Figure 3 from CLDN6 Expression Plasticity in Ovarian Cancer: Insights into Therapeutic Optimization for CLDN6-Targeted Immunotherapy

CLDN6 expression reversibly fluctuates in vitro depending on cell density, accompanied by changes in EMT-, ECM-, and stem cell–related genes. A, Schematic of in vitro culture experiments with different cell densities. NIH:OVCAR-3 cells were plated at low or high density for 1 week and used for gene expression analysis. Brightfield microscopy images show the cell confluency of NIH:OVCAR-3 cells after 7 days of culture at low and high cell densities. B, qPCR analysis of indicated gene expression in NIH:OVCAR-3 cells grown at low and high cell density for 7 days. Quantitative data are presented as the mean ± SD (n = 3). Two-tailed Student t test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. C and D, Heatmap of nCounter Analysis System results for NIH:OVCAR-3 cells grown at low or high cell density for 7 days (n = 3). The heatmap shows differential expression of genes involved in EMT (C) and ECM remodeling and ECM–receptor interaction (D) between cells with low and high cell densities. E, Schematic of in vitro culture experiments with reexpansion of NIH:OVCAR-3 cells at high cell density. On day 1, NIH:OVCAR-3 cells are plated at low, middle, and high cell densities. After 1 week of culture, cells are used for gene expression analysis (day 7 after first passage). Cells initially plated at high density are further seeded at low, middle, and high cell densities again. These cells were harvested after one additional week and used for expression analyses (day 7 after second passage). F, Representative flow cytometry plots of NIH:OVCAR-3 cells after first and second passages with different cell densities, assessed for the expression of CLDN6 and CD44. The plots of cells stained with isotype control are shown in Supplementary Fig. S6. G, Western blot analysis of CLDN6 protein levels in NIH:OVCAR-3 cells. NIH:OVCAR-3 cells were plated at low, middle, and high cell densities, and CLDN6 expression was examined after the first 7-day culture (first passage) and second 7-day culture (second passage). Densitometric analysis of CLDN6 normalized to GAPDH is shown below each lane.