Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

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.

The Drosophila homolog of human tumor suppressor TSC-22 promotes cellular growth, proliferation, and survival.

TSC22D1, which encodes transforming growth factor beta-stimulated clone 22 (TSC-22), is thought to be a tumor suppressor because its expression is lost in many glioblastoma, salivary gland, and prostate cancers. TSC-22 is the founding member of the TSC-22/DIP/Bun family of leucine zipper transcription factors; its functions have not been investigated in a multicellular environment. Genetic studies in the model organism Drosophila melanogaster often provide fundamental insights into mechanisms disrupted in carcinogenesis, because of the strong evolutionary conservation of molecular mechanisms between flies and humans. Whereas humans and mice have four TSC-22 domain genes with numerous isoforms, Drosophila has only one TSC-22 domain gene, bunched (bun), which encodes both large and small protein isoforms. Surprisingly, Drosophila Bun proteins promote cellular growth and proliferation in ovarian follicle cells. Loss of both large isoforms has the strongest phenotypes, including increased apoptosis. Cultured S2 cells depleted for large Bun isoforms show increased apoptosis and less frequent cell division, with decreased cell size. Altogether, these data indicate that Drosophila TSC-22/DIP/Bun proteins are necessary for cellular growth, proliferation, and survival both in culture and in an epithelial context. Previous work demonstrated that bun prevents recruitment of epithelial cells to a migratory fate and, thus, maintains epithelial organization. We speculate that reduced TSC22D1 expression generally reduces cellular fitness and only contributes to carcinogenesis in specific tissue environments.

Single-cell imaging of T cell immunotherapy responses in vivo.

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.