PDPN And CLEC-2Interaction Promotes Platelet Mediated Murine Melanoma Pulmonary Metastasis

Tumor metastasis is one of the leading cause of death in most of the patients withmalignant tumor. However, the exact mechanisms of tumor metastasis is still notcompletely clear. Melanoma is an aggressive and highly metastatic disease. It mainlyoccurs in the skin. Most of the tumor metastasis occurred at early stage of the disease.Pulmonary metastasis is the most common target organ for melanoma hematogenousspreading. Surgery, chemotherapy and radiotherapy are most commonly treatmentsfor melanoma now, but there still has no better treatment for the tumors whenmetastasis occurred. Metastatic melanoma develops when tumor cells dissociate fromthe primary lesion, migrate through the surrounding stroma, and invade blood vesselsand lymphatic to form a tumor at a distant site. The tumor cells were regulated by avarity of factors in vivo during the process of metastasis. Tumor cell-induced plateletaggregation (TCIPA) plays an important role in tumor metastasis. When metastasishappened, primary tumor cells in the circulation will contact with platelet and furtherdevelop secondary metastasis through multiple mechanisms. Platelet aggregationform a coat on the tumor cells enabling tumor cells to evade the immune system andfacilitating their adherence to the blood vessel wall. Activated platelet also releasegrowth factors and a variety of angiogenic factors that support tumor metastasis,facilitate the tumor cells transfer to the new metastatic sites.Podoplanin (PDPN) is a I type sialic acid transmembrane glycoprotein. It iscomposes of a highly O-glycosylated extracellular domain, a transmembrane portionand a short cytoplasmic tail. PDPN has been widly used as lymphatic endothelial cellmarker because it selectively expressed in lymphatic endothelial cells. In addition,PDPN has other variety of functions including regulation of blood-lymphatic vessel development, cell motility, and tumorigenesis and metastasis. Lots of different cancershave up-regulated podoplanin expression, including murine melanoma cell and humanmelanoma cell. PDPN expressed on the surface of some tumor cells has been reportedto contribute to cancer pathogenesis by promoting tumor cell invasion and spreading,but the detail mechanism still unclear. C-type lectin-like receptor2(CLEC-2) is a IItype transmembrane glycoprotein. It is a receptor for podoplanin, which expressed onthe surface of platelet and induce platelet activation by interacting with PDPN.Previous studies have shown that the platelet activation induced by PDPN andCLEC-2interaction is one of the important factors affecting the development oflymphatic and blood vessels during the embryonic statge. In recent years, more andmore opinion was arised on the effects of immunity, tumor and infection caused byPDPN and CLEC-2indued platelet aggregation.In our current study, we screen and isolate the murine melanomal cell line withdifferent podoplanin expressive level on cells. We set up bone marrow transplantationCLEC-2deficient mouse and obtain the CLEC-2deficient platelet. We establish theexperimental murine melanoma pulmonary metastasis models to investigated the roleof podoplanin and CLEC-2interaction in melanoma pulmonary metastasis and itsrelationship with platelet in vivo and in vitro. Furthmore, We also investigate the rloeof Syk-dependent signaling pathway in podoplanin and CLEC-2interaction andplatelet activation.1. Sorting B16-F0PDPN+and PDPN-melanoma cells and establishing bonemarrow transplantation CLEC-2deficient mouse.Flow cytometry profiles and Western blot analysis show B16-F0cells weresorted into PDPN+cells and PDPN-cells groups. The growth speed of these twopopulation cells has no difference in vitro. With1100cGy137Cs γ-ray irradiation andbone marrow transplantation, CLEC-2deficient mouse was established and the Flowcytometry data shows the CLEC-2delete efficiency.2. PDPN and CLEC-2affect the melanoma cells association with platelet throughactivating platelet in vitro.Flow cytometry, immunofluorescence and immunocytochemistry analysis of mixed tumor cells and platelet show podoplanin and CLEC-2activate the platelet andfacilitate the platelet and tumor cells aggregation in vitro. The result dedicate that theaggregation ratio of PDPN+cells with wildtype platelet is much higher than the othergroups. The platelet and tumor cell aggregated clots were observed under themicroscope.3. PDPN and CLEC-2interaction induces platelet activation in vivo and facilitate thetumor cells arrested in the lung through platelet-tumor cell aggergation.The equivalent PDPN+and PDPN-cells with different colour stained weremixed and were injected through tail vein into wild type mouse to establish murinemelanoma pulmonary metstasis models. Sacrificed the mice and remove the lungtissue30min,2h and6h after injection, we found PDPN+tumor cells injected intowild type mouse promote the tumor cells arrested in lung under immunoflrorescencemicroscope observation. We also injected PDPN+and PDPN-cells into wild type andCLEC-2knockout mouse, the resulte show more PDPN+cells arrested in WT mousethan the other groups. Platelet decrease ratio in whole blood after the tumor cellsinjection also indicated PDPN+cells activate the platelet and promote the reduce ofplatelet in blood. Results show that PDPN and CLEC-2interaction induce platletactivation and lead to the tumor cells and platlet aggregation in vivo.4. PDPN and CLEC-2interaction promotes murine melanoma pulmonary metastasis.The experimental pulmonary metastasis mouse models were established byintravenously inoculating PDPN+and PDPN-B16-F0cells into bone marrowtransplantation wild type and CLEC-2deficient C57BL/6J mice via lateral tail veininjection. The results show PDPN+cells injected into wild type mouse has higherpulmonary metastasis than other groups. Histology analysis also indicate that themetastasis number and mtastatsis area of PDPN+cells injecting into wild tpe mousegroup are higher than the other groups.5. Syk-dependent signaling pathway plays an important role in the action of plateletsaggregation and melanoma metastasis.To assess the role of Syk kinase in PDPN and CLEC-2interaction, we use Sykinhibitor R406to block the platelets binding to tumor cells in vivo and in vitro. Flow cytometry and immunofluorescence staining analysis indicate R406can partly blockthe aggregation of PDPN+cells and wild type platelet. Western blot data showPDPN+cells and WT platelet can activate the Syk kinase and promote the plateletaggregation. In vivo experiments also show R406partially block the murinemelanoma pulmonary metastasis.6. PDPN and CLEC-2interaction does not confer tumor growth in vivo.We established melanoma subcutaneous tumor model with PDPN+cells andPDPN-cells injecting into wild type mouse. Histology analysis data show there hasno significant difference in tumor growth and morphology of tumor tissue.In conclusion, this project use both FACS screening different PDPN expressivemurine melanoma cells and bone marrow transplantion CLEC-2gene defect mouse tostudy the PDPN and CLEC-2interaction in murine melanoma pulmonary metastasiswith in vivo and in vitro experiments. With tuomor cells and platlet experimentresearch in vitro and experimental murine melanoma pulmonary metastasis modelsstudies, we found that PDPN and CLEC-2interaction, both in vitro and in vivo, isable to induce platelet activation which promote the occurrence of melanomapulmonary metastasis. And the platelet activation in melanoma pulmonary metastasispartly depends on the Syk tyrosine kinase signaling pathway. So PDPN expressed onthe tumor cells and platelet CLEC-2are expected to be the new targets for tumormetastasis prediction and the treatment for antitumor metastasis.