Bioreactor-based Manufacturing of Glioblastoma Organoids

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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.