Cellular responses to pH and oxygen microenvironments in a new 3D tumor model

 描述(由申请人提供)：癌细胞对不断扩大的肿块中微环境的变化的适应是恶性进展的一个关键方面。为了增殖，细胞依赖于来自周围血管的营养和废物运输，随着肿瘤体积的增加，这些运输变得不那么有序，从而在细胞外pH(Phe)和氧气(O2)中产生化学梯度。缺氧诱导因子1α(HIF-1α)是一种受复杂网络调控的异二聚体转录因子的单体，介导细胞对血管生成、细胞增殖和存活、化疗敏感性和新陈代谢等生理和病理过程的反应。即使在氧气存在的情况下，糖酵解途径的激活也是许多癌细胞的特征，导致代谢消耗率增加，这可能与Phe和O2直接相关。缺氧诱导因子-1α通过激活糖酵解酶、调节酶和葡萄糖转运蛋白的基因表达来调节能量代谢。缺氧诱导因子-1α本身是通过抑制蛋白酶体的降解而被转录后调控的，因为它依赖于氧浓度。我们假设，Phe还通过影响转录因子的半衰期在调节HIF-1α中发挥作用，这可能解释了临床观察到即使在传统上被认为会导致化疗耐药的低氧条件下，化疗敏感性也会增加。目前，还没有实验平台提供工具来检验这样的假设。该奖学金培训计划通过三个具体目标，将开发一种新的体外3D细胞培养灌流系统，具有集成的荧光纳米传感能力，用于原位检测Phe和O2梯度。该仪器旨在通过使用简单的运输模型确定代谢消耗率，从而能够在空间上相关地研究细胞对测量的Phe和O2梯度的反应。从设备中指定的区域回收细胞和培养上清，可以对缺氧诱导因子-1α、缺氧诱导因子-1α调控的蛋白质、细胞增殖和存活以及不同微环境中细胞适应的血管内皮生长因子进行生化和分子分析。这三个目标最终将产生一个新的集成模型系统，该系统将在以下方面产生进展：1)肿瘤微环境的定量、可操作的体外3D模型；2)Phe和O2梯度与细胞适应的直接空间关联；3)了解肿瘤细胞对Phe和O2梯度的反应作为HIF-1α的函数。

 DESCRIPTION (provided by applicant): Adaptation of cancer cells to changes in the microenvironment in an expanding tumor mass is a crucial aspect of malignant progression. To proliferate, cells depend on nutrient and waste transport from surrounding vasculature that becomes less orderly as tumor volume increases, creating chemical gradients in extracellular pH (pHe) and oxygen (O2). Hypoxia inducible factor 1α (HIF-1α), a monomer of a heterodimeric transcription factor regulated by a complex network, mediates cellular responses to physiologic and pathologic processes that allow changes in angiogenesis, cell proliferation and survival, chemosensitivity, and metabolism. The activation of the glycolytic pathway even in the presence of oxygen is a characteristic of many cancer cells, leading to an increase in metabolic consumption rates that can be directly correlated to pHe and O2. HIF-1α regulates energy metabolism by activating the gene expression of glycolytic enzymes, regulatory enzymes, and glucose transporters. HIF-1α is itself post-transcriptionally regulated through blockage of proteosomal degradation as it depends on oxygen concentration. We hypothesize that pHe also plays a role in the regulation of HIF-1α by influencing the half-life of the transcription factor potentially explaining clinical observations of increased chemosensitivity even under hypoxic conditions traditionally thought to cause chemoresistance. Currently, there are no experimental platforms that provide the tools to test such a hypothesis. The fellowship-training plan, through three specific aims, will develop a new in vitro 3D cell culture perfusion system with integrated fluorescence nanosensing capabilities for in situ detection of pHe and O2 gradients. The instrument is designed to enable spatially correlated investigation of cellular responses to the measured pHe and O2 gradients by determination of metabolic consumption rates using a simple transport model. Recovering cells and supernatants from defined regions within the device allows for biochemical and molecular analysis of HIF-1α, proteins regulated by HIF-1α, cell proliferation and survival, and VEGF as adapted by cells in different microenvironments. The three aims will ultimately yield a new integrated model system that will produce advances in: 1) quantitative, manipulable in vitro 3D models of the tumor microenvironment; 2) direct spatial correlation of pHe and O2 gradients with cellular adaptations; and 3) understanding tumor cell response to gradients of pHe and O2 as a function HIF-1α.