An Oral DNA Vaccine Targeting VEGFR-2 Inhibits Murine Lewis Lung Cancer Growth And Metastasis

Objective: In our present study, we developed an oral VEGFR-2 DNA vaccinecarried by attenuated Salmonella typhimurium SL3261; demonstrated antitumoreffect on murine subcutaneous Lewis lung cancer model, lung cancer orthotopicmodel and experimental metastatic lung cancer model, recspectively; investigated theimmunotheraputic effect on Lewis lung cancer growth and metastasis in mice;focused on its antiangiogentic and anticancer mechanism.Methods:1．The encoding sequence of extracellular domains of VEGFR-2 was amplified by reverse transcriptase-polymerase chain reaction from C57BL/6 mice embryo and cloned into the Kpn1-Xba1 sites of pcDNA3．1．Then the recombinant plasmid pcDNA3．1-VR-2 was transfected into COS-7 cells and its protein expression was identified by Western blotting.2．The recombinant plasmid pcDNA3．1-VR-2 was transformed into attenuated Salmonella typhimurium SL3261 to develop an oral DNA vaccine against the extracellular domains of VEGFR-2．To assay for humoral and cellular immune responses, C57BL/6 mice were immunized with the oral vaccine. Serum titer of VEGFR-2-Ag specific antibody was detected by ELISA. We also used spenocytes in motified MTT assay to analysize T cell-mediated antigen-specific cytotoxicity against murine endothelium cells (Ms1) in vitro. In order to investigate the metabolic process of the recombinant attenuated S. typhimurium in vivo, mice were administered by SL3261 transfected with enhanced green fluorescence protein (EGFP) . And GFP positive cells were detected by flow cytometry(FCM) .3．In order to demonstrate if the vaccine can inhibit subcutaneous transplanted lung cancer growth, C57BL/6 mice were randomly divided into three groups: the DNA vaccine group, the empty vector group and the saline group. Mice were orally vaccinated with SL3261 transformed with pcDNA3．1-VR-2, or pcDNA3．1, or saline respectively 3 times. After the last immunization mice were challenged by subcutaneous injection of murine Lewis lung cancer cells (3LL) into the right front flank. Animals were examined every other day. Survial time of mice in each group was recorded. Tumor volume was measured in 2 dimensions and calculated as follows: length /2 (mm)×width~2 (mm) . CD3~+T cells, CD4~+T cells, and CD8~+ T cells were detected by flow cytometry (FCM) to evaluate T cell-mediated immune response. When all mice were killed, tumor weight was measured. Then tumor tissues were collected for the subsequent immunohistochemistry for microvessel density (MVD) .4．A lung cancer orthotopic mouse model was used to further investigate the inhibitive effects of the oral DNA vaccine on spontaneous lung metastasis. C57BL/6 mice were randomly divided into three groups: the DNA vaccine group, the empty vector group and the saline group. Mice were orally vaccinated with SL3261 transformed with pcDNA3．1-VR-2, or pcDNA3．1, or saline respectively. After the last immunization mice were challenged with 3LL lung cancer cells. Survial time of mice in each group was recorded. Orthotopic tumor tissues were fixed, embedded in paraffin, cross-sectioned, stained with hematoxylin and eosin, and identified the presence of tumor metastasis. Metastatic scores were evaluated by stereology.5．In order to examine the hypothesis that experimental pulmonary matastases can be suppressed by the vaccine, C57BL/6 mice were randomly divided into three groups: the DNA vaccine group, the empty vector group and the saline group. Mice were orally vaccinated with SL3261 transformed with pcDNA3．1-VR-2, or pcDNA3．1, or saline respectively. After the last immunization mice 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 analyized by stereology and histopathology.6．Statistic Analysis: For comparison of individual time points, ANOVA was used. Cumulative Survival was constructed according to the Kaplan-Meier method. Statistical significance was determined by the log-rank test. P values <0．05 were considered significant.Results:1．The encoding sequence of extracellular domains of VEGFR-2 was successfully cloned from C57BL/6 mice embryo. The recombinant plasmid pcDNA3．1-VR-2 was successfully constructed, and its protein expression was verified.2．We successfully developed an oral DNA vaccine against the extracellular domains of VEGFR-2 carried by attenuated Salmonella typhimurium SL3261．High levels of VEGFR-2-specific serum antibody were detected in all mice immunized with the oral vaccine and increased in a time-dependent manner. The VEGFR-2-specific IgG levels in all mice immunized with the oral vaccine were significantly elevated compared with those of the empty vector and salineimmunizations (P<0．05 ) , but there are no statistical difference between those ofthe empty vector and saline group (P>0．05) . The oral vaccine demonstratedspecific and efficient in vitro cytotoxicity against murine endothelium cells(Ms1) in a dose-dependent manner. Immunization with the oral vaccine led tosignificant lyses of target cells by effector cells (P<0．05) , in contrast to the emptyvector and saline immunizations. Immunizations with the empty vector and salineshowed no different killing of target cells by effector cells (P>0．05 ) . Using flowcytometry analysis, 7 days after the first vaccine administration GFP positive cells were detectable in speen and small intestine of mice treated with SL3261 haboring an expression vector encoding the GFP. In contrast, GFP positive cells were not detectable in speen and small intestine cells of mice receiving SL3261．3．We successfully developed murine subcutaneous Lewis lung cancer model, lung cancer orthotopic model and experimental metastatic lung cancer mode.4．In murine subcutaneous lung cancer model, C57BL/6 mice treated with the oral vaccine showed marked inhibition of subcutaneous tumor growth and an appreciable decrease in subcutaneous tumor volume, tumor weight andmicrovessel density (MVD) compared to those treated with the empty vector carried by SL3261or saline (P<0．05) . In contrast, mice received the empty vectorcarried by SL3261 or saline revealed no difference in subcutaneous tumor growth,tumor volume, tumor weight and microvessel density (MVD) (P>0．05) . Therewas no obvious difference in CD3~+ cells levels, CD4~+ cells levels, CD8~+ cellslevels, and CD4~+ / CD8~+ among three groups before vaccination (P>0．05) . Aftersuccessful vaccination, a marked increase was detected in CD3~+ cells levels, CD4~+ cells levels, CD8~+ cells levels, and CD4~+ / CD8~+ in the vaccine group comparedwith those in the empty group or the saline group ((P<0．05) . After inoculationwith 3LL cells, CD3~+ cells levels, CD4~+ cells levels, and CD8~+ cells levels obviously declined in the empty vector group and the saline group while highlevels were maintained in the vaccine group (P<0．05) . In comparison with theempty vector group or the saline group, the mice immunized with the DNAvaccine had significantly different cum survival (P<0．05) .5．The orthotopic tumor growth was markedly suppressed in mice treated with the oral DNA vaccine and mice have less spontaneous dissemination to the contralateral lung. In contrast, mice vaccinated with the empty vector or salinerevealed rapid tumor growth (P<0．05) . We noted a marked reduction in lungmetastatic foci in mice immunizated with the oral DNA vaccine (P<0．05) , while no significant difference was observed between the empty vector and saline group (P>0．05) .6．In experimental metastatic lung cancer model, we noted distinctly higher percentage of CD44v positive cells in the empty vector group and saline groupthan those in the DNA vaccine group (P<0．05) , but no obvious difference inpercentage of CD44v positive cells between the empty vector and saline group(P>0．05) . There was only few small lung metastatic scores in mice treated withthe vaccine, whereas all control animals treated with the empty vector or saline showed more pulmonary metastatic scores (P<0．05) . No significant difference of metastatic scores was observed between the empty vector and saline group (P>0．05) .Conclusions1．We successfully cloned DNA encoding the extracellular domains of murine VEGFR-2 and constructed the recombinant plasmid pcDNA3．1-VR-2, protein expression of which was demonstrated in COS-7 cells.2．We successfully developed an oral DNA vaccine against the extracellular domains of VEGFR-2 carried by attenuated Salmonella typhimurium SL3261, which can induce specific humoral and cellular immune responses.3．The oral DNA vaccine could significantly inhibit Lewis lung carcinoma growth and matastases by inhibiting angiogenesis.4．The oral DNA vaccine might induce humoral immune responses to block VEGF signal transduction to suppress tumor angiogenesis, or 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.