Elucidating tumor-vasculature interactions by patterning endothelial cells into vascular-like structures in 3D biomimetic hydrogels

摘要

Introduction: Cell-cell interactions between brain tumor and endothelial cells are key players in disease progression. To evaluate the effects of tumor-vasculature interactions in vitro, previous research has primarily utilized 2D Transwell plates in which both tumor and endothelial cells were plated in 2D and separated by a membrane. While previous 20 co-culture models offer valuable insights, such models fail to recapitulate the in vivo microanatomical architecture of vasculature. To overcome this limitation, we propose to recreate the in vivo endothelium architecture in 3D hydrogels for studying tumor-endothelial cell interactions and elucidating the effects of such interactions on tumor development and drug response. Materials and Methods: In this study, we utilized biomimetic hydrogels as we recently reported composed of PEG with CRGDS and MMP-degradable crosslinker to permit cell migration and with thiolated hyaluronic acid to mimic brain biochemical content. To deploy endothelial cells into vessel-like structures in 3D hydrogels, mouse brain endothelial cells (bEnd.3) were encapsulated in alginate microfibers, which were optimized to hydrolytically degraded within 24 hours. These microfibers were then used as porogens and mixed with hydrogel precursor before crosslinking. Hydrogels containing endothelial cell-lined microchannels were then cultured in the top well of Transwell plates with the bottom well containing one of the following: 1. base media (BM), 2. conditioned media (CM) from patient-derived tumor xenograft (PDTX) adult glioblastoma (GBM) cells, and 3. co-cultured (CC) with PDTX GBM cells plated in the bottom well. Endothelial cell fates were analyzed using bright field microscopy, confocal imaging, and gene expression. Results and Discussion: Two days after co-culture, endothelial cells were viable and displayed elongated morphology in all three groups. However, by Day 7, differential cell behavior and morphology was observed in CM and CC groups, specifically rounded morphology and monolayer disorganization. In contrast, BM group remained viable and confluent with extensive spreading. CD31 expression remained constant over time across all three groups, suggesting retention of endothelial cell phenotype. Decreases in ZO-1 expression in CM and CC groups implies downregulation of tight-junction proteins. These results suggest that paracrine signals from tumor cells induced endothelial cells to downregulate expression of cell-cell junction proteins and detach from the extracellular matrix. Furthermore, increases in VEGFa expression in CC groups supports previous evidence that tumor cells induce endothelial cells to participate in neovascularization. Conclusion: Here we report a highly reproducible and tunable in vitro model that allows spatial patterning of endothelial cells into vasculature-like structures in 3D biomimetic hydrogels for elucidating tumor/vasculature interactions. Our co-culture model suggests tumor cells downregulated intercellular junction proteins in endothelial cells and induced markers of neovascularization. We envision such tumor/vasculature co-culture models will empower mechanistic studies of tumor progression, as well as translational studies for screening novel cancer therapeutics.