Soluble rat tail collagen 1

HT1080 cells were embedded in 2 mg ml⁻¹ type I collagen gel as described previously by Fraley et al.31 (link) Briefly, cell suspensions containing 5,000 to 75,000 cells in 1:1 (v/v) ratio of cell culture media and reconstitution buffer were mixed with appropriate volume of soluble rat-tail collagen I (Corning Inc.) to obtain a final collagen I concentration of 2 mg ml⁻¹. A calculated amount of 1 M NaOH was added quickly and the final solution was mixed well to bring the pH to ∼7. The cell suspension was added to a 24-well coverslip-bottom cell-culture dish and immediately transferred to an incubator maintained at 37 °C to allow polymerization. Fresh medium was added 1 h before imaging. MDA-MB-231 and U-87 cells were embedded in 1 mg ml⁻¹ type I collagen matrix. The data represented for each cell line were obtained from three independent experiments.

HT1080 cells were embedded in 2 mg/ml type I collagen gel as described previously by Fraley et al. [30 (link)]. Briefly: cell suspensions containing 5,000 (low cell density environments) and 25,000 (high cell density environments) cells in 1:1 (v/v) ratio of cell culture medium and reconstitution buffer were mixed with the appropriate volume of soluble rat-tail collagen I (Corning Inc.) to obtain a final collagen I concentration of 2 mg/ml. A calculated amount of 1 M NaOH was quickly added, and the final solution was mixed well to bring the pH to ~7. The cell suspension was added to a 24-well coverslip-bottom cell-culture dish, and immediately transferred to an incubator maintained at 37° C to allow polymerization of the collagen. Fresh medium and medium containing specific concentrations of MMP inhibitors were added 1 h before imaging. MDA-MB-231 cells were embedded in 1 mg/ml type I collagen matrix with 5,000 cells/well to simulate low cell density environments, and 50,000 cells/well to simulate high cell density environments.

HT1080WT cells were embedded in type 1 3D collagen matrices in increasing cell numbers from 5,000 to 75,000 per well. Collagen gels were prepared to obtain a final collagen concentration of 2 mg ml⁻¹ using soluble rat-tail collagen I (Corning). Cell supernatants were extracted 24 hours after incubation at 37° C in a humidified incubator.
A Luminex High Performance Assay multiplex kit was obtained from R&D systems (Biotechne). All reagents were prepared following the instructions in the kit, specifically for cell supernatants. The assay plate was read using a Magpix Multiplexing system (Luminex). Data was analyzed using xPONENT software to determine secreted protein content (Luminex).