Elucidating the Role of Perivascular Niche in Glioblastoma Invasion and Therapeutic Resistance at Single Cell Resolution using Biomimetic Tumor Microenvironment Models

总结 胶质母细胞瘤（GBM）治疗中的关键挑战之一是存在高耐药细胞 具有干细胞样特性的胶质瘤干细胞（GSC），可以逃避手术切除，抵抗常规的 治疗，并主要负责肿瘤复发。GBM肿瘤内的血管周围小生境 微环境（TME）已被公认为是庇护GSC并促进其 干性、侵袭性和治疗抗性。 来自其他人和我们实验室的广泛研究，使用体外和体内模型，已经证明， 内皮细胞和GSC之间的相互作用调节GSC的增殖、致瘤性和自身免疫功能。 更新能力。然而，血管周围生态位是一个复杂的微环境， 还包括多种其它细胞类型，包括星形胶质细胞、周细胞和免疫细胞。细胞间的相互作用 血管周围小生境的各种细胞成分之间的相互作用调节GSC的行为（增殖对 静止和侵入对归巢）和治疗抗性的了解很少。为了解决这些未得到满足的 由于生物学知识的空白，迫切需要复杂和更现实的离体肿瘤模型， 更好地概括了GBM血管周围生态位的生理复杂性，以推进我们的基础研究。 了解疾病的生物学并预测治疗反应。 最近，我们建立并验证了GBM的芯片微流体肿瘤模型，具有独特的3D 器官型结构，研究血管周围生态位对GSC侵袭的影响。我们已经证明 星形胶质细胞的共培养增强了EC诱导的GSC侵袭，其中单培养物与 三重培养提供了一个机制的洞察受体配体对介导的相互作用， 细胞基于这些基础性的发展，在这项研究中，我们的目标是建立一个离体肿瘤模型， GBM，从天然血管周围生态位生物启发，与患者来源的细胞解剖细胞的作用， 在GSC生物学上的生态位内的组分和对单细胞分辨率的处理的响应。 在目标1中，我们的目标是确定血管周围小生境内的关键细胞类型对 GSC-EC相互作用。在目标2中，我们计划从机制上揭示放射治疗对GSC的影响- 血管周围生态位相互作用，而在目标3中，我们将通过破坏 小生境-肿瘤细胞相互作用。我们的研究设计独特地采用了跨学科的方法，包括 生物启发的离体肿瘤模型的微工程，单细胞水平分辨率分析，分子水平 转录组学和使用离体患者肿瘤样品的验证。成功完成这些研究将 不仅加深了我们对GSC与血管周围生态位相互作用的理解， 鉴定新的靶点以阻断疾病进展。

Summary One of the critical challenges in the treatment of Glioblastoma (GBM) is the presence of highly resistant cells with stem-like properties, called glioma stem cells (GSCs), that evade surgical resection, resist conventional treatments and are primarily responsible for tumor recurrence. The perivascular niche within the GBM tumor microenvironment (TME) has been well recognized as a critical site that shelters GSCs and promotes their stemness, invasion, and therapeutic resistance. Extensive studies from others and our lab, using in vitro and in vivo models, have demonstrated that the crosstalk between the endothelial cells (ECs) and GSCs regulates GSC proliferation, tumorigenicity and self- renewal capacity. However, the perivascular niche is a complex microenvironment comprised not only of ECs but multiple other cell types including astrocytes, pericytes, and immune cells. How the cell-cell interactions between the various cellular components of the perivascular niche modulate GSC behavior (proliferation vs. quiescence and invasion vs. homing) and therapy resistance is poorly understood. To address these unmet biological knowledge gaps, there is a critical need for sophisticated and more realistic ex vivo tumor models that better recapitulate the physiological complexities of the GBM perivascular niche to advance our fundamental understanding of the biology of the disease and predict therapeutic responses. Recently, we have established and validated an on-chip microfluidic tumor model of GBM, with a unique 3D organotypic architecture, to study the influence of the perivascular niche on GSC invasion. We have shown that co-culturing of astrocytes enhances EC-induced invasion of GSCs, where RNA-seq analysis of mono-culture vs. tri-culture provided a mechanistic insight into the receptor-ligand pairs that mediate the interactions between cells. Based on these foundational developments, in this study our goal is to develop an ex vivo tumor model of GBM, bioinspired from the native perivascular niche, with patient-derived cells to dissect the role of cellular components within the niche on GSC biology and response to treatment at single cell resolution. In Aim 1, our objective is to determine the influence of the key cell types within the perivascular niche on GSC-EC interactions. In Aim 2, we plan to mechanistically unveil the impact of radiation treatment on GSCs- perivascular niche interactions, while in Aim 3, we will blunt invasion and sensitize GSCs through disruption of niche-tumor cell interactions. Our study design uniquely employs an interdisciplinary approach including microengineering of a bioinspired ex vivo tumor model, single-cell level resolution analysis, molecular-level transcriptomics, and validation using ex vivo patient tumor samples. Successful completion of these studies will not only further our understanding of the interactions of GSCs with the perivascular niche but will also facilitate identification of novel targets to block disease progression.