(PQ5) Mitochondrial Heterogeneity in Melanoma Tumor and Immune Responses

项目摘要 本提案直接响应RFA-CA-17-017 PQ 5：线粒体异质性如何 影响肿瘤发生或进展？肿瘤的发生、生长、死亡和转移与 细胞代谢和信号传导的显著变化，其中心是线粒体。线粒体 是调节能量代谢、信号传导和凋亡的复杂细胞器， 线粒体DNA（mtDNA），从内部硬连接呼吸和ATP生产。人们往往低估了 不同细胞类型中的线粒体，甚至同一类型的单个细胞中的线粒体，功能不同， 动态（位置，形状和运动）基本上和对压力的反应。目前还不清楚 线粒体异质性的这些方面如何促进肿瘤发生。显著变化 在肿瘤中发生葡萄糖代谢和线粒体呼吸（“瓦尔堡效应”）;然而， 新陈代谢的适应性既不是统一的，也不是静止的。如线粒体与肿瘤代谢 细胞，浸润免疫细胞和基质细胞，是多样化的，并对不断变化的环境作出反应。 压力，包括营养和氧气的可用性，pH值和生长因子的变化。总体 该提案的主题是，线粒体呼吸、网络动力学和 mtDNA-干扰素（IFN）信号不仅影响癌细胞的代谢和生长， 他们对免疫反应和免疫疗法的敏感性。在目标1中，独特的小鼠黑色素瘤细胞系 将使用在免疫原性以及线粒体免疫原性方面表现出显著差异的 呼吸和mtDNA水平。在这些细胞中，线粒体呼吸将被激活或抑制， 分别敲除Mcj或Cox 10基因，以及肿瘤生长、免疫反应性和对 抗PD 1检查点阻断（免疫疗法）将被解决，以直接检查线粒体如何 异质性与免疫原性/免疫逃避有关。线粒体动力学中的异质性， 还将使用微流体、单细胞方法直接评估代谢。最后，作为一个潜在的 治疗途径，以增强抗肿瘤免疫力，T细胞将被工程化，以增加线粒体 呼吸和ATP的产生。在目标2中，重点将放在线粒体DNA应激介导的IFN信号转导的作用上。 以及它是否是肿瘤生长、免疫反应和免疫治疗敏感性差异的基础。在 目的3，将患者来源的异种移植物与其肿瘤浸润淋巴细胞配对的模型（PDX-TIL模型） 将进行分析，以探讨线粒体呼吸和线粒体DNA应激的异质性的相关性 人类癌症中的信号。这项工作有可能阐明肿瘤中新的候选途径， 免疫细胞来增强抗癌疗法和刺激抗肿瘤免疫。

PROJECT SUMMARY This proposal directly responds to RFA-CA-17-017 PQ5: How does mitochondrial heterogeneity influence tumorigenesis or progression? Tumor initiation, growth, death and metastasis are associated with significant changes in cell metabolism and signaling, central to which are mitochondria. Mitochondria are complex organelles that regulate energy metabolism, signaling and apoptosis, and contain mitochondrial DNA (mtDNA) that hardwires respiration and ATP production from within. It is often underappreciated that mitochondria in different cell types, and even within individual cells of the same type, vary in function and dynamics (location, shape and movement) basally and in response to stress. It is presently unclear how these aspects of mitochondrial heterogeneity contribute to tumorigenesis. Striking changes in glucose metabolism and mitochondrial respiration occur in tumors (the “Warburg effect”); however, these metabolic adaptations are neither uniform nor static. For example, mitochondria and metabolism of tumor cells, infiltrating immune cells and stromal cells, are diverse and responsive to ever-changing environmental stresses, including alterations in nutrient and oxygen availability, pH, and growth factors. The overarching theme of this proposal is that the heterogeneity in mitochondrial respiration, network dynamics and mtDNA-interferon (IFN) signaling not only affects cancer cell metabolism and growth, but also impacts their sensitivity to immune responses and immunotherapy. In Aim 1, unique mouse melanoma cell lines will be employed that exhibit significant differences in immunogenicity as well as mitochondrial respiration and mtDNA levels. In these cells, mitochondrial respiration will be activated or inhibited via knock-out of the Mcj or Cox10 genes, respectively, and tumor growth, immunoreactivity, and responses to anti-PD1 checkpoint blockade (immunotherapy) will be addressed to directly examine how mitochondrial heterogeneity links with immunogenicity/immunoevasion. Heterogeneity in mitochondrial dynamics and metabolism will also be assessed directly using microfluidic, single-cell approaches. Finally, as a potential therapeutic avenue to enhance anti-tumor immunity, T cells will be engineered to increase mitochondrial respiration and ATP production. In Aim 2, the focus will be on the role of mtDNA-stress mediated IFN signaling and whether it underlies differences in tumor growth, immune responses and sensitivity to immunotherapy. In Aim 3, models that pair patient-derived xenografts with their tumor infiltrating lymphocytes (PDX-TIL models) will be analyzed to probe the relevance of heterogeneity in mitochondrial respiration and mtDNA-stress signaling in human cancer. This work has the potential to illuminate new candidate pathways in tumor and immune cells to enhance anti-cancer therapies and stimulate anti-tumor immunity.