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，肿瘤的新陈代谢发生了变化，这表明 新陈代谢可以杀死肿瘤，同时保留健康的组织。然而，肿瘤的新陈代谢主要是 在体外测量，而最近的研究表明，肿瘤在体内的新陈代谢与体外不同 系统。因此，该领域需要测量体内肿瘤代谢通量的方法。在我的 博士后，我开发了测量糖酵解和三羧酸循环(TCA)通量的方法 活体使用动态输注同位素标记示踪剂。这些方法表明，肿瘤的发病率要低得多。 TCA流量高于健康组织(检测5种小鼠肿瘤模型)。尽管肿瘤有较高的糖酵解通量 与健康组织相比，糖酵解加TCA循环驱动的氧化产生的总ATP生成率 肿瘤组织中的磷酸化水平明显低于健康组织。此外，喂给老鼠一种高脂肪 生酮饮食增加肿瘤TCA流量，协同抑制肿瘤生长 化疗。 这些发现提出了两个关键问题。首先，由于体内的肿瘤是癌细胞和其他细胞的混合物 浸润性细胞，肿瘤中癌细胞与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通量能减缓肿瘤生长吗？我建议首先将我的糖酵解和TCA结合起来 具有免疫磁学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(目标1)。我将把这一策略应用于黑色素瘤，这是一种由CD8 T细胞浸润性的肿瘤类型 有助于控制肿瘤的细胞，以及胰腺癌，一种肿瘤类型，其中成纤维细胞和 髓系细胞甚至可以比癌细胞更丰富。下一步，我将直接上调肿瘤中的TCA通量 利用遗传学和药理学方法：过表达NADH解偶联蛋白有丝分裂 LbNOX，TCA抑制蛋白PDK的敲除，以及二氯乙酸酯对PDK的抑制。我会确认的 这些策略使用我开发的方法增加了TCA通量，并将测试是否增加了TCA通量 减缓原发和转移性乳腺肿瘤的肿瘤生长。这些目标的成功完成将揭示 不同细胞群在肿瘤微环境中的代谢，并将测试TCA上调作为一种 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.