A synthetic modular approach for modeling the role of the 3D microenvironment in tumor progression

Abstract

Here, we demonstrate the flexibility of peptide-functionalized poly(ethylene glycol) (PEG) hydrogels for modeling tumor progression. The PEG hydrogels were formed using thiol-ene chemistry to incorporate a matrix metalloproteinase-degradable peptide crosslinker (KKCGGPQG↓IWGQGCKK) permissive to proteolytic remodeling and the adhesive CRGDS peptide ligand. Tumor cell function was investigated by culturing WM239A melanoma cells on PEG hydrogel surfaces or encapsulating cells within the hydrogels, and either as monocultures or indirect (non-contact) cocultures with primary human dermal fibroblasts (hDFs). WM239A cluster size and proliferation rate depended on the shear elastic modulus for cells cultured on PEG hydrogels, while growth was inhibited by coculture with hDFs regardless of hydrogel stiffness. Cluster size was also suppressed by hDFs for WM239A cells encapsulated in PEG hydrogels, which is consistent with cells seeded on top of hydrogels. Notably, encapsulated WM239A clusters and single cells adopted invasive phenotypes in the hDF coculture model, which included single cell and collective migration modes that resembled invasion from human melanoma patient-derived xenograft tumors encapsulated in equivalent PEG hydrogels. Our combined results demonstrate that peptide-functionalized PEG hydrogels provide a useful platform for investigating aspects of tumor progression in 2D and 3D microenvironments, including single cell migration, cluster growth and invasion.

Figure 1. Schematic representation of poly(ethylene glycol) (PEG) hydrogels formed using “thiol-ene” chemistry.

(a) Hydrogel networks were formed by photopolymerization to crosslink 4-arm 20 kDa PEG-norbornene (PEG-NB) molecules with an MMP sensitive peptide crosslinker (KKCGGPQG↓IWGQGCKK). An adhesive peptide moiety (CRGDS) was also added to promote cell adhesion. (b) Changes in shear modulus as a function of PEG-norbornene concentration (with the molar ratio of crosslinker kept constant, 1:1 thiol:ene mole ratio).

Figure 2. The influence of matrix modulus and stromal cells on cluster growth for WM239A melanoma cells seeded on PEG hydrogels.

(a–f) Calcein/ethidium homodimer (Live/Dead) staining for WM239A clusters seeded on top of PEG hydrogels in (a–c) monoculture (−hDFs) or (d–f) cocultured with hDFs (+hDFs). PEG hydrogels were formed with the following shear moduli: (a,d) 520 Pa, (b,e) 700 Pa, and (c,f) 1150 Pa. Scale bars: 50 μm. (g) WM239A cluster size as a function of modulus after 31 days of culture on top of PEG hydrogels with (black bars) or without (white bars) hDFs (*p < 0.05; ***p < 0.001, n ≥ 20 clusters from 3 replicate hydrogels). See Supplementary Figure S1 for all statistical comparisons. (h) Proliferation as a function of modulus was measured by EdU assay for WM239A cells after 2 weeks of culture on PEG hydrogels (*p < 0.05; ***p < 0.001, n > 400 clusters from 3 replicate hydrogels).

Figure 3. The influence of stromal cells on cluster growth and invasion for WM239A cells encapsulated in PEG hydrogels.

(a) Schematic representation of cluster growth and coculture with hDFs. WM239A cells were encapsulated in PEG hydrogels and then surrounded by collagen with or without hDFs (+/− hDFs) on day 7. (b) Time-lapse images illustrating WM239A cluster growth from day 8 to day 11 (24 hrs/frame). WM239A cluster size after 11 days (c) as monocultures (−hDFs) or (d) after 7 days of monoculture followed by 4 days of coculture with hDFs (+hDFs). (e) Projected area for WM239A clusters cultured with or without hDFs (***p < 0.001; n > 200 clusters, ≥8 replicate hydrogels). (f) Time-lapse images (3 hrs/frame) beginning on day 12 for a WM239A cluster encapsulated in a PEG hydrogel and cocultured with hDFs (beginning at day 7). Scale bars: (b–d) 50 μm; (f) 25 μm.