The Influence of Hyaluronic Acid and Glioblastoma Cell Coculture on the Formation of Endothelial Cell Networks in Gelatin Hydrogels

Abstract

Glioblastoma (GBM) is the most common and deadly form of brain cancer. Interactions between GBM cells and vasculature in vivo contribute to poor clinical outcomes, with GBM-induced vessel co-option, regression, and subsequent angiogenesis strongly influencing GBM invasion. Here, elements of the GBM perivascular niche are incorporated into a methacrylamide-functionalized gelatin hydrogel as a means to examine GBM–vessel interactions. The complexity of 3D endothelial cell networks formed from human umbilical vein endothelial cells and normal human lung fibroblasts as a function of hydrogel properties and vascular endothelial growth factor (VEGF) presentation is presented. While overall length and branching of the endothelial cell networks decrease with increasing hydrogel stiffness and incorporation of brain-mimetic hyaluronic acid, it can be separately altered by changing the vascular cell seeding density. It is shown that covalent incorporation of VEGF supports network formation as robustly as continuously available soluble VEGF. The impact of U87-MG GBM cells on the endothelial cell networks is subsequently investigated. GBM cells localize in proximity to the endothelial cell networks and hasten network regression in vitro. Together, this in vitro platform recapitulates the close association between GBM cells and vessel structures as well as elements of vessel co-option and regression preceding angiogenesis in vivo.

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

“…We have previously demonstrated that endothelial cell networks can be sustained and tuned within gelatin hydrogels using a co-culture and endothelial cells and fibroblasts [40]. We also observed that including U87-MG GBM cells in the culture system leads to close association between GBM cells and the developing endothelial cell networks, followed by dissolution of these networks in a manner reminiscent of co-option [69].…”

“…Lithium acylphosphinate (LAP) was added as a photoinitiator at 0.1% w/v for GelMA-only gels and 0.02% w/v for GelMA/HAMA gels. The reduced photoinitiator concentration allowed for equivalent elastic moduli (~3 kPa) between the GelMA and GelMA + HA constructs [40]. Cell seeded hydrogels (GelMA only; GelMA+HA) were then formed in one of three configurations: GBM-only; Perivascular-only; or Tri-Culture.…”

“…Tri-Cultures were formed by re-suspending 1 × 10 6 HUVECs/mL, 2 × 10 6 NHLFs/mL, and 1 × 10 5 U87-MGs/mL in the GelMA or GelMA+HA polymer solution. Cell-laden solutions were subsequently pipetted into circular Teflon molds with a diameter of 5 mm and a thickness of 1 mm, and exposed to UV light at a wavelength of 365 nm and intensity of 5.69 mW/cm 2 for 30 s as previously described to form cell-seeded hydrogel disks [40]. Hydrogels were maintained for up to 14 days in 48-well plates in EGM-2 media with daily media changes.…”

“…Mixed cultures of tumor cells, endothelial cells, and stromal cells have been achieved within biomaterials for breast, lung, and prostate cancers, and these models have been used to investigate how non-tumor cells impact tumor proliferation, phenotype, and therapeutic response [35][36][37][38][39]. Our lab has recently described a tri-culture biomaterial platform for the purpose of modeling the perivascular niche of GBM [40]. We peer-reviewed) is the author/funder.…”

Biomimetic Strategies for the Glioblastoma Microenvironment

“…Using such integrated systems, microenvironmental adaptation was observed during GBM invasion (Cha et al, 2016); changes in the GBM invasion of the HA matrices were mediated by the focal adhesion kinase and MMP, within the fibrous structures, in response to blockade of a HA-mediated pathway of GBM invasion. Recently, other cellular components in the microenvironment were incorporated into 3D GBM models, revealing the roles of glial cells and the perivascular niche in GBM invasion (Grodecki et al, 2015;Iwadate et al, 2016;Gritsenko et al, 2017;Ngo and Harley, 2017).…”