Mechanisms of liver metastasis and associated resistance to immunotherapy

PROJECT SUMMARY Cancer immunotherapies have revolutionized the therapy of many cancers, including metastatic melanoma and clear cell renal cell carcinoma (ccRCC). Dating back 30-years, the first cancer immunotherapy, high-dose interleukin-2, was successfully tested in melanoma and ccRCC and provided a first proof-of-concept of a transformative therapeutic potential.1-3 In the last decade,

melanoma and ccRCC have represented archetypical diseases, in which a portion of patients experience durable responses to novel immunotherapies, such as immune checkpoint inhibitors (ICI), and these observations frequently extrapolated to other cancers as well (e.g., lung cancer). 4-8 However, most patients do not response to ICI and the underlying mechanisms are unclear.

In our initially funded grant (R37CA258829), we explore the role of liver metastasis and their role in conferring resistance to ICI, both locally and in concurrent metastatic sites (e.g., lung metastases). We leverage cutting-edge models and technologies, including single-cell genomics, to dissect this clinically relevant predictor for ICI resistance, and explore opportunities for pre-

clinical testing of combination therapies that may help overcoming resistance to ICI. In the current proposal, we will expand on these initial studies and study molecular predictors of response and resistance in patients with metastatic ccRCC who are treated with a combination of ICI and inhibitors of vascular endothelial growth factor receptors (VEGFR). For this purpose, we have

collected paired snap-frozen tissue specimens from patients before therapy and after a course of ICI and VEGFR inhibitor therapy. In preliminary studies, we applied cutting-edge technologies developed in our laboratory to enable multi-modal single-cell genomics from frozen tissues achieving excellent technical quality. Furthermore, we established panels for multiplexed

immunofluorescence (mIF) on matching tissues. In Aim 1, we will perform mIF on determining spatio-temporal effects of ICI + VEGFR inhibitor therapy, and correlate changes in immune infiltrates with clinical outcomes. In Aim 2, we will perform multi-modal single-cell genomics, including RNA-sequencing and T cell receptor sequencing (TCR) to determine both cancer cell

intrinsic and extrinsic molecular and clonal correlates of response and resistance at single-cell resolution. Together with the proposed work in the initial grant application, this supplement will further enhance our understanding of molecular and cellular correlates of response and resistance to immunotherapies.