Measuring and manipulating metabolic fluxes in the tumor microenvironment

测量和操纵肿瘤微环境中的代谢通量 与正常组织相比，肿瘤改变了新陈代谢，这表明药物治疗 新陈代谢可以杀死肿瘤而不伤害健康组织。然而，肿瘤代谢主要是 虽然最近的研究表明，体内的肿瘤代谢与体外不同， 系统.因此，该领域需要测量体内肿瘤代谢通量的方法。在我 作为博士后研究员，我开发了测量糖酵解和三羧酸循环（TCA）通量的方法， 使用同位素标记的示踪剂的动力学输注在体内进行。这些方法表明，肿瘤具有低得多的 TCA通量高于健康组织（检查了5种小鼠肿瘤模型）。虽然肿瘤有较高的糖酵解通量， 与健康组织相比，糖酵解加上TCA循环驱动的氧化 肿瘤中的磷酸化显著低于健康组织。此外，给小鼠喂食高脂肪 生酮饮食增加了肿瘤TCA通量，当与 化疗 这些发现提出了两个关键问题。首先，由于体内肿瘤是癌细胞和其他细胞的混合物， 在肿瘤浸润细胞中，癌细胞相对于免疫细胞或成纤维细胞的代谢是什么？第二、 直接上调肿瘤TCA流量是否能减缓肿瘤生长？我建议首先将糖酵解和TCA结合联合收割机 利用免疫磁性和分选策略的周期测量技术来测量癌细胞中的通量， 免疫细胞和成纤维细胞（Aim 1）。我将把这一策略应用于黑色素瘤，一种由CD8 T细胞浸润的肿瘤类型。 细胞，可以帮助控制肿瘤，和胰腺癌，一种肿瘤类型，其中成纤维细胞和 骨髓细胞甚至比癌细胞更丰富。接下来，我将直接上调肿瘤中的TCA流量， 细胞通过使用遗传和药理学方法：过表达NADH解偶联蛋白线粒体， LbNOX，敲除TCA抑制蛋白PDK，以及用二氯乙酸抑制PDK。我会确认 这些策略使用我开发的方法增加了TCA通量，并将测试增加的TCA通量是否 减缓原发性和转移性乳腺肿瘤的肿瘤生长。成功完成这些目标将揭示 肿瘤微环境中不同细胞群的代谢，并将测试TCA上调作为肿瘤微环境的一个指标。 癌症的治疗策略

Measuring and manipulating metabolic fluxes in the tumor microenvironment Tumors have altered metabolism compared to normal tissues, which suggests that drugging metabolism could kill tumors while sparing healthy tissues. However, tumor metabolism has chiefly been measured in vitro, while recent studies have showed that tumor metabolism in the body is distinct from in vitro systems. Therefore, the field needs approaches to measure tumor metabolic fluxes in vivo. During my postdoctoral fellowship, I developed methods to measure glycolytic and tricarboxylic acid cycle (TCA) flux in vivo using kinetic infusion of isotope-labeled tracers. These approaches revealed that tumors have much lower TCA flux than healthy tissues (5 mouse tumor models examined). Though the tumors had higher glycolytic flux than healthy tissues, the total ATP production rate from glycolysis plus TCA cycle-driven oxidative phosphorylation was significantly lower in tumors than in healthy tissues. Moreover, feeding mice a high-fat ketogenic diet increased tumor TCA flux and slowed tumor growth synergistically when combined with chemotherapy. These findings raise two key questions. First, since tumors in vivo are a mix of cancer cells and other infiltrating cells, what is the metabolism of cancer cells versus immune cells or fibroblasts in tumors? Second, can directly upregulating tumor TCA flux slow tumor growth? I propose first to combine my glycolysis and TCA cycle measuring techniques with immunomagnetic and sorting strategies to measure fluxes in cancer cells, immune cells, and fibroblasts (Aim 1). I will apply this strategy to melanoma, a tumor type infiltrated by CD8 T cells which can help control the tumor, and to pancreatic adenocarcinoma, a tumor type where fibroblasts and myeloid cells can be even more abundant than cancer cells. Next, I will directly upregulate TCA flux in tumor cells by using genetic and pharmacologic approaches: overexpressing the NADH uncoupler protein mito- LbNOX, knockout of the TCA suppressor protein PDK, and inhibition of PDK with dichloroacetate. I will confirm that these strategies increase TCA flux using the method I developed and will test whether increased TCA flux slows tumor growth in primary and metastatic breast tumors. Successful completion of these aims will reveal the metabolism of different cell populations in the tumor microenvironment and will test TCA upregulation as a therapeutic strategy in cancer.