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

项目摘要 黑色素瘤是最致命的皮肤癌，预计有超过10万例新病例和6,000例死亡 2020年在美国。虽然免疫检查点阻断的出现极大地改善了免疫系统的功能， 在晚期患者中，64%的患者在治疗后5年内进展，这表明 迫切需要提高无应答者的疗效。最近的证据表明，肿瘤浸润 淋巴细胞是阳性预后指标，也是预测免疫检查点应答的生物标志物 封锁因此，增加肿瘤的T细胞浸润的策略具有强大的治疗潜力。这里我 我建议使用内源性的免疫调节剂来阐明T细胞浸润的动力学和机制决定因素。 斑马鱼黑色素瘤模型，该模型证明了天然斑马鱼免疫系统的强大免疫应答 系统我假设肿瘤微环境中的细胞是肿瘤中T细胞浸润的关键调节因子 通过改变细胞因子环境和细胞外基质， 利基在目标1中，我试图描述内源性斑马鱼黑色素瘤中T细胞浸润的动态过程 使用标记细胞毒性T细胞的新型CD 8+报告细胞系和泛T细胞报告细胞系的模型。的能力 在斑马鱼中产生内源性非色素性黑色素瘤允许非侵入性地可视化黑色素瘤。 在整个肿瘤发展过程中，体内肿瘤微环境非常复杂。我还会将交互可视化 使用荧光报告分子标记肿瘤中的优势细胞， 微环境，包括基质细胞、内皮细胞和骨髓细胞。然后我会用单细胞ATAC 测序以表征不同的T细胞亚群，鉴定T细胞亚型特异性调节区，和 为免疫原性和免疫抑制性肿瘤的T细胞开发荧光报告基因。这些记者将 使我能够观察体内T细胞的活性，并建立一个系统来识别改变T细胞行为的因素 免疫原性和免疫抑制性肿瘤。在目标2中，我建议使用单细胞RNA测序， 斑马鱼黑色素瘤，以确定肿瘤微环境中转录不同的细胞亚群 T细胞浸润或未浸润的肿瘤。这将确定关键的微环境基因和途径， 区分T细胞浸润。我将在体内使用组织特异性 过表达或敲除相关基因的表达载体。总的来说，这些提议的实验 将促进对抗肿瘤反应机制的理解，以允许 开发治疗剂以诱导T细胞浸润并扩大应答者群体， 免疫疗法

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.