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. 2015 Feb;106(2):134-42.
doi: 10.1111/cas.12584. Epub 2015 Jan 16.

Cancer-associated fibroblast-targeted strategy enhances antitumor immune responses in dendritic cell-based vaccine

Yasuhiko Ohshio  1 , Koji Teramoto, Jun Hanaoka, Noriaki Tezuka, Yasushi Itoh, Tohru Asai, Yataro Daigo, Kazumasa Ogasawara
Affiliations

Cancer-associated fibroblast-targeted strategy enhances antitumor immune responses in dendritic cell-based vaccine

Yasuhiko Ohshio et al. Cancer Sci. 2015 Feb.

Abstract

Given the close interaction between tumor cells and stromal cells in the tumor microenvironment (TME), TME-targeted strategies would be promising for developing integrated cancer immunotherapy. Cancer-associated fibroblasts (CAFs) are the dominant stromal component, playing critical roles in generation of the pro-tumorigenic TME. We focused on the immunosuppressive trait of CAFs, and systematically explored the alteration of tumor-associated immune responses by CAF-targeted therapy. C57BL/6 mice s.c. bearing syngeneic E.G7 lymphoma, LLC1 Lewis lung cancer, or B16F1 melanoma were treated with an anti-fibrotic agent, tranilast, to inhibit CAF function. The infiltration of immune suppressor cell types, including regulatory T cells and myeloid-derived suppressor cells, in the TME was effectively decreased through reduction of stromal cell-derived factor-1, prostaglandin E2 , and transforming growth factor-β. In tumor-draining lymph nodes, these immune suppressor cell types were significantly decreased, leading to activation of tumor-associated antigen-specific CD8(+) T cells. In addition, CAF-targeted therapy synergistically enhanced multiple types of systemic antitumor immune responses such as the cytotoxic CD8(+) T cell response, natural killer activity, and antitumor humoral immunity in combination with dendritic cell-based vaccines; however, the suppressive effect on tumor growth was not observed in tumor-bearing SCID mice. These data indicate that systemic antitumor immune responses by various immunologic cell types are required to bring out the efficacy of CAF-targeted therapy, and these effects are enhanced when combined with effector-stimulatory immunotherapy such as dendritic cell-based vaccines. Our mouse model provides a novel rationale with TME-targeted strategy for the development of cell-based cancer immunotherapy.

Keywords: Cancer-associated fibroblasts; dendritic cell-based vaccine immunotherapy; suppressor immune cells; tranilast; tumor microenvironment.

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Figures

Fig 1
Fig 1
Reduction of infiltrative suppressor immune cells in tumor microenvironment through inhibition of cancer-associated fibroblast (CAF) function. Mice bearing E.G7 tumors were treated with both tranilast and a dendritic cell (DC)-based vaccine. Five days after the final treatment, tumor tissues were harvested from the mice. (a) Numbers of α-smooth muscle actin (α-SMA)+ CAFs in the tumor tissues were determined by immunohistochemical staining. (b) Expression of stromal cell-derived factor-1 (SDF-1) in tumor tissues was analyzed by Western blotting. The levels of prostaglandin E2 (PGE2) (c) and transforming growth factor-β1 (TGF-β1) (d) in tumor tissues were measured by ELISA. Infiltration of Foxp3+ regulatory T cells (black arrowheads) (e) and Gr-1+ myeloid-derived suppressor cells (white arrowheads) (f) in tumor tissues was analyzed by immunohistochemical staining. Bars represent mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. N.S., not significant.
Fig 2
Fig 2
Improvement of antitumor immune responses in tumor-draining lymph nodes (TDLNs) through inhibition of cancer-associated fibroblast (CAF) function. Mice bearing E.G7 tumors were treated with a dendritic cell (DC)-based vaccine in combination with or without anti-CAF therapy. Five days after the final treatment with tranilast, the populations of CD4+CD25+Foxp3+ regulatory T cells (a) and CD11b+Gr-1+ myeloid-derived suppressor cells (b) in TDLNs were analyzed by flow cytometry. (c) TDLNs cells were stimulated with ovalbumin (tumor-associated antigen)-derived MHC class I peptide in vitro, then the population of interferon-γ+CD8+ T cells was analyzed by flow cytometry (closed square, with tumor-associated antigen stimulation in vitro; open square, control). Bars represent mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001. N.S., not significant.
Fig 3
Fig 3
Improvement of systemic antitumor immune responses associated with inhibition of cancer-associated fibroblast (CAF) function. Mice bearing E.G7 tumors were treated with a dendritic cell (DC)-based vaccine in combination with or without anti-CAF therapy. Five days after the final treatment with tranilast, the populations of CD4+CD25+Foxp3+ regulatory T cells (a) and CD11b+Gr-1+ myeloid-derived suppressor cells (b) in spleen cells were analyzed by flow cytometry. (c) Spleen cells were stimulated with ovalbumin (tumor-associated antigen)-derived MHC class I peptide in vitro, then the population of interferon-γ+CD8+ T cells was analyzed by flow cytometry (closed square, with tumor-associated antigen stimulation in vitro; open square, control). Bars represent mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. N.S., not significant.
Fig 4
Fig 4
Enhancement of multiple components of antitumor immune systems by inhibition of cancer-associated fibroblast (CAF) functions in vivo. Mice bearing E.G7 tumors were treated with a dendritic cell (DC)-based vaccine in combination with or without anti-CAF therapy. Five days after the final treatment with tranilast, spleens and sera were harvested from the mice. (a) Spleen cells were stimulated with ovalbumin (tumor-associated antigen)-derived MHC class I peptide and interleukin-2 for 5 days in vitro, then were cocultured with E.G7 cells for 4 h. The cytotoxicity against E.G7 cells was measured by lactate dehydrogenase release assay. (b) Natural killer activity in spleen cells was evaluated by the lactate dehydrogenase release assay that targeted YAC-1 cells. (c) Levels of anti-ovalbumin antibody production in sera were measured by ELISA. Bars represent mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. N.S., not significant.
Fig 5
Fig 5
Enhancement of dendritic cell (DC)-based vaccine potency in combination with anti-cancerassociated fibroblast (CAF) therapy. Mice were inoculated s.c. with (5 × 105) tumor cells. Seven days after tumor inoculation, the mice were treated with a DC-based vaccine s.c. near the tumors on days 7, 13, and 19, in combination with or without intratumoral treatment with tranilast every day for 2 weeks. Twenty-four days after tumor inoculation, volumes of E.G7 (a, left), B16F1 (b), and LLC1 tumors (c) were measured. Tumor volumes in E.G7 tumor-bearing mice were monitored every 3–4 days (a, right). SCID mice bearing E.G7 tumors were administered tranilast into tumors every day for 2 weeks. Five days after the final treatment with tranilast, tumor tissues were harvested from the mice. (d) α-Smooth muscle actin+ CAFs in the tumor tissues were evaluated by immunohistochemical staining. (e) Twenty-four days after tumor inoculation, volumes of tumors were measured. Each group had 5–10 mice. Bars indicate the median tumor volume ± SD of the group. *P < 0.05, **P < 0.01, ***P < 0.001. N.S., not significant.
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