Inhibition of melanoma growth by subcutaneous administration of hTERTC27 viral cocktail in C57BL/6 mice

Abstract

Background: hTERTC27 is a 27 kDa C-terminal polypeptide of human telomerase reverse transcriptase that has previously been shown to reduce tumorigenicity of HeLa cells and suppress growth of xenografted glioblastoma in nude mice. Although ectopic expression of hTERTC27 upregulated genes that are involved in apoptosis, cell cycle, and immune response, the mechanism for hTERTC27-induced tumor suppression has not been completely elucidated. Since hTERT was identified as a universal tumor-associated antigen, we hypothesize that hTERTC27 inhibits tumor growth in vivo through activation of anti-tumor immune response.

Methodology/principal finding: Immunocompetent C57BL/6 mice were used for mouse B16 melanoma model. Mice bearing B16 melanoma were administered rAAV-/rAdv viral cocktail expressing hTERTC27, and tumor growth was monitored after viral cocktail treatment. Blood and splenocytes were used to determine the level of cytokines and the activity of immune cells, respectively. B16 tumor growth was significantly inhibited by subcutaneous administration of a single dose of 1.5×10(11) vg rAAV-hTERTC27 and 2.5×10(9) pfu rAdv-hTERTC27 viral cocktail (rAAV-/rAdv-hTERTC27). The population and cytotoxicity of NK cells in the mice were significantly augmented by rAAV-/rAdv-hTERTC27 treatment, and selective depletion of the NK cell population in mice by intraperitoneal injection of anti-GM1 antibody abrogated the growth suppression of melanoma induced by rAAV-/rAdv-hTERTC27 administration.

Conclusion: Activation of NK cells by administration of rAAV-/rAdv-hTERTC27 is critical for growth suppression of melanoma in mouse model.

Figure 1. rAAV-/rAdv-hTERTC27 treatment suppresses the growth of B16 melanoma in C57BL/6 mice.

(A) Anti-tumor efficacy of rAAV-hTERTC27, rAdv-hTERTC27 and rAAV-/rAdv-hTERTC27. Tumor-bearing mice (5mice/group) were subcutaneously injected with viruses as indicated. Tumor growth was monitored by measuring tumor size with calipers every other day till the day when mice sacrificed. *: P<0.05, compared with PBS treated group. (B) Antitumor efficacies of different administration routes of rAAV-/rAdv-hTERTC27 viral cocktail. Tumor-bearing mice were treated by injection of rAAV-/rAdv-hTERTC27, rAAV-/rAdv-EGFP, or PBS as indicated routes, i.e. intratumor (i.t.), intra-muscular (i.m.), intraperitoneal (i.p.), intravenous (i.v.) and subcutaneous (s.c.) injection (3mice/group). *: P<0.05, compared with PBS treated group. (C) Antitumor efficacy of subcutaneous administration of hTERTC27. Melanoma-bearing mice were subcutaneously received rAAV-/rAdv-hTERTC27 viral cocktail (5 mice), rAAV-/rAdv-EGFP viral cocktail (4 mice) or PBS (3 mice) around tumor on 7^th day after tumor cell injection, and were sacrificed on day 20 post-tumor cell injection. *: P<0.05, compared with PBS group.

Figure 2. rAAV-/rAdv-hTERTC27 treatment increases the population and cytotoxicity of NK cells.

(A) & (B) Flow cytometric analysis of PMBC from tumor-bearing mice treated with subcutaneous administration of rAAV-/rAdv-hTERTC27, rAAV-/rAdv-EGFP, or PBS. Data shown here represent one of several mice analyzed with similar results. (C) Lymphocyte profiles from A & B (data pooled from 3 mice/group treated with indicated viruses or PBS). *P<0.05, compared with EGFP group. (D) and (E): Cytotoxic activity of spleen NK cells isolated from mice treated with rAAV-/rAdv-hTERTC27, rAAV-/rAdv-EGFP or PBS was analyzed against YAC-1 (D) or B16 (E) cells. Lymphocytes isolated from each mouse were cultured with YAC-1 or B16 cells at various effector to target ratios (E/T). *: P<0.05, compared with EGFP group. (F): Profiles of NKT (NK1.1⁺/CD3⁺), NK (NK1.1⁺/CD3⁻) and T (NK1.1⁻/CD3⁺) cells in PMBC from tumor-bearing mice treated with rAAV-/rAdv-hTERTC27 or rAAV-/rAdv-EGFP. The numerical value presented with the bar graph indicates the percentage of the cell population in PMBC. The cell population without any marker (i.e. NK1.1⁻/CD3⁻) is not shown. Data pooled from 3 mice/group treated with indicated virus through flow cytometry analysis. *: P<0.05, compared with EGFP group.

Figure 3. NK cell activity is crucial for the anti-tumor effect of hTERTC27.

PMBC from mice intraperitoneally injected with anti-GM1 antibody were analyzed for populations of NK cells (A), helper T cell/cytotoxic T cell/B cell (B) and regulatory T cell (D) by flow cytometry. (C) Cytotoxicity of NK cells against YAC-1. Lymphocytes isolated from spleen of each mouse were cultured with YAC-1 cells at various E/T ratios, and the specific lysis was determined using CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit. (E) NK cell depletion abrogated the anti-tumor effects of rAAV-/rAdv-hTERTC27 in C57BL/6 mice. *: P<0.05, compared with EGFP group. (F) A representative photo of tumor morphology from different treatment groups. hTERTC27: hTERTC27 viral cocktail; EGFP: EGFP viral cocktail. *: P<0.05, compared with hTERTC27 group.

Figure 4. rAAV-/rAdv-hTERTC27 treatment increases plasma levels of Th1 cytokines.

Plasma from sacrificed mice on day 20 post tumor cell inoculation were analyzed to determine cytokine levels using mouse Th1/Th2 FlowCytomix assay kit. The numerical values presented with the bar graph indicate the fold differences of cytokine levels between mice treated with hTERTC27 and EGFP.

Figure 5. rAAV-/rAdv-hTERTC27 treatment has different effects on the activities of T cells and B cells.

(A) Proliferation activity of T cells and B cells isolated from mice treated with hTERTC27, EGFP or PBS. Data are expressed as means ± SD (n = 3). *: P<0.05, compared with EGFP group. (B) Cytotoxicity of T cells isolated from mice treated with hTERTC27, EGFP, or PBS. *: P<0.05, compared with EGFP group.