CCL21/CCR7 Axis Promotes A549 Cell Proliferation, Migration, Invasion In Vitro And Lymph Node Metastasis In Vivo

Background and objective:Lymph node metastasis is one of the most poor prognostic factors of non-small cell lung cancer(NSCLC). It is interesting to probe how lung cancer cells migrate directly to regional even remote lymph nodes. CCR7 is a member of CC chemokine family, and its ligand--CCL21 is found to be expressed specifically in high endothelial venules(HEV) and lymphatic endothelial cells of lymph nodes. CCL21/CCR7 axis plays a key role in the directed migration of lymphocytes in immunologic and inflammatory processes. Recently, CCL21/CCR7 was also found to be involved in metastatic process of many kinds of tumors such as melanoma, breast cancer, gastric cancer, esophageal cancer, hepatic cell cancer, colorectal cancer, thyroid cancer, cervical cancer and chronic lymphocytic leukemia, etc. It is not clear, however, whether CCL21/CCR7 axis is involved in the lymph node metastasis of NSCLC although the expression of CCR7 mRNA was found in cancer tissues of NSCLC by RT-PCR.Methods:IHC was used to detect the expression of CCR7 and VEGF-C(Vascular endothelial growth factor-C), and the relationship between CCR7 and VEGF-C expression and clinicopathological parameters of NSCLC was explored. The CCR7-expressing plasmid vector was constructed and stably expressed in human lung adenocarcinoma A549 cell line(named A549-CCR7) with A549-pEGFP as a negative control. The effects of CCL21 on proliferation, calcium influx, migration, and invasion of A549-CCR7 and A549-pEGFP cells in vitro were evaluated with WST-1, soft agar clone formation assay, laser scanning confocal microscope (LSCM) and Boyden chamber, respectively. Real-Time RT-PCR and Western blot were used to explore the relationship between the effects of CCL21/CCR7 axis and the expression of metastasis-ralated molecules such as VEGF-C, MMPs and TIMPs in A549-CCR7 cells and A549-pEGFP cells. Experimental lymph node metastasis, decrease of host survival was examined using footpad tumor xenografts model implanted with A549-CCR7 cells or A549-pEGFP cells in nude mice.Results:1. High-level expression of CCR7 and VEGF-C was found in lung tissues of NSCLC, with 56.9% and 60% respectively. The expression of CCR7 and VEGF-C was positively correlated (P<0.01). Their expression was also positively related with the stage of TNM and lymph node metastasis(P<0.01). Great accuracy to predict lymph node metastasis was found in combined examination of CCR7 and VEGF-C expression, with area under ROC curve 0.782.2. No significant difference was found in cell proliferation, calcium influx, migration and invasion between A549-CCR7 group and A549-pEGFP group (P>0.05) without CCL21 in culture medium. The ability of cell proliferation, calcium influx, migration and invasion was significantly increased in A549-CCR7 group compared with A549-pEGFP group after CCL21 was added into the culture medium (P<0.01). This suggested that CCL21/CCR7 axis could increase the proliferation, calcium influx, migration and invasion of A549 cells. With CCL21 existing in the culture medium, increased expression of VEGF-C, MMP-2, 9, 11(P<0.01 or 0.05) and decreased expression of TIMP-1, 2 were also found in A549-CCR7 cells(P<0.01).3. The proportions of A549-CCR7 cells in popliteal lymph nodes, iliac lymph nodes and renal hilar lymph nodes were all more than those of in situ cancer tissues (P<0.01). The futher lymph nodes were away from the footpad, the higher proportions of A549-CCR7 cells tended to be. Compared with A549-pEGFP group, the ratio of experimental lymph node metastasis in A549-CCR7 group was significantly increased in implanted nude mice(P<0.01), and the survival of host was significantly decreased(P<0.01). This suggested that CCL21/CCR7 might increase A549 cell lymph node metastasis through lymphatic vessels in nude mice.Conclusions:1.CCR7 might be a new marker in diagnosis of lymph node metastasis in NSCLC. Combined examination of CCR7 and VEGF-C might be beneficial for diagnosis of NSCLC lymph node metastasis and determining the area of surgery.2.CCL21/CCR7 axis can increase the proliferation, calcium influx, migration and invasion of A549 in vitro.3.CCL21/CCR7 axis might increase A549 metastasis to lymph nodes by regulating the expression of VEGF-C, MMPs and TIMPs.4.CCL21/CCR7 can increase lymph node metastasis of A549 cells in nude mice through lymphatic vessels.