Fig. 4

Ovarian cancer PGCCs can proliferate and form tumors on chick chorioallantoic membrane (CAM). SKOV3-Red and SKOV3-Green were mixed in co-culture with double-antibiotic. a and b Cell population (SKOV3-M) obtained after 3 weeks of double selection. a Visualization of double-positive mononucleated cells by fluorescent microscopy. Upper left: green signal (GFP). Upper right: red signal (mCherry). Lower left: Phase contrast. Lower right: Overlay. Scale bars represent 100 μm. b Quantification of red (mCherry), green (GFP), and double-positive cells by flow cytometry in SKOV3-Red, SKOV3-Green, SKOV3-M, and untransfected SKOV3 cells (ct-SKOV3). SKOV3-M cells were analyzed at passages 51, 63, and 72. c and d Cell cycle distribution was analyzed in SKOV3-Red, SKOV3-Green, and SKOV3-M cells by flow cytometry following Hoechst staining. Doublets cells have been removed from the analysis based on Hoescht W and Hoescht A signals. c Cell distribution depending on Hoechst signal intensity. d Percentages of cells in G0-G1 and G2 phases. e and f Proliferation capacities of SKOV3-Red, SKOV3-Green, and SKOV3-M cells were assessed by analyzing CellTrace™ far-red cytoplasmic signal dilution by flow cytometry. Cells were stained on day 0 (D0) and seeded in 10 cm culture dishes (5 × 10⁵ cells/dish). Far-red signal was analyzed for five consecutive days (D0 to D4). Doublets cells have been removed from the analysis based on SSC W and SSC A signals. e Evolution of far-red signal intensity from D0 to D4. f Evolution of far-red signal dilution expressed as a percentage of mean signal intensity ± SEM measured at D0. g and h Tumorigenic capacity of SKOV3-Red, SKOV3-Green, and SKOV3-M cells was assessed using the CAM model. Cells were inoculated on egg development day (EDD) 8, and tumor size was measured at EDD10.5 and EDD13.5. g Images of the tumors growing in CAM at EDD13.5 h Tumor growth (tumor size at EDD13.5 relative to tumor size at EDD10.5). Results are expressed as mean ± SEM