Investigating Amino Acid Depletion in the Tumor Microenvironment as a Metabolic Immune Checkpoint

项目概要/摘要 激活患者的免疫系统以靶向癌细胞已成为一种有效的治疗策略。 然而，许多癌症亚型，包括上皮恶性肿瘤，如胰腺癌、乳腺癌和结直肠癌， 癌症对现有的T细胞疗法的反应仍然很差。为什么基于免疫的策略表现如此糟糕 治疗这些常见癌症的成功率尚不清楚。除了表征抑制性检查点之外， 抑制T细胞活化，我们的实验室先前证明活化的T细胞依赖于 细胞外葡萄糖和氨基酸以维持其活力和效应子功能。在过去的几年里， 我们一直在研究肿瘤中多种细胞类型的代谢特性， 微环境除了肿瘤细胞消耗葡萄糖和游离氨基酸外，我们还发现， 肿瘤相关的成纤维细胞可以消耗谷氨酰胺和其他非必需氨基酸从细胞外 空间特别是，肿瘤和成纤维细胞依赖的谷氨酰胺和胱氨酸（半胱氨酸）消耗使其 肿瘤微环境中的细胞难以维持蛋白质翻译和氧化还原稳态。我们 已经揭示了肿瘤和基质细胞适应氨基酸耗尽的多种新机制， 包括通过巨胞饮作用利用细胞外蛋白质作为氨基酸的来源， 翻译的改变和转硫途径的激活以维持内源性半胱氨酸水平 和谷胱甘肽水平肿瘤浸润性免疫细胞是否可以使用任何或所有这些适应， 未知我们的初步证据表明，T细胞无法维持其活力和效应细胞。 当谷氨酰胺和/或半胱氨酸耗尽时起作用。因此，我们假设肿瘤 微环境通过消耗这些非必需氨基酸来驱动免疫抑制。为了解决这个 假设，我们提出了三个具体的目的：1）研究是否癌症相关的成纤维细胞有助于 通过消耗细胞外游离氨基酸进行免疫抑制，2）确定 谷氨酰胺耗竭损害抗肿瘤免疫功能，和3）检查肿瘤中半胱氨酸耗竭是否 浸润免疫细胞导致线粒体氧化还原稳态受损和/或效应子功能降低。 通过这些研究，我们希望证明谷氨酰胺和半胱氨酸的消耗如何驱动 肿瘤微环境中的免疫抑制，并确定这些氨基酸 可以恢复到维持抗肿瘤免疫反应的水平。

PROJECT SUMMARY/ABSTRACT Activating a patient's immune system to target cancer cells has emerged as an effective therapeutic strategy. However, many cancer subtypes, including epithelial malignancies such as pancreatic, breast, and colorectal cancers still respond poorly to existing T-cell therapies. Why immune-based strategies have shown such poor success in treating these common cancers is not known. In addition to characterizing the inhibitory checkpoints that restrain T-cell activation, our laboratory previously demonstrated that activated T-cells depend on extracellular glucose and amino acids to sustain their viability and effector function. Over the last several years, we have been studying the metabolic properties of the multiple cell types present in the tumor microenvironment. In addition to tumor cell consumption of glucose and free amino acids, we have found that tumor-associated fibroblasts can deplete glutamine and other nonessential amino acids from the extracellular space. In particular, the tumor and fibroblast-dependent depletion of glutamine and cystine (cysteine) makes it difficult for cells in the tumor microenvironment to maintain protein translation and redox homeostasis. We have uncovered multiple novel mechanisms by which tumor and stromal cells adapt to amino acid depletion, including utilizing extracellular proteins as a source of amino acids through macropinocytosis, adaptive alterations in translation, and activation of the transsulfuration pathway to maintain endogenous cysteine levels and glutathione levels. Whether tumor-infiltrating immune cells can use any or all of these adaptations is unknown. Our preliminary evidence suggest that T-cells are unable to maintain their viability and effector function when depleted of either glutamine and/or cysteine. Therefore, we hypothesize that the tumor microenvironment drives immunosuppression by depleting these non-essential amino acids. To address this hypothesis, we propose three Specific Aims: 1) Study whether cancer-associated fibroblasts contribute to immunosuppression by depleting extracellular free amino acids, 2) Determine the mechanism by which glutamine depletion impairs anti-tumor immune function, and 3) Examine whether cysteine depletion in tumor- infiltrating immune cells results in impaired mitochondrial redox homeostasis and/or reduced effector function. Through these studies, we hope to demonstrate how depletion of glutamine and cysteine drives immunosuppression within the tumor microenvironment and identify mechanisms by which these amino acids can be restored to levels that will sustain an anti-tumor immune response.