Innate Immune Mechanisms Contributing to Cancer Growth in Obesity

项目概要/摘要 结直肠癌（CRC）是美国癌症死亡率的第二大原因，而肥胖， 影响40%的人口，不仅增加了患CRC的风险，而且增加了CRC的死亡率 通过未知的机制。我们的初步研究表明，CRC在小鼠中生长得更快， 通过高脂饮食（HFD）肥胖，并且这些小鼠中的肿瘤相关巨噬细胞（TAM）表现出 酸敏感受体GPR 65的更高表达，已知其抑制免疫应答。 此外，在缺乏GRP 65的HFD诱导的肥胖小鼠中，TAM分泌更多的TNF-α，肿瘤生长受阻。 考虑到TAMs而非肥胖小鼠正常组织巨噬细胞表现出增加的GPR 65，我们检查了TAMs中的GPR 65。 这些小鼠肿瘤的pH值，发现它们更酸性。基于这些发现，我们假设 HFD中过量的脂质改变了肿瘤细胞的代谢，导致酸的产生增加， TAMs中的GPR 65表达和信号传导，导致它们成为免疫抑制剂并促进肿瘤 增长为了检验这一假设，我们将追求以下具体目标：1）确定 GPR 65信号传导对TAM功能和肥胖条件下CRC生长的影响，通过确定cAMP- 在肥胖小鼠和对照组的TAM中，GPR 65下游起作用的PKA信号传导轴被激活 TNF-α产生。我们还将分析巨噬细胞的GPR 65表达和细胞因子分泌能力， 和来自非肥胖和肥胖患者的CRC样品中的TAM; 2）鉴定 HFD促进CRC TAM中GPR 65信号传导的机制，通过测试HFD和油酸 脂肪酸，一种在HFD肿瘤中高度富集的膳食甘油三酯，以改变氧化电位， 通过流式细胞术、CyTOF和Seahorse测定法测定人肿瘤细胞的氧化能力和酸产生。 我们还将确定高油酸饮食是否足以改变GPR 65表达和细胞因子产生 通过TAM，并检查肿瘤酸度是否需要TAM的钝化炎症反应;并验证 GPR 65在人巨噬细胞中的作用和3）评估靶向GPR 65对肿瘤的作用 在患有CRC和其他肿瘤类型（即肝细胞癌）的肥胖和非肥胖小鼠中的免疫治疗 癌和黑色素瘤，并评估检查点阻断抗体对肿瘤生长和抗- 携带这些肿瘤的GPR 65+和GPR-/-小鼠的肿瘤免疫。这些研究的结果预计将 不仅揭示了肥胖症患者肿瘤生长加速的关键机制， 为这些癌症的治疗找到新的靶点。

Project Summary/Abstract Colorectal cancer (CRC) is the second leading cause of cancer mortality in the United States, and obesity, which affects 40% of the population, not only increases the risk of developing CRC, but also increases CRC mortality through unknown mechanisms. Our preliminary studies demonstrate that CRC grows faster in mice rendered obese through a high fat diet (HFD), and that the tumor associated macrophages (TAMs) in these mice exhibit higher expression of the acid sensing receptor, GPR65, which is known to dampen the immune response. Moreover, in HFD-induced obese mice lacking GRP65, TAMs secrete more TNF-α and tumor growth is retarded. Given that TAMs but not normal tissue macrophages of obese mice exhibit increased GPR65, we examined the pH of the tumors in these mice and found them to be more acidic. On the basis of these findings, we hypothesize that excess lipids in the HFD alter tumor cell metabolism resulting in increased acid production, which potentiates GPR65 expression and signaling in TAMs, causing them to become immunosuppressive and promote tumor growth. To test this hypothesis we will pursue the following specific aims: 1) Determine the contribution of GPR65 signaling to TAM function and CRC growth under conditions of obesity by determining if the cAMP- PKA signaling axis, which functions downstream of GPR65, is activated in TAMs of obese mice and controls TNF-α production. We will also analyze GPR65 expression and the cytokine secretion capacity of macrophages from healthy blood donors and TAMs in CRC samples from non-obese and obese patients; 2) Identify the mechanism by which HFD promotes GPR65 signaling in CRC TAMs by testing the ability of HFD and oleic acid, a dietary triglyceride that is highly enriched in HFD tumors, to alter the oxidative potential, fatty acid oxidation capacity and acid production of human tumor cells via flow cytometry, CyTOF and Seahorse assays. We will also determine if a high-oleic-acid diet is sufficient to modify GPR65 expression and cytokine production by TAMs, and examine if tumor acidity is required for the blunted inflammatory response of TAMs; and verify the role of GPR65 in human macrophages and 3) Assess the effects of targeting GPR65 for tumor immunotherapy in obese and nonobese mice with CRC and other tumor types, namely hepatocellular carcinoma and melanoma, and assess the effects of checkpoint blocking antibodies on tumor growth and anti- tumor immunity in GPR65+ and GPR-/- mice bearing these tumors. The results of these studies are expected to not only reveal a critical mechanism responsible for accelerated tumor growth in the setting of obesity, but also identify a novel target for the treatment of these cancers.