Use of Physiologic Media to Study Metabolic Regulation and Requirements in Cancer

7.项目摘要/摘要 新陈代谢改变几乎是癌细胞的普遍特征，癌细胞需要新陈代谢。 支持扩散和生存的适应。然而，决定多种新陈代谢的机制 癌症的易感性和表型仍然知之甚少。因此，人们对靶向癌症的兴趣重新抬头 为了患者的利益，代谢将需要更好地理解代谢调节和要求 在恶性细胞类型中。培养中的细胞通常被用于研究癌症新陈代谢和开发药物。 利用新陈代谢的弱点。然而，尽管人们越来越认识到环境因素 影响细胞新陈代谢，我们目前对癌症代谢重联的理解主要是基于发现 来自培养在不能很好地反映人体血浆代谢成分的培养液中的细胞。因此，我们的 最重要的假设是癌细胞新陈代谢的许多重要方面被忽视或 被误解为使用与生理条件不完全相似的模型系统的结果。 为了开始测试这一点，我们开发了一种新的培养液(类似人血浆的培养液；HPLM)，它含有 与成人血浆浓度相当的极性代谢物和盐。然后我们展示了 与传统介质相比，HPLM对细胞新陈代谢具有广泛且在很大程度上无法解释的影响。 我们的初步数据显示，当在传统培养液中培养时，某些细胞株会分泌丙氨酸， 但在HPLM中，这些细胞消耗丙氨酸的速度超过了大多数其他氨基酸。 因此，我们将检验丙氨酸在生理条件下培养的细胞中起关键作用的假设。 (目标1)。使用我们开发的恒化器技术，我们还将确定如何去除丙氨酸 来自HPLM的物质以集合方式影响40多个条形码血癌细胞系的生长。此外, 通过使用基于CRISPR的功能丧失筛查，我们鉴定了NME6，一个编码糟糕的 用HPLM研究了线粒体蛋白作为条件致死蛋白。因此，我们还将检验这一假设 NME6在生理性培养的细胞线粒体内稳态中起着关键的和不可预见的作用 条件(目标2)。最后，我们还发现HPLM显著影响细胞对抗癌药物的敏感性。 5-氟尿嘧啶不影响另一种常见化疗药物阿霉素的效力。因此， 使用1900种不同的肿瘤学相关小分子的文库，我们将进行高通量筛选 为了检验这一假设，相对于传统介质，HPLM改变了额外化合物的效力。我们 然后，将寻求对细胞系和原代细胞中不同药物表型的验证和机制洞察 模型(目标3)。我们提议的工作使用不同的方法来更好地理解环境因素如何 更能反映生理条件对血液癌细胞新陈代谢的影响，并具有 不仅有可能发现不可预见的生物学见解，而且还有可能发现新的治疗靶点和 可能与体内癌症治疗有更大相关性的方法。

7. Project Summary/Abstract Altered metabolism is a nearly universal characteristic of cancer cells, which require metabolic adaptations to support proliferation and survival. However, mechanisms that dictate the diverse metabolic liabilities and phenotypes of cancer remain poorly understood. Thus, the resurgence of interest to target cancer metabolism for patient benefit will require an improved understanding of metabolic regulation and requirements in malignant cell types. Cells in culture are commonly used to study cancer metabolism and to develop drugs that exploit metabolic vulnerabilities. However, while it is increasingly appreciated that environmental factors impact cell metabolism, our current understanding of metabolic rewiring in cancer is largely based on findings from cells cultured in media that poorly reflect the metabolic composition of human plasma. Therefore, our overarching hypothesis is that many important aspects of cancer cell metabolism have been overlooked or misconstrued as a consequence of utilizing model systems that inadequately resemble physiologic conditions. To begin to test this, we developed a new culture medium (human plasma-like medium; HPLM) that contains polar metabolites and salts at concentrations comparable to those of adult human plasma. We then showed that, relative to traditional media, HPLM has widespread and largely unexplained effects on cell metabolism. Our preliminary data reveal that, when cultured in traditional media, certain cell lines secrete alanine, but that in HPLM, those cells instead consume alanine at rates exceeding those for most other amino acids. Therefore, we will test the hypothesis that alanine has a key role for cells cultured in physiologic conditions (Aim 1). Using a chemostat technology that we developed, we will also determine how the removal of alanine from HPLM affects the growth of over 40 barcoded blood cancer cell lines in a pooled fashion. In addition, through the use of CRISPR-based loss-of-function screens, we identified NME6, a gene that encodes a poorly studied mitochondrial protein, as conditional lethal with HPLM. Thus, we will also test the hypothesis that NME6 serves a critical and unforeseen role in mitochondrial homeostasis for cells cultured in physiologic conditions (Aim 2). Lastly, we also found that HPLM dramatically influences cell sensitivity to the cancer drug 5-fluorouracil without affecting the potency of doxorubicin, another common chemotherapeutic. Therefore, using a library of > 1,900 diverse oncology-related small molecules, we will perform a high-throughput screen to test the hypothesis that, relative to traditional media, HPLM alters the potency of additional compounds. We will then pursue validation and mechanistic insights for differential drug phenotypes in cell line and primary cell models (Aim 3). Our proposed work uses distinct approaches to better understand how environmental factors that more closely reflect physiologic conditions impact the metabolism of blood cancer cells, and has the potential to not only identify unforeseen biological insights, but to also uncover new therapeutic targets and approaches that may have greater in vivo relevance for cancer therapy.