FIGURE 1.

Analysis of BCAM in OC ascites and comparative functional analysis of membrane‐bound and soluble BCAM. (A) Immunoblot of BCAM in eight different cell‐free ascites samples. For comparison, conditioned medium (containing sBCAM) and lysate from OVCAR4 cell was included (right‐most lanes). Quantitation of relative signal intensities as well as BCAM levels in ascites samples measured by ELISA are shown at the bottom. The band labelled with ‘?’ denotes an unspecific background band. The bottom panel shows the membrane after staining with the Pierce Reversible Total Protein Stain Kit as loading control. (B) Concentration of BCAM protein in the ascites from n = 70 high‐grade serous OC patients determined by ELISA. (C) Kaplan–Meier plot analysing the relapse‐free survival (RFS) of n = 65 evaluable patients analysed in panel B. Groups were split at the q = 0.7 quantile (best‐fit); p: logrank p value; HR: median hazard ratio; rfs: months to 50% RFS for patients with high/low BCAM levels. (D) Effect of BCAM on OC cell adhesion to LN‐511 on non‐adhesive microplates coated with LN‐511. Cell adhesion was quantified by RTCA. Left: BCAM‐overexpressing OVCAR8 cells (OVCAR8‐OE). Clones stably transfected with BCAM1 or BCAM2 (Figure S4) were compared with cells transduced with the empty expression vector (pcDNA‐6). Right: Adhesion of OVCAR8 cells was analysed in the presence of Fc‐BCAM or negative control (Fc) at equimolar concentration (1 μg/ml of Fc‐BCAM; 0.33 μg/ml of Fc). (s): solvent for Fc or Fc‐BCAM. (E) Effect of BCAM on two‐dimensional OC cell migration under the same conditions as in panel D, except that a further control clone (pcDNA3) was included. Transwell‐chamber microplates were coated with LN‐511 and cell migration was quantified by RTCA. The data in D and E are based on n = 3 biological replicates. *p < .05; **p < .01; ***p < .001; ns, not significant by unpaired t test.