Mimicking brain tumor-vasculature microanatomical architecture via co-culture of brain tumor and endothelial cells in 3D hydrogels

Abstract

Glioblastoma (GBM) is an aggressive malignant brain tumor with median survival of 12 months and 5-year survival rate less than 5%. GBM is highly vascularized, and the interactions between tumor and endothelial cells play an important role in driving tumor growth. To study tumor-endothelial interactions, the gold standard co-culture model is transwell culture, which fails to recapitulate the biochemical or physical cues found in tumor niche. Recently, we reported the development of poly(ethylene-glycol)-based hydrogels as 3D niche that supported GBM proliferation and invasion. To further mimic the microanatomical architecture of tumor-endothelial interactions in vivo, here we developed a hydrogel-based co-culture model that mimics the spatial organization of tumor and endothelial cells. To increase the physiological relevance, patient-derived GBM cells and mouse brain endothelial cells were used as model cell types. Using hydrolytically-degradable alginate fibers as porogens, endothelial cells were deployed and patterned into vessel-like structures in 3D hydrogels with high cell viability and retention of endothelial phenotype. Co-culture led to a significant increase in GBM cell proliferation and decrease in endothelial cell expression of cell adhesion proteins. In summary, we have developed a novel 3D co-culture model that mimics the in vivo spatial organization of brain tumor and endothelial cells. Such model may provide a valuable tool for future mechanistic studies to elucidate the effects of tumor-endothelial interactions on tumor progression in a more physiologically-relevant manner.

Keywords: Cancer model; Co-culture; Endothelial; Glioblastoma; Three-dimensional; Tumor.

Fig. 1.

(a). Schematic of experimental group design. Endothelial cells (mouse brain) were patterned into vessellike structures in 3D biomimetic hydrogels using hydrolytically degradable alginate microfibers as porogens. In GBM + ENDO group, single patient-derived glioblastoma xenograft (PDTX GBM) cells were co-cultured with patterned endothelial cells. In GBM + ONLY group, single PDTX GBM cells were co-encapsulated with acellular alginate microfibers. In ENDO ONLY group, patterned endothelial cells were cultured alone. (b). Increasing calcium bath stir speed (350–700 rpm) led to decreased microchannel diameter. Increasing alginate injection rate (0.25, 0.50 mL/min) led to increased microchannel diameter. (c). Cross-sectional views of encapsulated alginate microfibers in 3D hydrogels. Left = side view. Right =top view. Red =hydrogel edge. Scale bar = 2 mm.

Fig. 2.. Optimization of endothelial cell density in hydrolytically degradable alginate microfibers for microchannel formation in 3D hydrogels.

(a). Increasing initial endothelial cell density in alginate solution led to increased cell confluency and eventual cell aggregation after 2 days in culture. Scale bar = 250 μm. (b). Endothelial cells (4 M cells/mL) retained high cell viability and proliferated over time in microchannels. Scale bar =500 μm. Green =live. Red =dead. (c). Endothelial cells (4 M/mL) expressed high levels of CD31, along microchannel length and diameter after 7 days in culture. Scale bar = 100 μm. Blue =nuclei. Red = CD31. Maximal projection (left), orthogonal view (middle), single slice (right).

Fig. 3.. Effects of co-culture on cell morphology over time.

(a). High cell viability of tumor and endothelial cells was observed after encapsulation. Scale bar =500 μm. Green = live. Red = dead. (b),(c). Endothelial cells started with rounded morphology after release from alginate microfibers on day 1. In GBM + ENDO group, endothelial cells were more rounded and disorganized, compared to cells in ENDO ONLY group, after 14 days in culture. Tumor cells started with rounded morphology after encapsulation. In GBM ONLY group, tumor cells formed large cell aggregates with radial protrusions after 14 days in culture. When co-cultured with endothelial cells in GBM + ENDO group, tumor cells directly adjacent to endothelial microchannels had more spherical morphology. Scale bars = 500 μm (b), 125 μm (c).

Fig. 4.. Co-culture led to downregulation of endothelial cell adhesion protein expression.

(a). Expression of cell adhesion proteins in endothelial cells on day 7, normalized to day 1. Mouse specific primers were used to measure gene expression levels of claudin-5, ZO-1, and CD31 using RT-PCR. * p < 0.05. (b). Immunostaining of cell-cell junction proteins on day 14. Blue = nuclei. Red = human nuclear antigen. Green = claudin-5, ZO-1, or CD31. White dashed line = microchannel edge. Scale bar =100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5.. Co-culture with endothelial cells led to increased tumor cell proliferation.

(a). Immunostaining for proliferating (Ki67+) tumor cells on day 7. Scale bar = 100 μm. Red = human nuclear antigen. Green = Ki67. Blue = nuclei. (b). Percentage of proliferating (Ki67+) tumor cells on day 7 (n = 150). * p < 0.05. (c). Expression of CXCR4 in tumor cells on day 7, normalized to Day 1. Human specific primers were used to measure gene expression levels of CXCR4 using RT-PCR. * p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6.. Endothelial cells in GBM + ENDO and ENDO ONLY groups had comparable levels of cell proliferation.

(a). Immunostaining for proliferating (Ki67 + cells) on day 7. Scale bar = 100 μm. Green = Ki67. Blue = nuclei. (b). Percentage of proliferating cells (Ki67+) on day 7 (n = 150). * p < 0.05. (c). Expression of VEGFa in tumor cells on day 7, normalized to day 1. Human specific primers were used to measure gene expression levels of VEGFa using RT-PCR. (d). Expression of VEGFa in endothelial cells on day 7, normalized to day 1. Mouse specific primers were used to measure gene expression levels of VEGFa using RT-PCR. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)