All Publications


  • Single-cell imaging of T cell immunotherapy responses in vivo. The Journal of experimental medicine Yan, C., Yang, Q., Zhang, S., Millar, D. G., Alpert, E. J., Do, D., Veloso, A., Brunson, D. C., Drapkin, B. J., Stanzione, M., Scarfò, I., Moore, J. C., Iyer, S., Qin, Q., Wei, Y., McCarthy, K. M., Rawls, J. F., Dyson, N. J., Cobbold, M., Maus, M. V., Langenau, D. M. 2021; 218 (10)

    Abstract

    T cell immunotherapies have revolutionized treatment for a subset of cancers. Yet, a major hurdle has been the lack of facile and predicative preclinical animal models that permit dynamic visualization of T cell immune responses at single-cell resolution in vivo. Here, optically clear immunocompromised zebrafish were engrafted with fluorescent-labeled human cancers along with chimeric antigen receptor T (CAR T) cells, bispecific T cell engagers (BiTEs), and antibody peptide epitope conjugates (APECs), allowing real-time single-cell visualization of T cell-based immunotherapies in vivo. This work uncovered important differences in the kinetics of T cell infiltration, tumor cell engagement, and killing between these immunotherapies and established early endpoint analysis to predict therapy responses. We also established EGFR-targeted immunotherapies as a powerful approach to kill rhabdomyosarcoma muscle cancers, providing strong preclinical rationale for assessing a wider array of T cell immunotherapies in this disease.

    View details for DOI 10.1084/jem.20210314

    View details for PubMedID 34415995

    View details for PubMedCentralID PMC8383813

  • Single-cell imaging of human cancer xenografts using adult immunodeficient zebrafish NATURE PROTOCOLS Yan, C., Do, D., Yang, Q., Brunson, D. C., Rawls, J. F., Langenau, D. M. 2020; 15 (9): 3105-3128

    Abstract

    Zebrafish are an ideal cell transplantation model. They are highly fecund, optically clear and an excellent platform for preclinical drug discovery studies. Traditionally, xenotransplantation has been carried out using larval zebrafish that have not yet developed adaptive immunity. Larval engraftment is a powerful short-term transplant platform amenable to high-throughput drug screening studies, yet animals eventually reject tumors and cannot be raised at 37 °C. To address these limitations, we have recently developed adult casper-strain prkdc-/-, il2rgα-/- immunocompromised zebrafish that robustly engraft human cancer cells for in excess of 28 d. Because the adult zebrafish can be administered drugs by oral gavage or i.p. injection, our model is suitable for achieving accurate, preclinical drug dosing. Our platform also allows facile visualization of drug effects in vivo at single-cell resolution over days. Here, we describe the procedures for xenograft cell transplantation into the prkdc-/-, il2rgα-/- model, including refined husbandry protocols for optimal growth and rearing of immunosuppressed zebrafish at 37 °C; optimized intraperitoneal and periocular muscle cell transplantation; and epifluorescence and confocal imaging approaches to visualize the effects of administering clinically relevant drug dosing at single-cell resolution in vivo. After identification of adult homozygous animals, this procedure takes 35 d to complete. 7 days are required to acclimate adult fish to 37 °C, and 28 d are required for engraftment studies. Our protocol provides a comprehensive guide for using immunocompromised zebrafish for xenograft cell transplantation and credentials the model as a new preclinical drug discovery platform.

    View details for DOI 10.1038/s41596-020-0372-y

    View details for Web of Science ID 000561511100001

    View details for PubMedID 32826993

    View details for PubMedCentralID PMC8097243

  • PRL3 enhances T-cell acute lymphoblastic leukemia growth through suppressing T-cell signaling pathways and apoptosis LEUKEMIA Garcia, E. G., Veloso, A., Oliveira, M. L., Allen, J. R., Loontiens, S., Brunson, D., Do, D., Yan, C., Morris, R., Iyer, S., Garcia, S. P., Iftimia, N., Van Loocke, W., Matthijssens, F., McCarthy, K., Barata, J. T., Speleman, F., Taghon, T., Gutierrez, A., Van Vlierberghe, P., Haas, W., Blackburn, J. S., Langenau, D. M. 2021; 35 (3): 679-690

    Abstract

    T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes and is largely driven by the NOTCH/MYC pathway. Yet, additional oncogenic drivers are required for transformation. Here, we identify protein tyrosine phosphatase type 4 A3 (PRL3) as a collaborating oncogenic driver in T-ALL. PRL3 is expressed in a large fraction of primary human T-ALLs and is commonly co-amplified with MYC. PRL3 also synergized with MYC to initiate early-onset ALL in transgenic zebrafish and was required for human T-ALL growth and maintenance. Mass-spectrometry phosphoproteomic analysis and mechanistic studies uncovered that PRL3 suppresses downstream T-cell phosphorylation signaling pathways, including those modulated by VAV1, and subsequently suppresses apoptosis in leukemia cells. Taken together, our studies have identified new roles for PRL3 as a collaborating oncogenic driver in human T-ALL and suggest that therapeutic targeting of the PRL3 phosphatase will likely be a useful treatment strategy for T-ALL.

    View details for DOI 10.1038/s41375-020-0937-3

    View details for Web of Science ID 000544547300001

    View details for PubMedID 32606318

    View details for PubMedCentralID PMC8009053

  • Long-term Effects of an Evidence-based Guideline for Emergency Management of Pediatric Syncope. Pediatric quality & safety Shanahan, K. H., Monuteaux, M. C., Brunson, D., Guse, S. E., Alexander, M. E., Porter, J. J., Neuman, M. I., Fine, A. M. 2020; 5 (6): e361

    Abstract

    Variability exists in the management of childhood syncope as clinicians balance resource utilization with the need to identify serious diseases. Limited evidence exists regarding the long-term impact of evidence-based guidelines (EBGs) on clinical practices. This study's objective was to measure long-term changes in the management of syncope after implementing a syncope EBG in a single pediatric emergency department following the redistribution of resources to facilitate compliance over time.We included healthy patients aged 8-22 years, presenting to the pediatric emergency department with syncope between 2009 and 2017. Interrupted time series analysis compared testing rates and length of stay among the pre-EBG, short-term follow-up, and long-term follow-up periods.The study included 1,294 subjects. From the pre-EBG period to the long-term follow-up period, recommended electrocardiogram and urine pregnancy test rose significantly [level change odds ratio (95% confidence interval) 5.56 (1.73-17.91) and 3.15 (1.07-9.32), respectively]. Testing and management not recommended by the EBG decreased significantly, including complete blood count, electrolytes, point-of-care glucose, chest radiograph, and intravenous fluids [level change odds ratio (95% confidence interval) 0.19 (0.09-0.40), 0.15 (0.07-0.32), 0.38 (0.18-0.81), 0.17 (0.06-0.49), and 0.18 (0.08-0.39), respectively]. Length of stay declined significantly. No delayed diagnoses occurred.Sustained improvements in syncope management persisted during long-term follow-up of the EBG despite minimal resources. The EBG was associated with increased focused evaluation and decreased low yield testing. EBGs may be useful tools to influence sustained clinical practices to promote safe, cost-effective, and high-quality care.

    View details for DOI 10.1097/pq9.0000000000000361

    View details for PubMedID 33134761

    View details for PubMedCentralID PMC7591128

  • Adult immune compromised zebrafish for xenograft cell transplantation studies EBIOMEDICINE Yan, C., Yang, Q., Do, D., Brunson, D. C., Langenau, D. M. 2019; 47: 24-26

    View details for DOI 10.1016/j.ebiom.2019.08.016

    View details for Web of Science ID 000486976200013

    View details for PubMedID 31416720

    View details for PubMedCentralID PMC6796557

  • Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish CELL Yan, C., Brunson, D. C., Tang, Q., Do, D., Iftimia, N. A., Moore, J. C., Hayes, M. N., Welker, A. M., Garcia, E. G., Dubash, T. D., Hong, X., Drapkin, B. J., Myers, D. T., Phat, S., Volorio, A., Marvin, D. L., Ligorio, M., Dershowitz, L., McCarthy, K. M., Karabacak, M. N., Fletcher, J. A., Sgroi, D. C., Iafrate, J. A., Maheswaran, S., Dyson, N. J., Haber, D. A., Rawls, J. F., Langenau, D. M. 2019; 177 (7): 1903-+

    Abstract

    Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.

    View details for DOI 10.1016/j.cell.2019.04.004

    View details for Web of Science ID 000471256800026

    View details for PubMedID 31031007

    View details for PubMedCentralID PMC6570580