The Function And Mechanism Of Platelet And Podoplanin Over-expressing Cancer-associated Fibroblasts In The Development Of Breast Cancer

Part one: Podoplaninexpressing cancer-associated fibroblasts and platelet infiltration are associated with poor prognosis in invasive breast cancerObjective:To investigate the role of the presence and prognostic role of podoplanin(PDPN)expressing cancer-associated fibroblasts(CAFs)and the infiltration of platelets into tumors in invasive breast cancer.Methods:In this study,a total of 164 patients were included in the study at the time of primary surgery for invasive breast cancer between January 2007 and December 2008 from the Affiliated Hospital of Jiangnan University.The PDPN expressing in CAFs and the infiltration of platelets in tumor tissue was evaluated by immunohistochemistry respectively with mouse monoclonal anti-human PDPN(clone: D2-40)and mouse monoclonal anti-human CD61.The platelet count before surgery was also collected.The role of these indicators in the prognosis of invasive breast cancer were statistical analyzed later.Results: Seventy-two patients(44%)showed PDPN expressing in CAFs(CAF+),while fifty-seven patients(35%)showed the infiltration of platelets in tumor tissue(CD61+).Correlation analysis showed a significant correlation between multiple sets of data,including CAF+ with clinical stage(P=0.04),CD61+ with blood platelet count before surgery(P=0.01),and CAF+ with CD61+(P=0.04).Patients with CAF+,CD61+ or platelet count>280×109/L before surgery had a significantly shorter disease-free survival(DFS)according to univariate analysis.Multivariable analysis showed that CAF+ was an independent prognostic factor(Hazard ratio=3.928;p=0.005).Conclusions: CAF+ and CD61+ were found to be good prognosis factors for invasive breast cancer.CD61+ hassignificant correlation with blood platelet count before surgery.Part two: The roles and mechanism of platelet with PDPN over-expressing Human mammary fibroblasts in the in situ growth of breast cancerObjective: To confirm the role of PDPN over-expression Human mammary fibroblasts(HMF)in tumorigenesis of breast cancer cell line MDA-MB-231.At the same time,we studied the effect of reducing the peripheral platelet count to reduce the infiltration of platelet in the progress of PDPN over-expression Human mammary fibroblasts(HMF-P)induced tumor growth.Then we analyze the possible mechanism in these progress.Mrthods:we firstly constructed PDPN expressing HMF by lentivirus transfection;then we performed in vivoexperiments using HMF and HMF-P in conjunction with the cancer cell line implanted in the R2 mammary fat pad of female Bal B/c nu/nu mice to watch primary tumor development in different groups.Then we used platelet antibodies R-300 to construct mouse model of thrombocytopenia.The effect of HMF-P promoting tumor growth was observed again in Bal B/c mice with thrombocytopenia.Lastly,the expression of several tumor growth factors were detected by Western Blot in different treatment groups.Results: HMF-P had no significant difference with HMF in cell morphology and cell proliferation.HMF could promote the tumorigenesis of breast cancer cell lines in nude mice,while HMF-P could further promote tumorigenesis.The effect of HMF-P promoting tumorigenesis was inhibited in nude mice with thrombocytopenia,while the effect of HMF had no significant difference between normal and thrombocytopenianude mice.Western blot analysis showed that the expression of PDGF-D in HMF-P + MDA-MB-231 groupwas significantly higher,while in HMF-P + MDA-MB-231 + R300 group was significantly lower.Conclusions: HMF can promote the tumorigenesis of breast cancer cell lines,and overexpression of PDPN can further promote tumorigenesis.Its mechanism may be associated with PDPN expressed by HMF binding to CLEC-2 on infiltrating platelets to promote platelet activation and release PDGF-D.By reducing the peripheral blood platelet count to reduce platelet tumor infiltration can inhibit the promoting tumor growth effect of HMF-P.This study provides several new therapeutic targets for future breast cancer treatment strategies.