Engineered microenvironments as biomimetic culture platforms for studying the role of brain extracellular matrix in acquisition of resistance to EGFR inhibition across multiple biological scales

 产品说明：有大量证据表明，大脑中独特的细胞外环境，其中含有很少的纤维蛋白和大量的透明质酸（HA），是导致在归类为多形性胶质母细胞瘤（GBM）的脑肿瘤中观察到的特征性侵袭性和对常规治疗的抗性的原因。然而，肿瘤细胞与细胞外基质（ECM）之间的特异性相互作用及其与肿瘤生理学的关系在很大程度上是未知的。一般来说，这些机制一直具有挑战性的阐明，在很大程度上是因为缺乏生理学上可翻译的模型，可以在受控的情况下离体研究。为了满足这一需求，我们提出了一种基于生物材料的方法来创建原代GBM细胞的三维（3D）培养物，这些细胞准确地代表了复杂的体内微环境并保留了临床肿瘤的生理学。这些培养平台允许以前所未有的精度独立、正交地控制多个微环境参数（模量、肽含量、HA含量）。通过结合这种方法与微环境参数和转录因子（TF）活性的动态测量，我们建议在多个生物水平上研究GBM微环境与耐药性之间的关系。特别是，我们的目标是确定特定的细胞-ECM相互作用作为潜在的临床靶点，其破坏抑制获得耐药表皮生长因子受体（EGFR）抑制剂。本申请的目的1描述了一种计划，以1）充分表征临床GBM中的生物化学和生物物理景观，和2）鉴定传递这些微环境线索的特异性细胞-ECM相互作用，诱导GBM细胞对EGFR抑制剂治疗表现出不良反应。为了鉴定特异性相互作用，将在3D、基于HA的水凝胶内培养患者来源的GBM细胞，所述水凝胶代表受控实验系统，其中HA浓度、可用整联蛋白结合肽的密度和身份以及机械模量可以精确变化，并且它们的独立作用是不同的。同时，目标2描述了使用高通量方法动态监测活的3D培养物中TF活性的细胞内对EGFR抑制的反应的研究。由于脑中独特的ECM是耐药性的主要介质，因此目标1中概述的脑模拟水凝胶平台将提供进行这些研究的理想培养环境。这些实验将表征当非耐药GBM细胞首次暴露于EGFR抑制剂时发生的复杂转录事件，并实时定量测量对这些治疗的渐进反应，包括获得耐药性。总之，这些研究提供了一个充满希望的机会， 在多个生物学水平上鉴定用于EGFR抑制的辅助治疗的新药理学靶点（即，Aim 1中的细胞表面-ECM界面和Aim 2中的TF与细胞核中的基因启动子结合）。考虑到GBM肿瘤内细胞的显著异质性以及这些细胞可能采用多种机制来获得对化疗药物的抗性[46，47，50，51]，对所涉及的信号传导网络的系统水平理解将是该领域用于鉴定GBM治疗的有效药理学靶点的宝贵资产。

 DESCRIPTION: There is a large body of evidence that suggests that the unique extracellular environment in the brain, which contains few fibrous proteins and high amounts of hyaluronic acid (HA), is responsible for the characteristic aggressiveness and resistance to conventional treatments observed in brain tumors categorized as glioblastoma mulitforme (GBM). However the specific interactions between tumor cells and the extracellular matrix (ECM) and their relationship to tumor physiology are largely unknown. In general, these mechanisms have been challenging to elucidate, in large part because of the lack of physiologically translatable models that can be studied in controlled context ex vivo. To fulfill this need, we propose a biomaterials-based approach to create three-dimensional (3D) cultures of primary GBM cells that accurately represent the complex, in vivo microenvironment and preserve physiology of clinical tumors. These culture platforms permit independent, orthogonal control of multiple micro environmental parameters (modulus, peptide content, HA content) with unprecedented precision. By combining this approach with dynamic measurements of micro environmental parameters and transcription factor (TF) activity, we propose to investigate the relationships between the GBM microenvironment and drug resistance on multiple biological levels. In particular, we aim to identify specific cell-ECM interactions as potential clinical targets whose disruption inhibits acquisition of resistance epidermal growth factor receptor (EGFR) inhibitors. Aim 1 of this application describes a plan to 1) fully characterize the biochemical and biophysical landscape in clinical GBM and 2) identify specific cell-ECM interactions that relay these microenvironment cues, inducing GBM cells to exhibit poor response to treatment with EGFR inhibitors. To identify specific interactions, patient- derived GBM cells will be cultured within 3D, HA-based hydrogels, which represent a controlled experimental system where HA concentration, density and identity of available integrin-binding peptides, and mechanical modulus can be precisely varied and their independent effects distinguished. In parallel, Aim 2 describes studies in which the intracellular response to the EGFR inhibition using a high-throughput approach to dynamically monitor TF activity in live, 3D cultures. As the unique ECM in the brain is a major mediator of drug resistance, the brain-mimetic hydrogel platforms outlined in Aim 1 will provide an ideal culture environment in which to perform these studies. These experiments will characterize the complex transcriptional events that occur when non-resistance GBM cells are first exposed to EGFR inhibitors and quantitative measure the progressive response to these treatments, including acquisition of resistance, in real-time. Together, these studies provide a promising opportunity to identify new pharmacological targets for adjunct treatments to EGFR inhibition at multiple biological levels (i.e., cell surface-ECM interface in Aim 1 and TF binding to gene promoters in the nucleus in Aim 2). Given the significant heterogeneity of cells within GBM tumors and the variety of mechanisms that these cells may adopt to gain resistance to chemotherapeutic drugs [46,47,50,51], a systems-level understanding of the signaling networks involved would be a valuable asset to the field for identifying potent pharmacological targets for GBM treatment.