| Background and purposeNewcasle disease virus (NDV) treatment of cancer as a potential biological treatment, has been gradually used in cancer treatment of the foundation and clinical research. We isolated NDV wild strains in Jiangxi Poyang Lake wild duck (named WDK/JX/7793/2004, abbreviated as NDV 7793), based on multiplex RT-PCR electrophoresis and gene sequence analysis, NDV7793 was identified as a member of the weak strains. Our preliminary results showed that, NDV7793 intratumoral injection could inhibit obviously the growth of human small cell lung cancer transplanted subcutaneously in nude mice, the effect maybe relate to the upregulation of tumor cells Bax protein expression; It was also found that NDV7793 intratumoral injection in mice knot colorectal cancer liver metastasis also had obvious inhibitory effect, which perhaps by correcting the tumor microenvironment of Th1/Th2 cell ratio imbalance and increasing thymus index or other ways to inhibit colon cancer liver metastasis.However, the change of subcutaneous transplantation tumor microenvironment by NDV7793 intratumoral injection besides had the inhibitory effect on tumor growth, whether or not there were the same on abdominal metastatic tumor effect on tumor invasion and metastasis, it was not yet clear. The purpose of this research is to study the effect of NDV7793 intratumoral injection in the treatment of subcutaneous transplantation tumor of lung adenocarcinoma in mice; Preliminary study the effect on invasion and metastasis of NDV7793 intratumoral injection of subcutaneous transplantation tumor of lung adenocarcinoma in mice and the related mechanisms.Contents and Methods1. The killing effect of NDV7793 on LLC cells were detected by CCK-8 assay.LLC cells were cultured in vitro, respectively different titer of NDV7793 and DDP at different concentrations for packet processing, time end, added CCK-8 reagent, assayed by OD values of each group.2.The establishment of mouse lung adenocarcinoma subcutaneous transplantation tumor model.Cultured mouse lung cancer Lewis cells (referred as the LLC). 1×106 LLC, cells were injected into C57BL/6 mice subcutaneously to establish the mouse model of the lung adenocarcinoma subcutaneously transplanted tumor.3.The curative effect of NDV7793 to mouse lung adenocarcinoma subcutaneously transplanted tumor.Group administration:lung adenocarcinoma subcutaneously transplanted tumor model mice were divided into PBS negative control group, DDP positive control group, NDV7793 group. Used repeat-measures to analysis the impact of drugs on tumor volume,did survival analysis, calculated the inhibition rate.4. Observe the tumor infiltration and metastasis.Compared mouse right axillary lymph nodes, subcutaneous cancerous adjacent tissue infiltration and the stuation of intrathoracic tumor metastasis.5. Detect the CCL19, CCL21 expression levels in tumor tissue.ELISA was used to detect tumor tissue CCL19, CCL21 expression levels.6. The effect of NDV7793 to lung adenocarcinoma subcutaneously transplanted tumor model mouse T cells infiltration, CCR7 protein expression of T cells and LLC cells.FCM was used to detect tumor T cells/TILs ratio, CCR7+LLC cells/LLC cells ratio and CCR7+T cells/T cells ratio.7. Analysis the relationship between the tumor tissue CCL21 expression and T cells infiltration, T cells and LLC cells CCR7 protein expression.The CCL21 levels of tumor tissues in all groups of mice were respectively correlated with the proportion of T cells/TILs, the proportion of CCR7+LLC cells/LLC cells and the proportion of CCR7+T cells/T cells.8. Analysis the relationship between the tumor tissue LLC cells CCR7 expression and T cells infiltration and T cells CCR7 expression.The proportion of CCR7+LLC cells/LLC cells in tumor tissue were respectively correlated with the proportion of CCR7+T cells/T cells and the proportion of T cells/TILs.9. Detect the expression levels of CEACAM1 protein in tumor tissue.FCM was used to detect the tumor tissue CEACAM1 positive cells percentage of tumor cells; Immunohistochemistry was used to compare the tumor tissue in mice CEACAM1 expression levels.10. Analysis the relationship between the CEACAM1 expression levels of tumor tissue and CCL21 protein expression, T cells infiltration, CCR7 protein expression of T cells and LLC cells.The the proportion of CEACAMl+cells/tumor tissue cells were respectively correlated with the CCL21 levels of tumor tissues in all groups of mice, the proportion of T cells/TILs, the proportion of CCR7+LLC cells/LLC cells and the proportion of CCR7+T cells/T cells.Research results1. The reaction time was 48 hours, different titers NDV7793 and vary concentrations of DDP had the killing effect on LLC cells, which NDV7793 group (the minimum titer of 1:64) and DDP group (the lowest concentration 0.0025mg/mL) OD values were less than the Blank control group, P<0.05, and the difference was statistically significant.2.Successfully constructed mouse lung adenocarcinoma subcutaneously transplanted tumor model, the rate of successed model was 96% (48/50).3. NDV7793 therapeutic effect on mouse lung adenocarcinoma subcutaneously transplanted tumor.1) Analysised to the tumor volume growth could be found that:the administration period, compared to the PBS negative control group, NDV7793 group and DDP positive control group could slow the growth rate of tumor volume, P<0.05, the difference was statistically significant, and the tumor volume growth inhibition of NDV7793 group were more stronger than DDP positive control group.2) Each group of mice survival analysis available:the lifetime of NDV7793 group were significantly longer than PBS negative control group, P<0.05, the difference was statistically significant; but the survival of DDP positive control group compared with PBS negative control group, P>0.05, the difference was not statistically significant.3)Inhibition rate of each group statistical analysis available:the administration period, compared with the PBS negative control group, NDV7793 group and DDP positive control group both inhibited tumor growth in mice, and the inhibitory of NDV7793 group was higher than DDP positive control group.4. Observe adjacent tissues and right axillary lymph node tumor infiltration.Pathological observation:PBS negative control group, cancer cells had infiltrated into the adjacent tissue and some mouse cancer cells had infiltrated into the right axillary lymph nodes; DDP positive control group, some mouse cancer cells had infiltrated into the adjacent tissue, but had not infiltrated into the right axillary lymph nodes; NDV7793 group, cancer cells had not infiltrated into the adjacent tissue and the right axillary lymph nodes; three groups of mice did not occur the pleural metastasis.5. CCL21 expression levels in tumor tissue showed that:CCL21 expression levels in tumor tissues of NDV7793 group was significantly higher than PBS negative control group, P<0.05, the difference was statistically significant, DDP positive control group were similar to PBS negative control group, P>0.05, the difference was not statistically significant.6. CCL19 expression levels in tumor tissue showed that:PBS negative control group, NDV7793 group and DDP positive control group, the CCL19 expression levels had not difference, and the CCL19 expression levels were lower than the CCL21 expression levels.7. FCM was used to detect tumor LLC cells CCR7 expression results showed that:compared with the PBS negative control group, NDV7793 group CCR7+LLC cells/LLC cells was decreased, P<0.05, the difference was statistically significant, the proportions of DDP positive control group were similar to PBS negative control group, P>0.05, the difference was not statistically significant, it was indicating that CCR7 expression levels in tumor LLC cells of NDV7793 group were weaker than PBS negative control group and DDP positive control group.8. FCM was used to detect tumor tissue T cells infiltration and T cells CCR7 expression showed that:NDV7793 group the T cells/TILs ratio greater than PBS negative control groupand DDP positive control group, P＜0.05, the difference was statistically significant; NDV7793 group and DDP positive control group CCR7+ T cells/T cells ratio was significantly lower than PBS negative control group, P<0.05, the difference was statistically significant.9. Analysis the relationship between the tumor tissue CCL21 expression and T cell infiltration, T cells and LLC cells CCR7 protein expression:The results showed that the tumor tissue CCL21 expression and T cells/TILs ratio, CCR7+T cells/T cells ratio had nothing, with the CCR7+ LLC cells/LLC cells ratio had a significant negative correlation, r=-0.589, P<0.01, the correlation coefficient was statistically significant.10. Analysis the relationship between the tumor tissue LLC cells CCR7 expression and T cells infiltration, T cells CCR7 protein expression:The results showed that the tumor tissue CCR7+ LLC cells/LLC cells ratio and CCR7+T cells/T cells ratio had nothing, with the T cells/TILs ratio had a negatively correlation, r=-0.503, P＜0.05, the correlation coefficient was statistically significant.11. FCM used to detect tumor tissue CEACAM1 expression levels displaied that: NDV7793 group tumor tissue CEACAMl+cells/tumor tissue cells ratio were smaller than PBS negative control group (P>0.05) and DDP positive control group (P<0.05), the difference was not statistically significant.12. The immunohistochemical examination of tumor tissue showed expression levels of CEACAM1:the CEACAM1 expression levels in tumor tissue of DDP positive control group were stronger than NDV7793 group and PBS negative control group, and NDV7793 group compared with PBS negative control group were weaker.13. Analysis the relationship between CEACAM1 expression and CCL21 expression, T cells infiltration, T cells and LLC cells CCR7 protein expression: The results showed that the tumor tissue CEACAM1+ cells/tumor cells ratio with the T cells/TILs ratio had a negatively correlation(r=-0.601, P＜0.01) and had a positively correlation with the proportion of CCR7+LLC cells/LLC cells (r=0.584, P<0.01), the correlation coefficient was statistically significant, were unrelated with the CCL21 expression levels and the CCR7+T cells/T cells ratio.Conclusions1. NDV7793 and DDP both had killing effect on LLC cells.2.The curative effect of NDV7793 on lung adenocarcinoma subcutaneously transplanted tumor was better than DDP, not only to extend the survival time of mice and inhibit tumor growth, but also inhibit tumor invasion and metastasis.3. NDV7793 inhibited lung adenocarcinoma subcutaneously transplanted tumor growth, invasion and metastasis, it might though inducing CCL21 expression in the tumor microenvironment, inhibiting the expression of CEACAM1, promoting T cells infiltration into the tumor tissue, inhibiting LLC cells and T cells CCR7 expression. |
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