Kevin Liu

All Publications

• Field-effect-informed urine liquid biopsy for bladder cancer. Cell Shi, W. Y., Liu, K. J., Esfahani, M. S., Mach, K. E., Phillips, N. A., Almanza, D., Bajpai, R. K., Schroers-Martin, J. G., Trabanino, L., Lee, T. J., La, V., Rodriguez, G., Holton, G., Chen, S. B., Mullane, P., Wu, D. J., Nesselbush, M. C., Sugio, T., Cheng, J. C., Jabara, I., Hamilton, E. G., Alig, S. K., Liu, C. L., Peterson, D. J., Prado, K., Shkolyar, E., Thong, A., Shah, J. B., Gill, H., Kunder, C. A., Chan, E., Khaki, A. R., Skinner, E. C., Alizadeh, A. A., Liao, J. C., Diehn, M. 2026

  Abstract

  Only some non-muscle-invasive bladder cancer (NMIBC) patients benefit from intravesical Bacillus Calmette-Guérin (BCG), and predictive biomarkers remain lacking. While urine tumor DNA (utDNA) analysis is promising, mutations in tumor-adjacent normal urothelium, namely the field effect, limit specificity. Here, we show that the prevalence of somatic mutations in the urine increases with age. We introduce an improved utDNA minimal residual disease (MRD) approach that increases specificity by removing field-effect mutations. Applying this field-effect-informed MRD approach to 261 samples from NMIBC patients undergoing surgery and adjuvant BCG, we identify three molecular response classes, including surgical responders, BCG responders, and non-responders. Molecular predictors of response to the two treatments differ, with pre-existing immune activation and higher mutation burden enriched in BCG but not surgery responders. These findings highlight the potential of field-effect-informed liquid biopsy methods for guiding personalized therapy and uncovering biomarkers for individual components of multimodal treatments.

  View details for DOI 10.1016/j.cell.2025.12.054

  View details for PubMedID 41605210

• An ultrasensitive method for detection of cell-free RNA. Nature Nesselbush, M. C., Luca, B. A., Jeon, Y. J., Jabara, I., Meador, C. B., Garofalo, A., Binkley, M. S., Hui, A. B., van 't Erve, I., Xu, N., Shi, W. Y., Liu, K. J., Sugio, T., Kastelowitz, N., Hamilton, E. G., Liu, C. L., Olsen, M., Bonilla, R. F., Wang, Y. P., Jiang, A., Lau, B., Eichholz, J., Banwait, M., Schroers-Martin, J., Boegeholz, J., King, D. A., Luikart, H., Esfahani, M. S., Mehrmohamadi, M., Stehr, H., Raclin, T., Tibshirani, R., Khush, K., Srinivas, S., Yu, H., Rogers, A. J., Nair, V. S., Isbell, J. M., Li, B. T., Piotrowska, Z., Sequist, L. V., Hata, A. N., Neal, J. W., Wakelee, H. A., Gentles, A. J., Alizadeh, A. A., Diehn, M. 2025

  Abstract

  Sensitive methods for detection of cell-free RNA (cfRNA) could facilitate non-invasive gene expression profiling and monitoring of diseases1-6. Here we describe RARE-seq (random priming and affinity capture of cfRNA fragments for enrichment analysis by sequencing), a method optimized for cfRNA analysis. We demonstrate that platelet contamination can substantially confound cfRNA analyses and develop an approach to overcome it. In analytical validations, we find RARE-seq to be approximately 50-fold more sensitive for detecting tumour-derived cfRNA than whole-transcriptome RNA sequencing (RNA-seq), with a limit of detection of 0.05%. To explore clinical utility, we profiled 437 plasma samples from 369 individuals with cancer or non-malignant conditions and controls. Detection of non-small-cell lung cancer expression signatures in cfRNA increased with stage (6 out of 20 (30%) in stage I; 5 out of 8 (63%) in stage II; 10 out of 15 (67%) in stage III; 80 out of 96 (83% sensitivity) in stage IV at 95% specificity) and RARE-seq was more sensitive than tumour-naive circulating tumour DNA (ctDNA) analysis. In patients with EGFR-mutant non-small-cell lung cancer who developed resistance to tyrosine kinase inhibitors, we detected both histological transformation and mutation-based resistance mechanisms. Finally, we demonstrate the potential utility of RARE-seq for determination of tissue of origin, assessing benign pulmonary conditions and tracking response to mRNA vaccines. These results highlight the potential value of ultrasensitive cfRNA analysis and provide proof of concept for diverse clinical applications.

  View details for DOI 10.1038/s41586-025-08834-1

  View details for PubMedID 40240612

  View details for PubMedCentralID 8060291

• Metabolically purified human stem cell-derived hepatocytes reveal distinct effects of Ebola and Lassa viruses. bioRxiv : the preprint server for biology Prescott, J. B., Liu, K. J., Lander, A., Pek, N. M., Jha, S. K., Bokelmann, M., Begur, M., Koh, P. W., Yang, H., Lim, B., Red-Horse, K., Weissman, I. L., Loh, K. M., Ang, L. T. 2025

  Abstract

  Ebola and Lassa viruses require biosafety-level-4 (BSL4) containment, infect the liver, and cause deadly hemorrhagic fevers. The cellular effects of these viruses, and whether different families of hemorrhagic-fever viruses elicit similar effects, remain fundamental questions in BSL4 virology. Here, we introduce a new metabolic selection approach to create nearly-pure hepatocytes from human pluripotent stem cells, killing non-liver cells by withholding essential nutrients. Unexpectedly, Ebola and Lassa exerted starkly different effects on human hepatocytes. Ebola infection activated the integrated stress response (ISR) and WNT pathways in hepatocytes in vitro and killed them, whereas Lassa did not. Within non-human primates, Ebola likewise infected hepatocytes and activated ISR signaling in vivo . In summary, we present a single-cell transcriptional and chromatin accessibility roadmap of human hepatocyte differentiation, purification, and viral infection.

  View details for DOI 10.1101/2025.02.17.638665

  View details for PubMedID 40027809

  View details for PubMedCentralID PMC11870522

• Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses. Cell Ang, L. T., Nguyen, A. T., Liu, K. J., Chen, A., Xiong, X., Curtis, M., Martin, R. M., Raftry, B. C., Ng, C. Y., Vogel, U., Lander, A., Lesch, B. J., Fowler, J. L., Holman, A. R., Chai, T., Vijayakumar, S., Suchy, F. P., Nishimura, T., Bhadury, J., Porteus, M. H., Nakauchi, H., Cheung, C., George, S. C., Red-Horse, K., Prescott, J. B., Loh, K. M. 2022

  Abstract

  Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

  View details for DOI 10.1016/j.cell.2022.05.024

  View details for PubMedID 35738284

• Genetic elements promote retention of extrachromosomal DNA in cancer cells. Nature Sankar, V., Hung, K. L., Gnanasekar, A., Wong, I. T., Shi, Q., Kraft, K., Jones, M. G., He, B. J., Yan, X., Belk, J. A., Liu, K. J., Agarwal, S., Wang, S. K., Henssen, A. G., Mischel, P. S., Chang, H. Y. 2025

  Abstract

  Extrachromosomal DNA (ecDNA) is a prevalent and devastating form of oncogene amplification in cancer1,2. Circular megabase-sized ecDNAs lack centromeres, stochastically segregate during cell division3-6 and persist over many generations. It has been more than 40 years since ecDNAs were first observed to hitchhike on mitotic chromosomes into daughter cell nuclei, but the mechanism underlying this process remains unclear3,7. Here we identify a family of human genomic elements, termed retention elements, that tether episomes to mitotic chromosomes to increase ecDNA transmission to daughter cells. Using Retain-seq, a genome-scale assay that we developed, we reveal thousands of human retention elements that confer generational persistence to heterologous episomes. Retention elements comprise a select set of CpG-rich gene promoters and act additively. Live-cell imaging and chromosome conformation capture show that retention elements physically interact with mitotic chromosomes at regions that are mitotically bookmarked by transcription factors and chromatin proteins. This activity intermolecularly recapitulates promoter-enhancer interactions. Multiple retention elements are co-amplified with oncogenes on individual ecDNAs in human cancers and shape their sizes and structures. CpG-rich retention elements are focally hypomethylated. Targeted cytosine methylation abrogates retention activity and leads to ecDNA loss, which suggests that methylation-sensitive interactions modulate episomal DNA retention. These results highlight the DNA elements and regulatory logic of mitotic ecDNA retention. Amplifications of retention elements promote the maintenance of oncogenic ecDNA across generations of cancer cells, and reveal the principles of episome immortality intrinsic to the human genome.

  View details for DOI 10.1038/s41586-025-09764-8

  View details for PubMedID 41261124

  View details for PubMedCentralID 9470669

• A human arteriovenous differentiation roadmap reveals vein developmental mechanisms and vascular effects of viruses. bioRxiv : the preprint server for biology Ang, L. T., Zheng, S. L., Liu, K. J., Masaltseva, A., Winters, J., von Creytz, I., Jha, S. K., Yin, Q., Qian, C., Xiong, X., Dailamy, A., Xi, E., Alcocer, J. C., Sorensen, D. W., She, R., Smolyar, K., Szumska, D., Nornes, S., Martin, R. M., Lesch, B. J., Restrepo, N. K., Sun, W., Weissman, J. S., Lickert, H., Porteus, M. P., Skylar-Scott, M. A., Mosimann, C., Sumanas, S., De Val, S., Prescott, J. B., Red-Horse, K., Loh, K. M. 2025

  Abstract

  We map human artery and vein endothelial cell (EC) differentiation from pluripotent stem cells, and employ this roadmap to discover new mechanisms of vascular development (vein differentiation) and disease (viral infection). We discovered vein development unfolds in two steps driven by opposing signals: VEGF differentiates mesoderm into "pre-vein" ECs, but surprisingly, VEGF/ERK inhibition subsequently specifies vein ECs. Pre-vein ECs co-expressed certain arterial (SOX17) and venous (APLNR) markers, harbored poised chromatin at future venous genes, but completed venous differentiation only upon VEGF inhibition. Intersectional lineage tracing revealed that early Sox17+ Aplnr+ ECs also formed veins in vivo. Next, we compared how Ebola, Andes, and Nipah viruses infect artery and vein ECs under biosafety-level-4 containment. Each virus distinctly affected ECs. Interestingly, artery and vein ECs also responded divergently to the same virus, thus revealing that developmentally-specified cell identity impacts viral infection. Collectively, this arteriovenous differentiation roadmap illuminates vascular development and disease.

  View details for DOI 10.1101/2025.10.11.681838

  View details for PubMedID 41279810

  View details for PubMedCentralID PMC12632389

• A microfluidic platform for anterior-posterior human endoderm patterning via countervailing morphogen gradients in vitro ISCIENCE Engel, L., Liu, K. J., Cui, K. W., de la Serna, E. L., Vachharajani, V. T., Dundes, C. E., Zheng, S., Begur, M., Loh, K. M., Ang, L., Dunn, A. R. 2025; 28 (3)

  View details for DOI 10.1016/j.isci.2025.111744

  View details for Web of Science ID 001430645400001

• A microfluidic platform for anterior-posterior human endoderm patterning via countervailing morphogen gradients in vitro. iScience Engel, L., Liu, K. J., Cui, K. W., de la Serna, E. L., Vachharajani, V. T., Dundes, C. E., Zheng, S. L., Begur, M., Loh, K. M., Ang, L. T., Dunn, A. R. 2025; 28 (3): 111744

  Abstract

  Understanding how morphogen gradients spatially pattern tissues is a fundamental question in developmental biology but can be difficult to directly address using conventional approaches. Here, we expose hPSC-derived endoderm cells to countervailing gradients of anteriorizing and posteriorizing signals using a widely available microfluidic device. This approach yielded spatially patterned cultures comprising anterior foregut (precursor to the thyroid, esophagus, and lungs) and mid/hindgut (precursor to the intestines) cells, whose identities were confirmed using single-cell RNA sequencing (scRNA-seq). By exposing stem cells to externally applied signaling gradients, this widely accessible microfluidic platform should accelerate the production of spatially patterned tissues, complementing internally self-organizing organoids. Applying artificial morphogen gradients in vitro may also illuminate how developing tissues interpret signaling gradients in systems that are not readily accessible for in vivo studies.

  View details for DOI 10.1016/j.isci.2025.111744

  View details for PubMedID 40040808

  View details for PubMedCentralID PMC11879597

• Protocol for efficient generation of human artery and vein endothelial cells from pluripotent stem cells. STAR protocols Loh, K. M., Zheng, S. L., Liu, K. J., Yin, Q., Amir-Ugokwe, Z. A., Jha, S. K., Qi, Y., Wazny, V. K., Nguyen, A. T., Chen, A., Njunkeng, F. M., Cheung, C., Spiekerkoetter, E., Red-Horse, K., Ang, L. T. 2024; 6 (1): 103494

  Abstract

  Blood vessels permeate all organs and execute myriad roles in health and disease. Here, we present a protocol to efficiently generate human artery and vein endothelial cells (ECs) from pluripotent stem cells within 3-4 days of differentiation. We delineate how to seed human pluripotent stem cells and sequentially differentiate them into primitive streak, lateral mesoderm, and either artery or vein ECs. We differentiate stem cells in defined, serum-free culture media in monolayers, without feeder cells or genetic manipulations. For complete details on the use and execution of this protocol, please refer to Ang et al. 1.

  View details for DOI 10.1016/j.xpro.2024.103494

  View details for PubMedID 39705146

• Protease Activated Probes for Real-Time Ratiometric Imaging of Solid Tumors ACS CENTRAL SCIENCE Faucher, F. F., Liu, K. J., Cosco, E. D., Widen, J. C., Sorger, J., Guerra, M., Bogyo, M. 2023: 1059-1069

  Abstract

  Surgery is the preferred treatment option for most solid tumors. However, inaccurate detection of cancer borders leads to either incomplete removal of malignant cells or excess excision of healthy tissue. While fluorescent contrast agents and imaging systems improve tumor visualization, they can suffer from low signal-to-background and are prone to technical artifacts. Ratiometric imaging has the potential to eliminate many of these issues such as uneven probe distribution, tissue autofluorescence, and changes in positioning of the light source. Here, we describe a strategy to convert quenched fluorescent probes into ratiometric contrast agents. Conversion of the cathepsin-activated probe, 6QC-Cy5, into a two-fluorophore probe, 6QC-RATIO, significantly improved signal-to-background in vitro and in a mouse subcutaneous breast tumor model. Tumor detection sensitivity was further enhanced using a dual-substrate AND-gate ratiometric probe, Death-Cat-RATIO, that fluoresces only after orthogonal processing by multiple tumor-specific proteases. We also designed and built a modular camera system that was coupled to the FDA-approved da Vinci Xi robot, to enable real-time imaging of ratiometric signals at video frame rates compatible with surgical workflows. Our results demonstrate that ratiometric camera systems and imaging probes have the potential to be clinically implemented to improve surgical resection of many types of cancer.

  View details for DOI 10.1021/acscentsci.3c00261

  View details for Web of Science ID 000985613600001

  View details for PubMedID 37252358

  View details for PubMedCentralID PMC10214504

• Controversies surrounding the origin of hepatocytes in adult livers and the in vitro generation or propagation of hepatocytes. Cellular and molecular gastroenterology and hepatology Qian Pek, N. M., Liu, K. J., Nichane, M. n., Ang, L. T. 2020

  Abstract

  Epithelial cells in the liver (known as hepatocytes) are high-performance engines of myriad metabolic functions and versatile responders to liver injury. As hepatocytes metabolize amino acids, alcohol, drugs and other substrates, they produce and are exposed to a milieu of toxins and harmful byproducts that can damage themselves. In the healthy liver, hepatocytes generally divide slowly. However, after liver injury, hepatocytes can ramp up proliferation to regenerate the liver. Yet, upon extensive injury, regeneration falters and liver failure ensues. It is therefore critical to understand the mechanisms underlying liver regeneration, and in particular, which liver cells are mobilized during liver maintenance and repair. Controversies continue to surround the very existence of hepatic stem cells and, if they exist, their spatial location, multipotency, degree of contribution to regeneration, ploidy, and susceptibility to tumorigenesis. This review discuss these controversies. Finally, we highlight how insights into hepatocyte regeneration and biology in vivo can inform in vitro studies to propagate primary hepatocytes with signals in liver regeneration and to generate hepatocytes de novo from pluripotent stem cells.

  View details for DOI 10.1016/j.jcmgh.2020.09.016

  View details for PubMedID 32992051

• Circulating tumor cells exhibit metastatic tropism and reveal brain metastasis drivers. Cancer discovery Klotz, R. n., Thomas, A. n., Teng, T. n., Han, S. M., Iriondo, O. n., Li, L. n., Restrepo-Vassalli, S. n., Wang, A. n., Izadian, N. n., MacKay, M. n., Moon, B. S., Liu, K. J., Ganesan, S. K., Lee, G. n., Kang, D. S., Walmsley, C. S., Pinto, C. J., Press, M. F., Lu, W. n., Lu, J. n., Juric, D. n., Bardia, A. n., Hicks, J. n., Salhia, B. n., Attenello, F. J., Smith, A. D., Yu, M. n. 2019

  Abstract

  Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTCs) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from breast cancer patients are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D (SEMA4D) as a regulator of tumor cell transmigration through the blood-brain-barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis.

  View details for DOI 10.1158/2159-8290.CD-19-0384

  View details for PubMedID 31601552