Figure 1.

The conditioned medium from Meso 301 induces ovarian cancer cell motility through LPA. Subconfluent Meso 301 cells were allowed to condition in SFM for 24 hours, and the collected conditioned medium (heated or not) was used as a chemoattractant (600 µl in the bottom chambers of transwell inserts) for SKOV3 (A) and OVCAR3 (B) (1 x 10⁵ cells/100 µl of SFM-0.4% BSA in the upper chamber) in the presence or in the absence of SPARC (10 µg/ml). Results are expressed as the mean ± SEM of the fold change in cell migration induced by a complete medium in the bottom chamber as controls. FN invasion by SKOV3 (C) and OVCAR3 (D) cells was tested in response to the conditioned medium of Meso 301 (heated or not) added with the cells to the upper chamber of transwell inserts, in the presence or in the absence of SPARC (10 µg/ml). LPA production by Meso 301 was inhibited by PLA₂ inhibitor (AACOCF₃). The conditioned medium of Meso 301 serum-starved for 24 hours was collected in the absence (control conditioned medium) or in the presence of AACOCF₃ (25 µM) added at serum starvation (AACOCF₃-CM) or after the conditioned medium had been generated (conditioned medium + AACOCF₃). All collected conditioned media were heat-inactivated and added to the bottom chamber of transwell inserts, and the chemotactic activity of SKOV3 (E) and OVCAR3 (F) was tested in the aforementioned conditioned medium and after replenishment with LPA (50 µM; AACOCF₃-CM + LPA). Results are expressed as the mean ± SEM of the fold change of the chemotactic activity of heated conditioned medium under experimental conditions, compared to the conditioned medium (control conditioned medium) of Meso 301 heated for 24 hours. The effect of the above-mentioned conditioned medium on FN invasion by SKOV3 (G) and OVCAR3 (H) was tested when they were added with the cells to the top chambers of the inserts, whereas the bottom chambers contained a complete growth medium. Results are expressed as the mean ± SEM of the fold change of the invasive activity of SKOV3 and OVCAR3 induced by different experimental conditions, relative to that induced by the conditioned medium (control conditioned medium) of Meso 301 heated for 24 hours. Data summarized are representative of an experiment performed in triplicate and repeated thrice with similar results. *P < .05, compared to unheated Meso-CM. **P < .05, compared to heated Meso-CM. #P < .05, compared to heated Meso-CM. ^¶P < .05, compared to AACOCF₃-CM.