Bioreactor-based Manufacturing of Glioblastoma Organoids

多形性胶质母细胞瘤(GBM)是最致命的脑肿瘤。患者通常在首次诊断后只存活12-15个月。因此，迫切需要新的治疗方法。然而，寻找治疗GBM的新疗法一直是具有挑战性的，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，因为这种疾病具有高度的异质性。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有机体”，这是一种模仿微型GBM肿瘤行为的三维细胞团。这些有机体将使用直接来自患者的GBM干细胞(专门的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。这种生物制造的有机类化合物的潜在长期结果是减少了对癌症研究动物模型的依赖，并为未来为GBM患者提供新的治疗方法提供了框架。该项目还将为代表不足的少数族裔本科生和研究生提供研究和教育机会。计划将研究成果整合到夏季海外留学课程中，以帮助培养下一代更具全球能力的工程师。该项目的目标是开发保留GBM肿瘤三个主要特征的GBM(胶质母细胞瘤)有机化合物：肿瘤间异质性(即分子亚型)、肿瘤内异质性(即肿瘤内部/坏死核心的细胞表现出与肿瘤外围细胞非常不同的特征)和癌症干细胞(CSCs)。这一目标将通过使用GBM CSCs创建大小均匀但在体内保留类似肿瘤内异质性的GBM有机化合物来实现。该研究计划旨在证明该项目的生物反应器培养的GBM有机体可以重现固有的肿瘤微环境，该计划组织在三个目标下，每个目标都有一个科学假设。第一个目标是在生物反应器中可重复生物制造GBM有机化合物。Subaims的目的是：a)证明亚致死性流体剪切促进GBM类有机物的均质产生；b)阐明剪切诱导的EPCAM(内皮细胞黏附分子)阳性的EVS(细胞外血管)在类有机物产生中的作用。所获得的结果将被用来检验搅拌槽中的亚致死剪应力通过EpCAM阳性的细胞外小泡(EVS)导致GBM类有机物产生的假设。第二个目标是制造具有血管周围细胞外基质(ECM)和微血管的GBM有机化合物。Subaim的目的是：a)证明在富含透明质酸(HA)的条件下，GSCs在GBM有机体中产生CD31+微血管样细胞；b)利用3D打印的HA水凝胶产生GBM有机体；c)与共培养的微血管产生GBM有机体；d)证明PDX(患者来源的异种移植)组织的病理特征在生物制造的有机体中重现。所获得的结果将用于检验这样的假设，即当有机物直径为500μm时，GSC发生肿瘤组织样血管生成。第三个目的是描述生物制造的GBM有机化合物的遗传组成和化疗敏感性。Subaim将：a)描述患者特定的生物制造有机物的遗传亚型(神经(PN)、神经(N)、经典(C)和间充质(M))，以及b)表征有机物对化疗药物TMZ(替莫唑胺)和Sal(盐霉素)的反应。所获得的结果将被用来检验这一假设，即病理上相关的有机化合物概括了在肿瘤组织中发现的肿瘤内和肿瘤间的多样性，因此可以作为更好的体外药物反应模型。这一奖项反映了NSF的法定使命，并通过使用基金会的智力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.