Yasuhiko Ohshio

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

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.

Tranilast Inhibits the Function of Cancer-Associated Fibroblasts Responsible for the Induction of Immune Suppressor Cell Types

Cancer‐associated fibroblasts (CAFs) are the dominant stromal component in the tumour microenvironment (TME), playing critical roles in generation of pro‐tumourigenic TME; however, their contribution to suppression of antitumour immune responses has not been fully understood. To elucidate the interaction between CAFs and immune suppressor cells, we examined whether inhibition of CAFs function would impair the induction of immune suppressor cell types in vitro. In this study, we applied an anti‐allergic and antifibrotic agent tranilast, which is used clinically, and evaluated a potential of tranilast to serve as a CAFs inhibitor. CAFs that had been isolated from E.G7 or LLC1 tumour‐bearing mice were cultured in the presence of tranilast, and thereafter, CAFs functions on the secretion of some soluble factors as well as the induction of immune suppressor cells were evaluated. As a result, tranilast inhibited the proliferation of CAFs and reduced the levels of stromal cell‐derived factor‐1, prostaglandin E2 and transforming growth factor‐β1 from CAFs in a dose‐dependent manner. On the other hand, tranilast exerted no inhibitory effects on immune cells at doses under 100 μm. The induction of regulatory T cells and myeloid‐derived suppressor cells from their progenitor cells was suppressed in the medium that CAFs had been cultured in the presence of tranilast; however, these findings were not observed when those progenitor cells were cultured in the medium containing tranilast alone. These data demonstrate that tranilast inhibits CAFs function, which is responsible for the induction of immune suppressor cells, and possesses a potential to serve as a specific CAFs inhibitor.

Inhibition of transforming growth factor-β release from tumor cells reduces their motility associated with epithelial-mesenchymal transition

The high level of transforming growth factor‑β (TGF‑β) in tumor tissue, which is primarily released from tumor cells, helps maintain their metastatic nature and exacerbates the creation of a pro-tumor microenvironment. Although the strategy of targeting TGF‑β in cancer therapy has shown promise, its effects remain limited. In the present study, we focused on tumor cells as sources of TGF‑β release, and hypothesized that inhibition of their TGF‑β release could suppress their epithelial-mesenchymal transition (EMT)-associated metastatic nature and inactivate the induction of suppressor immune cells. To investigate this hypothesis, LLC1 cells, a mouse lung cancer cell line, were cultured with the TGF‑β release inhibitor tranilast and the motility of LLC1 cells was examined. Furthermore, to examine whether inhibition of TGF‑β release influences the induction of regulatory T (Treg) cells, spleen cells from normal mice were cultured in medium in which LLC1 cells had been cultured with tranilast. The results showed that tranilast inhibited the release of TGF‑β1 from LLC1 cells without affecting their proliferation. Inhibition of TGF‑β1 release suppressed the invasive activity of LLC1 cells, but enhanced their activity to adhere. mRNA levels of Slug and Twist were decreased in LLC1 cells, whereas levels of E‑cadherin were recovered. Treg cells were less frequently induced by medium in which LLC1 cells had been cultured with tranilast. Taken together, inhibition of TGF‑β1 release dampens the metastatic nature of LLC1 cells through the downregulation of EMT and possesses the possibility to improve antitumor immune responses through suppression of Treg cell induction. These findings provide a new rationale for development of TGF‑β‑targeted molecular immunotherapy against cancer.

Simultaneous activation of T helper function can augment the potency of dendritic cell-based cancer immunotherapy

PurposeSimultaneous activation of T helper 1 (Th1) cell function has crucial roles in induction of potent cytotoxic T lymphocyte (CTL) responses in cancer immunotherapy. Here, we investigated whether dendritic cell (DC)-based vaccines loaded with both tumor-associated antigen (TAA)-derived MHC class I and pan-MHC class II peptides could elicit more potent CTL responses through simultaneous activation of Th1 function and reduction in CD4+ regulatory T (Treg) cell proliferation.MethodsC57BL/6 mice bearing LLC1, a mouse Lewis lung cancer cell line, were subcutaneously administered DCs loaded with both LLC-derived MHC class I (MUT1&2) and LLC-unrelated pan-MHC class II (PADRE) peptides (DC-MUT1&2-PADRE). In assays using samples from advanced lung cancer patients, peripheral blood mononuclear cells were stimulated with autologous DCs loaded with both MUC1 MHC class I and PADRE peptides (DC-MUC1-PADRE) in vitro. Subsequently, TAA-specific CTL responses and the population of CD4+ Treg cells were analyzed.ResultsThe population of spleen CD4+ PADRE-specific cells producing interferon-gamma (IFNγ) was significantly increased by DC-MUT1&2-PADRE administration. Vaccinations with DC-MUT1&2-PADRE decreased the population of CD4+ Treg cells in spleen and augmented CTL responses, effectively leading to suppression of tumor growth. In assays with human samples, CD4+ Treg cells were induced less frequently, and MUC1-specific cytotoxicity was enhanced by stimulation with DC-MUC1-PADRE compared with that by stimulation with DC-MUC1 alone.ConclusionsSimultaneous activation of Th1 function by DCs loaded with both TAA-derived MHC class I and PADRE peptides augments TAA-specific CTL responses while reducing Treg cell proliferation.

Pulmonary venous aneurysm: Forming a glove balloon-like shape.

Highlights • Pulmonary venous aneurysm is a rare venous abnormality and a candidate for surgery because of the risk of rupture and continued growth.• Preoperative information is very important to choose an appropriate surgical approach, and 3D-CT is the optimum modality to evaluate PVA.• If the aneurysm forms a “glove balloon-like shape,” lobectomy is necessary because end-to-end anastomosis is difficult to perform.

Abstract 5080: HIF-1 is responsible for the induction of cancer-associated fibroblasts in hypoxic tumor microenvironment

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA In tumor tissues, tumor cells and tumor stromal cells interact with each other, generating the complex tumor microenvironment (TME). Whereas cancer-associated fibroblasts (CAFs) are dominant cells among tumor stromal cell types, the induction of CAFs in TME still remains unclear. Given that TME turns to be hypoxic while tumor is growing, we hypothesized that hypoxia in TME would contribute to induction of CAFs. In this study, we focused on a role of hypoxia-inducible factor-1 (HIF-1), a key transcription factor in hypoxic responses, and examined the implication of HIF-1 in the induction of CAFs in TME. The mouse lymphoma cell line, E.G7 cells, which had been transfected with HIF-1α shRNA (E.G7- HIF-1α shRNA) were cultured under hypoxia with an O2 concentration of 1.0%. As controls, E.G7 cells or those which had been transfected with control shRNA (E.G7-control shRNA) were also cultured under the same hypoxic condition as in case of E.G7- HIF-1α shRNA. Three days later, the culture medium was harvested for following experiments. To evaluate a role of HIF-1α in migration of CAFs progenitor cells into TME, mouse bone marrow cells as CAFs progenitor cells were put in the upper compartment, and hypoxic culture medium was added in the lower compartment of a transwell chamber. We observed that the number of α-smooth muscle actin (α-SMA)-positive cells that had migrated was decreased when hypoxic culture medium from E.G7- HIF-1α shRNA was added in the lower compartment. Transforming growth factor-beta (TGF-β) has been reported to be one of soluble factors which are responsible for induction of CAFs. Thus, we examined the level of TGF-β in hypoxic culture medium by ELISA, demonstrating that it was decreased in the medium from E.G7- HIF-1α shRNA as compared with E.G7-control shRNA or E.G7 cells. Next, we examined the association between HIF-1α inhibition and CAFs induction in a tumor-bearing mouse model. C57BL/6 mice were subcutaneously administered E.G7-HIF-1α shRNA, E.G7-control shRNA, or E.G7 cells at the flank. Three weeks later, tumor tissues were harvested from the mice, and α-SMA-positive cells in tumor tissues were examined by immunohistochemistry. The data showed that α-SMA-positive CAFs were decreased in E.G7-HIF-1α shRNA tumor tissues as compared with E.G7-control shRNA, or E.G7 tumor tissues. In addition, tumor growth was significantly suppressed in mice bearing E.G7-HIF-1α shRNA, while inhibition of HIF-1α did not exert a suppressive effect on the proliferation of E.G7 cells in vitro. These data indicated that HIF-1α would play a critical role in the induction of CAFs via TGF-β in hypoxic TME. Inhibition of HIF-1α could have contributed to reduction of CAFs in tumor tissues, leading to suppression of tumor growth. These results provide a novel rationale with TME-targeted strategy against cancer. Citation Format: Koji Teramoto, Yoko Kataoka, Tomoyuki Igarashi, Yasuhiko Ohshio, Jun Hanaoka, Yataro Daigo. HIF-1 is responsible for the induction of cancer-associated fibroblasts in hypoxic tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5080. doi:10.1158/1538-7445.AM2015-5080

Superior Sulcus Tumor Resection With Multiple Pulmonary Arteriovenous Fistulas

The authors present a case of a 66-year-old male presenting with a superior sulcus tumor and severe hypoxemia due to bilateral multiple pulmonary arteriovenous fistulas. The unilateral pulmonary arterial occlusion test was useful before surgery because it enabled evaluation of the feasibility and safety of intraoperative pulmonary artery clamp and one-lung ventilation during lung resection. Results facilitated safe resection of the superior sulcus tumor using the modified transmanubrial osteomuscular sparing approach, providing an excellent surgical field.

Abstract 1173: HIF-1 promotes the infiltration of regulatory T cells into the tumor via TGF-β

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In the growing tumor under hypoxia, a transcription factor, hypoxia-inducible factor-1 (HIF-1) plays a critical role in regulating expressions of various genes associated with tumor growth. In order to elucidate the immunosuppression promoted by the hypoxic tumor microenvironment, we focused on a relationship between HIF-1 and an immunosuppressive cytokine, transforming growth factor-beta (TGF-β). TGF-β which is released from either tumor cells or tumor stromal cells and suppresses anti-tumor immune responses in tumor-bearing hosts may be regulated by HIF-1 in the hypoxic tumor microenvironment. In this report, we hypothesized that inhibition of HIF-1 in tumor cells would improve an immunosuppression to augment anti-tumor immune responses, and examined the possibilities in the tumor-bearing mice model. The transfected mouse lymphoma cells E.G7 with HIF-1 shRNA was administered to C57BL/6 mice subcutaneously at the flank. Three weeks later, anti-tumor immune responses elicited in the mice were examined. As results, in mice bearing the transfected E.G7 with HIF-1 shRNA, the protein level of TGF-β in the tumor tissue was decreased as compared with that in mice bearing the transfected E.G7 with control shRNA or the non-transfected E.G7. The infiltration of Foxp3+ cells in the tumor tissue was decreased in mice bearing the transfected E.G7 with HIF-1 shRNA. When spleen cells from those tumor-bearing mice were stimulated with E.G7-derived antigen, cytotoxic activity against target E.G7 cells were enhanced in mice bearing the transfected E.G7 with HIF-1 shRNA. While there were no significant differences in the cell proliferation between the transfected E.G7 cells with HIF-1 shRNA and those with control shRNA or non-transfected E.G7 cells in vitro, tumor growth was effectively suppressed in mice bearing the transfected E.G7 with HIF-1 shRNA. Inhibition of HIF-1 in tumor cells decreased the level of TGF-β in the hypoxic tumor microenvironment, leading to suppression of infiltrating Foxp3+ cells and improvement of anti-tumor immune responses. These results demonstrate that hypoxia in the tumor microenvironment is responsible for the induction of immunosuppression through the infiltration of regulatory T cells by TGF-β. Citation Format: Koji Teramoto, Yasuhiko Ohshio, Jun Hanaoka, Atsushi Takano, Yataro Daigo. HIF-1 promotes the infiltration of regulatory T cells into the tumor via TGF-β. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1173. doi:10.1158/1538-7445.AM2014-1173

Abstract 4945: The cancer-associated fibroblasts-targeted strategycan augment the potency of the dendritic cell-based vaccine immunotherapy.

The dendritic cell (DC) -based vaccine immunotherapy has been a promising cancer immunotherapy, but has been insufficient to eradicate the tumor in patients with advanced cancer. This can result from the complicated tumor microenvironment (TME) that is implicated in suppression of anti-tumor immune responses. Several immune cell types in TME, such as Tregs, myeloid derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs), have been reported to regulate anti-tumor immune responses negatively. Cancer-associated fibroblasts (CAFs) are also primary stromal cells in TME, and contribute to tumor growth and metastases through the secretion of TGF-β and stromal cell-derived factor-1 (SDF-1). We considered that TME-targeted strategies should be innovated for the development of the potent cancer immunotherapy. On the basis of these viewpoints, we focused on the role of CAFs in TME, and hypothesized that inhibition of CAFs would lead to improvement of systemic anti-tumor immune responses and enhancement of the potency of the DCs-based vaccine immunotherapy. In this study, we applied tranilast in order to inhibit CAFs, the anti-fibrotic and -allergic agent that is used clinically and has been shown to inhibit fibroblast in the scar tissue. In in vitro studies, we examined effects of tranilast on CAFs that were isolated from established EG7 (mouse lymphoma cells) tumors. As results, tranilast was able to suppress the proliferation of CAFs, and decrease the production of SDF-1 as well as TGF-β from CAFs. Regarding the effect on Tregs, tranilast was able to decrease the induction of them from spleen cells of normal mice. Based on these results, we confirmed that tranilast could inhibit the function of CAFs. Next, we examined the association between inhibition of CAFs and anti-tumor immune responses in tumor-bearing mouse model. C57BL/6 mice bearing EG7 were administered tranilast into the established tumor in combination with tumor antigen-loaded DCs vaccination, and were evaluated anti-tumor immune responses. As results, the population of CAFs was decreased by targeting them, leading to lower expressions of TGF-β as well as SDF-1 in TME. Inhibition of CAFs in TME resulted in the decreased distributions of Tregs in TME and tumor-draining lymph nodes (TDLs). On the induction of effector cells, antigen-specific CD8+ cells producing IFN-γ were significantly increased in TDLs and spleen through inhibition of CAFs in TME. In these mice, systemic antigen-specific cytotoxic responses were augmented, leading to suppression of tumor growth as compared with mice in control groups. These results demonstrate that CAFs are associated with immune suppression, and inhibition of CAFs functions in TME can augment systemic anti-tumor immune responses. Our mouse models provide a new rationale with TME-targeted strategies for enhancing the potency of the DCs-based vaccine immunotherapy. Citation Format: Yasuhiko Ohshio, Ryosuke Kaku, Keiko Ishida, Masayuki Hashimoto, Shoji Kitamura, Koji Teramoto, Jun Hanaoka, Noriaki Tezuka. The cancer-associated fibroblasts-targeted strategycan augment the potency of the dendritic cell-based vaccine immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4945. doi:10.1158/1538-7445.AM2013-4945

Abstract 1253: Improvement of the potency of the dendritic cell-based vaccine immunotherapy through local inhibition of TGF-β in tumor-draining lymph nodes.

The dendritic cell (DC) -based vaccine immunotherapy has been a promising anti-cancer immunotherapy, but has been unable to elicit anti-tumor immune responses enough to eradicate the tumor in patients with advanced cancer. In order to develop the potent DC-based vaccine immunotherapy, it should be required to focus on the unique environment implicated in the induction of anti-tumor immune responses. Transforming growth factor-β (TGF-β), an immunosuppressive cytokine, is produced in the tumor microenvironment, and flows into tumor-draining lymph nodes (TDLs), primary priming sites for generation of anti-tumor immune responses. In TDLs, TGF-β plays a critical role in suppressing the induction of anti-tumor immune responses. In this study, we focused on TGF-β-mediated immunosuppression in TDLs, and examined whether local inhibition of TGF-β in TDLs could improve the potency of the DC-based vaccine immunotherapy in tumor-bearing mouse model. C57BL/6 mice bearing Lewis lung carcinoma (LLC1) subcutaneously were vaccinated with DCs loading LLC1-derived antigen. In order to inhibit TGF-β in TDLs, those mice were administered the plasmid DNA encoding the extracellular domain of TGF-β type II receptor fused to the human IgG heavy chain(TGFR DNA) near the established tumor intramuscularly. Soluble TGF-β type II receptor secreted from TGFR DNA-transfected cells is expected to function to attenuate TGF-β level in TDLs. After this combination therapy, anti-tumor immune responses elicited in those mice were evaluated. As results, the level of TGF-β in TDLs was decreased by administrations of TGFR DNA near the established tumor. The attenuation of TGF-β level in TDLs by TGFR DNA was able to suppress the proliferative activity of Foxp3 + regulatory T cells in TDLs. Regarding the induction of effector cells in TDLs, tumor antigen-specific CD8 + cells producing interferon-gamma were significantly increased by this combination therapy. Next, we evaluated the association between the local inhibition of TGF-β in TDLs and systemic anti-tumor immune responses. Tumor antigen-specific cytotoxic responses as well as natural killer activities were significantly enhanced through the local inhibition of TGF-β in TDLs, leading to effective suppression of tumor growth. Such potent anti-tumor immune responses were not achieved in mice that had been administered DC-based vaccine alone. Improvement of the induction of anti-tumor immune responses in TDLs by the local inhibition of TGF-β contributed to enhancement of systemic anti-tumor immune responses. In conclusion, inhibition of TGF-β-mediated immunosuppression in TDLs can enhance the potency of DC-based vaccine immunotherapy. This animal model provides for a novel rationale with DC-based vaccine immunotherapy. Citation Format: Koji Teramoto, Yasuhiko Ohshio, Takuya Fujita, Jun Hanaoka, Yataro Daigo. Improvement of the potency of the dendritic cell-based vaccine immunotherapy through local inhibition of TGF-β in tumor-draining lymph nodes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1253. doi:10.1158/1538-7445.AM2013-1253