Paracrine Signaling By VEGF-C Promotes Non-small Cell Lung Cancer Cell Metastasis Via Recruitment Of Tumor-associated Macrophages

Objectives:1.To investigate whether VEGF-C regulates the migration of tumor-associated macrophages.2.To explore the mechanism by which VEGF-C regulates tumor-associated macrophage migration.3.To investigate whether VEGF-C-mediated macrophage infiltration promotes the metastasis of non-small cell lung cancer(NSCLC).Methods:1.Real-time RT-PCR(qRT-PCR)In normal human lung epithelial cells BEAS-2B and various lung cancer cell lines(A549,H460,H358,H441,HCC827),total cellular RNA was extracted for RT-qPCR,and the expression levels of EGF-C mRNA in different cell lines were detected.2.In vitro migration assayTranswell cell migration assay was used to detect the effect of tumor onditioned medium on macrophage migration.3.Western blottingIn RAW264.7 cell line,VEGF-C recombinant protein was added and the macrophage M2 polarization marker CD206 was detected by Western blotting.Further,in the A549 cell line,after treatment with a pathway inhibitor,the expression of p-Src and p-p38 were examined.4.ELISA assayThe expression of cytokines IL12,IL23 and IL10 in macrophage M1and M2 were detected by ELISA kit after uesd VEGF-C recombinant protein.5.In vivo metastasis modelA549 or A549 cells overexpressing VEGF-C were injected into the tail vein of nude mice,for approximately 5×10 ~6 cells.In the drug-administered group,SAR131675(100 mg/Kg/day)was intragastrically administered 5 days after the injection of tumor cells.The other group was injected with chlorophosphate liposome in the tail vein and then injected into A549 cells expressing VEGF-C.After4 weeks,the nude mice were treated with neck removal,and the liver tissues were taken to observe the number of liver metastases.6.Immunohistochemistry(IHC)The liver tissue in the tumor metastasis experiment was taken and immunohistochemical staining was performed to detect the expression of macrophage marker F4/80.Results:1.VEGF-C is highly expressed in a variety of non-small cell lung cancer cell lines;the addition of VEGF-C recombinant protein can promote tumor-associated macrophage migration,but does not affect the polarization of tumor-associated macrophages.2.Overexpression of VEGF-C in A549 and H441 cell lines significantly increased the number of macrophage migration.VEGFR-2/3 inhibitors can significantly reverse macrophage migration caused by VEGF-C overexpression in tumor cells.3.In the RAW264.7 cell line,the VEGF-C recombinant protein was used o activate the Src/p38 pathway,and VEGFR-2/3 inhibitor treatment ignificantly inhibited the activation of the Src/p38 pathway by EGF-C.At the same time,the number of macrophage migration can e significantly reduced after the use of the Src/p38 pathway inhibitor.4.In the A549 cell nude mice metastasis model,after using the VEGFR-3inhibitor SAR131675,the number of tumor metastases in liver tissue as significantly reduced,and the degree of macrophage infiltration in issues was also reduced.At the same time,it was found that in the overexpressed VEGF-C group,the number of tumor metastases in liver tissue increased significantly,and macrophage infiltration also increased.In the experimental group that depleted macrophages in nude mice in advance,the number of tumor metastases and the degree of macrophage infiltration decreased.Conclusions:1.VEGF-C is highly expressed in NSCLC cell lines and can promote tumor-associated macrophage migration.2.VEGF-C activates Src/p38 MAPK signaling by paracrine action on VEGFR-2/3 on macrophages,thereby promoting macrophage migration.3.NSCLC cells-derived VEGF-C can promote macrophage recruitment,and resultantly leads to tumor metastasis.