Mechanisms of liver metastasis and associated resistance to immunotherapy

项目摘要 癌症免疫疗法已经彻底改变了许多癌症的治疗，包括转移性癌症。 黑色素瘤和透明细胞肾细胞癌（ccRCC）。30年前，第一个癌症 免疫疗法，高剂量白细胞介素-2，在黑色素瘤和ccRCC中成功测试， 提供了第一个概念验证的变革治疗潜力。1 -3在过去的十年中， 黑色素瘤和ccRCC代表了典型的疾病，其中一部分患者 对新型免疫疗法（如免疫检查点抑制剂）产生持久反应 (ICI)，并且这些观察结果也经常外推到其他癌症（例如，肺癌）。 4-8然而，大多数患者对ICI无反应，其潜在机制尚不清楚。 在我们最初资助的基金（R37 CA 258829）中，我们探讨了肝转移的作用及其在 在赋予对ICI的抗性方面，在局部和并发转移部位（例如，肺 转移）。我们利用尖端的模型和技术，包括单细胞基因组学， 分析ICI抵抗的临床相关预测因素，并探索预治疗的机会。 联合治疗的临床试验，可能有助于克服ICI的耐药性。在当前 我们将扩大这些初步研究，研究反应的分子预测因子， 在接受ICI和ICSI联合治疗的转移性ccRCC患者中， 血管内皮生长因子受体（VEGFR）抑制剂。为此，我们 从治疗前和治疗后的患者中收集配对的速冻组织标本， ICI和VEGFR抑制剂治疗。在初步研究中，我们应用了尖端技术 在我们的实验室开发，使多模态单细胞基因组学从冷冻组织 达到优良的技术质量。此外，我们还建立了多重 免疫荧光（mIF）匹配的组织。在目标1中，我们将在确定 ICI + VEGFR抑制剂治疗的时空效应，以及免疫相关变化 与临床结果密切相关。在目标2中，我们将进行多模态单细胞基因组学， 包括RNA测序和T细胞受体测序（TCR），以确定两种癌细胞 单细胞反应和抗性内在和外在分子和克隆相关性 分辨率连同最初资助申请中的拟议工作，该补充将 进一步增强我们对反应的分子和细胞相关性的理解， 对免疫疗法的抵抗。

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.