Mechanisms of tumor microenvironmental regulation of T-cell infiltration in melanoma

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Project Summary Melanoma is the deadliest form of skin cancer, with over 100,000 new cases and 6,000 deaths predicted in the United States in 2020. While the advent of immune checkpoint blockade has greatly improved the prognoses of advanced-stage patients, 64% of patients progress within 5 years post-treatment, indicating a critical need to improve efficacy in non-responders. Recent evidence has demonstrated that tumor-infiltrating lymphocytes are a positive prognostic indicator and a biomarker predictive of response to immune checkpoint blockade. Strategies to increase T-cell infiltration of tumors therefore have strong therapeutic potential. Here, I propose to elucidate the dynamics and mechanistic determinants of T-cell infiltration using an endogenous zebrafish melanoma model that demonstrates a robust immune response by the native zebrafish immune system. I hypothesize that cells in the tumor microenvironment are critical regulators of T-cell infiltration in tumors by alteration of the cytokine milieu and extra-cellular matrix which create an immunogenic or immunosuppressive niche. In aim 1, I seek to describe the dynamic process of T-cell infiltration in an endogenous zebrafish melanoma model using a novel CD8+ reporter line marking cytotoxic T cells and a pan-T cell reporter line. Our ability to generate endogenous, non-pigmented melanomas in the zebrafish allows for non-invasive visualization of the complex, in vivo tumor microenvironment throughout tumor development. I will additionally visualize interactions between T cells and the tumor niche using fluorescent reporters that label prevalent cells in the tumor microenvironment, including stromal cells, endothelial cells, and myeloid cells. I will then use single-cell ATAC- sequencing to characterize distinct T-cell subpopulations, identify T-cell subtype-specific regulatory regions, and develop fluorescent reporters for T cells of immunogenic and immunosuppressive tumors. These reporters will enable me to observe T-cell activity in vivo and establish a system to identify factors that modify T-cell behavior in immunogenic and immunosuppressed tumors. In aim 2, I propose to use single-cell RNA-sequencing of zebrafish melanomas to identify transcriptionally-distinct cellular subpopulations in the tumor microenvironment of T cell-infiltrated or non-infiltrated tumors. This will identify key microenvironmental genes and pathways that demarcate T-cell infiltration. I will functionally evaluate candidate pathways in vivo using tissue-specific expression vectors overexpressing or knocking out relevant genes. Collectively, these proposed experiments will advance understanding of the mechanisms underlying the anti-tumor response in order to allow for the development of therapeutics to induce T-cell infiltration and expand the population of responders to immunotherapy.