Perivascular signals alter global gene expression profile of glioblastoma and response to temozolomide in a gelatin hydrogel

Abstract

Glioblastoma (GBM) is the most common primary malignant brain tumor, with patients exhibiting poor survival (median survival time: 15 months). Difficulties in treating GBM include not only the inability to resect the diffusively-invading tumor cells but also therapeutic resistance. The perivascular niche within the GBM tumor microenvironment contributes significantly to tumor cell invasion, cancer stem cell maintenance, and has been shown to protect tumor cells from radiation and chemotherapy. In this study, we describe the development of an in vitro artificial perivascular niche (PVN), culturing endothelial and stromal cells along with GBM cells within a methacrylamide-functionalized gelatin hydrogel, in order to examine howthe presence of the PVN alters global genomic expression profiles. Using RNA-seq, we demonstrate that the inclusion of perivascular niche cells with GBM cells upregulates genes related to angiogenesis and remodeling, while downregulated genes are related to cell cycle and DNA damage repair.Signaling pathways and genes commonly implicated in GBM malignancy, such as MGMT, EGFR, PI3K-Akt signaling, and Ras/MAPK signaling are also upregulated in the presence of perivascular niche cells. We describe the kinetics of gene expression within the PVN hydrogels over a course of 14 days, observing the patterns associated with previously-observed GBMmediated endothelial network co-option and regression that are altered in the presence of covalently-bound hyaluronic acid. We finally examine the effect of PVN culture on GBM cell responsiveness to temozolomide, a frontline chemotherapy used clinically against GBM.Notably, the presence of a PVN leads to significantly increased TMZ resistance compared to hydrogels containing only GBM cells. Overall, these results demonstrate that inclusion of cellular and matrix-associated elements of the PVN within in vitro models of GBM provide critical signals to regulate the phenotype and therapeutic responsiveness of GBM cells.

Multidimensional hydrogel models reveal endothelial network angiocrine signals increase glioblastoma cell number, invasion, and temozolomide resistance

“…Prior literature demonstrates that IGFBP2 and HGF support GBM proliferation, which supports the results obtained here [51,57]. In a previous study, we used RNA-seq to demonstrate that transcriptomic signatures are altered by interactions between GBM cells and endothelial networks cultured in the same hydrogel [31]. Differential gene analysis revealed that tumor-vascular interactions, which in this case encompassed paracrine, juxtacrine, and reciprocal ECM changes, downregulated genes related to metabolism and cell cycle progression.…”

“…Our findings that angiocrine signaling supports TMZ chemoresistance in glioblastoma are consistent with previous efforts from our group that showed that direct co-culture of GBM cells within endothelial cell networks in GelMA hydrogels are less responsive to temozolomide compared to GBM cells alone. That work also suggested that tumor-vascular interactions modulated MGMT and mismatch repair gene expression profiles, which are DNA repair mechanisms responsible for temozolomide resistance [31]. As a result, ongoing efforts are examining if the other proteins identified in this study are individual mediators of temozolomide resistance in order to identify underlying signaling mechanisms that lead to this response.…”

“…Our choice of gelatin as the building block for GelMA hydrogel was inspired by the diversity of fibrillar ECM proteins (e.g., collagens and fibronectin) in the perivascular niche [46]. We have shown that GelMA hydrogels can be fabricated such that their elastic modulus is tunable within the 1 -10 kPa range [31,32] reported by Miroshnikova et al as the elastic modulus of human glioblastoma specimens via atomic force microscopy [47].…”

“…We have previously described a gelatin hydrogel platform for co-culturing endothelial cells, fibroblasts, and GBM tumor cells to model the GBM PVN [31]. Here, we specifically investigate the role of the secretome of 3D self-assembled endothelial networks in influencing the behavior of U87-MG tumor cells encapsulated in gelatin hydrogels.…”

Perivascular Secretome Influences Hematopoietic Stem Cell Maintenance in a Gelatin Hydrogel

“…43 More recently, we showed 20 paracrine signals from marrow-derived mesenchymal stem cells could enhance the retention of quiescent HSCs, 26 though this effect was also strongly dependent on the relative ratio of HSCs to MSCs as well as on processes of MSC-mediated hydrogel remodeling. Separately, a wide range of studies have focused on developing microphysiological models of the perivascular environment to interrogate processes associated with disease progression, 9,29,54,55 cancer invasion or metastasis, 4,37,50,51,66 and even dormancy of cancer stem cells. 10,24 These studies provide an illustrative example of the power of the perivascular environment and motivate our study of the role of perivascular signals on murine HSC culture.…”