2D). In addition, invasion of Huh7 cells through Matrigel was www.selleckchem.com/products/PF-2341066.html increased in the presence of PTFs and was blocked when BrP-LPA was added. The invasive capability of Huh7 cells was enhanced in the presence of both PTFs and CAFs and was inhibited in the presence of BrP-LPA (Fig. 2E). Conversely, PLC/PRF/5 showed a poor invasive capacity through Matrigel in the presence of either PTFs or CAFs. Therefore, BrP-LPA did not display any effect on these cells (Fig. 2F). No toxicity effect was observed at used concentration on Huh7 cells, PLC/PRF/5 cells, PTFs, or CAFs as evaluated by MTT assay (Supporting Fig. 3). In conclusion, PTFs and CAFs increased the aggressive
phenotype in Huh7 cells but not in PLC/PRF/5 cells. To gain a better insight into the molecular mechanisms underlying the paracrine cross-talk between stromal and HCC cells, we studied
the paracrine action of LPA. We first measured the concentrations of secreted LPA in conditioned medium from PTFs and CAFs and in two different HCC cell lines (Huh7 and PLC/PRF/5). High levels of LPA were detected in Huh7 cells compared with PLC/PRF/5 cells, CAFs, and PTFs (P < 0.0001) (Fig. 3A). We then analyzed the messenger RNA (mRNA) expression levels of LPA receptors 1-5 in the same cells. We found that among the LPA receptors investigated, LPA Selleck JAK inhibitor receptor 1 was the most strongly expressed, being mainly expressed by CAFs and PTFs compared with Huh7 cells (Fig. 3B). In agreement with the
LPA levels, ATX expression levels were more abundant in Huh7 compared with PLC/PRF/5 cells, CAFs, and PTFs (P < 0.0001) (Supporting Fig. 4A). In conclusion, Huh7 cells produced LPA and ATX, whereas PLC/PRF/5cells, PTFs, and CAFs did not, and CAFs and PTFs only expressed LPA receptors. To investigate the functional role of PTFs or CAFs in the cross-talk between stromal and HCC cells, we challenged PTFs to migrate in the presence of Huh7- and PLC/PRF/5-conditioned medium (CM). In the presence of Huh7-CM, PTFs migrated efficiently to the same extent as in the presence of LPA. This effect was already evident after those 12 hours but was stronger after 72 hours. BrP-LPA blocked this migration (Fig. 3C). On the contrary, no PTF migration was observed in the presence of PLC/PRF/5-CM. Therefore, BrP-LPA did not display any effect on this coculture, whereas the addition of LPA still promoted strong migration (Fig. 3D). Moreover, the number of α-SMA–positive cells was increased in PTFs migrating in the presence of Huh7-CM and LPA, but was strongly reduced by BrP-LPA (P < 0.05). This effect was particularly evident after 72 hours (Fig. 3E). On the contrary, the number of α-SMA–positive cells was not increased in PTFs migrating in the presence of PLC/PRF/5-CM, but was strongly increased when exogenous LPA was added (Fig. 3F). Similar results were obtained with Hep3B and HLE, LPA-producing and nonproducing, respectively, as shown in Supporting Fig. 5.