Fig. 6: Conditioned medium from invasive cells makes non-invasive cells degradative and invasive.
From: Intercellular transfer of cancer cell invasiveness via endosome-mediated protease shedding
a HeLa cells were seeded on Oregon GreenTM 488 labelled gelatin and treated with conditioned medium (CM) from MDA-MB-231-TKS5-GFP-mCh-MT1-MMP cells or culture medium for 24 h. Cells were labelled with rhodamine-Phalloidin (actin) and Hoechst 33342 (nuclei). Representative of 10 images per condition. Illustration created with BioRender.com. b HeLa cells were seeded on Oregon GreenTM 488 labelled gelatin and treated with conditioned medium from MDA-MB-231 cells or regular cell culture medium for 24 h. Graph represents the area of gelatin degradation per cell, measured automatically by NIS-Elements and shown as mean +/− SD from n = 3 independent experiments. In total >300 cells were analysed per condition. Unpaired two-sided t-test. c MCF7 cells were seeded on Oregon GreenTM 488 labelled gelatin and treated with CM from HT1080 cells or regular cell culture medium for 24 h. Representative of 25 images per condition from two independent experiments. d HeLa cells embedded in type I collagen were incubated with culture medium or CM from MDA-MB-231-TKS5-GFP-mCh-MT1-MMP cells and analysed by live cell microscopy. Graph shows the relative mean velocity per cell (µm min −1) +/−SD. Dots represent the mean value from n = 10 wells (each comprising 35–80 cells) collected from three independent experiments. Cells tracked in total, culture medium: 367, CM: 497. Unpaired two sided t-test. Rose plots represent cell tracks from one well per condition. Culture medium 55 cells; CM 58 cells. e Inverted invasion assay of HeLa cells into a collagen-I matrix. Co-culturing with invasive (MDA-MB-231-TKS5-GFP-mCh-MT1-MMP) cells stimulates invasive migration of HeLa cells (labelled with Calcein AM, green) when compared to co-culturing them with non-invasive cells (HeLa). Graphs represent the relative number of cells in the collagen plugs (right) and the amount of cells which invaded more than 40 µm into the collagen-I matrix (left). Error bars denote mean +/− SD from n = 3 independent experiments, each comprising three collagen plugs with 5–6 confocal z-stacks per condition. One-sample two-sided t-test. Example images from one representative image stack per condition are displayed. Images marked with a yellow frame are magnifications of the 50 µm z slice. Illustration created with BioRender.com.
