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. 2008 Dec;99(12):2477-84.
doi: 10.1111/j.1349-7006.2008.00967.x. Epub 2008 Nov 17.

Differential effector mechanisms induced by vaccination with MUC1 DNA in the rejection of colon carcinoma growth at orthotopic sites and metastases

Daisuke Sugiura  1 , Satoshi Aida, Kaori Denda-Nagai, Kazuyoshi Takeda, Mika Kamata-Sakurai, Hideo Yagita, Tatsuro Irimura
Affiliations

Differential effector mechanisms induced by vaccination with MUC1 DNA in the rejection of colon carcinoma growth at orthotopic sites and metastases

Daisuke Sugiura et al. Cancer Sci. 2008 Dec.

Abstract

The effects of MUC1 DNA vaccination on the orthotopic growth and liver metastasis of colon carcinoma cells were investigated in mice. Vaccination with MUC1 DNA resulted in immune responses that were effective in suppressing mouse colon carcinoma cells transfected with MUC1 cDNA. CD4+ T cells but not CD8+ T cells mediated this antitumor response as shown by the in vivo depletion of lymphocyte subpopulations with the use of anti-CD4 or anti-CD8 antibody. The effects of neutralizing antibodies in vivo revealed that the predominant effector molecule in preventing orthotopic tumor growth was FasL, whereas the effector molecule effective in preventing liver metastasis was tumor necrosis factor-alpha. Colon carcinoma cells isolated from tumors growing in the ceca, spleens, and livers were shown to be equally sensitive to FasL and tumor necrosis factor-alpha. The results strongly suggest that elimination of tumor cells initiated by DNA vaccination in the present protocol is mediated by antigen-specific CD4+ T cells and the effector mechanisms in the cecum and in the liver are distinct due to a unique organ microenvironment.

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Figures

Figure 1
Figure 1
MUC1 was expressed on the cell surfaces of MUC1‐transfected but not mock‐transfected SL4 cells. Mock‐transfected and MUC1‐transfected cells, (a) SL4‐P36 and (b) SL4‐M11 for the orthotopic model and (c) SL4‐P46 and (d) SL4‐M8 for the liver metastasis model, were stained with the anti‐MUC1 monoclonal antibody MY.1E12 and analyzed by flow cytometry. Gray‐filled histograms represent the controls and open histograms represent the binding of anti‐MUC1 monoclonal antibody.
Figure 2
Figure 2
MUC1 DNA vaccine effectively suppressed tumor growth at orthotopic sites and in the liver metastases in a MUC1‐specific manner. One week after the final MUC1 DNA immunization, SL4‐P36 or SL4‐M11 cells (1 × 106 cells/50 µL) were injected into the space under the cecal serosa of mice. (a) Twenty‐one days after the challenge, the mice were killed and the weight of the cecum was examined. In the liver metastasis model, SL4‐P46 or SL4‐M8 cells (5 × 105 cells/50 µL) were injected into the spleen. Nineteen days after the challenge, the weights of the (b) spleen and (c) liver were measured. Open circles represent tumor‐free mice and filled circles represent tumor‐bearing mice. n.s., not significant; *P < 0.005.
Figure 3
Figure 3
Effector cells were CD4+ in both the orthotopic and liver metastasis models. One week after the final MUC1 DNA immunization, the mice were treated with anti‐CD4 (GK1.5) or CD8 (53–6.72) monoclonal antibody and injected with (a) SL4‐M11 or (b,c) SL4‐M8 as described in Figure 1. Vaccine effects were assessed by tumor growth in the (a) cecum, (b) spleen, and (c) liver. Open circles represent tumor‐free mice and filled circles represent tumor‐bearing mice. n.s., not significant; *P < 0.05; **P < 0.005.
Figure 4
Figure 4
Tumor cells from the ceca, spleens, and livers expressed low but detectable levels of MHC class I and II molecules on their surfaces. The cells grown (a,c,f,h) in vitro in cultures and obtained from tumors growing in (a,f) ceca, (d,i) spleens, and (e,j) livers were stained with the anti‐MUC1 monoclonal antibody MY.1E12 and anti‐MHC class I or II monoclonal antibody. Using flow cytometric analysis, MUC1+ cells regarded as tumor cells were gated and analyzed further for MHC expression. The gray‐filled histograms represent the isotype controls and the open histograms represent (a–e) anti‐MHC class I and (f–j) anti‐MHC class II monoclonal antibody binding.
Figure 5
Figure 5
The antitumor effector mechanism induced by the MUC1 DNA vaccine was independent of interferon (IFN)‐γ. After MUC1 DNA immunization, the mice were treated with anti‐IFN‐γ monoclonal antibody (R4‐6A2) and injected with MUC1‐transfected SL4 cells. Vaccine effects were assessed by tumor growth in the (a) cecum, (b) spleen, and (c) liver. Open circles represent tumor‐free mice and filled circles represent tumor‐bearing mice. n.s., not significant; *P < 0.005.
Figure 6
Figure 6
The antitumor effector mechanism at the orthotopic sites was distinct from that in the liver metastasis. After MUC1 DNA immunization, the mice were treated with anti‐tumor necrosis factor (TNF)‐α (MP6‐XT22) or anti‐FasL (MFL4) monoclonal antibody and injected with MUC1‐transfected SL4 cells. Vaccine effects were assessed by tumor growth in the (a) cecum, (b) spleen, and (c) liver. Open circles represent tumor‐free mice and filled circles represent tumor‐bearing mice. n.s., not significant; *P < 0.05; **P < 0.005.
Figure 7
Figure 7
Tumor cells from the ceca, spleens, and livers expressed Fas and were sensitive to FasL‐induced apoptosis. Fas expression and sensitivity to FasL of (c–e,h–j) SL4‐M8 and (a,b,f,g) SL4‐M11 cells were examined. The cells were grown (a,c,f,h) in vitro and in vivo and were collected from (a,f) ceca, (d,i) spleens, and (e,j) livers. (a–e) The cell surface expression of Fas was examined by anti‐Fas monoclonal antibody binding by flow cytometric analysis. MUC1+ cells that were regarded as in vivo‐growing tumor cells were gated and analyzed. The gray‐filled histograms represent the isotype controls and the open histograms represent anti‐Fas monoclonal antibody binding. (f–j) FasL killing assays were carried out. Target tumor cells were collected from ceca, spleens, and livers by cell sorting using anti‐MUC1 monoclonal antibody (MY.1E12) binding. Parental L5178Y cells (open circle) and mFasL/L5178Y cells (filled circle) were used as effector cells. The percentage of specific 51Cr release was calculated according to the following formula: 51Cr release (%) = 100 × ([cpm experiment – cpm spontaneous release]/[cpm maximum – cpm spontaneous release]). Spontaneous release was obtained from target cells incubated with medium alone and the maximum release was obtained from target cells incubated with 1 M HCl instead of effector cells. Data are represented as the mean ± SD (*P < 0.005). E:T, effector:target.
Figure 8
Figure 8
Tumor cells from the ceca, spleens, and livers were sensitive to tumor necrosis factor (TNF)‐α‐induced apoptosis. MUC1‐transfected SL4 cells injected into ceca (SL4‐M11 cells) or spleens (SL4‐M8 cells) were grown in vivo at the injection sites and as liver metastases. One week later, tumors were collected from the ceca, spleens, and livers and MUC1‐positive cells were obtained. Tumor cells grown (a) in vivo and (b) in vitro were incubated with recombinant mouse TNF‐α and cell viability and proliferation were evaluated using WST‐1 cell proliferation assays. The viability of cells cultured without TNF‐α was regarded as 100%. Data are represented as the mean ± SD. *P < 0.005.
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