Background: Anti-tumor effects of radiation therapy (RT) largely depend on host immune function. Adenosine with its strong immunosuppressive properties is an important immune checkpoint molecule. Method: We examined how intra-tumoral adenosine levels modify anti-tumor effects of RT in a murine model using an anti-CD73 antibody which blocks the rate-limiting enzyme to produce extracellular adenosine. We also evaluated CD73 expression in irradiated human rectal cancer tissue. Results: LuM-1, a highly metastatic murine colon cancer, expresses CD73 with significantly enhanced expression after RT. Subcutaneous (sc) transfer of LuM-1 in Balb/c mice developed macroscopic sc tumors and microscopic pulmonary metastases within 2 weeks. Adenosine levels in the sc tumor were increased after RT. Selective RT (4Gyx3) suppressed the growth of the irradiated sc tumor, but did not affect the growth of lung metastases which were shielded from RT. Intraperitoneal administration of anti-CD73 antibody (200 μg × 6) alone did not produce antitumor effects. However, when combined with RT in the same protocol, anti-CD73 antibody further delayed the growth of sc tumors and suppressed the development of lung metastases presumably through abscopal effects. Splenocytes derived from RT+ CD73 antibody treated mice showed enhanced IFN-γ production and cytotoxicity against LuM-1 compared to controls. Immunohistochemical studies of irradiated human rectal cancer showed that high expression of CD73 in remnant tumor cells and/or stroma is significantly associated with worse outcome. Conclusion: These results suggest that adenosine plays an important role in the anti-tumor effects mediated by RT and that CD73/adenosine axis blockade may enhance the anti-tumor effect of RT, and improve the outcomes of patients with locally advanced rectal cancer.

We report the case of a patient with Crohn's disease who initially presented with a ceco-urachal fistula. The patient was a 31-year-old female who underwent an appendectomy 6 years before presenting to our institution. She had a one-year history of diarrhea, and had recently developed polyuria and a sensation of residual urine. She was admitted with fever and lower abdominal pain. Endoscopy and computed tomography revealed a ceco-urachal fistula, which was consistent with Crohn's disease. An urachal resection was performed, which included partial cystectomy and ileocecal resection. A ceco-urachal fistula is a rare initial symptom of Crohn's disease. During the surgical management of such cases, it is necessary to resect the urachus, the affected portion of the bladder, the fistula, and the affected part of the digestive tract in order to avoid recurrence.

Although preoperative chemoradiation therapy can down-stage locally advanced rectal cancer (LARC), it has little effect on distant metastases. Metformin exerts an anti-cancer effect partly through the activation of host immunity. LuM1, a highly lung metastatic subclone of colon 26, was injected subcutaneously (sc) in BALB/c mice and treated with metformin and/or local radiation (RT). Lung metastases and the primary tumors were evaluated and the phenotypes of immune cells in the spleen and lung metastases were examined with flow cytometry and immunohistochemistry. Local RT, but not metformin, partially delayed the growth of sc tumor which was augmented with metformin. Lung metastases were unchanged in metformin or RT alone, but significantly reduced in the combined therapy. The ratios of splenic T cells tended to be low in the RT group, which were increased by the addition of metformin. IFN-γ production of the splenic CD4(+) and CD8(+) T cells was enhanced and CD49b (+) CD335(+) activated NK cells was increased after combined treatment group. Density of NK cells infiltrating in lung metastases was increased after combination treatment. Metformin effectively enhances local and abscopal effects of RT though the activation of cell-mediated immunity and might be clinically useful for LARC.

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Background: Tumor-associated macrophages (TAMs) play pivotal roles to regulate tumor cell behavior in tumor microenvironment and a high density of TAMs is associated with poor prognosis in various cancers. Recent studies have demonstrated that metformin, the first-line medication for the treatment of type 2 diabetes, have suppressive effects on tumor growth. Here, in this study, we investigated the effect of metformin on monocyte differentiation to TAMs. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and CD14+ peripheral blood monocytes(PBMo) were purified by positive selection using the magnetic cell separation system. The PBMo were co-cultured with Panc-1, a human pancreas cancer cell, with metformin (10μM~10mM) using double chamber inserts of 0.4-μm sized pores at 37°C in 5% CO2 conditions for 5 days. Separately, PBMo were cultured with M-CSF for 5 days and then with IL-10 (20ng/ml)+TGF-β(20ng/ml) for addition 2 days with or without metformin. The differentiation to TAM were evaluated by the expression of M1/M2 markers as well as PD-L1/L2 by flowcytometry. The macrophages were co-cultured with autologous T cells stimulated with anti-CD3 and anti-CD3 and anti-CD28 microbeads and the effect on T cell proliferation was evaluated with CFSE dilution assay. Results: PBMo co-cultured with Panc-1 showed significantly enhanced expression of CD163, CD206, as well as markedly increased side scatter, suggesting the differentiation to M2-type macrophages. The macrophage also highly expressed PD-L1 and strongly inhibited the proliferation of autologous T cells. However, the expression of these antigens and inhibition of T cell growth were significantly suppressed by the presence of metformin with dose dependent manner. Metformin was effective at 100μM and almost nullified the effects of Panc-1 at 1mM. Metformin did not affect the antigen expression pattern during the treatment by M-CSF, but did downregulate the expression of CD206 and PD-L1 if added together with IL-10 +TGF-β. Conclusion: During the co-culture with tumor cells, metformin suppresses the monocyte differentiation to TAM at relatively late stage. Metformin can restore T cell mediated-tumor immunity in tumor microenvironment, at least partly, through the effects on TAM, that may result in tumor inhibition. Citation Format: Akira Saito, Hideyuki Ohzawa, Mineyuki Tojo, Yuko Kumagai, Rihito Kanamaru, Hidenori Tsukui, Satomi Shiba, Homare Ito, Naohiro Sata, Joji Kitayama. Metformin inhibits monocyte differentiation to tumor associated macrophage (TAM) and may restore anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 516.