abstract: Glioblastoma Multiforme (GBM) is a grade IV astrocytoma and the most aggressive form of cancer that begins within the brain. The two-year average survival rate of GBM in the United States of America is 25%, and it has a higher incidence in individuals within the ages of 45 - 60 years. GBM Tumor formation can either begin as normal brain cells or develop from an existing low-grade astrocytoma and are housed by the perivascular niche in the brain microenvironment. This niche allows for the persistence of a population of cells known as glioma stem cells (GSC) by supplying optimum growth conditions that build chemoresistance and cause recurrence of the tumor within two to five years of treatment. It has therefore become imperative to understand the role of the perivascular niche on GSCs through in vitro modelling in order to improve the efficiency of therapeutic treatment and increase the survival rate of patients with GBM. In this study, a unique three dimensional (3D) microfluidic platform that permitted the study of intercellular interactions between three different cell types in the perivascular niche of the brain was developed and utilized for the first time. Specifically, human endothelial cells were embedded in a fibrin matrix and introduced into the vascular layer of the microfluidic platform. After spontaneous formation of a vascular layer, Normal Human Astrocytes and Patient derived GSC were embedded in a Matrigel® matrix and incorporated in the stroma and tumor regions of the microfluidic device respectively. Using the established platform, migration, proliferation and stemness of GSCs studies were conducted. The findings obtained indicate that astrocytes in the perivascular niche significantly increase the migratory and proliferative properties of GSCs in the tumor microenvironment, consistent with previous in vivo findings. The novel GBM tumor microenvironment developed herein, could be utilized for further in-depth cellular and molecular level studies to dissect the influence of individual factors within the tumor niche on GSCs biology, and could serve as a model for developing targeted therapies. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2020

Glioblastoma multiforme (GBM) is the most deadly and common form of brain cancer and is responsible for over 50% of adult brain tumors. A specific region within the GBM environment is known as the perivascular niche (PVN). We have designed a 3D in vitro model of the PVN comprised of either collagen Type 1 or HyStem-C®, human umbilical vein endothelial cells (HUVECs) or human brain microvascular endothelial cells (HBMECs), and LN229 (GBM) cells. A synergistic response between HUVECs and LN229 cells was observed in co-culture, including 10 – 16-fold increased cell proliferation, a decrease in the height of hydrogels of up to 68%, as well as elevated secretion of TGF-β and CXCL12 up to 2.6-fold from Day 8 to 14. These trends correlated with cell colocalization, indicating a chemotactic role for CXCL12 in enabling the migration of LN229 cells towards HUVECs in co-cultures. Von Willebrand factor (vWF) was co-expressed with glial fibrillary acidic protein (GFAP) in up to 40% of LN229 cells after 14 days in co-culture in collagen (2.2 mg/mL) and HyStem-C® gels. The expression of vWF indicates the early stages of trans-differentiation of LN229 cells to an endothelial cell phenotype. We then investigated the effect of chemotherapeutic drugs temozolomide (TMZ) and Avastin® on EC networks, LN229 cell morphology and alignment, cytotoxicity, colocalization, and trans-differentiation. TMZ was observed to primarily affect LN229 cells, with treatment at high concentrations resulting in up to 2.3-fold reduced alignment as well as an increase in cell circularity. Cytotoxicity of up to 94% was also observed up to in LN229 monocultures, and was significantly higher in collagen (1.1 mg/mL) gels. Avastin® treatment resulted in changes to ECs. Network features were significantly reduced and EC cellular proliferation decreased up to 69% with Avastin® treatment. Significant increases in percentages of colocalized and GFAP+/vWF+ cells were also observed when treated with 8 µg/mL Avastin®. This suggests that chemotactic signaling may have been altered. TGF-β secretion was reduced in co-cultures when 150 µM TMZ or 8 µg/mL Avastin® were administered. / Doctor of Philosophy / Glioblastoma (GBM) is the most common and deadly form of brain cancer and is responsible for over 50% of adult brain tumors. A specific region within the GBM environment of particular interest is located near the vasculature, known as the perivascular niche (PVN). We have designed a 3D in vitro model of the PVN consisting of either collagen type 1 or HyStem-C®, a material made of primarily hyaluronic acid. Human umbilical vein endothelial cells (HUVECs), an immortalized cell line, or primary human brain microvascular endothelial cells (HBMECs) as well as LN229 (GBM) cells were used. A synergistic response was observed between HUVECs and LN229 cells in co-culture, including changes to the extracellular matrix, and signaling factor secretion. Further supporting this data, colocalization between LN229 cells and HUVECs was observed. Colocalization is a phenomenon where two cell types come into physical contact after one moves toward another. This indicated preferential migration, specifically in response to CXCL12. Endothelial cell marker von Willebrand factor (vWF) was co-expressed with glial fibrillary acidic protein (GFAP), commonly used to identify GBM cells. This percentage was increased in co-cultures with HBMECs, pointing to differences in the response of primary cells to immortalized cell lines. The expression of vWF indicates the early stages of trans-differentiation of LN229 cells to an endothelial cell phenotype. We then investigated the effect of chemotherapeutic drugs temozolomide (TMZ) and Avastin® in the PVN model. TMZ was observed to primarily affect LN229 cells, by reducing their alignment as well as causing cell death. Avastin® treatment resulted in changes to ECs. Networks and cell growth were significantly reduced after Avastin® treatment. When either TMZ or Avastin® was administered, the secretion of TGF-β, was reduced.

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