Anti-tumor angiogenesis effect of genetic fusion vaccine encoding murine beta-defensin 2 and tumor endothelial marker-8 in a CT-26 murine colorectal carcinoma model

Abstract

Tumor endothelial marker 8 (TEM8) is an endothelial-specific marker that is upregulated during tumor angiogenesis. We previously demonstrated that DNA-based vaccine encoding xenogeneic TEM8 can potentiate anti-angiogenesis immunotherapy of malignancy; nevertheless, it remains to be improved in minimizing immune tolerance. Recently, it has been reported that murine beta-defensin 2 (MBD2) is chemotactic for immature dendritic cells and plays a pivotal role in breaking immune tolerance. Herein, we constructed a genetic fusion vaccine encoding murine TEM8 and MBD2 to investigate whether the novel vaccine preferentially elicits therapeutic antitumor immune responses and suppresses cancerous angiogenesis in mouse models. The anti-angiogenesis effect was determined by microvessel density (MVD) using immunohistochemical staining. The efficacy of the fusion vaccine was primarily assessed by detecting cytotoxic T lymphocyte activity ((51)Cr-release assay). Enzyme-linked immunosorbent spot (ELISpot) assay was used to detect TEM8-specific INF-γ production, and the activity of CTL was further verified by a depletion of CD8(+) T cells via anti-CD8 monoclonal antibody. Our results showed that the DNA fusion vaccine possessed an enhanced therapeutic antitumor immunity through anti-angiogenesis in BALB/c mice inoculated with CT26 cells, and this effect was generally attributed to stimulation of an antigen specific CD8(+) T-cell response against mTEM8. In conclusion, our study demonstrated that the fusion vaccine based on mTEM8 and MBD2 induced autoimmunity against endothelial cells, resulting in deceleration of tumor growth, and could be potential therapeutical application in clinic.

Keywords: MBD2-mTEM8; anti-tumor effect; fusion vaccine; immunotherapy.

Figure 1

A. PCR products of fusion gene. Lane 1, marker; lane 2, PCR products of MBD2-mTEM. B. Expression of the fusion gene by Western Blot. The plasmids DNA were transfected into COS-7 cells with different plasmids. Lane 1, COS-7 cells transfected with pmTEM8, (MW, 71kD); lane 2, COS-7 cells transfected with pcDNA3.1; lane 3, COS-7 cells transfected with pMBD2-mTEM8 (MW, 80 kD).

Figure 2

The examination of antitumor efficiency. BALB/c mice (n=10) were inoculated with s.c. injection of CT-26 cells (5×10⁵), and 7 d later they received vaccination of PBS, pcDNA3.1, pmTEM8, mix, or pMBD2-mTEM8 vaccine once a week for four times. The tumor volume and the lifespan of mice were observed to assess antitumor efficiency (A); The average tumor growth volume was calculated as 0.5× length× width². There was a significant difference in tumor volume (P<0.01) in BALB/c mice with pMBD2-mTEM8 immunized when compared with other controls (C); The mean lifespan of mice in the pMBD2-mTEM8 group was prolonged remarkably (B).

Figure 3

Inhibition of angiogenesis of tumors. BALB/c mice were immunized with PBS (A), pcDNA3.1 vector (B), pmTEM8 (C), mixture (D) or pMBD2-mTEM8 (E). (E) Three days after the last vaccination, the tumor tissues were fixed in acetone, and stained with an antibody reactive to CD31. (F) Vessel density of tumor tissues from pMBD2-mTEM8 immunized mice indicated a significant decrease compared with control groups (**, P<0.01).

Figure 4

Decreased antitumor activity by the depletion of CD8⁺ T lymphocytes via corresponding mAbs in BALB/c mice. BALB/c mice were treated and immunized as described in materials and methods. Depletion of CD8⁺ T lymphocytes impaired the antitumor activity of the pMBD2-mTEM8 vaccine in CT26 model. The immunized group of pMBD2-mTEM8 resulted in significantly high CTL activity with a ~60% of specificlysis at an effect-to-target ratio of 100:1 (A). Similarly, the mice vaccinated with pMBD2-mTEM8 showed the highest production of IFN-γ among all the groups (B). Compared with the control groups, the antitumor effect of pMBD2-mTEM8 obviously decreased by depleting of CD8⁺ T cells (C) (*, P<0.05).