| Objective: To investigate an oral DNA vaccine in VEGFR-2 target against tumor angiogenesis, and observe the inhibitive effects of Lewis lung cancer growth and metastasis bearing C57BL/6 mice, and explore mechanism of the vaccine on tumor development and metastasis in vivo.Methods: C57BL/6 mice were divided into three groups and every group was divided into three sub-groups randomly i.e DNA vaccine group, empty vector group and normal saline group. Mice were immunized with SL3261 transformed with pcDNA3．1-VR-2, or pcDNA3．1, or saline respectively 3 times. Two weeks after the last immunization , Mouse models of lung carcinoma were established by injection of murine Lewis lung cancer cells (3LL) .Then mice in the first group were challenged by subcutaneous (s.c.) injection of murine Lewis lung cancer (3LL) line into the right front flank. Survival time of mice in each group was observed. Mice in the second group were challenged by subcutaneous injection of 3LL cells into the right front flank. The volume of subcutaneously implanted tumors was observed regularly; CD3~+ T cells, CD4~+ T cells, and CD8~+T cells were measured by flow cytometry (FCM) .After all mice were killed, tumor weight and volume ex vivo were measured. Microvessel density (MVD) was detected by immunohistochemistry. Mice in the third group were challenged by tail intravenous injection of 3LL cells. CD44v positive cells in each group were detected by flow cytometry (FCM) .The lung metastatic scores were analyzed by stereology and histopathology.Results: 1．We successfully developed mouse subcutaneous Lewis lung cancermodels and experimental metastatic lung cancer models. 2．In comparison with the empty vector group or the saline group, survival rate of the mice immunized with the DNA vaccine had significantly difference (P<0．05 ) .The median survival time of the mice in the DNA vaccine group was 48 days, in the empty vector group, 32 days, and in the saline group, 31 days. 3．In murine subcutaneous lung cancer models, tumor growth in vaccine group was slower than those in empty vector group or in saline group. Tumor volume was smaller in vaccine group than those in empty vector group and in saline group (448．41±194．00vs1355．39±468．91, and 1280．15±264．49, P<0．05) . Tumor weight was lighter in vaccine group than those in empty vector group and in saline group (2．05±1．32vs4．83±1．47, and 5．12±1．02, P<0．05) . MVD was decreased in vaccine group than those in empty vector group and in saline group (1．75±1．07vs6．89±2．52, and 7．57±3．75, P<0．05) . There was no obvious difference in CD3~+,CD4~+,CD8~+cells levels, and CD4~+/CD8~+ among three groups before vaccination (P<0．05) . After successful vaccination, a marked increase was measured in CD3~+,CD4~+,CD8~+ cells levels, and CD4~+/CD8~+ in the vaccine group compared with those in the empty vector group or the saline group (P<0．05) . After inoculation with 3LL cells, CD3~+,CD4~+,and CD8~+cells levels obviously declined in the empty vector group and the saline group while high levels were maintained in the vaccine group (P<0．05) . In experimental metastatic lung cancer models, Lung metastasis scores was decreased in vaccine group than those in empty vector group and in saline group (0．7±0．48vs2．10±0．74, and 2．3±0．68, P<0．05) . After inoculation with 3LL cells, we noted higher percentage of CD44v positive cells in empty vector group and in saline group than those in DNA vaccine group (P<0．05 ) , but no obvious difference in percentage of CD44v positive cells between the empty vector and saline group (P>0．05 ) .Conclusions:T he oral DNA vaccine could prolong the median survival time Lewis lung cancer of mouse. The oral DNA could inhibit lung cancer growth by inhibiting angiogenesis. The oral DNA vaccine could inhibit metastasis scores of Lewis lung carcinoma. Mechanism of the vaccine may stimulate cellular immune responses by breaking of peripheral immune tolerance against the self antigen to kill proliferating endothelial cells overexpressing VEGFR-2 in the tumor vasculature, resulting in suppression of tumor growth and metastasis.
|
PDF Full Text Request