Figure 4

Evaluation of anti-angiogenetic effect by CD31 immunohistochemistry and alginate assay. Sections of frozen CT26 tumor tissues obtained from mice treated with sFlt-1-bearing BMSCs, GFP-expressing BMSCs, unmodified BmSCs and 0.9% NaCl solution were stained with CD31 antibody and Peroxidase-DAB (Figure 4-A). Vessel density was determined by counting the number of microvessels per high-power field (×200) in sections. (Figure4-B). Alginate beads containing different ratio of sFlt-1-bearing BMSCs and LLC (1/10, 1/100, 1/1000, LLC only) were implanted subcutaneously into C57BL/6 mice. Three weeks later, beads were surgically removed, and FITC-dextran was quantified. FITC-dextran uptake decrease and photograph of alginate implants showed the reduction of vascularization in beads containing relatively more sFlt-1-expressing BMSCs. (Figure4-C and D). Alginate beads containing MethA, BMSCs and mixture of both with ratio of 1/10 were prepared. Beads-MethA and beads-mixture were served as positive and negative controls. The third team of mice was implanted with two different beads containing MethA or BMSCs respectively in different sites of mice, which served as systemic sFlt-1 model to show the non-targeted effect of viruses. The fourth team of mice was implanted with beads containing MethA and hence injected intravenously with BMSCs in the same number of the former team, which served as local sFlt-1 model to show the targeted effect of viruses loaded in BMSCs. Compared with the controls, the effect of systemic production of sFlt-1 was weaker than local one. (Figure 4-E and F).