The Role And Mechanism Of PDGF-D In The Progression, Metastasis, And Chemo-drug Delivery In Breast Carcinoma

Part OneExpression of PDGF-D in breast carcinoma tissues, and overexpression of PDGF-D in breast cancer cell line.Objective:To detect the expression level of PDGF-D in benign breast tumors and breast carcinomas and different breast cancer cell lines; And to estabilish a human breast cancer cell line that overexpresses PDGF-D.Methods:Immunocytochemistry staining was used to detect the PDGF-D expression level in breast tissue array sections. RT-PCR was used to determine the PDGF-D and PDGFR?expression levels in different human breast cancer cell lines. Full-length human Pdgf-d cDNA was cloned and transfected into MDA-MB-231 cells in virto. Overexpression of PDGF-D in MDA-MB-231 cells was confirmed by both RT-PCR and western blot.Results:PDGF-D has no expression or weak expression in normal breast tissues and benign breast tumor tissues, but moderate to strong expression in breast carcinoma tissues. Single-factor correlation analysis showed PDGF-D positively correlated with progesterone receptor (PR) and HER-2 expression (P<0.01). MCF-7, MDA-MB-231, MDA-MB-468 and BT474 all express PDGF-D and PDGFR?. Overexpression of PDGF-D in MDA-MB-231 cells was confirmed by both RT-PCR and western blot, and PDGF-D could activate PDGFRp in MDA-MB-231-PDGFD cells.Conclusion:PDGF-D has high expression level in human breast carcinoma tissues and cell lines; overexpression of PDGF-D in MDA-MB-231 cell lines activates PDGFR?signaling pathway. Part Two Functions and mechanisms of PDGF-D on breast cancer progression and metastasis.Objective:To determine the effects of PDGF-D in breast cancer progression and metastasis and its specific molecular mechanisms.Methods:Overexpression and down-regulation of PDGF-D in orthotopic breast cancer mice models were established, whole-body imaging was used to study the tumor growth and lymph node metastasis. PDGFR inhibitor Gleevec was used to treat 231 and 231-PDGFD tumor bearing mice. Immunofluorescent staining was used to detect the tumor cell proliferation and apoptosis and CXCR4 expression; and western blot was used to detect the activation of MAPK and Akt signaling pathway. CXCR4 antagonist was used as treatment to study the mechanism.Results:Overexpression of PDGF-D enhances breast tumor growth and lymph node metastasis, while down-regulation of PDGF-D inhibits breast cancer growth and lymph node metastasis. Gleevec could significantly inhibit 231-PDGFD tumor growth, but had no obvious effect in 231 tumors. PDGF-D increases tumor cell proliferation and inhibits tumor cell apoptosis via activation of MAPK and Akt signaling pathways. PDGF-D might induce lymph node metastasis through activation of SDF-la/ CXCR4 chemokine axis.Conclusions:PDGF-D promotes breast cancer growth and lymph node metastasis, and the specific mechanisms are activation of MAPK and Akt signaling pathways and SDF-1?/CXCR4 chemokine axis. Part ThreePDGF-D's effect on vascular normalization and drug deliveryObjective:To determine whether PDGF-D has effect on chemotherapeutic drug delivery in an orthotopic breast cancer nude mice model.Methods:Double immunofluorescent staining was used to detect the pericyte coverage of tumor blood vessels. Muti-photon laser scanning microscope was used to study the morphology and diameters of tumor vasculature in the mammary fat pad window model. Fluorescent doxorubicin and FITC-lectin were used to study the doxorubicin delivery and efficacy in an orthotopic breast carcinoma model.Results:In the parental 231 group, we found that most of the vessels were not covered by NG2-positive perivascular cells, whereas in the 231-PDGFD group, NG2-positive coverage fraction increased significantly. Muti-photon vascular images showed that blood vessels of 231 tumors seemed abnormal and tortuous, while 231-PDGFD tumors had normalized vasculature. Doxorubicin had better penetration in 231-PDGFD tumors compared to parental 231 tumors. Also doxorubicin treatment was significantly more effective in inhibiting the primary tumor growth and lymph node metastasis of 231-PDGFD tumors than those of 231 tumors.Conclusion:PDGF-D can improve the chemotherapeutic drug delivery and efficacy via vascular normalization in an orthotopic breast carcinoma model.