Anti-angiogenesis therapy based on the bone marrow-derived stromal cells genetically engineered to express sFlt-1 in mouse tumor model - PubMed (original) (raw)

doi: 10.1186/1471-2407-8-306.

J-L Yang, H Teng, Y-Q Jia, R Wang, X-W Zhang, Y Wu, Y Luo, X-C Chen, R Zhang, L Tian, X Zhao, Y-Q Wei

Affiliations

Anti-angiogenesis therapy based on the bone marrow-derived stromal cells genetically engineered to express sFlt-1 in mouse tumor model

M Hu et al. BMC Cancer. 2008.

Abstract

Background: Bone marrow-derived stromal cells (BMSCs) are important for development, tissue cell replenishment, and wound healing in physiological and pathological conditions. BMSCs were found to preferably reach sites undergoing the process of cell proliferation, such as wound and tumor, suggesting that BMSCs may be used as a vehicle for gene therapy of tumor.

Methods: Mouse BMSCs were loaded with recombinant adenoviruses which express soluble Vascular Endothelial Growth Factor Receptor-1 (sFlt-1). The anti-angiogenesis of sFlt-1 in BMSCs was determined using endothelial cells proliferation inhibition assay and alginate encapsulation assay. The anti-tumor effects of BMSCs expressing sFlt-1 through tail-vein infusion were evaluated in two mouse tumor metastases models.

Results: BMSCs genetically modified with Adv-GFP-sFlt-1 could effectively express and secret sFlt-1. BMSCs loaded with sFlt-1 gene could preferentially home to tumor loci and decrease lung metastases and prolong lifespan in mouse tumor model through inducing anti-angiogenesis and apoptosis in tumors.

Conclusion: We demonstrated that BMSCs might be employed as a promising vehicle for tumor gene therapy which can effectively not only improve the concentration of anticancer therapeutics in tumors, but also modify the tumor microenvironment.

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Figures

Figure 1

Figure 1

BMSCs infection and verification of secreted sFlt-1. After the BMSCs were confirmed by flow cytometric analysis, BMSCs were grown to 90% confluence and infected with adenoviruses at a multiple of infection of 3000 for 2 hours. 24 hours later, BMSCs were harvested and ready for use. Fluorescence microscope showed up to 100% GFP-positive cells. (Figure 1-A). Supernatants deposits from sFlt-1 bearing BMSCs could be recognized by antibodies reactive to NH2 terminus of mouse Flt-1, but negative staining in supernatants deposits from control BMSCs in Western blot analysis. (Figure 1-B). Conditioned media from Adv-sFlt-1 infected BMSCs was shown to inhibit the VEGF-driven mouse endothelial cells proliferation by about 50% compared with controls (Figure 1-C, P < 0.05).

Figure 2

Figure 2

Anti-tumor effect in mouse CT26 colon adenocarcinoma model. Female BALB/c mice received i.v. injection of 2×105 CT26 cells in 100 μl of PBS. Treatment with 1×106 Adv-GFP-sFlt-1 infected BMSCs(◆, black rhombus), 1×106 Adv-GFP infected BMSCs (▪, black quadrilateral), 1×106 uninfected BMSCs (▲, black triangle) and 100 μl of 0.9% NaCl solution alone (●, black circularity) were administered i.v. from tail vein. Treatment with sFlt-1-bearing BMSCs could decrease the number of and growth of surface metastases (Figure 2-A, D) and abolish the tumor burden by rendering the weight of lungs similar to that of normal mice (Figure 2-C). Mean numbers of lung tumor nodules in each group are shown (Figure 2-B). Values are plotted as means ± SEM (P < 0.05). The percentage of metastatic foci > 3 mm are marked as solid bars. A significant increase of survival rate in sFlt-1-bearing BMSCs treated mice, compared with the controls (Figure 2-E, P < 0.05. By log-rank test), was found in the tumor model.

Figure 3

Figure 3

Anti-tumor effect in mouse LLC lung carcinoma model. Female C57BL/6 mice received i.v. injection of 2×105 LLC cells in 100 μl of PBS. Treatment with 1×106 Adv-GFP-sFlt-1 infected BmSCs (◆, black rhombus), 1×106 Adv-GFP infected BmSCs (▪, black quadrilateral), 1×106 uninfected BmSCs (▲, black triangle) and 100 μl of 0.9% NaCl solution alone (●, black circularity) were administered i.v. from tail vein. A significant increase of survival and decrease of metastases in sFlt-1-expressing BMSCs treated mice, compared with the controls (P < 0.05. By log-rank test), was found in this model.

Figure 4

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).

Figure 5

Figure 5

Apoptosis Assay by TUNEL staining. Paraffin sections from CT26 tumor tissues obtained from mice systemically administered with sFlt-1-expressing BMSCs, GFP expressing BMSCs, unmodified BMSCs and NaCl solution were stained using an in situ cell death detection kit (Roche) following the manufacturer's protocol. Apoptosis was determined by counting the number of the positive cells per high-power field in tumor sections. Original magnification, ×200. (Figure 5-A and B, P < 0.05).

Figure 6

Figure 6

Fluorescent in situ hybridization. Frozen sections of lung metastases were tested for the presence of Y chromosome which is indication of male originated BMSCs by Cy3 labeled Y chromosome probe (STAR*FISH; Cambio, Cambridge, England). Positive result could be seen in the tumor area obviously, whereas the relatively normal parts of the sections were totally negative for fluorescent signals.

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