Administrative Appointments


  • Co-lead, Cancer Biology Program, Stanford Cancer Center (2012 - Present)
  • Vice Chair, Department of Medicine (2015 - Present)

Honors & Awards


  • Maureen Lyles D'Ambrogio Professor of Medicine, Stanford University School of Medicine (2015)
  • Fellow, AAAS (2015)
  • Member, American Academy of Physicians (2016)
  • Consulting Editor, JCI (2012)
  • American Heart Association Innovative Science Award, AHA (2012)
  • Research Chair, NIH Intestinal Stem Cell Consortium, NIH (2009)
  • Transformative R01 Award, NIH (2009)
  • Member, American Society for Clinical Investigation, American Society for Clinical Investigation (2007)
  • Samantha Janower Research Chair, Brain Tumor Society (2005)
  • Merck Faculty Development Award, Merck (2003)
  • Kimmel Foundation Scholar in Translational Science, Kimmel Foundation (2002)
  • Burroughs Wellcome Foundation New Investigator in Pharmacological Sciences, Burroughs Wellcome Foundation (2001)
  • HHMI Physician-Scientist Fellowship, HHMI (1998)
  • Summa cum laude, Harvard College (1987)

Boards, Advisory Committees, Professional Organizations


  • Scientific Advisory Board, AP Giannini Foundation (2008 - Present)
  • Advisory Board, American Heart Association Silicon Valley Chapter (2013 - Present)

Professional Education


  • Fellowship: Brigham and Women's Hospital Harvard Medical School (2000) MA
  • Residency: Brigham and Women's Hospital Harvard Medical School (1997) MA
  • Medical Education: Stanford University School of Medicine (1994) CA
  • A.B., Harvard College, Biochemical Sciences (1987)
  • M.D./Ph.D., Stanford University, Cancer Biology (1994)
  • Internship/ Residency, Brigham and Women's Hospital, Internal Medicine (1997)
  • Fellowship, Dana-Farber/Partners, Adult Oncology (2000)

Community and International Work


  • American Heart Association Silicon Valley Chapter

    Topic

    President-Elect

    Ongoing Project

    No

    Opportunities for Student Involvement

    No

Current Research and Scholarly Interests


Organoid modeling of cancer cells and the tumor immune microenvironment.
We have successfully established primary 3D organoid cultures of diverse tissues and used them to achieve the first in vitro conversion of primary intestine, stomach and pancreas tissue to adenocarcinoma (Ootani et al, Nat Med 2009; Li et al, Nat Med 2014) amongst others. These organoid systems comprise an robust in vitro system which we are exploiting for the functional validation of putative oncogenic loci which are identified in whole-genome cancer surveys such as TCGA. In a new direction, we have developed organoid methods to preserve tumor cells along with a diversity of endogenous infiltrating immune cells (T, B, NK, macrophages) and demonstrated that such organoids are responsive to checkpoint inhibitor therapy (Neal et al, Cell 2018). Further, we have established large biobanks of organoids from clinical cancer biopsies with relevance to tumor modeling and predication of patient responses to therapeutics.

Organoids for regenerative medicine.
We are also interested in using organoids as a method to grow mini-organs that can be transplanted into recipients for regenerative medicine purposes. We are establishing proof-of-principle for human or mouse organoid transplantation, ultimately to effect phenotypic correction of diseases.

Intestinal stem/progenitor biology.
The complete regeneration of the epithelial lining of the intestine every 5-7 days renders the intestine a model system for studying stem cell behaviors. We are investigating the regulation of the intestinal stem cell (ISC) compartment by extracellular signals such as Wnts, using adenoviral and conditional knockout approaches. We have defined R-spondins as dominant regulators of the ISC niche with Wnts playing a more permissive role using lineage tracing, bioengineered Wnts and single cell RNA-seq approaches (Yan et al., Nature, 2017a; Janda et al, Nature 2017b). We have found that Bmi1+ ISC are strongly injury-inducible versusthe homeostatic function of Lgr5+ ISC (c.f. Yan et al, PNAS 2012, Barry et al, Nature 2013) and have enteroendocrine characteristics (Yan et al., Stem Cell, 2017). Further, we have derived robust organoid methods for prolonged culture of and ex vivo expansion of primary intestine and other GI organs, with preservation of ISCs and recapitulation of the Wnt- and Notch-dependent ISC niche, even allowing peristalsis (Ootani et al, Nat Med 2009; Li et al Nat Med 2014).

Angiogenesis and the blood-brain barrier.
We are interested in determining functions of novel molecules regulating angiogenesis including receptors such as GPCRs, microRNAs and secreted molecules. We found that GPR124 is essential for developmental brain angiogenesis (Kuhnert et al, Science 2010) that GPR124 is critical for maintaining blood-brain barrier integrity during stroke and brain tumor growth (Chang et al, Nat Med 2017) and that the GPR124-associated protein RECK is a Wnt7 receptor (Vallon et al, Cell Reports, 2018). We have several active projects in stroke and blood-brain barrier (BBB) basic biology and therapeutic development. We have previously exploring the functions of the endothelial miRNA miR-126 in adults using conditional ko mice (Kuhnert et al, Development 2008) and have extensive interests in pharmacologic inhibition of novel targets for anti-angiogenic therapy of cancer and ocular disorders.

2023-24 Courses


Stanford Advisees


All Publications


  • 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

  • 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

  • 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

  • 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

  • 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

  • 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
  • Models for Immuno-oncology Research CANCER CELL Kuo, C. J., Voest, E., Parrini, M., Zou, W., Teng, M. 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

  • 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

  • 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
  • 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

  • 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
  • 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
  • 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-+
  • 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-+
  • 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

  • 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
  • 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. Y., Davis, M. M., Kuo, C. J. 2018; 175 (7): 1972-+
  • The Intestinal Stem Cell Niche: Homeostasis and Adaptations TRENDS IN CELL BIOLOGY Santos, A. M., Lo, Y., Mah, A. T., Kuo, C. J. 2018; 28 (12): 1062–78
  • 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-+
  • 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

  • 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
  • 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

  • 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
  • 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
  • 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
  • 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
  • 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

  • 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 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

  • 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
  • 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
  • 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

  • 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

  • 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

  • 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
  • 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

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

  • 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
  • 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