Bioreactor-based Manufacturing of Glioblastoma Organoids

多形性胶质母细胞瘤（GBM）是最致命的脑肿瘤。患者在最初诊断后通常只能存活12-15个月。因此，迫切需要新的GBM治疗方法。然而，寻找GBM的新疗法一直具有挑战性，主要是因为缺乏充分模拟患者病情的模型，这一点尤其重要，因为这种疾病是高度异质性的。也就是说，肿瘤的病理特征差异很大，对治疗的反应也不同。为了解决这些问题，该项目将开发“GBM类器官”，这是一种模拟微型GBM肿瘤行为的三维细胞团。这些类器官将使用直接从患者身上提取的GBM干细胞（特化的GBM细胞，在GBM肿瘤中产生所有类型的细胞）来制造。细胞将使用3D打印机定位并在生物反应器中培养。这种生物制造类器官的潜在长期结果是减少了对癌症研究动物模型的依赖，并为未来GBM患者提出新治疗方法提供了框架。该项目还将为未被充分代表的少数民族本科生和研究生提供研究和教育机会。计划提供国际机会，将研究成果融入暑期海外学习课程，以帮助培养更具全球能力的下一代工程师。该项目的目标是开发保留GBM（胶质母细胞瘤）肿瘤三个主要特征的类器官：肿瘤间异质性（即分子亚型），肿瘤内异质性（即内/坏死核心细胞与肿瘤周围细胞表现出非常不同的特征）和癌症干细胞（CSCs）。该目标将通过使用GBM CSCs创建大小均匀但保留体内样肿瘤内异质性的GBM类器官来实现。该研究计划旨在证明该项目的生物反应器培养的GBM类器官可以概括先天肿瘤微环境，它分为三个目标，每个目标都由一个科学假设驱动。首要目标是在生物反应器中可再生地制造GBM类器官。子目标是：a)证明亚致死流体剪切改善了GBM类器官的均匀生产，b)阐明剪切诱导的内皮细胞粘附分子（Endothelial Cell Adhesion Molecule, EpCAM）阳性的细胞外血管（extracellular vascular, EVs）在类器官生产中的作用。获得的结果将用于验证搅拌槽中亚致死剪切应力通过epcam阳性细胞外囊泡（ev）导致GBM类器官产生的假设。第二个目标是生产具有血管周围龛样ECM（细胞外基质）和微血管的GBM类器官。子目标是：a)证明透明质酸（HA）丰富条件下的GSCs在GBM类器官内产生CD31+微血管样细胞；b)用3d打印透明质酸水凝胶生成GBM类器官；c)用共同培养的微血管产生GBM类器官，d)证明PDX（患者来源的异种移植物）组织的病理特征在生物制造的类器官中得到重现。所得结果将用于验证当类器官直径为500 μm时，由GSCs产生肿瘤组织样血管生成的假设。第三个目标是分析生物制造的GBM类器官的基因组成和化疗敏感性。子目标是：a)描述生物制造类器官的患者特异性遗传亚型(前神经（PN），神经（N)，经典（C)和间充质(M)）， b）表征类器官对化疗药物TMZ（替莫唑胺）和SAL（盐碱霉素）的反应。获得的结果将用于验证病理相关的类器官概括肿瘤组织中发现的肿瘤内和肿瘤间多样性的假设，因此可以作为更好的体外药物反应模型。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor. Patients typically survive only 12-15 months after their initial diagnosis. Therefore, new treatments for GBM are urgently needed. However, finding new therapies for GBM has been challenging, largely due to the lack of models that adequately mimic the patient’s condition, which is particularly important since the disease is highly heterogeneous. That is, tumors differ widely in their pathological features and respond differently to treatments. To address these issues, this project will develop "GBM organoids," which are three-dimension masses of cells that mimic the behavior of miniature GBM tumors. These organoids will be manufactured using GBM stem cells (specialized GBM cells that give rise to all types of cells in a GBM tumor) derived directly from patients. Cells will be positioned using 3D printers and cultured in bioreactors. The potential long-term outcomes of such biomanufactured organoids are reduced reliance on animal models for cancer research and a framework for suggesting novel treatments for GBM patients in the future. This project will also provide research and educational opportunities to underrepresented minority undergraduate and graduate students. International opportunities are planned that integrate the research results into summer study-abroad courses in order to help prepare the next generation of more globally competent engineers.The goal of this project is to develop GBM (glioblastoma) organoids that retain three main features of GBM tumors: intertumoral heterogeneity (i.e., molecular subtypes), intratumoral heterogeneity (i.e., cells in the inner/necrotic core exhibiting very different characteristics to those at the tumor periphery), and cancer stem cells (CSCs). The goal will be achieved by creating GBM organoids that are homogeneous in size but retain in vivo-like intratumoral heterogeneity by using GBM CSCs. The Research Plan, designed to demonstrate that the project’s bioreactor-cultured GBM organoids can recapitulate the innate tumor microenvironment, is organized under three aims, each motivated by a scientific hypothesis. The FIRST Aim is to reproducibly biomanufacture GBM organoids in bioreactors. Subaims are to: a) demonstrate that sublethal fluid shear improves homogenous production of GBM organoids and b) elucidate the role of shear-induced EpCAM (Endothelial Cell Adhesion Molecule)-positive EVs (extracellular vessels) in organoid production. Results obtained will be used to test the hypothesis that sublethal shear stress in stirred-tanks leads to GBM organoid production via EpCAM-positive extracellular vesicles (EVs). The SECOND Aim is to produce GBM organoids with perivascular niche-like ECM (extracellular matrix) and microvessels. Subaims are to: a) demonstrate that GSCs in hyaluronic acid (HA)-rich conditions generate CD31+microvessel-like cells within GBM organoids; b) generate GBM organoids with 3D-printed HA hydrogels; c) produce GBM organoids with co-cultured microvessels and d) demonstrate that pathological features of PDX (Patient-derived xenograft) tissues are recapitulated in biomanufactured organoids. Results obtained will be used to test the hypothesis that tumor tissue-like angiogenesis by GSCs occurs when organoids are 500 μm diameter. The THIRD Aim is to profile the genetic composition and chemotherapy sensitivity of biomanufactured GBM organoids. Subaims are to: a) profile the patient-specific genetic subtypes (proneural (PN), neural (N), classical (C), and mesenchymal (M)) of biomanufactured organoids and b) characterize organoids’ responses to chemotherapeutic drugs TMZ (temozolomide) and SAL(salinomycin). Results obtained will be used to test the hypothesis that pathologically relevant organoids recapitulate the intratumoral and intertumoral diversity found in tumor tissues, and thus, can serve as better in vitro models for drug response.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.