FIGURE 1:
Tumorigenic cancer cells exhibit a basal-like phenotype that is fostered by ECM stiffness and EMT. (a) Schematic of the 3D collagen bioreactor. Collagen I hydrogels are made in a media reservoir as described previously (Cassereau et al., 2015), and the application of 10% strain leads to an increase in effective gel stiffness from 0.5 to 2.5 kPa. (b) SEM images of representative 2.5 mg/ml collagen hydrogels in strained and unstrained conditions. Porosity and fiber orientation are unaltered at 10% strain; increased stiffness may reflect fiber bundling. Scale bar, 2 µm. (c) A 3D collagen invasion assay of multicellular spheroids composed of tumorigenic PyMT cells in 2.5 mg/ml 3D collagen gels that are unstrained (soft, 0.5 kPa) or strained (stiffened, 2.5 kPa) for 72 h. Tumorigenic PyMT cells recapitulate invasive behavior when induced to undergo EMT via treatment with TGFβ. Scale bar, 100 µm. (d) Quantification of invasion parameters, total cross-sectional area of spheroid and protrusions, number of protrusions, and number of migrating cells normalized to the unstrained, untreated condition. Mean ± SEM (*p < 0.05; ***p < 0.001; >20 cells). (e) Immunofluorescence imaging of PyMT cell spheroids in 3D collagen gel stained for β-catenin (green) and DNA (DAPI, blue). β-Catenin strongly localizes to cell–cell junctions in cells cultured in soft 3D gels, whereas it is found more diffusely within the cytoplasm and nucleus in strain-stiffened gels and TGFβ-treated cells. (f) PyMT cell spheroids in a 3D collagen bioreactor express basal epithelial marker K14 at the leader cells when cultured in strain-stiffened gels but maintain luminal epithelial K8/18 in the noninvasive core. Induction of EMT via TGFβ treatment also leads to expression of K14 in leader cells. (g) Snail transcription visualized by Snail-YFP is up-regulated in leader cells in strain-stiffened gels and TGFβ-treated cells. Scale bar, 50 µm (e–g), 10 µm (inset).