Study On The Mechanism Of Lung Pre-metastatic Niche Formation Induced By Caveolin-1 Secretion In Breast Cancer

Objective:Caveolin-1(Cav-1)is a cell membrane functional protein that plays an important role in the primary microenvironment of breast cancer.Whether it affects the microenvironment of tumor metastasis through exosomes means has not been studied.The research results of the research group in the early stage of the cell level found that that Cav-1 promotes the metastasis of breast cancer cells by enhancing the stemness of tumor cells and EMT.At the same time,animal experiments have found that Cav-1can also promote breast cancer lung metastasis through exosomes.The project focuses on the shaping of pre-metastasis niche(PMN).Exosomes derived from breast cancer can be used as carriers to transport Cav-1 in the primary tumor microenvironment to lung tissue to form PMN,thus promoting the occurrence of lung metastasis,providing theoretical and practical basis for exploring innovative therapeutic targets for lung metastasis of breast cancer.Methods:1.To explore the effects of breast cancer cell derived exosomes containing Cav-1on breast cancer metastasis,cell models were constructed,and exosomes were extracted to detect the physicochemical characteristics of exosomes(particle size analysis,electron microscope observation and Western Blot were used to detect the expression of exosome marker protein and target protein Cav-1);Transwell experiment was used to detect MDA-MB-231 biological effects of breast cancer cell derived exosomes containing Cav-1 on parental MDA-MB-231 cells and non-metastatic MCF-7 cells;Mouse breast cancer orthotopic tumor models were constructed,and exosomes in each group were injected into the mice through the tail veins.One month later,from HE staining and immunohistochemistry,we observed the lung metastasis of breast cancer in each group of mice.2.To study the mechanism of the formation of lung PMNs induced by breast cancer cell-derived exosomes containing Cav-1,Western Blot and RT-q PCR were used to detect the expression of exosomes containing Cav-1 on the PMN marker genes CCL2 and s100a8 of Beas2B in lung epithelial cells;Besides,Western Blot,RT-q PCR and immunofluorescence methods were used to detect the expression of Tn C driven by exosomes containing Cav-1 in lung fibroblasts HFL1.Finally,we used flow cytometry to detect the function of exosomes containing Cav-1 in the polarization of M2(CD68~+/CD206~+)macrophages THP-1.3.To study the mechanism of breast cancer cell-derived exosomes containing Cav-1 in promoting the formation of lung PMNs by transporting Tn C and inhibiting PTEN/CCL2/VEGF-A signaling pathway,RT-q PCR and Western Blot methods were used to detect the expression levels of Tn C in the lysate of lung fibroblasts HFL1stimulated by exosomes containing Cav-1.We also detected the expression levels of PTEN,CCL2 and VEGF-A in the lysate of lung macrophage THP-1 cell lysate;After knocking out the Tn C gene in lung fibroblasts HFL1,RT-q PCR and Western Blot methods were used to detect expression levels of Tn C;In addition,we also detected the expression levels of CCL2 and VEGF-B in lung macrophages THP-1 after transfected with PTEN overexpressed pc DNA.Results:1.The results of Transwell invasion and migration assay showed that breast cancer cell-derived Exo containing Cav-1 could increase the migration and invasion ability of breast cancer invasive cells MDA-MB-231 cells and non-invasive cells MCF-7(p<0.01);The results of HE staining showed that the mice treated with Exo containing Cav-1 formed lung metastases.Under the microscope,cancer cells with large and deep nuclei with atypical mitoses were seen,and some tumor emboli were also seen.Immunohistochemical results showed that the breast cancer cell-derived Exo containing Cav-1 promotes the expression of FN and CD31 in vivo,which is beneficial to the formation of lung metastases.2.Marker gene expression of breast cancer cell-derived exosomes containing Cav-1 educating the formation of lung PMN:(1)In lung epithelial cells Beas2B,Western Blot results showed that breast cancer cell-derived Exo containing Cav-1 would increase the expression of CCL2,and RT-q PCR results showed that breast cancer cell-derived Exo containing Cav-1would increase the expression of S100a8(p<0.05);(2)In lung fibroblast HFL1,the expression of ECM components in breast cancer cell-derived exosomes containing Cav-1 increased(p<0.01),and immunofluorescence results showed that breast cancer cell-derived Exo containing Cav-1 increased the expression of Tn C(p<0.05);(3)In lung macrophage THP-1,flow cytometry results showed that CD206expression increased and the CD68~+/CD206~+ratio decreased after stimulation of THP-1 by Cav-1 containing exosomes from breast cancer(p<0.05).3.Expression of pathway proteins related to the education of lung PMN by exosomes containing Cav-1 derived from breast cancer cells:(1)In lung fibroblasts HFL1,the expression level of Tn C in breast cancer cell-derived Exo containing Cav-1 was higher(p<0.05).(2)The expression levels of CCL2 and VEGF-A in lung macrophage THP-1 were higher,and the expression level of PTEN was lower(p<0.05).(3)Knockout of Tn C gene in lung fibroblasts HFL1 and overexpression of PTEN gene in lung macrophages THP-1:In lung fibroblast HFL1,the expression level of Tn C in breast cancer cell derived exosomes containing Cav-1 was higher(p<0.05);In lung macrophage THP-1,the expression level of PTEN in breast cancer cell-derived exosomes containing Cav-1 was lower,and the expression level of CCL2 and VEGF-B was higher(p<0.05);Conclusion:1.MDA-MB-231 cell-derived Exo containing Cav-1 can enhance the invasion and migration ability of recipient cells MDA-MB-231 cells,enable MCF-7 cells to acquire invasion and migration ability,and accelerate breast cancer lung metastasis.2.Exo containing Cav-1 derived from MDA-MB-231 cells can promote the expression of PMN marker genes in lung epithelial cells,ECM deposition,and M2-type polarization of macrophages.3.The arrival of exosomes containing Cav-1 and Tn C derived from tumor supernatant and breast cancer cells to the lung can promote the deposition of Tn C fiber matrix in lung tissue,and the arrival of exosomes derived from MDA-MB-231containing Cav-1 can inhibit the PTEN in macrophages and promote the polarization of M2 macrophages and the angiogenesis in lung tissue,forming PMN.