Perivascular tissue models to overcome MGMT-mediated temozolomide resistance in glioblastoma

摘要 胶质母细胞瘤（GBM）是最常见和致命的脑癌形式。标准治疗是手术切除 随后用烷化剂替莫唑胺（TMZ）处理。然而，两大挑战使GBM 目前无法治疗的：1）它的弥漫性侵犯超出手术边缘;和2）TMZ耐药， 与DNA损伤修复蛋白MGMT的表达有关。而血管周围壁龛（PVN）从 肿瘤进入周围实质被认为是调节侵袭、复发和生存不良的因素， 大多数动物胶质瘤模型对TMZ敏感，并且大多数不表达MGMT，这使得难以 在不显示TMZ耐药性的动物模型中评估新的治疗方法。肿瘤组织工程 合作项目将开发和彻底表征多维工程PVN生物材料， 研究驱动GBM侵袭和TMZ耐药性的病理生理过程，并加速评估 新的TMZ衍生物被创建为靶向弥漫性GBM细胞，而不管MGMT状态。我们将使用先进的 微流体技术，以创建包含边缘模拟透明质酸（HA）的小型化明胶水凝胶库 和嵌入的血管周围网络。我们还使用一种新的合成管道来制造TMZ衍生物， 产生替代的DNA修饰，不能被MGMT去除，我们假设在MGMT中起作用， 独立时尚结合这些技术，我们将对使用以下技术形成的工程PVN平台进行基准测试： 原代脑神经血管细胞用于快速评估GBM侵袭、MGMT表达和TMZ抗性 适用于分析具有不同MGMT谱的细胞系和患者来源的GBM标本。要执行此操作， 我们将首先构建并彻底表征工程化血管周围小生境（目标1）。我们将使用这个 新的生物材料，用于对GBM细胞系中的侵袭模式和MGMT表达进行基准测试（Aim 2）。最后， 我们将建立TMZ变体在工程化血管周围小生境中的预测功效（目标3）。一起我们 将开发，表征和基准组织工程PVN，以检查微环境的作用， 肿瘤边缘对侵袭相关行为的选择压力，MGMT介导的TMZ抗性， 复发和生存率低。与CTEC计划的分数驱动标准一致，我们将开发和 彻底表征工程PVN生物材料，表明它适合现有癌症的连续性 模型，用它来检查无法实现持久生存的现象，并获得可操作的 关于新型TMZ衍生物的见解，其具有有效靶向边缘GBM细胞的潜力， MGMT状态。

ABSTRACT Glioblastoma (GBM) is the most common and lethal form of brain cancer. Standard of care is surgical resection followed by treatment with the alkylating agent temozolomide (TMZ). However, two major challenges make GBM currently untreatable: 1) its diffuse invasion beyond the surgical margin; and 2) TMZ resistance that is tightly linked to expression of the DNA damage repair protein MGMT. While perivascular niches (PVNs) extending from the tumor into the surrounding parenchyma are believed to regulate invasion, recurrence, and poor survival, the majority of animal glioma models are sensitive to TMZ and most do not express MGMT, making it difficult to assess novel therapeutics in animal models that don’t display TMZ resistance. This Cancer Tissue Engineering Collaborative project will develop and thoroughly characterize a multidimensional engineered PVN biomaterial, study pathophysiological processes driving GBM invasion and TMZ resistance, and accelerate the evaluation of novel TMZ derivatives created to target diffuse GBM cells regardless of MGMT status. We will use advanced microfluidics to create libraries of miniaturized gelatin hydrogels containing margin-mimetic hyaluronic acid (HA) and an embedded perivascular network. We also use a novel synthetic pipeline to create TMZ derivatives that generate alternate DNA modifications that cannot be removed by MGMT that we hypothesize work in an MGMT- independent fashion. Merging these technologies, we will benchmark an engineered PVN platform formed using primary brain neurovascular cells for rapid evaluation of GBM invasion, MGMT expression, and TMZ resistance amenable to analysis of cell lines and patient-derived GBM specimens with disparate MGMT profiles. To do this, we will first construct and thoroughly characterize an engineered perivascular niche (Aim 1). We will use this novel biomaterial to benchmark patterns of invasion and MGMT expression in GBM cell lines (Aim 2). Finally, we will establish predictive efficacy of TMZ variants in an engineered perivascular niche (Aim 3). Together, we will develop, characterize, and benchmark a tissue engineered PVN to examine the role of microenvironmental selection pressures in the tumor margin on behaviors related to invasion, MGMT-mediated TMZ resistance, recurrence, and poor survival. Consistent with score-driving criteria of the CTEC program, we will develop and thoroughly characterize an engineered PVN biomaterial, show it fits within the continuum of existing cancer models, use it to examine phenomena underlying the failure to achieve durable survival, and gain actionable insight regarding novel TMZ derivatives with potential to effectively target GBM cells in the margins independent of MGMT status.