| Objective:To study the influence of MCP-1on vascular endothelial growth inhibitor (VEGI, TNFSF15, tumor necrosis factor superfamily-15) expression in HUVEC. Furthermore, we evaluate the effects of VEGI on tumor angiogenesis and tumor growth of ovarian cancer in mice and mean to explore a novel strategy for the treatment of ovarian cancer.Methods:1. We used immunohischemistry to detect the infiltration of CD4+cells in normal ovary and ovarian cancer specimens, and used immunofluorescence assay to detect the distribution of CD4+CD25+FOXP3+Treg cells in stage III&IV ovarian cancer specimens.2. We used magnetic absorbance cell sorting (MACS) to isolate CD4+CD25+Treg cells from healthy human peripheral blood, CD4+CD25+Treg cells were used for the related following experiment.3. We used ELISA to evaluate the effect of MCP-1(0,5,10,20,40ng/mL) or CD4+CD25+Treg-CM on VEGI expression in HUVECs.4. We used RayBio Human Cytokine Antibody Array to detect cytokines secreted by untreated Treg cells or Treg treated with OVCAR3-CM.5. The effect of recombinant VEGI on ovarian cancer growth in C57BL/6mice: Six-week-old female C57BL/6mice were injected subcutaneously on the flank with ID8cells (1x107).Tumor-bearing animals were randomized on day6into two groups. The experimental group received intraperitoneal injection of VEGI (125μg/0.5mL PBS/mouse) on the same day and every other day thereafter. The control group received PBS (0.5mL/mouse).The animals were observed and weighed and tumor sizes were measured daily prior to next treatment. All mice were sacrificed on the11d after the last injection of VEGI.The tumors were retrieved for pathologic analysis.6. The effect of VEGI-shRNA on ovarian cancer growth in C57BL/6mice: Six-week-old female C57BL/6mice were randomized into two groups. Animals in the experimental group were injected subcutaneously on the flank with VEGI-shRNA(1.5×105IFU/100LμPBS/mouse). The control group were injected control-shRNA. After3days, all animals were inoculated subcutaneously with5x106ID8cells at the same sites. The same dose of VEGI-shRNA or control-shRNA were repeatedly injected on day-6post-cancer cell inoculation. The animals were observed, weighed, and the tumor sizes were measured as described above. The animals were euthanized at the day10after the second injection of VEGI-shRNA and the tumors were retrieved for pathologic analysis as described above.Results:1. Compared to normal ovary tissue, infiltration of CD4+T cells increased notably in ovarian cancer, and the degree of CD4+T cells accumulation in III&IV were more than I&II.80%of these specimens were Treg+in ovarian cancer FIGO III&IV. In the Treg+specimens,30-60%(median=45%) of the CD4+cells were also CD25+FOXP3+, about95%of the CD4+CD25+cells were CD4+CD25+FOXP3+Treg.2. MCP-1can downmodulate the expression of VEGI in HUVECs in a dose-dependent manner and MCP-1mAb can block the influence of MCP-1on VEGI production. When treated with CM of CD4+CD25+Treg treated with OVCAR3-CM, the VEGI production in HUVECs reduced markedly.3. CD4+CD25+Treg cells produced a detectable amount of MCP-1.However, when treated with OVCAR3-CM, MCP-1produced by the CD4+CD25+Treg cells sharply increased.4. System injection of recombinant VEGI markedly decreased the tumor MVD and inhibit the growth of ID8tumors.5. Local VEGI-shRNA injection could increase MVD of the tumor and accelerate the growth of ID8tumors.Conclusion:1. The degree of CD4+T cell accumulation in ovarian cancer specimens increased markedly as the disease progresses based on FIGO staging. In FIGO III&IV, infiltration rate of Treg+(CD4+CD25+FOXP3+/CD4+) increased.2. MCP-1treatments led to a signifcant decrease of VEGI production by HUVEC in a dose-dependent manner. A neutralizing antibody against MCP-1specically blocked the inhibitory effect.3. When treated with OVCAR3-CM, MCP-1produced by the CD4+CD25+Treg cells sharply increased.4. In ID8tumor model, increased VEGI in the cancer microenvironment results in significant inhibition of tumor angiogenesis and ovarian cancer growth, whereas decreased VEGI led to greatly facilitated tumor angiogenesis and ovarian cancer growth. |
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