduced proliferation and increased apoptosis. Our results support a critical role for KLF4 in modulating BMI1(+) intestinal stem cell fate in both homeostasis and the regenerative response to radiation injury.

  View details for DOI 10.1016/j.stemcr.2016.04.014

  View details for Web of Science ID 000378032600005

  View details for PubMedID 27237377

  View details for PubMedCentralID PMC4911500

• Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis EMBO JOURNAL Lange, C., Garcia, M. T., Decimo, I., Bifari, F., Eelen, G., Quaegebeur, A., Boon, R., Zhao, H., Boeckx, B., Chang, J., Wu, C., le Noble, F., Lambrechts, D., Dewerchin, M., Kuo, C. J., Huttner, W. B., Carmeliet, P. 2016; 35 (9): 924-941

  Abstract

  Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo-spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel-specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia-inducible factor (HIF)-1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.

  View details for PubMedID 26856890

• The Wnt7's Tale: A story of an orphan who finds her tie to a famous family CANCER SCIENCE Noda, M., Vallon, M., Kuo, C. J. 2016; 107 (5): 576-582

  Abstract

  The transformation suppressor gene RECK was isolated by cDNA expression cloning (1998), and GPR124/TEM5 was detected as a tumor endothelial marker by differential screening (2000). The importance of Wnt7a/b and Gpr124 in brain angiogenesis was demonstrated by reverse genetics in mice (2008-2010). A series of recent studies using genetically engineered mice and zebrafish as well as luciferase reporter assays in cultured cells led to the discovery of functional interactions among Reck, Gpr124, and Wnt7a/b in triggering canonical Wnt signaling with relevance to embryonic brain angiogenesis and blood-brain barrier formation.

  View details for DOI 10.1111/cas.12924

  View details for Web of Science ID 000378714600002

  View details for PubMedID 26934061

  View details for PubMedCentralID PMC4970824

• Home Sweet Home: a Foxl1(+) Mesenchymal Niche for Intestinal Stem Cells CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY Mah, A. T., Kuo, C. J. 2016; 2 (2): 116–17

  View details for PubMedID 28174709

• Patient-Derived Organoids as an In Vitro Model of Neuroendocrine Tumors Liu, I. H., Neal, J. T., Zemek, A. J., Kunz, P. L., Kuo, C. J. LIPPINCOTT WILLIAMS & WILKINS. 2016: 467

  View details for Web of Science ID 000370956600026

• Oligodendrocyte precursors migrate along vasculature in the developing nervous system. Science (New York, N.Y.) Tsai, H. H., Niu, J., Munji, R., Davalos, D., Chang, J., Zhang, H., Tien, A. C., Kuo, C. J., Chan, J. R., Daneman, R., Fancy, S. P. 2016; 351 (6271): 379-84

  Abstract

  Oligodendrocytes myelinate axons in the central nervous system and develop from oligodendrocyte precursor cells (OPCs) that must first migrate extensively during brain and spinal cord development. We show that OPCs require the vasculature as a physical substrate for migration. We observed that OPCs of the embryonic mouse brain and spinal cord, as well as the human cortex, emerge from progenitor domains and associate with the abluminal endothelial surface of nearby blood vessels. Migrating OPCs crawl along and jump between vessels. OPC migration in vivo was disrupted in mice with defective vascular architecture but was normal in mice lacking pericytes. Thus, physical interactions with the vascular endothelium are required for OPC migration. We identify Wnt-Cxcr4 (chemokine receptor 4) signaling in regulation of OPC-endothelial interactions and propose that this signaling coordinates OPC migration with differentiation.

  View details for DOI 10.1126/science.aad3839

  View details for PubMedID 26798014

  View details for PubMedCentralID PMC5472053

• An Air-Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary Gastrointestinal Tissues. Methods in molecular biology (Clifton, N.J.) Li, X., Ootani, A., Kuo, C. 2016; 1422: 33-40

  Abstract

  Conventional in vitro analysis of gastrointestinal epithelium usually relies on two-dimensional (2D) culture of epithelial cell lines as monolayer on impermeable surfaces. However, the lack of context of differentiation and tissue architecture in 2D culture can hinder the faithful recapitulation of the phenotypic and morphological characteristics of native epithelium. Here, we describe a robust long-term three-dimensional (3D) culture methodology for gastrointestinal culture, which incorporates both epithelial and mesenchymal/stromal components into a collagen-based air-liquid interface 3D culture system. This system allows vigorously expansion of primary gastrointestinal epithelium for over 60 days as organoids with both proliferation and multilineage differentiation, indicating successful long-term intestinal culture within a microenvironment accurately recapitulating the stem cell niche.

  View details for DOI 10.1007/978-1-4939-3603-8_4

  View details for PubMedID 27246020

• Organoids as Models for Neoplastic Transformation ANNUAL REVIEW OF PATHOLOGY: MECHANISMS OF DISEASE, VOL 11 Neal, J. T., Kuo, C. J. 2016; 11: 199-220

  Abstract

  Cancer models strive to recapitulate the incredible diversity inherent in human tumors. A key challenge in accurate tumor modeling lies in capturing the panoply of homo- and heterotypic cellular interactions within the context of a three-dimensional tissue microenvironment. To address this challenge, researchers have developed organotypic cancer models (organoids) that combine the 3D architecture of in vivo tissues with the experimental facility of 2D cell lines. Here we address the benefits and drawbacks of these systems, as well as their most recent advances. In particular, we focus on the application of such models to the discovery of novel cancer drivers, the study of tumor biology, and the development of novel therapeutic approaches for the treatment of cancer. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease Volume 11 is May 23, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

  View details for DOI 10.1146/annurev-pathol-012615-044249

  View details for Web of Science ID 000377037200009

  View details for PubMedID 26907527

• Fluorescence Imaging In Vivo at Wavelengths beyond 1500 nm ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Diao, S., Blackburn, J. L., Hong, G., Antaris, A. L., Chang, J., Wu, J. Z., Zhang, B., Cheng, K., Kuo, C. J., Dai, H. 2015; 54 (49): 14758-14762

  Abstract

  Compared to imaging in the visible and near-infrared regions below 900 nm, imaging in the second near-infrared window (NIR-II, 1000-1700 nm) is a promising method for deep-tissue high-resolution optical imaging in vivo mainly owing to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single-walled carbon nanotubes with large diameters were used for in vivo fluorescence imaging in the long-wavelength NIR region (1500-1700 nm, NIR-IIb). With this imaging agent, 3-4 μm wide capillary blood vessels at a depth of about 3 mm could be resolved. Meanwhile, the blood-flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR-IIb tumor imaging of a live mouse was explored. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700 nm for high-performance in vivo optical imaging.

  View details for DOI 10.1002/anie.201507473

  View details for Web of Science ID 000367723400025

• Novel TIA biomarkers identified by mass spectrometry-based proteomics. International journal of stroke : official journal of the International Stroke Society George, P. M., Mlynash, M., Adams, C. M., Kuo, C. J., Albers, G. W., Olivot, J. M. 2015; 10 (8): 1204-11

  Abstract

  Transient ischemic attacks remain a clinical diagnosis with significant variability between physicians. Finding reliable biomarkers to identify transient ischemic attacks would improve patient care and optimize treatment.Our aim is to identify novel serum TIA biomarkers through the use of mass spectroscopy-based proteomics.Patients with transient neurologic symptoms were prospectively enrolled. Mass spectrometry-based proteomics, an unbiased method to identify candidate proteins, was used to test the serum of the patients for biomarkers of cerebral ischemia. Three candidate proteins were found, and serum concentrations of these proteins were measured by enzyme-linked immunosorbent assay in a second cohort of prospectively enrolled patients. The Student's t-test was used for comparison. The Benjamini-Hochberg false discovery rate controlling procedure for multiple comparison adjustments determined significance for the proteomic screen.Patients with transient ischemic attacks (n = 20), minor strokes (n = 15), and controls (i.e. migraine, seizure, n = 12) were enrolled in the first cohort. Ceruloplasmin, complement component C8 gamma (C8γ), and platelet basic protein were significantly different between the ischemic group (transient ischemic attack and minor stroke) and the controls (P = 0·0001, P = 0·00027, P = 0·00105, respectively). A second cohort of patients with transient ischemic attack (n = 22), minor stroke (n = 20), and controls' (n = 12) serum was enrolled. Platelet basic protein serum concentrations were increased in the ischemic samples compared with control (for transient ischemic attack alone, P = 0·019, for the ischemic group, P = 0·046). Ceruloplasmin trended towards increased concentrations in the ischemic group (P = 0·127); no significant difference in C8γ (P = 0·44) was found.Utilizing mass spectrometry-based proteomics, platelet basic protein has been identified as a candidate serum biomarker for transient ischemic attack. This unbiased proteomic approach may be a promising method to identify novel biomarkers to more precisely diagnose transient ischemic attacks.

  View details for DOI 10.1111/ijs.12603

  View details for PubMedID 26307429

• Novel TIA biomarkers identified by mass spectrometry-based proteomics INTERNATIONAL JOURNAL OF STROKE George, P. M., Mlynash, M., Adams, C. M., Kuo, C. J., Albers, G. W., Olivot, J. 2015; 10 (8): 1204-1211

  Abstract

  Transient ischemic attacks remain a clinical diagnosis with significant variability between physicians. Finding reliable biomarkers to identify transient ischemic attacks would improve patient care and optimize treatment.Our aim is to identify novel serum TIA biomarkers through the use of mass spectroscopy-based proteomics.Patients with transient neurologic symptoms were prospectively enrolled. Mass spectrometry-based proteomics, an unbiased method to identify candidate proteins, was used to test the serum of the patients for biomarkers of cerebral ischemia. Three candidate proteins were found, and serum concentrations of these proteins were measured by enzyme-linked immunosorbent assay in a second cohort of prospectively enrolled patients. The Student's t-test was used for comparison. The Benjamini-Hochberg false discovery rate controlling procedure for multiple comparison adjustments determined significance for the proteomic screen.Patients with transient ischemic attacks (n = 20), minor strokes (n = 15), and controls (i.e. migraine, seizure, n = 12) were enrolled in the first cohort. Ceruloplasmin, complement component C8 gamma (C8γ), and platelet basic protein were significantly different between the ischemic group (transient ischemic attack and minor stroke) and the controls (P = 0·0001, P = 0·00027, P = 0·00105, respectively). A second cohort of patients with transient ischemic attack (n = 22), minor stroke (n = 20), and controls' (n = 12) serum was enrolled. Platelet basic protein serum concentrations were increased in the ischemic samples compared with control (for transient ischemic attack alone, P = 0·019, for the ischemic group, P = 0·046). Ceruloplasmin trended towards increased concentrations in the ischemic group (P = 0·127); no significant difference in C8γ (P = 0·44) was found.Utilizing mass spectrometry-based proteomics, platelet basic protein has been identified as a candidate serum biomarker for transient ischemic attack. This unbiased proteomic approach may be a promising method to identify novel biomarkers to more precisely diagnose transient ischemic attacks.

  View details for DOI 10.1111/ijs.12603

  View details for Web of Science ID 000367673700011

• Fluorescence Imaging In Vivo at Wavelengths beyond 1500 nm. Angewandte Chemie (International ed. in English) Diao, S., Blackburn, J. L., Hong, G., Antaris, A. L., Chang, J., Wu, J. Z., Zhang, B., Cheng, K., Kuo, C. J., Dai, H. 2015; 54 (49): 14758-62

  Abstract

  Compared to imaging in the visible and near-infrared regions below 900 nm, imaging in the second near-infrared window (NIR-II, 1000-1700 nm) is a promising method for deep-tissue high-resolution optical imaging in vivo mainly owing to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single-walled carbon nanotubes with large diameters were used for in vivo fluorescence imaging in the long-wavelength NIR region (1500-1700 nm, NIR-IIb). With this imaging agent, 3-4 μm wide capillary blood vessels at a depth of about 3 mm could be resolved. Meanwhile, the blood-flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR-IIb tumor imaging of a live mouse was explored. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700 nm for high-performance in vivo optical imaging.

  View details for DOI 10.1002/anie.201507473

  View details for PubMedID 26460151

• Personalizing pancreatic cancer organoids with hPSCs NATURE MEDICINE Zhang, H., Kuo, C. J. 2015; 21 (11): 1249–51

  View details for PubMedID 26540385

• Chemodetection and Destruction of Host Urea Allows Helicobacter pylori to Locate the Epithelium CELL HOST & MICROBE Huang, J. Y., Sweeney, E. G., Sigal, M., Zhang, H. C., Remington, S. J., Cantrell, M. A., Kuo, C. J., Guillemin, K., Amieva, M. R. 2015; 18 (2): 147-156

  Abstract

  The gastric pathogen Helicobacter pylori interacts intimately with the gastric mucosa to avoid the microbicidal acid in the stomach lumen. The cues H. pylori senses to locate and colonize the gastric epithelium have not been well defined. We show that metabolites emanating from human gastric organoids rapidly attract H. pylori. This response is largely controlled by the bacterial chemoreceptor TlpB, and the main attractant emanating from epithelia is urea. Our previous structural analyses show that TlpB binds urea with high affinity. Here we demonstrate that this tight binding controls highly sensitive responses, allowing detection of urea concentrations as low as 50 nM. Attraction to urea requires that H. pylori urease simultaneously destroys the signal. We propose that H. pylori has evolved a sensitive urea chemodetection and destruction system that allows the bacterium to dynamically and locally modify the host environment to locate the epithelium.

  View details for DOI 10.1016/j.chom.2015.07.002

  View details for Web of Science ID 000359601800007

  View details for PubMedID 26269952

• Engineering Gastrointestinal Cancer in Organoid Cultures Kuo, C. SPRINGER. 2015: S1

  View details for Web of Science ID 000355594000002

• Keynote symposium. In vitro cellular & developmental biology. Animal Kuo, C. 2015; 51 Suppl 1: 1

  View details for DOI 10.1007/s11626-015-9900-4

  View details for PubMedID 25967471

• Oligodendrocyte precursors migrate along vasculature in the developing nervous system SCIENCE Tsai, H., Niu, J., Munji, R., Davalos, D., Chang, J., Zhang, H., Tien, A., Kuo, C. J., Chan, J. R., Daneman, R., Fancy, S. P. 2015; 351 (6271): 379-384

  Abstract

  Oligodendrocytes myelinate axons in the central nervous system and develop from oligodendrocyte precursor cells (OPCs) that must first migrate extensively during brain and spinal cord development. We show that OPCs require the vasculature as a physical substrate for migration. We observed that OPCs of the embryonic mouse brain and spinal cord, as well as the human cortex, emerge from progenitor domains and associate with the abluminal endothelial surface of nearby blood vessels. Migrating OPCs crawl along and jump between vessels. OPC migration in vivo was disrupted in mice with defective vascular architecture but was normal in mice lacking pericytes. Thus, physical interactions with the vascular endothelium are required for OPC migration. We identify Wnt-Cxcr4 (chemokine receptor 4) signaling in regulation of OPC-endothelial interactions and propose that this signaling coordinates OPC migration with differentiation.

  View details for DOI 10.1126/science.aad3839

  View details for Web of Science ID 000368440500039

  View details for PubMedCentralID PMC5472053

• Organoid modeling for cancer precision medicine. Genome medicine Cantrell, M. A., Kuo, C. J. 2015; 7 (1): 32-?

  Abstract

  Three-dimensional organotypic culture models show great promise as a tool for cancer precision medicine, with potential applications for oncogene modeling, gene discovery and chemosensitivity studies.

  View details for DOI 10.1186/s13073-015-0158-y

  View details for PubMedID 25825593

  View details for PubMedCentralID PMC4377844

• Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids BIOMATERIALS SCIENCE Dimarco, R. L., Dewi, R. E., Bernal, G., Kuoc, C., Heilshorn, S. C. 2015; 3 (10): 1376-1385

  Abstract

  Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air-liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.

  View details for DOI 10.1039/c5bm00108k

  View details for Web of Science ID 000361194900004

  View details for PubMedID 26371971

• 3-Dimensional air-liquid interface organoid culture of primary human tumor biopsies Neal, J. T., Cantrell, M., Rack, P., Kuo, C. J. AMER ASSOC CANCER RESEARCH. 2014

  View details for DOI 10.1158/1538-7445.AM2014-LB-34

  View details for Web of Science ID 000349910205368

• Through-skull fluorescence imaging of the brain in a new near-infrared window. Nature photonics Hong, G., Diao, S., Chang, J., Antaris, A. L., Chen, C., Zhang, B., Zhao, S., Atochin, D. N., Huang, P. L., Andreasson, K. I., Kuo, C. J., Dai, H. 2014; 8 (9): 723-730

  Abstract

  To date, brain imaging has largely relied on X-ray computed tomography and magnetic resonance angiography with limited spatial resolution and long scanning times. Fluorescence-based brain imaging in the visible and traditional near-infrared regions (400-900 nm) is an alternative but currently requires craniotomy, cranial windows and skull thinning techniques, and the penetration depth is limited to 1-2 mm due to light scattering. Here, we report through-scalp and through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy utilizing the intrinsic photoluminescence of single-walled carbon nanotubes in the 1.3-1.4 micrometre near-infrared window. Reduced photon scattering in this spectral region allows fluorescence imaging reaching a depth of >2 mm in mouse brain with sub-10 micrometre resolution. An imaging rate of ~5.3 frames/s allows for dynamic recording of blood perfusion in the cerebral vessels with sufficient temporal resolution, providing real-time assessment of blood flow anomaly in a mouse middle cerebral artery occlusion stroke model.

  View details for DOI 10.1038/nphoton.2014.166

  View details for PubMedID 27642366

  View details for PubMedCentralID PMC5026222

• Developmental and pathological angiogenesis in the central nervous system. Cellular and molecular life sciences Vallon, M., Chang, J., Zhang, H., Kuo, C. J. 2014; 71 (18): 3489-3506

  Abstract

  Angiogenesis, the formation of new blood vessels from pre-existing vessels, in the central nervous system (CNS) is seen both as a normal physiological response as well as a pathological step in disease progression. Formation of the blood-brain barrier (BBB) is an essential step in physiological CNS angiogenesis. The BBB is regulated by a neurovascular unit (NVU) consisting of endothelial and perivascular cells as well as vascular astrocytes. The NVU plays a critical role in preventing entry of neurotoxic substances and regulation of blood flow in the CNS. In recent years, research on numerous acquired and hereditary disorders of the CNS has increasingly emphasized the role of angiogenesis in disease pathophysiology. Here, we discuss molecular mechanisms of CNS angiogenesis during embryogenesis as well as various pathological states including brain tumor formation, ischemic stroke, arteriovenous malformations, and neurodegenerative diseases.

  View details for DOI 10.1007/s00018-014-1625-0

  View details for PubMedID 24760128

  View details for PubMedCentralID PMC4165859

• Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture. Nature medicine Li, X., Nadauld, L., Ootani, A., Corney, D. C., Pai, R. K., Gevaert, O., Cantrell, M. A., Rack, P. G., Neal, J. T., Chan, C. W., Yeung, T., Gong, X., Yuan, J., Wilhelmy, J., Robine, S., Attardi, L. D., Plevritis, S. K., Hung, K. E., Chen, C. Z., Ji, H. P., Kuo, C. J. 2014

  Abstract

  The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

  View details for DOI 10.1038/nm.3585

  View details for PubMedID 24859528

• Engineering of three-dimensional microenvironments to promote contractile behavior in primary intestinal organoids. Integrative biology Dimarco, R. L., Su, J., Yan, K. S., Dewi, R., Kuo, C. J., Heilshorn, S. C. 2014; 6 (2): 127-142

  Abstract

  Multiple culture techniques now exist for the long-term maintenance of neonatal primary murine intestinal organoids in vitro; however, the achievement of contractile behavior within cultured organoids has thus far been infrequent and unpredictable. Here we combine finite element simulation of oxygen transport and quantitative comparative analysis of cellular microenvironments to elucidate the critical variables that promote reproducible intestinal organoid contraction. Experimentally, oxygen distribution was manipulated by adjusting the ambient oxygen concentration along with the use of semi-permeable membranes to enhance transport. The culture microenvironment was further tailored through variation of collagen type-I matrix density, addition of exogenous R-spondin1, and specification of culture geometry. "Air-liquid interface" cultures resulted in significantly higher numbers of contractile cultures relative to traditional submerged cultures. These interface cultures were confirmed to have enhanced and more symmetric oxygen transport relative to traditional submerged cultures. While oxygen availability was found to impact in vitro contraction rate and the orientation of contractile movement, it was not a key factor in enabling contractility. For all conditions tested, reproducible contractile behavior only occurred within a consistent and narrow range of collagen type-I matrix densities with porosities of approximately 20% and storage moduli near 30 Pa. This suggests that matrix density acts as a "permissive switch" that enables contractions to occur. Similarly, contractions were only observed in cultures with diameters less than 15.5 mm that had relatively large interfacial surface area between the compliant matrix and the rigid culture dish. Taken together, these data suggest that spatial geometry and mechanics of the microenvironment, which includes both the encapsulating matrix as well as the surrounding culture device, may be key determinants of intestinal organoid functionality. As peristaltic contractility is a crucial requirement for normal digestive tract function, this achievement of reproducible organoid contraction marks a pivotal advancement towards engineering physiologically functional replacement tissue constructs.

  View details for DOI 10.1039/c3ib40188j

  View details for PubMedID 24343706

• Partial Proteasome Inhibitors Induce Hair Follicle Growth by Stabilizing ß-Catenin. Stem cells Yucel, G., Van Arnam, J., Means, P. C., Huntzicker, E., Altindag, B., Lara, M. F., Yuan, J., Kuo, C., Oro, A. E. 2014; 32 (1): 85-92

  Abstract

  The activation of tissue stem cells from their quiescent state represents the initial step in the complex process of organ regeneration and tissue repair. While the identity and location of tissue stem cells are becoming known, how key regulators control the balance of activation and quiescence remains mysterious. The vertebrate hair is an ideal model system where hair cycling between growth and resting phases is precisely regulated by morphogen signaling pathways, but how these events are coordinated to promote orderly signaling in a spatial and temporal manner remains unclear. Here, we show that hair cycle timing depends on regulated stability of signaling substrates by the ubiquitin-proteasome system. Topical application of partial proteasomal inhibitors (PaPIs) inhibits epidermal and dermal proteasome activity throughout the hair cycle. PaPIs prevent the destruction of the key anagen signal β-catenin, resulting in more rapid hair growth and dramatically shortened telogen. We show that PaPIs induce excess β-catenin, act similarly to the GSK3β antagonist LiCl, and antagonize Dickopf-related protein-mediated inhibition of anagen. PaPIs thus represent a novel class of hair growth agents that act through transiently modifying the balance of stem cell activation and quiescence pathways. Stem Cells 2014;32:85-92.

  View details for DOI 10.1002/stem.1525

  View details for PubMedID 23963711

• Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer GENOME BIOLOGY Nadauld, L. D., Garcia, S., Natsoulis, G., Bell, J. M., Miotke, L., Hopmans, E. S., Xu, H., Pai, R. K., Palm, C., Regan, J. F., Chen, H., Flaherty, P., Ootani, A., Zhang, N. R., Ford, J. M., Kuo, C. J., Ji, H. P. 2014; 15 (8)

  Abstract

  Gastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis.Both the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity.We document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.

  View details for DOI 10.1186/s13059-014-0428-9

  View details for Web of Science ID 000346604100009

  View details for PubMedID 25315765

  View details for PubMedCentralID PMC4145231

• A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Magness, S. T., Puthoff, B. J., Crissey, M. A., Dunn, J., Henning, S. J., Houchen, C., Kaddis, J. S., Kuo, C. J., Li, L., Lynch, J., Martin, M. G., May, R., Niland, J. C., Olack, B., Qian, D., Stelzner, M., Swain, J. R., Wang, F., Wang, J., Wang, X., Yan, K., Yu, J., Wong, M. H. 2013; 305 (8): G542-G551

  Abstract

  Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM(+)/CD44(+)) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.

  View details for DOI 10.1152/ajpgi.00481.2012

  View details for Web of Science ID 000325809200002

  View details for PubMedID 23928185

  View details for PubMedCentralID PMC3798732

• A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Magness, S. T., Puthoff, B. J., Crissey, M. A., Dunn, J., Henning, S. J., Houchen, C., Kaddis, J. S., Kuo, C. J., Li, L., Lynch, J., Martin, M. G., May, R., Niland, J. C., Olack, B., Qian, D., Stelzner, M., Swain, J. R., Wang, F., Wang, J., Wang, X., Yan, K., Yu, J., Wong, M. H. 2013; 305 (8): G542-G551

  Abstract

  Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.

  View details for DOI 10.1152/ajpgi.00481.2012

  View details for PubMedID 23928185

• Design and Chemical Synthesis of a D-Protein Antagonist against VEGF-A that Inhibits Proliferation of Human Umbilical Vein Endothelial Cells Lee, D., Mandal, K., Uppalapati, M., Kosinski, C., Kuo, C., Ault-Riche, D., Sidhu, S., Kent, S. B. H. WILEY-BLACKWELL. 2013: 240

  View details for Web of Science ID 000336530200058

• Colorectal cancer stem cells and intestinal stem cells: The two faces of janus Annual Meeting of the Society-of-Academic-and-Research-Surgery Yeung, T. M., Kuo, C. J., Bodmer, W. F. WILEY-BLACKWELL. 2012: 1–1

  View details for Web of Science ID 000303151900003

• The HIF Signaling Pathway in Osteoblasts Directly Modulates Erythropoiesis through the Production of EPO CELL Rankin, E. B., Wu, C., Khatri, R., Wilson, T. L., Andersen, R., Araldi, E., Rankin, A. L., Yuan, J., Kuo, C. J., Schipani, E., Giaccia, A. J. 2012; 149 (1): 63-74

  Abstract

  Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.

  View details for DOI 10.1016/j.cell.2012.01.051

  View details for Web of Science ID 000302235400010

  View details for PubMedID 22464323

  View details for PubMedCentralID PMC3408231

• PDGF-B exploits stromal EPO NATURE MEDICINE McGinnis, L. M., Kuo, C. J. 2012; 18 (1): 22-24

  View details for Web of Science ID 000299018600015

  View details for PubMedID 22227660

• Reversible cell-cycle entry in adult kidney podocytes through regulated control of telomerase and Wnt signaling NATURE MEDICINE Shkreli, M., Sarin, K. Y., Pech, M. F., Papeta, N., Chang, W., Brockman, S. A., Cheung, P., Lee, E., Kuhnert, F., Olson, J. L., Kuo, C. J., Gharavi, A. G., D'Agati, V. D., Artandi, S. E. 2012; 18 (1): 111-119

  View details for DOI 10.1038/nm.2550

  View details for Web of Science ID 000299018600036

• Reversible cell-cycle entry in adult kidney podocytes through regulated control of telomerase and Wnt signaling. Nature medicine Shkreli, M., Sarin, K. Y., Pech, M. F., Papeta, N., Chang, W., Brockman, S. A., Cheung, P., Lee, E., Kuhnert, F., Olson, J. L., Kuo, C. J., Gharavi, A. G., D'Agati, V. D., Artandi, S. E. 2012; 18 (1): 111-119

  Abstract

  Mechanisms of epithelial cell renewal remain poorly understood in the mammalian kidney, particularly in the glomerulus, a site of cellular damage in chronic kidney disease. Within the glomerulus, podocytes--differentiated epithelial cells crucial for filtration--are thought to lack substantial capacity for regeneration. Here we show that podocytes rapidly lose differentiation markers and enter the cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression in mice induces marked upregulation of Wnt signaling and disrupts glomerular structure, resulting in a collapsing glomerulopathy resembling those in human disease, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased expression of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Silencing transgenic TERT expression or inhibiting Wnt signaling through systemic expression of the Wnt inhibitor Dkk1 in either TERT transgenic mice or in a mouse model of HIVAN results in marked normalization of podocytes, including rapid cell-cycle exit, re-expression of differentiation markers and improved filtration barrier function. These data reveal an unexpected capacity of podocytes to reversibly enter the cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt-β-catenin pathways in podocyte proliferation and disease.

  View details for DOI 10.1038/nm.2550

  View details for PubMedID 22138751

  View details for PubMedCentralID PMC3272332

• A Novel Method of Local Gene Delivery and Noninvasive Imaging of Transgene Expression in the Mouse Endometrium 44th Annual Meeting of the Society-for-the-Study-of-Reproduction (SSR) Fan, X., Dhal, S., Wu, J. C., Kuo, C. J., Druzin, M. L., Nayak, N. R. SOC STUDY REPRODUCTION. 2011

  View details for Web of Science ID 000310746200585

• MAINTENANCE BEVACIZUMAB MONOTHERAPY INCREASES HEMOGLOBIN (HGB) IN PATIENTS WITH ADVANCED NON-SMALL CELL LUNG ADENOCARCINOMA (NSCLC-AD) Riess, J. W., Logan, A., Krupitskaya, Y., Clement-Duchene, C., Kuo, C., Wakelee, H. LIPPINCOTT WILLIAMS & WILKINS. 2011: S962–S963

  View details for Web of Science ID 000291769800090

• Development and Characterization of a Novel Long-Term Human Endometrial Slice Culture System Fan, X., Ootani, A., Dhal, S., Vo, K. C., Giudice, L. C., Druzin, M. L., Kuo, C. J., Nayak, N. R. SAGE PUBLICATIONS INC. 2011: 225A–226A

  View details for Web of Science ID 000291721701109

• Targeting Endothelium-Pericyte Cross Talk by Inhibiting VEGF Receptor Signaling Attenuates Kidney Microvascular Rarefaction and Fibrosis AMERICAN JOURNAL OF PATHOLOGY Lin, S., Chang, F., Schrimpf, C., Chen, Y., Wu, C., Wu, V., Chiang, W., Kuhnert, F., Kuo, C. J., Chen, Y., Wu, K., Tsai, T., Duffield, J. S. 2011; 178 (2): 911-923

  Abstract

  Microvascular pericytes and perivascular fibroblasts have recently been identified as the source of scar-producing myofibroblasts that appear after injury of the kidney. We show that cross talk between pericytes and endothelial cells concomitantly dictates development of fibrosis and loss of microvasculature after injury. When either platelet-derived growth factor receptor (R)-β signaling in pericytes or vascular endothelial growth factor (VEGF)R2 signaling in endothelial cells was blocked by circulating soluble receptor ectodomains, both fibrosis and capillary rarefaction were markedly attenuated during progressive kidney injury. Blockade of either receptor-mediated signaling pathway prevented pericyte differentiation and proliferation, but VEGFR2 blockade also attenuated recruitment of inflammatory macrophages throughout disease progression. Whereas injury down-regulated angiogenic VEGF164, the dys-angiogenic isomers VEGF120 and VEGF188 were up-regulated, suggesting that pericyte-myofibroblast differentiation triggers endothelial loss by a switch in secretion of VEGF isomers. These findings link fibrogenesis inextricably with microvascular rarefaction for the first time, add new significance to fibrogenesis, and identify novel therapeutic targets.

  View details for DOI 10.1016/j.ajpath.2010.10.012

  View details for Web of Science ID 000287264400045

  View details for PubMedID 21281822

  View details for PubMedCentralID PMC3070546

• Novel Receptor-Mediated Endothelial Cell Chemotaxis Shamloo, A., Kuhnert, F., Choksi, V., Kuo, C., Heilshorn, S. CELL PRESS. 2010: 497A

  View details for DOI 10.1016/j.bpj.2009.12.2705

  View details for Web of Science ID 000208762004500

• Signaling in Normal and Pathological Angiogenesis SIGNAL TRANSDUCTION: PATHWAYS, MECHANISMS AND DISEASES Mancuso, M. R., Kuo, C. J. edited by Sitaramayya, A. 2010: 159–80

  View details for DOI 10.1007/978-3-642-02112-1_9

  View details for Web of Science ID 000273693600009

• G Protein-Coupled Receptor 124 (GPR124) Gene Polymorphisms and Risk of Brain Arteriovenous Malformations American-Association-International-Stroke Conference 2009 Weinsheimer, S., Pawlikowska, L., Brettman, A., Mancuso, M. R., Kuhnert, F., Kuo, C., Sidney, S., Young, W. L., Kim, H. LIPPINCOTT WILLIAMS & WILKINS. 2009: E135–E135

  View details for Web of Science ID 000264709500208

• Endochondral ossification is required for haematopoietic stem-cell niche formation NATURE Chan, C. K., Chen, C., Luppen, C. A., Kim, J., DeBoer, A. T., Wei, K., Helms, J. A., Kuo, C. J., Kraft, D. L., Weissman, I. L. 2009; 457 (7228): 490-U9

  Abstract

  Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.

  View details for DOI 10.1038/nature07547

  View details for PubMedID 19078959

• Attribution of vascular phenotypes of the murine Egfl7 locus to the microRNA miR-126 DEVELOPMENT Kuhnert, F., Mancuso, M. R., Hampton, J., Stankunas, K., Asano, T., Chen, C., Kuo, C. J. 2008; 135 (24): 3989-3993

  Abstract

  Intronic microRNAs have been proposed to complicate the design and interpretation of mouse knockout studies. The endothelial-expressed Egfl7/miR-126 locus contains miR-126 within Egfl7 intron 7, and angiogenesis deficits have been previously ascribed to Egfl7 gene-trap and lacZ knock-in mice. Surprisingly, selectively floxed Egfl7(Delta) and miR-126(Delta) alleles revealed that Egfl7(Delta/Delta) mice were phenotypically normal, whereas miR-126(Delta/Delta) mice bearing a 289-nt microdeletion recapitulated previously described Egfl7 embryonic and postnatal retinal vascular phenotypes. Regulation of angiogenesis by miR-126 was confirmed by endothelial-specific deletion and in the adult cornea micropocket assay. Furthermore, miR-126 deletion inhibited VEGF-dependent Akt and Erk signaling by derepression of the p85beta subunit of PI3 kinase and of Spred1, respectively. These studies demonstrate the regulation of angiogenesis by an endothelial miRNA, attribute previously described Egfl7 vascular phenotypes to miR-126, and document inadvertent miRNA dysregulation as a complication of mouse knockout strategies.

  View details for DOI 10.1242/dev.029736

  View details for Web of Science ID 000261151000002

  View details for PubMedID 18987025

• Use of R-spondin1, An Intestinotrophic Mitogen, in the Treatment of Murine Graft-Versus-Host Disease 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Zambricki, E. A., Ootani, A., Mancuso, M. R., Zeiser, R., Kuo, C. J., Negrin, R. S. AMER SOC HEMATOLOGY. 2008: 1206–

  View details for Web of Science ID 000262104704138

• Increased Hemoglobin Associated with VEGF Inhibitors in Advanced Renal Cell Carcinoma 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Harshman, L. C., Kuo, C. J., Wong, B. Y., Vogelzang, N. J., Srinivas, S. AMER SOC HEMATOLOGY. 2008: 1185–85

  View details for Web of Science ID 000262104704071

• Systemic VEGF Inhibition Induces Hepatic EPO Production and Erythrocytosis Via HIF-2a-Dependent and -Independent Mechanisms 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Wei, K., Logan, A. C., Wakelee, H., Simon, M. C., Kuo, C. J. AMER SOC HEMATOLOGY. 2008: 183–84

  View details for Web of Science ID 000262104700483

• Soluble receptor-mediated selective inhibition of VEGFR and PDGFR beta signaling during physiologic and tumor angiogenesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kuhnert, F., Tam, B. Y., Sennino, B., Gray, J. T., Yuan, J., Jocson, A., Nayak, N. R., Mulligan, R. C., McDonald, D. M., Kuo, C. J. 2008; 105 (29): 10185-10190

  Abstract

  The simultaneous targeting of both endothelial cells and pericytes via inhibition of VEGF receptor (VEGFR) and PDGFbeta receptor (PDGFRbeta) signaling, respectively, has been proposed to enhance the efficacy of antiangiogenic tumor therapy. Clinical and preclinical modeling of combined VEGFR and PDGFRbeta signaling inhibition, however, has used small molecule kinase inhibitors with inherently broad substrate specificities, precluding detailed examination of this hypothesis. Here, adenoviral expression of a soluble VEGFR2/Flk1 ectodomain (Ad Flk1-Fc) in combination with a soluble ectodomain of PDGFRbeta (Ad sPDGFRbeta) allowed highly selective inhibition of these pathways. The activity of Ad sPDGFRbeta was validated in vitro against PDGF-BB and in vivo with near-complete blockade of pericyte recruitment in the angiogenic corpus luteum, resulting in prominent hemorrhage, thus demonstrating an essential function for PDGF signaling during ovarian angiogenesis. Combination therapy with Ad PDGFRbeta and submaximal doses of Ad Flk1-Fc produced modest additive antitumor effects; however, no additivity was observed with maximal VEGF inhibition in numerous s.c. models. Notably, VEGF inhibition via Ad Flk1-Fc was sufficient to strongly suppress tumor endothelial and pericyte content as well as intratumoral PDGF-B mRNA, obscuring additive Ad sPDGFRbeta effects on pericytes or tumor volume. These studies using highly specific soluble receptors suggest that additivity between VEGFR and PDGFRbeta inhibition depends on the strength of VEGF blockade and appears minimal under conditions of maximal VEGF antagonism.

  View details for DOI 10.1073/pnas.0803194105

  View details for Web of Science ID 000257913200061

  View details for PubMedID 18632559

  View details for PubMedCentralID PMC2474564

• Recombinant adenovirus as a methodology for exploration of physiologic functions of growth factor pathways JOURNAL OF MOLECULAR MEDICINE-JMM Wei, K., Kuhnert, F., Kuo, C. J. 2008; 86 (2): 161-169

  Abstract

  The use of recombinant adenoviruses (Ad) to express secreted antagonists of growth factors represents a powerful strategy for studying physiologic functions of growth factor pathways in experimental animals. Indeed, a single adenoviral injection can produce characteristic high-level and persistent plasma expression of soluble receptor ectodomains or secreted protein antagonists, allowing highly stringent conditional inactivation of target pathways in vivo. In this review, we describe our experience using recombinant Ad to inactivate growth factor pathways in vivo and discuss their advantages and limitations. Using our studies on vascular endothelial growth factor and Wnt systems as examples, we further describe how recombinant Ad can unveil previously unknown physiological roles of signaling pathways. Finally, we discuss the potential physiological and therapeutic relevance of our findings.

  View details for DOI 10.1007/s00109-007-0261-7

  View details for Web of Science ID 000252799300004

  View details for PubMedID 17891365

• Augmented Wnt signaling in a mammalian model of accelerated aging SCIENCE Liu, H., Fergusson, M. M., Castilho, R. M., Liu, J., Cao, L., Chen, J., Malide, D., Rovira, I. I., Schimel, D., Kuo, C. J., Gutkind, J. S., Hwang, P. M., Finkel, T. 2007; 317 (5839): 803-806

  Abstract

  The contribution of stem and progenitor cell dysfunction and depletion in normal aging remains incompletely understood. We explored this concept in the Klotho mouse model of accelerated aging. Analysis of various tissues and organs from young Klotho mice revealed a decrease in stem cell number and an increase in progenitor cell senescence. Because klotho is a secreted protein, we postulated that klotho might interact with other soluble mediators of stem cells. We found that klotho bound to various Wnt family members. In a cell culture model, the Wnt-klotho interaction resulted in the suppression of Wnt biological activity. Tissues and organs from klotho-deficient animals showed evidence of increased Wnt signaling, and ectopic expression of klotho antagonized the activity of endogenous and exogenous Wnt. Both in vitro and in vivo, continuous Wnt exposure triggered accelerated cellular senescence. Thus, klotho appears to be a secreted Wnt antagonist and Wnt proteins have an unexpected role in mammalian aging.

  View details for DOI 10.1126/science.1143578

  View details for Web of Science ID 000248624500040

  View details for PubMedID 17690294

• Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis SCIENCE Brack, A. S., Conboy, M. J., Roy, S., Lee, M., Kuo, C. J., Keller, C., Rando, T. A. 2007; 317 (5839): 807-810

  Abstract

  The regenerative potential of skeletal muscle declines with age, and this impairment is associated with an increase in tissue fibrosis. We show that muscle stem cells (satellite cells) from aged mice tend to convert from a myogenic to a fibrogenic lineage as they begin to proliferate and that this conversion is mediated by factors in the systemic environment of the old animals. We also show that this lineage conversion is associated with an activation of the canonical Wnt signaling pathway in aged myogenic progenitors and can be suppressed by Wnt inhibitors. Furthermore, components of serum from aged mice that bind to the Frizzled family of proteins, which are Wnt receptors, may account for the elevated Wnt signaling in aged cells. These results indicate that the Wnt signaling pathway may play a critical role in tissue-specific stem cell aging and an increase in tissue fibrosis with age.

  View details for DOI 10.1126/science.1144090

  View details for Web of Science ID 000248624500041

  View details for PubMedID 17690295

• VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis NATURE MEDICINE Tam, B. Y., Wei, K., Rudge, J. S., Hoffman, J., Holash, J., Park, S., Yuan, J., Hefner, C., Chartier, C., Lee, J., Jiang, S., Niyak, N. R., Kuypers, F. A., Ma, L., Sundram, U., Wu, G., Garcia, J. A., Schrier, S. L., Maher, J. J., Johnson, R. S., Yancopoulos, G. D., Mulligan, R. C., Kuo, C. J. 2006; 12 (7): 793-800

  Abstract

  Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.

  View details for DOI 10.1038/nm1428

  View details for Web of Science ID 000238862800066

  View details for PubMedID 16799557

• Apc tumor suppressor gene is the "zonation-keeper" of mouse liver DEVELOPMENTAL CELL Benhamouche, S., Decaens, T., Godard, C., Chambrey, R., Rickman, D. S., Moinard, C., Vasseur-Cognet, M., Kuo, C. J., Kahn, A., Perret, C., Colnot, S. 2006; 10 (6): 759-770

  Abstract

  The molecular mechanisms by which liver genes are differentially expressed along a portocentral axis, allowing for metabolic zonation, are poorly understood. We provide here compelling evidence that the Wnt/beta-catenin pathway plays a key role in liver zonation. First, we show the complementary localization of activated beta-catenin in the perivenous area and the negative regulator Apc in periportal hepatocytes. We then analyzed the immediate consequences of either a liver-inducible Apc disruption or a blockade of Wnt signaling after infection with an adenovirus encoding Dkk1, and we show that Wnt/beta-catenin signaling inversely controls the perivenous and periportal genetic programs. Finally, we show that genes involved in the periportal urea cycle and the perivenous glutamine synthesis systems are critical targets of beta-catenin signaling, and that perturbations to ammonia metabolism are likely responsible for the death of mice with liver-targeted Apc loss. From our results, we propose that Apc is the liver "zonation-keeper" gene.

  View details for DOI 10.1016/j.devcel.2006.03.015

  View details for Web of Science ID 000238244700010

  View details for PubMedID 16740478

• VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Kamba, T., Tam, B. Y., Hashizume, H., Haskell, A., Sennino, B., Mancuso, M. R., Norberg, S. M., O'Brien, S. M., Davis, R. B., Gowen, L. C., Anderson, K. D., Thurston, G., Joho, S., Springer, M. L., Kuo, C. J., McDonald, D. M. 2006; 290 (2): H560-H576

  Abstract

  Unlike during development, blood vessels in the adult are generally thought not to require VEGF for normal function. However, VEGF is a survival factor for many tumor vessels, and there are clues that some normal blood vessels may also depend on VEGF. In this study, we sought to identify which, if any, vascular beds in adult mice depend on VEGF for survival. Mice were treated with a small-molecule VEGF receptor (VEGFR) tyrosine kinase inhibitor or soluble VEGFRs for 1-3 wk. Blood vessels were assessed using immunohistochemistry or scanning or transmission electron microscopy. In a study of 17 normal organs after VEGF inhibition, we found significant capillary regression in pancreatic islets, thyroid, adrenal cortex, pituitary, choroid plexus, small-intestinal villi, and epididymal adipose tissue. The amount of regression was dose dependent and varied from organ to organ, with a maximum of 68% in thyroid, but was less in normal organs than in tumors in RIP-Tag2-transgenic mice or in Lewis lung carcinoma. VEGF-dependent capillaries were fenestrated, expressed high levels of both VEGFR-2 and VEGFR-3, and had normal pericyte coverage. Surviving capillaries in affected organs had fewer fenestrations and less VEGFR expression. All mice appeared healthy, but distinct physiological changes, including more efficient blood glucose handling, accompanied some regimens of VEGF inhibition. Strikingly, most capillaries in the thyroid grew back within 2 wk after cessation of treatment for 1 wk. Our findings of VEGF dependency of normal fenestrated capillaries and rapid regrowth after regression demonstrate the plasticity of the adult microvasculature.

  View details for DOI 10.1152/ajpheart.00133.2005

  View details for Web of Science ID 000234531000012

  View details for PubMedID 16172168

• Cellular changes in normal blood capillaries undergoing regression after inhibition of VEGF signaling AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Baffert, F., Le, T., Sennino, B., Thurston, G., Kuo, C. J., Hu-Lowe, D., McDonald, D. M. 2006; 290 (2): H547-H559

  Abstract

  The vasculature of the embryo requires vascular endothelial growth factor (VEGF) during development, but most adult blood vessels lose VEGF dependence. However, some capillaries in the respiratory tract and selected other organs of adult mice regress after VEGF inhibition. The present study sought to identify the sequence of events and the fate of endothelial cells, pericytes, and vascular basement membrane during capillary regression in mouse tracheas after VEGF signaling was blocked with a VEGF-receptor tyrosine kinase inhibitor AG-013736 or soluble receptor construct (VEGF Trap or soluble adenoviral VEGFR-1). Within 1 day, patency was lost and fibrin accumulated in some tracheal capillaries. Apoptotic endothelial cells marked by activated caspase-3 were present in capillaries without blood flow. VEGF inhibition was accompanied by a 19% decrease in tracheal capillaries over 7 days and 30% over 21 days. During this period, desmin/NG2-immunoreactive pericytes moved away from regressing capillaries onto surviving vessels. Empty sleeves of basement membrane, left behind by regressing endothelial cells, persisted for about 2 wk and served as a scaffold for vascular regrowth after treatment ended. The amount of regrowth was limited by the number of surviving basement membrane sleeves. These findings demonstrate that, after inhibition of VEGF signaling, some normal capillaries regress in a systematic sequence of events initiated by a cessation of blood flow and followed by apoptosis of endothelial cells, migration of pericytes away from regressing vessels, and formation of empty basement membrane sleeves that can facilitate capillary regrowth.

  View details for DOI 10.1152/ajpheart.00616.2005

  View details for Web of Science ID 000234531000011

  View details for PubMedID 16172161

• Cotargeting tumor and tumor endothelium effectively inhibits the growth of human prostate cancer in adenovirus-mediated antiangiogenesis and oncolysis combination therapy CANCER GENE THERAPY Jin, F. S., Xie, Z. H., Kuo, C. J., Chung, L. W., Hsieh, C. L. 2005; 12 (3): 257-267

  Abstract

  Tumor-endothelial interaction contributes to local prostate tumor growth and distant metastasis. In this communication, we designed a novel approach to target both cancer cells and their "crosstalk" with surrounding microvascular endothelium in an experimental hormone refractory human prostate cancer model. We evaluated the in vitro and in vivo synergistic and/or additive effects of a combination of conditional oncolytic adenovirus plus an adenoviral-mediated antiangiogenic therapy. In the in vitro study, we demonstrated that human umbilical vein endothelial cells (HUVEC) and human C4-2 androgen-independent (AI) prostate cancer cells, when infected with an antiangiogenic adenoviral (Ad)-Flk1-Fc vector secreting a soluble form of Flk1, showed dramatically inhibited proliferation, migration and tubular formation of HUVEC endothelial cells. C4-2 cells showed maximal growth inhibition when coinfected with Ad-Flk1-Fc and Ad-hOC-E1, a conditional replication-competent Ad vector with viral replication driven by a human osteocalcin (hOC) promoter targeting both prostate cancer epithelial and stromal cells. Using a three-dimensional (3D) coculture model, we found that targeting C4-2 cells with Ad-hOC-E1 markedly decreased tubular formation in HUVEC, as visualized by confocal microscopy. In a subcutaneous C4-2 tumor xenograft model, tumor volume was decreased by 40-60% in animals treated with Ad-Flk1-Fc or Ad-hOC-E1 plus vitamin D3 alone and by 90% in a combined treatment group, compared to untreated animals in an 8-week treatment period. Moreover, three of 10 (30%) pre-established tumors completely regressed when animals received combination therapy. Cotargeting tumor and tumor endothelium could be a promising gene therapy strategy for the treatment of both localized and metastatic human prostate cancer.

  View details for DOI 10.1038/sj.cgt.7700790

  View details for Web of Science ID 000227026700005

  View details for PubMedID 15565180

• Angiopoietin-1 expression in the primate endometrium: Potential role in spiral artery growth. 52nd Annual Meeting of the Society-for-Gynecologic-Investigation Nayak, N. R., Brenner, R. M., Mah, K., Kuo, C. J., Giudice, L. C. ELSEVIER SCIENCE INC. 2005: 325A–325A

  View details for Web of Science ID 000227329101294

• The axonal attractant Netrin-1 is an angiogenic factor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Park, K. W., Crouse, D., Lee, M., Karnik, S. K., Sorensen, L. K., Murphy, K. J., Kuo, C. J., Li, D. Y. 2004; 101 (46): 16210–15

  Abstract

  Blood vessels and nerves often follow parallel trajectories, suggesting that distal targets use common cues that induce vascularization and innervation. Netrins are secreted by the floor plate and attract commissural axons toward the midline of the neural tube. Here, we show that Netrin-1 is also a potent vascular mitogen. Netrin-1 stimulates proliferation, induces migration, and promotes adhesion of endothelial cells and vascular smooth muscle cells with a specific activity comparable to vascular endothelial growth factor and platelet-derived growth factor. Our evidence indicates that the netrin receptor, Neogenin, mediates netrin signaling in vascular smooth muscle cells, but suggests that an unidentified receptor mediates the proangiogenic effects of Netrin-1 on endothelial cells. Netrin-1 also stimulates angiogenesis in vivo and augments the response to vascular endothelial growth factor. Thus, we demonstrate that Netrin-1 is a secreted neural guidance cue with the unique ability to attract both blood vessels and axons, and suggest that other cues may also function as vascular endothelial growth factors.

  View details for DOI 10.1073/pnas.0405984101

  View details for Web of Science ID 000225226200024

  View details for PubMedID 15520390

  View details for PubMedCentralID PMC528958

• The cardiovascular regulator apelin is an angiogenic factor in vivo Kundu, R. K., Eichhorn, J., Chen, M., Ho, Y. D., Ashley, E., Varner, J., Kuo, C., Quertermous, T. LIPPINCOTT WILLIAMS & WILKINS. 2004: 173

  View details for Web of Science ID 000224783500824

• Adenoviral gene transfer with soluble VEGF receptors impairs angiogenesis and arteriogenesis in a murine model of hindlimb ischaemia ESC Congress 2004 Jacobi, J., Tam, B. Y., Cooke, J. P., Kuo, C. J. OXFORD UNIV PRESS. 2004: 253–253

  View details for Web of Science ID 000224056501006

• Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kuhnert, F., DAVIS, C. R., Wang, H. T., Chu, P., Lee, M., Yuan, J., Nusse, R., Kuo, C. J. 2004; 101 (1): 266-271

  Abstract

  Whereas the adult gastrointestinal epithelium undergoes tremendous self-renewal through active proliferation in crypt stem cell compartments, the responsible growth factors regulating this continuous proliferation have not been defined. The exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. Dickkopf-1 (Dkk1) is a potent secreted Wnt antagonist that interacts with Wnt coreceptors of the LRP family. To address the contribution of Wnt signaling to gastrointestinal epithelial proliferation, adenoviral expression of Dkk1 was used to achieve stringent, conditional, and reversible Wnt inhibition in adult animals. Adenovirus Dkk1 (Ad Dkk1) treatment of adult mice repressed expression of the Wnt target genes CD44 and EphB2 within 2 days in both small intestine and colon, indicating an extremely broad role for Wnt signaling in the maintenance of adult gastrointestinal gene expression. In parallel, Ad Dkk1 markedly inhibited proliferation in small intestine and colon, accompanied by progressive architectural degeneration with the loss of crypts, villi, and glandular structure by 7 days. Whereas decreased Dkk1 expression at later time points (>10 days) was followed by crypt and villus regeneration, which was consistent with a reversible process, substantial mortality ensued from colitis and systemic infection. These results indicate the efficacy of systemic expression of secreted Wnt antagonists as a general strategy for conditional inactivation of Wnt signaling in adult organisms and illustrate a striking reliance on a single growth factor pathway for the maintenance of the architecture of the adult small intestine and colon.

  View details for PubMedID 14695885

• Adenovirus-mediated delivery of a soluble form of the VEGF receptor Flk1 delays the growth of murine and human pancreatic adenocarcinoma in mice Tseng, J. F., Farnebo, F. A., Kisker, O., Becker, C. M., Kuo, C. J., Folkman, J., Mulligan, R. C. MOSBY-ELSEVIER. 2002: 857-865

  Abstract

  Because pancreatic adenocarcinoma is poorly responsive to chemotherapy and radiation therapy, novel treatments such as antiangiogenic gene therapy may have use in the adjuvant treatment of this malignancy. We evaluated the antitumor effects of the in vivo administration of an adenovirus vector encoding a soluble form of Flk1 (Flk1-Fc), a receptor for vascular endothelial growth factor, in 3 murine models of pancreatic adenocarcinoma.In a first model, immunocompetent C57Bl/6 mice were injected subcutaneously with Panc02 murine pancreatic adenocarcinoma cells before treatment. In a second model, immunodeficient severe combined immunodeficiency mice were injected subcutaneously with BxPc-3 human pancreatic adenocarcinoma cells before treatment. In a third model, C57Bl/6 mice were injected with Panc02 cells through an intrasplenic route before treatment, in an effort to model metastatic disease. In each model, half the tumor-bearing mice were injected intravenously with 10(9) Flk1-Fc adenovirus particles and half with control adenovirus.In subcutaneous tumor models, Ad Flk1-Fc-treated animals were found to have 75% smaller murine and 78% smaller human pancreatic tumor volumes, relative to tumor volumes of Ad Fc-treated animals, 6 weeks after vector administration. In animals injected with tumor through the intrasplenic route, pathologic and histologic analyses made 10 days after injection of tumor revealed hepatic, pancreatic, and splenic tumors, together with a desmoplastic response consistent with pathologic findings in human pancreatic cancer. Cohorts of these tumor-bearing mice treated with Ad Flk1-Fc demonstrated significantly longer survival and decreased liver replacement with tumor at the time of death, relative to animals treated with Ad Fc.A recombinant adenovirus encoding soluble Flk-1 inhibited pancreatic tumor growth in mice. These studies suggest that the delivery of gene products such as Flk1-Fc through in vivo gene transfer may be useful in the future treatment of patients with pancreatic cancer.

  View details for DOI 10.1067/msy.2002.127680

  View details for Web of Science ID 000179764300014

  View details for PubMedID 12464871

• Gene therapy of prostate cancer with the soluble vascular endothelial growth factor receptor Flk1 CANCER BIOLOGY & THERAPY Becker, C. M., Farnebo, F. A., Iordanescu, Behonick, D. J., Shih, M. C., Dunning, P., Christofferson, R., Mulligan, R. C., Taylor, G. A., Kuo, C. J., Zetter, B. R. 2002; 1 (5): 548-553

  Abstract

  A variety of novel therapeutic approaches have emerged recently for the treatment of human cancers. We have coupled two of these therapeutic approaches, gene therapy and antiangiogenic therapy and tested them in two murine prostate cancer models Recombinant adenovirus encoding the ligand-binding ectodomain of the VEGF receptor 2 (Flk1) fused to an Fc domain was administered to SCID mice carrying orthotopic human LNCaP tumors as well as to transgenic (TRAMP) mice with spontaneous prostate tumors. Ad Flk1-Fc injection reduced tumor growth by 66% for orthotopic LNCaP tumors and by 42% for spontaneous tumors in TRAMP mice. Microvessel density in the primary tumors was reduced by 68% and 40% in the two models respectively. A decrease in microvessel density was also observed in lymphatic metastases in Ad Flk1-Fc-treated TRAMP mice and was correlated with a decrease in the frequency of regional metastases in the treated animals. Survival time was also extended in the Ad Flk1-Fc-treated TRAMP mice relative to the control-treated animals. Our results suggest that adenoviral delivery of soluble Flk1 receptor can reduce vascular density and prostate tumor growth and prolong survival time in orthotopically implanted tumors as well as in spontaneous prostate tumors in transgenic animals.

  View details for DOI 10.4161/cbt.1.5.176

  View details for Web of Science ID 000180996500020

  View details for PubMedID 12496487

• Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kuo, C. J., Farnebo, F., Yu, E. Y., Christofferson, R., Swearingen, R. A., Carter, R., von Recum, H. A., Yuan, J., Kamihara, J., Flynn, E., D'Amato, R., Folkman, J., Mulligan, R. C. 2001; 98 (8): 4605-4610

  Abstract

  Although the systemic administration of a number of different gene products has been shown to result in the inhibition of angiogenesis and tumor growth in different animal tumor models, the relative potency of those gene products has not been studied rigorously. To address this issue, recombinant adenoviruses encoding angiostatin, endostatin, and the ligand-binding ectodomains of the vascular endothelial growth factor receptors Flk1, Flt1, and neuropilin were generated and used to systemically deliver the different gene products in several different preexisting murine tumor models. Single i.v. injections of viruses encoding soluble forms of Flk1 or Flt1 resulted in approximately 80% inhibition of preexisting tumor growth in murine models involving both murine (Lewis lung carcinoma, T241 fibrosarcoma) and human (BxPC3 pancreatic carcinoma) tumors. In contrast, adenoviruses encoding angiostatin, endostatin, or neuropilin were significantly less effective. A strong correlation was observed between the effects of the different viruses on tumor growth and the activity of the viruses in the inhibition of corneal micropocket angiogenesis. These data underscore the need for comparative analyses of different therapeutic approaches that target tumor angiogenesis and provide a rationale for the selection of specific antiangiogenic gene products as lead candidates for use in gene therapy approaches aimed at the treatment of malignant and ocular disorders.

  View details for Web of Science ID 000168059700062

  View details for PubMedID 11274374

  View details for PubMedCentralID PMC31881

• Antiangiogenic gene therapy using soluble VEGF receptors. Kuo, C. J., Farnebo, F. A., Christofferson, R., Yu, E., Folkman, J., Mulligan, R. AMER SOC HEMATOLOGY. 2000: 211A–211A

  View details for Web of Science ID 000165256100903

• RAPAMYCIN SELECTIVELY INHIBITS INTERLEUKIN-2 ACTIVATION OF P70 S6 KINASE NATURE Kuo, C. J., Chung, J. K., Fiorentino, D. F., Flanagan, W. M., Blenis, J., Crabtree, G. R. 1992; 358 (6381): 70-73

  Abstract

  The macrolide rapamycin induces cell cycle G1 arrest in yeast and in mammalian cells, which suggests that an evolutionarily conserved, rapamycin-sensitive pathway may regulate entry into S phase. In mammals, rapamycin inhibits interleukin-2 receptor-induced S phase entry and subsequent T-cell proliferation, resulting in immunosuppression. Here we show that interleukin-2 selectively stimulates the phosphorylation and activation of p70 S6 kinase but not the erk-encoded MAP kinases and rsk-encoded S6 kinases. Rapamycin completely and rapidly inhibits interleukin-2-induced phosphorylation and activation of p70 S6 kinase at concentrations comparable to those blocking S phase entry of T cells (0.05-0.2 nM). The structurally related macrolide FK506 competitively antagonizes the actions of rapamycin, indicating that these effects are mediated by FKBP, which binds the transition-state mimic structure common to both rapamycin and FK506 (refs 4, 6, 9-11). The selective blockade of the p70 S6 kinase activation cascade by the rapamycin-FKBP complex implicates this signalling pathway in the regulation of T cell entry into S phase.

  View details for Web of Science ID A1992JB34100056

  View details for PubMedID 1614535

• A TRANSCRIPTIONAL HIERARCHY INVOLVED IN MAMMALIAN CELL-TYPE SPECIFICATION NATURE Kuo, C. J., Conley, P. B., Chen, L., Sladek, F. M., Darnell, J. E., Crabtree, G. R. 1992; 355 (6359): 457-461

  Abstract

  Although transcriptional hierarchies have been extensively studied in invertebrates, their involvement in mammalian cell-type specification is poorly understood. Here we report a hepatocyte transcriptional cascade suggested by the expression patterns of hepatic transcription factors in dedifferentiated hepatomas and hepatocyte: fibroblast hybrids in which the liver phenotype was extinguished. These results indicated that the homeoprotein hepatocyte nuclear factor-1 alpha (HNF-1 alpha), and HNF-4, a member of the steroid hormone receptor superfamily, were regulated coordinately or in a hierarchy by a higher-order locus, independently of other hepatic transactivators. HNF-4 was implicated as an essential positive regulator of HNF-1 alpha, as deletion of an HNF-4 binding site in the HNF-1 alpha promoter abolished promoter activity, and HNF-4 potently transactivated the HNF-1 alpha promoter in cotransfection assays. Moreover, genetic complementation of dedifferentiated hepatomas with HNF-4 complementary DNA rescued expression of endogenous HNF-1 alpha messenger RNA and DNA-binding activity. Our studies therefore define an HNF-4----HNF-1 alpha (4----1 alpha) transcriptional hierarchy operative in differentiated hepatocytes but selectively inhibited by an extinguishing locus and somatic mutations which antagonize the liver phenotype.

  View details for Web of Science ID A1992HB53000073

  View details for PubMedID 1734282