Matrix Stiffness Modulates Patient-Derived Glioblastoma Cell Fates in Three-Dimensional Hydrogels

Abstract

Cancer progression is known to be accompanied by changes in tissue stiffness. Previous studies have primarily employed immortalized cell lines and 2D hydrogel substrates, which do not recapitulate the 3D tumor niche. How matrix stiffness affects patient-derived cancer cell fate in 3D remains unclear. In this study, we report a matrix metalloproteinase-degradable poly(ethylene-glycol)-based hydrogel platform with brain-mimicking biochemical cues and tunable stiffness (40-26,600 Pa) for 3D culture of patient-derived glioblastoma xenograft (PDTX GBM) cells. Our results demonstrate that decreasing hydrogel stiffness enhanced PDTX GBM cell proliferation, and hydrogels with stiffness 240 Pa and below supported robust PDTX GBM cell spreading in 3D. PDTX GBM cells encapsulated in hydrogels demonstrated higher drug resistance than 2D control, and increasing hydrogel stiffness further enhanced drug resistance. Such 3D hydrogel platforms may provide a valuable tool for mechanistic studies of the role of niche cues in modulating cancer progression for different cancer types.

Keywords: glioblastoma; hydrogels; in vitro cancer models; patient-derived cells; stiffness.

FIG. 1.

Hydrogels with brain-mimicking biochemical cues and tunable stiffness for encapsulating PDTX GBM cells. (a) Schematic of hydrogel platform structure and design. Hydrogel mechanical and degradation properties were modulated by multiarm PEG-norbonene (8-arm PEG-NB), linear PEG-dithiol (PEG-SH) crosslinker, and MMP-cleavable crosslinker. To permit cell adhesion, CRGDS peptide was chemically incorporated. Thiolated hyaluronic acid (HA-SH) was chemically conjugated to mimic brain extracellular matrix content. Hydrogel crosslinking was achieved through thiol-ene UV photopolymerization. (b) Hydrogels with tunable stiffnesses were fabricated as 3D niche for PDTX GBM cells by varying PEG concentration (w/v %). Stiffness of hydrogels was measured on day 1 and day 14. *p < 0.05 (compared to hydrogel of same PEG concentration on day 1). Data are presented as mean ± SD (n = 3/group). (c) PDTX GBM cells showed high cell viability in all groups following encapsulation. Live = green. Dead = red. Scale bar = 500 μm. PDTX GBM, patient-derived glioblastoma xenograft; PEG, poly(ethylene-glycol); MMP, matrix metalloproteinase. Color images are available online.

FIG. 2.

Decreasing hydrogel stiffness enhanced PDTX GBM cell proliferation and spreading in 3D, as observed using bright-field microscopy. (a) PDTX GBM cell growth in hydrogels with varying stiffness on days 1, 7, and 14. Scale bar = 500 μm. (b) PDTX GBM cell morphology in hydrogels with varying stiffness on days 1, 7, and 14. Scale bar = 50 μm.

FIG. 3.

Decreasing hydrogel stiffness led to significant increase in the fold of PDTX GBM cell proliferation after 14 days in culture; increasing hydrogel stiffness to 26,600 Pa significantly upregulated gene expression of key markers. (a) Fold of cell proliferation on day 14 over day 1, as calculated from cell DNA content in hydrogels on days 1 and 14. *p < 0.05. Data are presented as mean ± SD (n = 3 samples/group). (b–f) Gene expression of PDTX cells in hydrogels on day 7 (n = 3) for mechanotransduction proteins RhoA (b) and ROCK1 (c), HIF1α (d), VEGFa (e), and MMP2 (f) (Normalized to day 1). *p < 0.05.

FIG. 4.

Decreasing hydrogel stiffness enhanced PDTX GBM cell spreading and formation of interconnected cell network in 3D hydrogels. Confocal Z-stack projection of F-actin cytoskeletal staining on day 14. Scale bar = 100 μm. Red = F-actin. Blue = nuclei. Z-stack projection generated from 200 μm stack with 2 μm step size. Color images are available online.

FIG. 5.

PDTX GBM cells cultured in 3D hydrogels exhibited higher chemotherapeutic drug resistance compared to 2D control; increasing hydrogel stiffness further enhanced drug resistance in 3D. TMZ drug resistance, as measured by fraction of viable cells normalized to no drug control (0 μM), for tumor cells were cultured in either 3D hydrogels with varying stiffness or in 2D dish. *p < 0.05. Data are presented as mean ± SD (n = 3 samples/group). TMZ, temozolomide.