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

黑色素瘤T细胞浸润的肿瘤微环境调节机制

• 批准号: 10358533
• 负责人: Georgia Stirtz
• 金额: $ 3.45万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10358533
• 关键词: Aftercare; Antitumor Response; Bioinformatics; CD8B1 gene; Cancer Patient; Candidate Disease Gene; Cell Communication; Cell Mobility; Cells; Cessation of life; Chromatin; Complex; Cytotoxic T-Lymphocytes; Data; Development; Endothelial Cells; Extracellular Matrix; Future; Genes; Genetic; Genetic Transcription; Genomic Segment; Immune; Immune response; Immune system; Immunotherapy; Infiltration; Knock-out; Label; Modeling; Myeloid Cells; Nucleic Acid Regulatory Sequences; Pathway interactions; Patients; Population; Process; Prognosis; Regulatory Element; Reporter; Research; Skin Cancer; Stromal Cells; System; T cell regulation; T cell response; T-Lymphocyte; Testing; Therapeutic; Tissues; Transgenic Organisms; Tumor Immunity; Tumor-Associated Process; Tumor-Infiltrating Lymphocytes; Tumor-infiltrating immune cells; United States; Visualization; XCL1 gene; Zebrafish; cancer immunotherapeutics; cell behavior; cytokine; experimental study; expression vector; immune checkpoint blockade; immunogenic; immunosuppressed; improved; in vitro Model; in vivo; in vivo fluorescence imaging; lymphatic vessel; melanoma; neoplastic cell; novel; overexpression; predicting response; predictive marker; prognostic indicator; recruit; response biomarker; single-cell RNA sequencing; survival outcome; therapeutic development; transplant model; tumor; tumor microenvironment

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.

项目概要 黑色素瘤是最致命的皮肤癌，预计将有超过 100,000 例新病例和 6,000 人死亡 2020年在美国。虽然免疫检查点封锁的出现大大改善了 晚期患者的预后，64%的患者在治疗后5年内病情进展，表明 迫切需要提高无反应者的疗效。最近的证据表明，肿瘤浸润 淋巴细胞是积极的预后指标和预测免疫检查点反应的生物标志物 封锁。因此，增加肿瘤 T 细胞浸润的策略具有强大的治疗潜力。在这里，我 提议利用内源性阐明 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展示了天然斑马鱼免疫的强大免疫反应 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，产生免疫原性或免疫抑制性 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 该模型使用标记细胞毒性 T 细胞的新型 CD8+ 报告基因系和泛 T 细胞报告基因系。我们的能力 在斑马鱼中产生内源性、非色素性黑色素瘤，可以对斑马鱼进行非侵入性可视化 整个肿瘤发育过程中复杂的体内肿瘤微环境。我还会将交互可视化 使用标记肿瘤中常见细胞的荧光报告基因在 T 细胞和肿瘤微环境之间进行检测 微环境，包括基质细胞、内皮细胞和骨髓细胞。然后我将使用单细胞 ATAC- 测序以表征不同的 T 细胞亚群，识别 T 细胞亚型特异性调节区域，以及 开发免疫原性和免疫抑制性肿瘤 T 细胞的荧光报告基因。这些记者将 使我能够观察体内 T 细胞活性并建立一个系统来识别改变 T 细胞行为的因素 在免疫原性和免疫抑制性肿瘤中。在目标 2 中，我建议使用单细胞 RNA 测序 斑马鱼黑色素瘤识别肿瘤微环境中转录不同的细胞亚群 T细胞浸润或非浸润的肿瘤。这将确定关键的微环境基因和途径 标定 T 细胞浸润。我将使用组织特异性功能评估体内候选途径 过度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 将增进对抗肿瘤反应潜在机制的理解，以便能够 开发诱导 T 细胞浸润并扩大反应者群体的疗法 免疫疗法。