An Oral DNA Vaccine PcDNA3.1+/flk-1_nl-7Against Angiogenesis In Colorectal Cancer

Objective To construct an oral DNA vaccine pcDNA3.1+/flk-1_n1-7 against tumor angiogenesis and investigate the effects and mechanism of the vaccine on tumor development and metastasis in vivo.Methods 1.Total RNA was extracted from BALB/c mice embryo. Extracellular domain of flk-1 was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). After confirmed by DNA sequence analysis, flk-1_n1-7 cDNA was inserted into eucaryotic expression vector pcDNA3.1+ containing CMV promoter. The recombinant plasmids pcDNA3.1+/flk-1_n1-7 was constructed and transfected into eucaryotic expression system COS-7 cells by lipofectamine. Then detecting protein expression of flk-1_n1-7 by Western blot. 2.The recombinant plasmid pcDNA3.1+/ flk-1_n1-7 was transformed into attenuated Salmonella typhimurium SL3261 to develop an oral DNA vaccine against the extracellular domain of flk-1. 3.Mice weredivided into 3 groups (DNA vaccine group, vector group and saline group) and were orally immunized with SL3261 transformed with the recombinant plasmid, pcDNA3.1 (+), or saline respectively 3 times at 2-wk intervals. Then mice was challenged by subcutaneous (s.c.) injection of colonic adenocarcinoma cells (CT-26) into the right armpit 2 weeks after the last immunization and survival time of the animals was recorded. 4.Mice were divided into 3 groups(DNA vaccine group, vector group and saline group)and were orally immunized with SL3261 transformed with the recombinant plasmid, pcDNA3.1(+), or saline respectively 3 times at 2-wk intervals. Then mice was challenged by subcutaneous (s.c.) injection of colonic adenocarcinomacells (CT-26) into the right armpit 2 weeks after the last immunization. Flow cytometry (FCM) was used to detect CD3+ cells and CD8+ T cells before vaccination, after vaccination and after the inoculation of CT-26 cells respectively . Twenty-eight days after tumor cells inoculation, mice were sacrificed and analyzed for tumor growth.The tumors were studied histologically and microvessel density (MVD) was investigated with immunohistochemical method . Transmission electron microscope (TEM) was used to survey the ultrastructure of the tumors.5.Mice were divided into 3 groups randomly (DNA vaccine group,vector group and saline group) and were orally immunized with SL3261 transformed with the recombinant plasmid, pcDNA3.1(+), or saline respectively 3 times at 2-wk intervals. Then mice were challenged by intrasplenic injection of colonic adenocarcinoma cells (CT-26) to develop a liver metastasis 2 weeks after the last immunization. Flow cytometry (FCM) was used to detect CD44v before vaccination, after vaccination and after the inoculation of CT-26 cells respectively. Fifteen days after tumor cell inoculation, mice were sacrificed and analyzed for liver tumor growth. The liver metastasises were studied by stereological method.Results l.We successfully constructed a recombinant plasmid DNA encoding extracellular domain of VEGFR2 which was proved by DNA sequencing and comparison with GenBank data.Protein expression of flk-l(ni.7) was detected in eucaryotic expression system COS-7 cells. 2. We developed an oral DNA vaccine against the extracellular domain of flk-1 carried by attenuated Salmonella typhimurium SL3261. 3. In subcutaneous animal models, the median survival time of the animals in the DNA vaccine group was 60 days, in the vector group, 35 days, and in the saline group, 32 days . 4 . In subcutaneous animal models , there was no obvious difference in CD3+ levels in the three groups before and aftervaccination(F>0.05),but after inoculation of CT-26 cells, CD3+ levels obviously declined in the vector group and the saline group while high levels were maintained in the DNA vaccine group (P<0.05).There was no obviously difference in CD8+ T levels in the three groups before vaccination (P>0.05),but after vaccination and after inoculation of CT-26 cells 3 weeks later , CDS'T levels was obviously higher in the DNA vaccine group than that of the vector group and the saline group (P<0.05). Tumor size, weight and microvessel density (MVD) were significantly greater in the vector group and saline group than that of the DNA vaccine group (P<0.05). No significant difference in control groups (P>0.05). We found the tumors in the vector group and saline group accorded with poorly differentiated colonic adenocarcinoma while the tumors in DNA vaccine group accorded with better differentiated colonic adenocarcinoma by the use of transmission electron microscope (TEM). 5.1n liver metastasis animal models , we found no obvious difference in CD44v levels was in the three groups before and after vaccination (P>0.05),but after inoculation of CT-26 cells, CD44v levels were obviously higher in the vector group and saline group than that of the DNA vaccine group (P<0.05) by flow cytometry. We found all animals inoculated with CT26 cell lines developed liver metastasis but the liver metastasises were markedly more in the vector group and saline group than that of the DNA vaccine group (P<0.05) by stereological method. No significant difference in the control group (P>0.05).Conclusions 1. We successfully amplified the extracellular domain of flk-1 which was proved by DNA sequencing and comparison with GenBank data. 2. We successfully constructed the recombinant plasmid pcDNA3.1+/flk-l(nl.7), the protein expression of which was detected in eucaryotic expression system COS-7 cells. 3. We successfully developed an oral DNA vaccine against the extracellular domain of flk-1 carried by attenuated Salmonella typhimurium SL3261. 4. The oral DNA vaccineagainst extracellular domain of flk-1 can prolong the median survival time of mice challenged with lethal CT-26 cells. 5.The oral DNA vaccine against mouse extracellular domain of flk-1 can markedly inhibit angiogenesis of tumor which was induced by colonic adenocarcinoma cells (CT-26). 6.The oral DNA vaccine against mouse extracellular domain of flk-1 can markedly reduce liver metastasises and inhibit their growth. 7. The oral DNA vaccine may have the ability of inducing cell differentiation. 8. The oral DNA vaccine can inhibit tumor angiogenesis and metastasis by stimulating cellular immunity .