Fig. 6

Using the pipeline to characterize the escape and colonization of SK-MEL-28 melanoma cells. Melanoma cells were introduced into four pipeline assays, providing the opportunity to compare and contrast cell behaviour in each context. In the 2D/3D assay, melanoma cells adopted an elongated morphology on 2D plastic, at border zones (white B) and on top of the 3D matrix. Collagen was used at two different concentrations: a, 1 mg/ml and c, 2 mg/ml and with 10 μg/ml fibronectin: b, 1 mg/ml collagen + fibronectin and d, 2 mg/ml collagen + fibronectin. When melanoma cells were encapsulated in collagen (g, 1 mg/ml, h 2 mg/ml) or collagen with 10 μg/ml fibronectin (i, 1 mg/ml + fibronectin, j, 2 mg/ml + fibronectin), they proliferated to form small tight colonies in the 2 mg/ml gels and looser spread structures at 1 mg/ml. In a 2 mg/ml compressed collagen matrix, e1 - e2, melanoma cells partially colonized the matrix (blue arrows show uncolonized matrix) before escaping into the surrounding lower density matrix (LC). Melanoma cells within the compressed collagen (CC) adopted ovoid groupings (yellow arrows) cells becoming elongated with narrow filopodia upon escape (green arrows). Similar behaviours were observed in compressed collagen with fibronectin (CCF), f1 - f2. Melanoma cells seeded onto dCAM, cultured for 10 days proliferated to form layers. Ki67 staining (green) indicated that cells were actively dividing within each layer – white arrows. Phalloidin – red. Dapi – blue. Images a, b, e1–2, f1–2 were generated using Nikon TiE timelapse system and Plan × 10/0,25 Ph1 DL lens and NIS Nikon Elements software. Scale bars = 100 μm. Images c, d, g-j were generated using a Leica DMi8 inverted microscope, Leica DF33000G camera, Tokai Hit STR stage top incubator, ND × 4/0.10 PH0 HI PLAN and × 10/0.25 PH1 N PLAN achromatic objective lenses. Scale bars = 50 μm. Image k was generated using Nikon A1-R confocal microscope with a × 60 1.40 Plan Apo ∞/0.17 WD 0.13, NA 1.4 lens. Scale bar = 25 μm