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

摘要 胶质母细胞瘤(GBM)治疗的关键挑战之一是高度耐药细胞的存在 具有干细胞样的特性，称为胶质瘤干细胞(GSCs)，可以逃避手术切除，抵抗传统的 治疗，并对肿瘤复发负有主要责任。肾小球基底膜肿瘤内的血管周围龛 微环境(TME)已被公认为是保护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(ECs)和GSCs之间的串扰调节GSC的增殖、致瘤性和自身 续订能力。然而，血管周围的壁龛是一个复杂的微环境，不仅由内皮细胞组成。 但还有多种其他类型的细胞，包括星形胶质细胞、周细胞和免疫细胞。细胞与细胞的相互作用 血管周围壁龛的不同细胞成分之间调节GSC的行为(增殖与 静止期和侵袭期与归巢相比)和治疗抵抗力知之甚少。来解决这些未得到满足的问题 生物学知识差距，迫切需要复杂和更现实的体外肿瘤模型 更好地概括基底膜血管周围壁龛的生理复杂性，以推进我们的基础 了解疾病的生物学并预测治疗反应。 最近，我们建立并验证了基底膜的芯片上微流控肿瘤模型，具有独特的3D 器官型构筑，研究血管周围生态位对GSC侵袭的影响。我们已经证明了 共培养的星形胶质细胞增强了EC诱导的GSCs的侵袭，其中单一培养的RNA-SEQ分析与。 三层培养提供了一种对受体-配体对的机械性洞察，受体-配体对介导了 细胞。在这些基础性进展的基础上，本研究的目标是建立一种体外肿瘤模型。 GBM，生物灵感来自于天然的血管周围壁龛，用患者来源的细胞来剖析细胞的作用 在GSC生物学和单细胞分辨率下的治疗反应的利基成分。 在目标1中，我们的目标是确定血管周围壁龛内的关键细胞类型对 GSC-EC相互作用。在目标2中，我们计划从机械上揭示放射治疗对GSC的影响- 血管周围生态位相互作用，而在目标3，我们将钝化入侵和敏化GSCs通过破坏 壁龛与肿瘤细胞的相互作用。我们的研究设计独特地采用了跨学科的方法，包括 生物启发的体外肿瘤模型的微工程，单细胞水平分辨率分析，分子水平 转录学，并使用体外患者肿瘤样本进行验证。成功完成这些研究将 不仅加深了我们对GSCs与血管周围壁龛的相互作用的了解，而且还将促进 确定阻止疾病进展的新靶点。

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.