CTL-killing capacity and cancer stiffness in cancer immunity and therapy

项目总结/摘要：CD 8 + T细胞（CTL）介导保护性肿瘤免疫。肿瘤的目标 免疫治疗是产生长期的保护性效应T细胞免疫，并导致肿瘤放射， 癌症患者。为此，肿瘤细胞必须是可接受的并且对CTL介导的肿瘤杀伤敏感。 然而，除了CTL的质量和数量，是什么决定了肿瘤细胞对CTL的感受性？ 介导的肿瘤杀伤知之甚少。因此，有必要进行全面的分子和功能研究， 研究人类肿瘤微环境中肿瘤细胞对CTL的感受性的性质。 肿瘤生物学家一直在研究肿瘤代谢如何使肿瘤细胞增殖，存活， 没有全面考虑免疫参与的侵袭性。近期免疫学 研究已经开始检查肿瘤代谢如何影响不同免疫细胞的表型和功能， 肿瘤微环境中的细胞亚群。然而，肿瘤代谢如何影响肿瘤细胞对CTL的感受性， 杀人基本上是未知的。建立了光镊显微镜（OTM）技术和原子能 力显微镜（AFM）用于细胞弹性和刚度测量，并应用气流辅助 解吸电喷雾电离质谱成像（AFANOM-MSI），我们能够初步 证明人和小鼠乳腺肿瘤细胞具有特定的氨基酸摄取能力， 降低肌球蛋白II介导的收缩激活和肿瘤细胞僵硬。肿瘤硬度的丧失导致 肿瘤细胞膜对穿孔素钻孔力和CTL孔形成的抵抗，导致T细胞功能受损。 细胞介导的肿瘤杀伤。从机制上讲，肿瘤细胞对某些氨基酸上瘾，包括 谷氨酰胺，通过高表达SLC 6A 14，谷氨酰胺转运蛋白。有趣的是，肿瘤SLC 6A 14表达 是由缺氧控制的这些数据揭示了以前未知的机制之间的联系具体 肿瘤微环境中的氨基酸代谢、缺氧和T细胞免疫，从而鉴定 癌症谷氨酰胺转运蛋白作为潜在的新型免疫代谢检查点靶点。 基于这些令人惊讶和新颖的发现，我们假设癌细胞硬度决定肿瘤细胞 对CTL杀伤的感受性，以及缺氧和特定氨基酸摄取之间的相互作用是一种新的 肿瘤微环境中的免疫逃避机制。我们提出了两个具体目标和8个分目标， 测试我们的中心假设，即缺氧靶向肿瘤氨基酸转运蛋白，例如SLC 6A 14， 改变肿瘤细胞对CTL杀伤和肿瘤免疫和治疗的感受性。我们的具体目标是： 目的1是验证我们的假设，即缺氧代谢减弱肿瘤对CTL杀伤的感受性。 目的2是检验我们的假设，即缺氧机械地削弱肿瘤对CTL杀伤的感受性。

Project Summary/Abstract: CD8+ T cells (CTLs) mediate protective tumor immunity. The goal of tumor immune therapy is to engender long-term protective effector T cell immunity and cause tumor eradiation in patients with cancer. To this end, tumor cells must be receptive and susceptible to CTL-mediated tumor killing. However, in addition to the quality and quantity of CTLs, what determines tumor cell receptivity to CTL- mediated tumor killing is poorly understood. It is essential to conduct comprehensive molecular and functional research on the nature of tumor cell receptivity to CTLs in the human tumor microenvironment. Tumor biologists have been working on how tumor metabolism enables tumor cell proliferation, survival, and invasiveness without a comprehensive consideration of the immune involvement. Recent immunological studies have started to examine how tumor metabolism affects the phenotype and function of different immune cell subsets in the tumor microenvironment. Yet, how tumor metabolism affects tumor cell receptivity to CTL- killing is basically unknown. Having established an Optical Tweezers Microscope (OTM) technique and Atomic Force Microscope (AFM) for cell elasticity and stiffness measurements and applied an airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI), we are able to preliminarily demonstrate that human and mouse breast tumor cells empowered with particular amino acid uptake resulted in reduced myosin II-mediated contractile activation and tumor cell stiffness. Loss of tumor stiffness led to tumor cell membrane resistance to perforin drilling force and pore formation by CTLs, resulting in impaired T cell-mediated tumor killing. Mechanistically, tumor cells were addicted to certain amino acids, including glutamine, via high expression of SLC6A14, a glutamine transporter. Interestingly, tumor SLC6A14 expression was controlled by hypoxia. These data reveal previously unknown mechanisms of connection between specific amino acid metabolism, hypoxia, and T cell immunity in the tumor microenvironment, thereby identifying cancer glutamine transporter(s) as a potential novel immune metabolic checkpoint target. Based on these surprising and novel findings, we hypothesize that cancer cell stiffness determines tumor cell receptivity to CTL-killing, and the interplay between hypoxia and particular amino acid uptake is a novel immune evasion mechanism in the tumor microenvironment. We propose two specific aims and 8 subaims to test our central hypothesis that hypoxia targets tumor amino acid transporters, exemplified by SLC6A14, to alter tumor cell receptivity to CTL-killing and tumor immunity and therapy. Our specific aims are: Aim 1 is to test our hypothesis that hypoxia metabolically weakens tumor receptivity to CTL-killing. Aim 2 is to test our hypothesis that hypoxia mechanically weakens tumor receptivity to CTL-killing.