SUMMARY Tumors are complex systems composed of genetically and transcriptionally heterogeneous cells, and this heterogeneity has been implicated as a cause of drug resistance and overall mortality. Understanding intra- tumor heterogeneity is therefore likely to have widespread impact, both fundamental and clinical. The advent of single-cell RNA-Seq has led to the detection of transcriptionally distinct states among cancer cells across a wide range of tumor types and stages. However, the field lacks robust computational and experimental technologies to functionally identify and characterize these cancer cell states. In this project, we take a gene module-centric view to define cell states in a rigorous and widely applicable manner. We will validate the importance of each cell state using human samples and the zebrafish melanoma model, which has exceptional capabilities for imaging and perturbation of cell states throughout tumor progression and metastasis. To systematically characterize these cell states experimentally, we propose methods to assay their interdependencies within the tumor and with elements of the microenvironment. Our genetically engineered zebrafish will mark each cancer cell state with a fluorescent reporter and an ablation cassette, providing a flexible experimental platform to study and perturb each cell state. The reporter will enable us to sort cancer cell states and study them individually, with a particular emphasis on their plasticity. By systematically disrupting cancer cell states, we will further elucidate their individual contributions to tumor initiation, progression and metastasis. Using a spatial transcriptomics approach and integrating with single-cell RNA-seq data, we will map cancer cell states in relation to their microenvironment to screen for putative interactions. Finally, we will directly test predicted interactions between specific cancer cell states and the immune compartment using T cell-deficient fish. Throughout our three Aims, we adopt a systems biology workflow that iteratively cycles through modes of observations, perturbations, and refinement of our model of the functional role of cancer cell states during tumor progression. Our proposal collectively integrates the complementary expertise of the White and Yanai labs and sets out to significantly improve our understanding of intratumoral heterogeneity through the lens of cancer cell states.

NARRATIVE In this proposal, we describe a systems biology approach to investigate tumor progression and metastasis through the lens of cancer cell states. The proposal involves a strongly complementary collaboration between Dr. White who is an oncologist with expertise in zebrafish cancer modeling, and Dr. Yanai who has expertise in analyzing transcriptional heterogeneity at the single-cell level. Overall this proposal is innovative in its integration of today’s most cutting-edge molecular and cellular methods for the analysis of transcriptomic data, and uses a tractable genetic system that allows us to functionally study and perturb tumorigenesis over space and time.