Study On The Mechanisms Of Platelet Promoting Tumor Cell Metastasis

Tumor metastasis is the main reason of high mortality and challenge for cancer treatment in patients with cancer. Platelets increase adherence of tumor cell to endothelium and promote tumor cell metastasis and growth, while anti-platelet therapy could inhibit tumor metastasis. However, the exact mechanisms of platelet promoting tumor cell adherence and metastasis have not been well understood. Therefore, it will have important significance for tumor therapy to investigate the molecular mechanisms of platelet promoting tumor metastasis.Objective To investigate the effects of platelet on adherence of tumor cell-endothelial cell (EC) and on tumor metastasis, to analyze the related molecular mechanisms.Methods Human liver sinusoidal EC (LSEC) and five tumor cell lines (human colon carcinoma HCT116, human breast cancer MDA-MB-231, human lung cancer A549, human bone osteosarcoma HOS and MG-63) were used for this study. The five tumor cell lines were activated by culture under conditions of hypoxia for 24h and followed by re-oxygenation for 2h, and then co-incubated with BCECF-labeled platelets, respectively. The adherence of platelets to the five tumor cell lines was determined by fluorescent intensity measured in a fluorescence plate reader. To analyze the platelet adhesion molecules involved in platelet-tumor cell adherence, the monoclonal antibodies against different platelet adhesion molecules were used for blocking experiments. LSEC was also activated by culture under conditions of hypoxia for 24h and followed by re-oxygenation for 2h, and were then co-incubated with BCECF-labeled platelets. The adherence of platelets to LSEC and the involved adhesion molecules in this process were evaluated by the same methods as above. Then tumor cell adherence to LSEC was investigated in which LSEC was hypoxia-reoxygenated as above and pre-incubated with or without platelets. The role of platelets in the adhesion of tumor cells to LSEC was analyzed by antibody block experiments using adhesion molecules. The fusion between platelet and LSEC was demonstrated by platelet labeled dye transfer, flow cytometry and transmission electron microscopy, respectively. The role of platelet in mediating tumor-endothelial cells adhesion was further demonstrated by the laser scanning confocal microscope (LSCM) and transmission electron microscope. In order to evaluate the role of platelets on tumor metastasis, tumor cells (B16) alone or together with platelets were injected into nude mice by tail vein, respectively. After 2 weeks, lungs surface metastatic foci were counted under a dissecting microscope. Tumor cell pulmonary metastasis was further analyzed by pathologic examination of lung tissue. To investigate the role of platelet in tumor growth, tumor cells (HCT116) alone or together with platelets were respectively subcutaneously inoculated into nude mice. The tumor growth was determined in 2 weeks and the micro-vessel density (MVD) in tumor tissue was compared by immunohistochemistry staining with vWF.Results Adhesion of platelet was significantly increased to the five tumor cell lines suffered hypoxia-reoxygenation as compared with that to resting tumor cells and the differences between the two groups were statistically significant. Interestingly, the adherence of platelets to the five tumor cell lines was inhibited by monoclonal antibodies against different platelet adhesion molecules. The adhesion of platelets to A549 and HCT116 were inhibited by monoclonal antibodies against gpⅠb, gpⅡb and gpⅢa; platelet-MG63 adhesion was reduced by monoclonal antibodies against gpⅡb and CD36; the adhesion of platelets to MDA-MB-231 were inhibited by monoclonal antibodies against gpⅠb, gpⅡb, gpⅢa and CD31; while platelet-HOS adhesion was inhibited by monoclonal antibodies against gpⅠb and gpⅡb, respectively. Adhesion of platelets to LSEC was also significantly increased after LSEC hypoxia-reoxygenation and the difference of platelet adhesion between resting and hypoxia-reoxygenated LSEC is statistically significant. The platelet-LSEC adhesion was inhibited by monoclonal antibodies against gpⅠb, gpⅡb, gpⅢa, P-selectin and CD31, respectively. Adherence of tumor cells was significantly increased on hypoxia-reoxygenated LSEC when LSEC was pre-incubated with platelets compared with the relative controls. And the increase of tumor cells adherence positively correlates with the amount of platelets. The increased of tumor cell adherence was selectively reduced by antibodies against platelet adhesion molecules. Using dye transfer, flow cytometry and transmission electron microsope, we found that fluorescent dye was transferred from labeled platelets into LSEC cytoplasm; platelet specific marker gpⅡb was transferred from platelets to LSEC; and adhered platelets fused into LSEC after adherence of platelet to hypoxia-reoxygenated LSEC. Furthermore, we found the direct evidence that adhesion of tumor cells to LSEC was mediated by platelets by the laser scanning confocal microscope and transmission electron microscope. Animal experiments showed that tumor cells adhered with platelets exhibited higher pulmonary metastasis capacity as compared with tumor cell alone after injection from tail vein in nude mice. Compared with the group of subcutaneously inoculating tumor cells without platelets, tumor growth was significantly faster and MVD in tumor tissue was also higher in the group of subcutaneously inoculating tumor cells with platelets. These results of animal experiments indicated that platelets promoted tumor cell growth and metastasis in vivo.Conclusion Platelets exist those adhesion molecules that mediate tumor cell adherence and endothelial cell adherence. After adhering or fusing to endothelial cells, platelets increase tumor-endothelium adhesion by its surface adhesion molecules and enhance tumor cell growth and metastasis.