Experimental Study Of The Induction Of Lymphangiogenesis By Alternatively Activated Macrophages In Mouse Lung Adenocarcinoma

Nowadays, activated macrophages are considered as complicated groups which can execute distinct functions. Among them, alternatively activated macrophages (aaMphi) participate in the process of cell growth, angiogenesis, immunosuppression, tissue repair and stroma formation. There is a great quantity of tumor-associated macrophages (TAM) infiltrating in the stroma of human lung adenocarcinoma, which are closely related to tumor development and metastasis. Lymphatic metastasis is one of the major routes of lung adenocarcinoma dissemination. However, the activated phenotypes of TAM and their roles in lymphangiogenesis remain unknown. This thesis will i) identify TAM in human lung adenocarcinoma to be aaMphi, ii) investigate the roles of aaMphi in lymphangiogenesis of lung adenocarcinoma based on the models of mouse Lewis lung carcinoma (LLC) transplantation tumors, iii) explore the possible mechanisms of the induction of lymphangiogenesis by aaMphi in lung adenocarcinoma. This research shows new approach for the lymphangiogenesis in lung adenocarcinoma and thus provides a new target for the inhibition of the metastasis of this kind of malignant carcinoma.ObjectiveTo identify the activated phenotypes of TAM in lung adenocarcinoma, and to explore the possible mechanisms by which aaMphi induce lymphangiogenesis in mouse lung adenocarcinoma.Methods1. The expression of CD68, vascular endothelial growth factor (VEGF)-C and vascular endothelial growth factor receptor (VEGFR)-3 was detected by immunohistochemistry (SP method) in human lung adenocarcinoma, respectively, and then CD68 positive TAM counts, VEGF-C positive indexes and VEGFR-3 positive lymphatic microvessel density (LMVD) were counted for the analysis of the correlations among them and their relationships with clinicopathological features.2. Since CD68 being the marker of TAM, co-expression of CD68 and macrophage mannose receptor (MMR) was used as the marker of aaMphi, and co-expression of CD68 and inducible nitric oxide synthase (iNOS) was used as the marker of classically activated macrophages (caMphi). The activated phenotypes of TAM in human lung adenocarcinoma were detected by double-labelled immunofluorescence staining and observed by laser confocal microscopy.3. To establish the models of caMphi or aaMphi, RAW264.7 macrophages were treated by IFN-γ+LPS or IL-4, respectively. Then, whether the reversion of aaMphi phenotype by CpG+IL-10RA or not was investigated. The models of mice LLC transplantation tumors were established via subcutaneous injection of LLC cells and aaMphi. The LYVE-1+ LMVD were detected by immunohistochemistry (SP method), meanwhile, the numbers of metastatic lymph nodes and nodes in distant organs were detected by HE staining. Furthermore, another 50 mice were monitored for survival rates.4. Mice lymphangiomas in abdominal cavity were induced by intraperitoneal injection of incomplete Freund's adjuvant (IFA). LEC were obtained from the induced lymphangiomas after disruption and digestion, and then were cultured in the flask or plate previously coated with self-made rat-tail collagen. Expressions of VEGFR-3 and LYVE-1 were identified by immunofluorescence. The proliferation activity of LEC was evaluated by 3H-TdR incorporation efficiency. The capability of LEC to form lymphatic vessel-like structures was assessed by the in vitro lymphatic vessel formation assay.5. Based on the co-culture systems of aaMphi and murine LEC, cell growth curve of LEC was drawn, and migration assay and matrigel assay were used to observe the effect of aaMphi on the migration and tube-like structure formation of LEC. Expressions of VEGF-C and VEGF-D mRNA in aaMphi, VEGFR-3 and Prox1 mRNA in LEC were detected by real time quantitative RT-PCR. Then, 100ng/ml VEGF-C was selected for establishing the aaMphi transdifferentiation system. The mRNA expression of VEGFR-3, Prox1 and Fizz1 in aaMphi were detected by real time quantitative RT-PCR on the day 0, 7, 14 and 28 following VEGF-C stimulation, respectively. During the 28 days, tube-like structure formation was observed by inverted phase contrast microscope continuously. Finally, based on the aaMphi-LLC cells co-culture, VEGF-C expressions in LLC cells were detected by immunocytochemsitry. Besides, 3H-TdR incorporation and transwell invasion model were used to observe the effect of aaMphi on the proliferation and invasion of LLC cells, respectively.Results1. TAM counts and VEGF-C positive indexes in lung adenocarcinoma were (103.36±15.31) and (20.60±11.96), which were both higher than those in lung benign lesion (32.15±5.48, 3.82±3.21), respectively (P<0.01). There was no significant difference in VEGFR-3 positive rates between lung adenocarcinoma (54.7%) and lung benign lesion (50.0%) (P>0.05), but VEGFR-3 positive LMVD was significantly higher in the former (11.56±10.73) than in the latter (4.29±4.18) (P<0.01). CD68 positive TAM counts, VEGF-C positive indexes were related to lymph node metastasis and P-TNM staging, but VEGFR-3 positive LMVD was only related to lymph node metastasis. Close correlations were found between TAM counts and VEGF-C positive indexes, VEGFR-3 positive LMVD, respectively (r=0.338, P<0.01; r=0.410, P<0.01), and also found between VEGF-C positive indexes and VEGFR-3 positive LMVD (r=0.653, P<0.01).2. Co-exprssion of CD68 and MMR of TAM were detected in all the 40 lung adenocarcinoma specimens, and the percentages of MMR positive macrophages in TAM were 74.7~98.2%. Among the 40 cases, co-exprssion of CD68 and iNOS of TAM were detected in 3 cases, and the percentages of iNOS positive macrophages in TAM were only 2.4~12.7%. Among the 10 cases with lung benign lesion, 8 cases co-expressed CD68 and iNOS, 2 cases only expressed CD68, and there was no case which co-expressed CD68 and MMR.3. Mouse caMphi and aaMphi models were established successfully, and aaMphi could be reversed into caMphi by CpG+IL-10RA. Compared to blank control group, aaMphi group and RAW264.7 group have increased LMVD in transplantation tumors (14.80±3.64, 10.50±3.17), enhanced number of metastatic lymph nodes (10.80±1.32, 7.30±0.95) (P<0.01), incremented quantity of metastatic nodes in double lungs (32.50±4.91, 29.30±4.42) (P<0.01), and decreased survival rates of tumor-bearing mice (P<0.01). Besides, the number of metastatic lymph nodes and LMVD in aaMphi group were higher than those in RAW264.7 group (P<0.05), but there was no significant difference in the metastatic nodes in double lungs and the survival rates of mice between them (P>0.05). In caMphi group and aaMphi+CpG+IL-10RA group, LYVE-1 expression in transplantation tumors and lymph node metastasis were hardly detected, the metastatic nodes in double lungs were less (5.80±1.15, 5.17±1.28) (P<0.01), and the survival rates of tumor-bearing mice increased (P<0.01).4. Mice lymphangiomas in abdominal cavity were induced successfully by IFA, and more than 98% living LEC were harvested. The expression of VEGFR-3 and LYVE-1 were positive in LEC. In the rat-tail collagen coated flask or plate, LEC grew well, with 3H-TdR incorporation efficiency increasing obviously. In the gel formed by rat-tail collagen, LEC could form lymphatic vessel-like structures.5. After co-culture, the ability of LEC to proliferate, migrate and form tube-like structure in aaMphi group was significantly stronger than that in blank control, caMphi, and RAW264.7 groups. The expression of VEGF-C mRNA in aaMphi, VEGFR-3 and Prox1 mRNA in LEC increased significantly after co-culture (P<0.05~0.01), however, there was no significant change in VEGF-D mRNA level in aaMphi (P>0.05). After the aaMphi transdifferentiation system was established, the expression of VEGFR-3 and Prox1 mRNA in aaMphi incresesd greatly, while the Fizz1 mRNA in aaMphi decreased. On day 14, VEGFR-3 and Prox1 mRNA reached the peak value, and Fizz1 mRNA reduced to the minimum level. There was no significant difference in the expression of VEGFR-3, Prox1 and Fizz1 mRNA between on the day 28 and day 14. With the time passed, aaMphi could form tube-like structure in matrigel. Among the four macrophages-LLC cells co-culture groups, aaMphi group has the highest VEGF-C expression, fastest growth velocity, and strongest invasion capability of LLC cells, with RAW264.7 group ranking the secondary. Compared to blank control group, VEGF-C expression in LLC cells of caMphi group has no change, and the proliferation and invasion of LLC cells were inhibited.Conclusions1. TAM are positively correlated to VEGF-C expression and lymphangiogenesis, and probably contribute to lymph node metastasis in human lung adenocarcinoma.2. TAM in human lung adenocarcinoma is proved to be one kind of aaMphi.3. aaMphi can promote the lymphangiogenesis, lymph node and lung metastasis of LLC transplantation tumors, and reduce the tumor-bearing mice survival. In the progression of transplantation tumors, the function of RAW264.7 cells may be polarized toward that of aaMphi. CpG+IL-10RA can inhibit aaMphi-induced lymphangiogenesis by the reversion of aaMphi phenotype.4. Mouse LEC culture system using rat-tail collagen as adhesives is a convenient, cheap and stable culture system.5. aaMphi can induce lymphangiogenesis in lung adenocarcinoma by the means of promoting the proliferation, migration and tube-like structure formation of LEC, transdifferentiating into LEC, and promoting the expression of VEGF-C in LLC cells.