A) Overview of collagen invasion live imaging assay. EOC cells are fluorescently tagged with RFP or GFP via lentiviral transduction and applied as either individual cells or pre-formed MCAs on top of a 3D collagen gel (1.5mg/ml collagen concentration in complete medium) inside a glass-bottom dish. Continuous z-stack imaging of cell/MCA dynamics (green or red) and collagen (blue) was performed using confocal fluorescence and reflectance modes, respectively. B–F) Imaging and analysis of collagen invasion. Multiple representative z-stack snapshots of (C) OvCa433-RFP (Ecad+) cells, (D) DOV13-GFP (Ncad+) cells, (E) OvCa433-RFP (Ecad+) MCAs, (F) DOV13-GFP MCAs, and (Suppl. fig. 2) control fluorescent beads were obtained for up to 72h, and depth of penetration depth quantified (Mean±SD, N=100 in three assays);scale bar: 100μm. Statistical significance shown between the penetration depth of each cell line relative to fluorescent beads; p<0.05, Mann-Whitney U test.