Dynamically Crosslinked Poly(ethylene-glycol) Hydrogels Reveal a Critical Role of Viscoelasticity in Modulating Glioblastoma Fates and Drug Responses in 3D

Abstract

Glioblastoma multiforme (GBM) is the most prevalent and aggressive brain tumor in adults. Hydrogels have been employed as 3D in vitro culture models to elucidate how matrix cues such as stiffness and degradation drive GBM progression and drug responses. Recently, viscoelasticity has been identified as an important niche cue in regulating stem cell differentiation and morphogenesis in 3D. Brain is a viscoelastic tissue, yet how viscoelasticity modulates GBM fate and drug response remains largely unknown. Using dynamic hydrazone crosslinking chemistry, a poly(ethylene-glycol)-based hydrogel system with brain-mimicking stiffness and tunable stress relaxation is reported to interrogate the role of viscoelasticity on GBM fates in 3D. The hydrogel design allows tuning stress relaxation without changing stiffness, biochemical ligand density, or diffusion. The results reveal that increasing stress relaxation promotes invasive GBM behavior, such as cell spreading, migration, and GBM stem-like cell marker expression. Furthermore, increasing stress relaxation enhances GBM proliferation and drug sensitivity. Stress-relaxation induced changes on GBM fates and drug response are found to be mediated through the cytoskeleton and transient receptor potential vanilloid-type 4. These results highlight the importance of incorporating viscoelasticity into 3D in vitro GBM models and provide novel insights into how viscoelasticity modulates GBM cell fates.

Keywords: 3D disease models; drug responses; glioblastoma multiforme; hydrogels; stress relaxation; viscoelasticity.

Figure 1.

Dynamically crosslinked PEG hydrogels demonstrate tunable stress relaxation with brain-mimicking stiffness. a) Schematic representing 8-arm PEG macromers functionalized with reactive groups that enable hydrazone crosslinking. b) Chemical structures of dynamic alkyl-hydrazone (blue) and stable benzyl-hydrazone (red) crosslinks. c) Schematic depicting how varying the molar ratio of alkyl-hydrazone:benzyl-hydrazone (AH:BH) crosslinks allows formation of three hydrogel groups with tunable stress relaxation. Collagen-I (green) is incorporated at a constant concentration to provide cell adhesion. d-g) Characterizing the mechanical property of the three hydrogel formulations using shear rheology. Mouse brain was included as a positive control. d) Representative stress relaxation profiles of fast-, medium-, and slow-relaxing hydrogels and mouse brain. e) Time for the normalized modulus to reduce to half its original value, τ_½, from stress relaxation tests. f) Loss tangent measurements. g) Young’s modulus. One-way ANOVA with Dunnett’s multiple comparisons test was used for analysis of the data in e-g, comparing with mouse brain: ns, not significant; ****p< 0.0001; n = 6, 5, 5, and 5 independent samples (fast, medium, slow, and mouse brain). Data reported in e-g represent mean value ± s.d.

Figure 2.

Stress relaxation promotes GBM cell spreading, migration, and proliferation in 3D. a) Representative maximum intensity projection images of membrane stained (R18) D-270 MG cells cultured within fast-, medium-, and slow-relaxing hydrogels on day 3. Scale bar, 100 µm. b) Roundness quantification of D-270 MG cells in the three hydrogel groups on day 3. One-way ANOVA with Tukey’s multiple comparisons test was used for analysis of the data: ****p< 0.0001; n = 498, 343, and 216 (fast, medium, and slow) cells across 3 independent biological replicates. The dashed lines in the violin plots represent median values. c) Brightfield time-lapse imaging of a single cell within fast- and slow-relaxing hydrogels over 90 minutes. Times are indicated in min:s. Scale bar, 20 µm. d) Representative 3D track reconstructions for cell migration in the three hydrogel groups from time-lapse imaging. 80 randomly selected cell migration track trajectories are shown for each condition. Grid size, 10 µm. e-g) Analysis of cell migration track data from time-lapse imaging: e) probability of cell migration, f) mean speed, and g) migration track length of cells tracked within the three hydrogel groups. One-way ANOVA with Tukey’s multiple comparisons test was used for analysis of the data: *p<.05, ***p<.001, ****p < 0.0001; n = 855, 500, and 556 (fast, medium, and slow) cells tracked across 3 independent biological replicates. Bars indicate mean value ± s.d. The dashed lines in the violin plots represent median values. h) Representative immunostaining images of D-270 MG cells for EdU staining (green) and nucleus (blue) on day 3. Scale bar, 50 µm. i) Fraction of EdU-positive D-270 MG cells on day 3. One-way ANOVA with Tukey’s multiple comparisons test was used for analysis of the data: **p<.01, ****p< 0.0001; n = 212, 191, and 153 (fast, medium, and slow) cells across 3 independent biological replicates. Data reported represent mean value ± s.d.

Figure 3.

Stress relaxation promotes glioblastoma stem-like cell (GSC) marker expression in 3D. a) Representative immunostaining images of D-270 MG cells for GSC marker expression (Nestin, CD133, or Sox2; green), F-actin (red), and nucleus (blue) in fast-, medium-, and slow-relaxing hydrogels on day 3. Scale bar, 10 µm. b) Western blot analysis of GSC marker expression by GBM cells in fast-, medium-, and slow-relaxing hydrogels. c) Quantification of western blot images by normalizing each marker to GAPDH. One-way ANOVA with Tukey’s multiple comparisons test was used for statistical analysis: ns, not significant; *p<.05, ***p<.001, ****p < 0.0001; n = 3 independent biological replicates per group. Data reported represent mean value ± s.d.

Figure 4.

Stress relaxation increases drug sensitivity of GBM cells in 3D. Two model chemotherapeutic drugs were used, including (a,b) Temozolomide (TMZ) and (c, d) Carmustine (BCNU), to treat two GBM cell lines (D-270 MG and U87-MG). Relative cell viability was reported by normalizing treated to untreated GBM cells in fast (blue), medium (purple), and slow (red) stress relaxing hydrogels. Both GBM cell lines demonstrate enhanced chemosensitivity to both TMZ and BCNU as stress relaxation increases. Two-way ANOVA with Tukey’s multiple comparisons test was used for statistical analysis: *p<.05, **p<.01, ***p<.001, ****p< 0.0001; n = 3 independent biological replicates per condition. Data reported represent mean value ± s.d.

Figure 5.

Stress relaxation enhances cytoskeletal formation in GBM cells in 3D, and pharmacologically disrupting the cytoskeleton abrogates stress relaxation-induced GBM spreading, migration and drug sensitivity. a-c) Representative immunostaining images of D-270 MG cells for myosin IIa (green), F-actin (red), and nucleus (blue) within fast-, medium-, and slow-relaxing hydrogels on day 3: a) vehicle-alone (control), b) a myosin inhibitor (+ Blebbistatin), or c) an inhibitor of actin polymerization (+ Cytochalasin D). Scale bar, 10 µm. d) Representative 3D track reconstructions for cell migration in fast-relaxing hydrogels with vehicle alone, Blebbistatin, or Cytochalasin D treatment from time-lapse imaging. 80 randomly selected cell migration track trajectories are shown for each condition. Grid size, 10 µm. e-g) Analysis of cell migration track data from time-lapse imaging: e) probability of cell migration, f) mean speed, and g) migration track length of cells tracked within fast-relaxing hydrogels. One-way ANOVA with Tukey’s multiple comparisons test was used for data analyses: ns, not significant; ****p< 0.0001; n = 670, 845, and 710 (control, BLEB, and CytoD) cells tracked across 3 independent biological replicates. Bars indicate mean value ± s.d. The dashed lines in the violin plots represent median values. h) Relative cell viability of D-270 MG cells treated with Temozolomide (TMZ only), TMZ with Blebbistatin (TMZ + BLEB), or TMZ with Cytochalasin D (TMZ + CytoD). Two-way ANOVA with Tukey’s multiple comparisons test was used for analysis of the data: **p<.01, ****p< 0.0001; comparison with TMZ only: #p<.01, ##p<.0001; n = 3 independent biological replicates per condition. Data reported represent mean value ± s.d.

Figure 6.

Stress relaxation promotes TRPV4 expression in 3D, and modulating TRPV4 activity impacts GBM cytoskeleton, migration and drug response. a) Representative immunostaining images of D-270 MG cells for TRPV4 (green), F-actin (red), and nucleus (blue) within fast-, medium-, and slow-relaxing hydrogels on day 3. Scale bar, 10 µm. b) Western blot analysis of TRPV4 for cells in fast-, medium-, and slow-relaxing hydrogels. c) Quantification of TRPV4 expression normalized to GAPDH from western blot. One-way ANOVA with Tukey’s multiple comparisons test was used for analysis of the data: ns, not significant; **p<.01, ***p<.001, ****p< 0.0001; n = 3 independent biological replicates per group. Data reported represent mean value ± s.d. d) Representative immunostaining images of D-270 MG cells for TRPV4 (green), F-actin (red), and nucleus (blue) within fast-relaxing hydrogels treated with vehicle-alone (control), a TRPV4 agonist (GSK101), or a TRPV4 antagonist (GSK205). Scale bar, 10 µm. e) Representative 3D track reconstructions for cell migration in fast-relaxing hydrogels with vehicle-alone (control), GSK101, or GSK205 treatment from time-lapse imaging. 80 randomly selected cell migration track trajectories are shown for each condition. Grid size, 10 µm. f-h) Analysis of cell migration track data from time-lapse imaging: e) probability of cell migration, f) mean speed, and g) migration track length of cells. One-way ANOVA with Tukey’s multiple comparisons test was used for analysis of the data: ns, not significant; ****p< 0.0001; n = 670, 913, and 777 (control, GSK101, and GSK205) cells tracked across 3 independent biological samples. Bars indicate mean value ± s.d. The dashed lines in the violin plots represent median values. i) Relative cell viability of D-270 MG cells treated with TMZ only, TMZ + GSK101, or TMZ + GSK205. One-way ANOVA with Tukey’s multiple comparisons test was used for analysis of the data: *p<.05, ***p<.001, ****p< 0.0001; n = 3 independent biological replicates per condition. Data reported represent mean value ± s.d. j) Intracellular calcium level in D-270 MG cells in fast-relaxing hydrogels treated with vehicle-alone (control), GSK101, or GSK205 treatment. One-way ANOVA with Tukey’s multiple comparisons test was used for analysis of the data: *p<.05, ****p < 0.0001; n = 43, 37, and 46 (control, GSK101, and GSK205) cells across 3 independent biological samples. The dashed lines in the violin plots represent median values.

Figure 7.

Stress relaxation reduces nascent protein deposition by GBM cells in 3D, which correlates with higher drug sensitivity. Pharmacological inhibition of nascent protein deposition increases GBM drug sensitivity in slow-relaxing hydrogels. a-c) Representative immunostaining images of D-270 MG cells for ECM proteins (Laminin, Fibronectin, or Collagen-IV; green), F-actin (red), and nucleus (blue) on day 3 within fast-, medium-, and slow-relaxing hydrogels. Samples were treated with a) vehicle-alone (control), b) an inhibitor of nascent protein deposition (+ Exo-1), or c) an inhibitor for matrix remodeling (+ TIMP-3). Scale bar, 10 µm. d) Relative cell viability of D-270 MG cells treated with Temozolomide (TMZ only), TMZ with Exo-1 (TMZ + Exo-1), or TMZ with TIMP-3 (TMZ + TIMP-3). Two-way ANOVA with Tukey’s multiple comparisons test was used for analysis of the data, comparing with TMZ only condition: ns, not significant; **p<.01; n = 3 independent biological replicates per condition. Data reported represent mean value ± s.d.

Figure 8.

A summary schematic of the key findings on the effect of stress relaxation on GBM cell fates and drug responses in 3D. Brain-mimicking, fast-relaxing hydrogels (left panel) promote GBM drug sensitivity and enhances GBM invasive phenotype including cell spreading, migration, and proliferation. Stress relaxation induced drug sensitivity is associated with enhanced actomyosin cytoskeletal formations, GSC marker expression, TRPV4 activity, and reduced nascent protein deposition. Conversely, slow-relaxing hydrogels (right panel) reduce GBM drug sensitivity and induces opposite trends in corresponding cell fates.