大澤 英之

BACKGROUND: Repeat intraperitoneal (IP) chemotherapy has been successfully used for treatment of peritoneal metastases (PM) from gastric cancer (GC). Exosomes play important roles not only in tumor progression but also in chemoresistance via transfer of microRNAs (miRNAs). However, there is little evidence of an effect of miRNAs in peritoneal exosomes on chemosensitivity of peritoneal lesions. METHODS: In 74 patients with advanced GC who underwent staging laparoscopy, exosomes were isolated from peritoneal fluid and expression levels of miR-21-5p, miR-223-3p, and miR-29b-3p determined using TaqMan Advanced miRNA assays. In 43 patients with PM treated with combination chemotherapy, S-1 plus Oxaliplatin together with IP Paclitaxel, the relationship between their relative expression levels and outcomes was examined. RESULTS: The ratios of miR-21-5p/miR-29b-3p and miR-223-3p/miR-29b-3p were significantly upregulated in patients with PM, especially in patients with high serum CA125 levels. They showed a mild association with Peritoneal Cancer Index (PCI) score and ascites. More impressively, the ratios were significantly higher in 16 patients with progression of PM within 1 year compared with 27 patients with an excellent tumor response (miR-21-5p/miR-29b-3p: median 17.49, range 1.83-50.90 vs. median 4.64, range 0.40-38.96, p = 0.0015, miR-223-3p/miR-29b-3p: median 1.02, range 0.23-25.85 vs. median 0.21, range 0.01-50.07, p = 0.0006). Overall survival of patients with high miR-21/miR-29b or miR-223/miR-29b ratios was significantly worse than in patients with low ratios (p = 0.0117, p = 0.0021). CONCLUSIONS: The ratios of miRNAs in peritoneal exosome correlate with survival of the patients with PM from GC and suggest the possibility that they modify the chemosensitivity against IP chemotherapy.

Accumulating evidence suggests that metformin reduces the incidence and mortality of colorectal cancer (CRC). However, underlying mechanisms have not been fully clarified. The aim of this study was to examine the pathological characteristics of resected CRC from patients treated with metformin for type 2 diabetes mellitus (DM). In total, 267 patients with DM underwent curative colectomy for Stage I-III CRC and 53 (19.9%) patients had been treated medically including metformin. Pathological N-stage was significantly lower in metformin-treated patients (P < .05) with prolonged disease-free survival (DFS) (P < .05). Immunohistochemistry showed that the densities of CD3(+) and CD8(+) tumor-infiltrating lymphocytes (TILs) in the invasive front area were significantly higher in 40 patients treated with metformin compared with propensity score matched cases without metformin (P < .05). The density of tertiary lymphoid structures (TLS) in tumor stroma was markedly increased in metformin-treated patients (P < .001). In those tumors, there were more CD68(+) tumor-associated macrophages (TAM) infiltrated (P < .05), while the ratio of CD163(+) M2-phenotype was markedly reduced (P < .001). Stromal fibrosis tended to be suppressed by metformin intake (P = .051). These findings suggested that metformin drastically changes the characteristics of infiltrating immune cells in CRC and reprograms the tumor microenvironment from immunosuppressive to immunocompetent status, which may lead to suppression of microscopic tumor spread and improve the outcomes of patients with CRC and type 2 DM.

＜文献概要＞はじめに 腹膜播種は消化器がん,特に胃がんや膵がんにおいて最も頻度の高い転移・再発形式で,最大の予後規定因子である。近年のがん薬物療法の進歩によって,根治切除不能・再発がん患者の治療成績は著しく向上した。その一方で,腹膜播種に関してはいまだに顕著な予後改善はみられておらず,実臨床での大きな課題となっている。播種治療を困難にする要因の1つとして,治療効果を反映する適切なバイオマーカーがないことが挙げられる。一般に,個々の播種病変のサイズは数mm程度と小さく,CTスキャンなどの画像診断では検出されないことから,他の固形がんとは異なりRECIST(Response Evaluation Criteria in Solid Tumors)による治療効果の判定は困難である。腹膜播種の状況を最も正確に評価できるのは全身麻酔下での審査腹腔鏡であるが,侵襲を伴う手技であり頻回に行うことはできない。したがって,自覚症状や末梢血中腫瘍マーカーの値などに頼らざるを得ないが,これらの情報は必ずしも腹膜病変を正確に反映するわけではない。エクソソームは細胞が放出する30〜150nmの細胞外&#12073;胞で,さまざまな生理活性を有する蛋白質やDNA,マイクロRNA(miRNA)などの核酸成分を含有し,細胞間コミュニケーションに重要な役割を果たしている。近年の研究で,がん患者の血中エクソソームはがん特異的な機能分子を多量に含有しており,標的臓器の微&#12073;環境をがん細胞の生育に適したものに変換することによって,遠隔転移の成立を促進している事実が報告されてきている。腹膜播種をきたした患者では,原発巣から腹膜表面に播種したがん細胞が腹腔というフリースペースに直接接している。末梢血中の循環がん細胞が血液中の白血球106〜108個に対して数個のレベルで検証されているのに対し,播種患者の腹腔内液中の浮遊がん細胞は少なくとも104個の白血球に対して数個の割合で存在する。したがって,腹膜播種患者の腹腔内液は,血中に比べて圧倒的に多量のがん細胞特異的蛋白質やmiRNAを含んだエクソソームが放出されていることが予想される。体液中のエクソソームを対象とした研究では血液を使用したものが多いが,最近は腹水中のエクソソームを使用した報告も増えつつある。漿膜浸潤胃がんの腹腔内洗浄液中のエクソソームでmiR-21とmiR-1225-5pの発現が高いことや,卵巣がんの腹水中に含まれるエクソソーム中のMMP1のmRNAが腹膜中皮に作用して腹膜播種を引き起こすことが報告されている。われわれの研究室では,腹腔内液中に含まれるエクソソームに内包されるmiRNAに着目して,腹膜播種の成立や進展に関わる機序を明らかにし,腹膜播種患者の診療に応用できるバイオマーカーを開発することを目指している。

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.

The peritoneal surface is the most frequent site of metastasis disease in patients with gastric cancer. Even after curative surgery and adjuvant chemotherapy, peritoneal recurrences often develop. Exosomes play pivotal roles in tumor metastasis via the transfer of microRNAs (miRNAs). In the present study, exosomes were isolated from peritoneal lavage fluid or ascites in 85 patients with gastric cancer and the relative expression levels of miR‑29s were examined. The expression of miR‑29a‑3p, miR‑29b‑3p and miR‑29c‑3p in peritoneal exosomes were all downregulated in patients with peritoneal metastases (PM) compared to those without PM. In 30 patients who underwent curative gastrectomy with serosa‑involved (T4) gastric cancer, 6 patients exhibited recurrence in the peritoneum within 12 months. The expression levels of miR‑29s at gastrectomy tended to be lower in these 6 patients than in the other 24 patients with significant differences in miR‑29b‑3p (P=0.003). When the patients were divided into two groups based on median levels of miR‑29s, peritoneal recurrence developed more frequently in patients with low expression of miR‑29b‑3p, and lower expression of miR‑29s were related with worse overall survival. miR‑29s are thought to play a suppressive role in the growth of disseminated peritoneal tumor cells. Reduced expression of miR‑29b in peritoneal exosomes is a strong risk factor of developing postoperative peritoneal recurrence.

Aim: Peritoneal metastases (PM) frequently occur in patients with gastric cancer and result in a poor prognosis. Exosomes play pivotal roles in tumor metastasis through the transfer of microRNAs (miRNAs). We examined the exosomal miRNA profile in peritoneal fluids to identify novel biomarkers to reflect tumor burden in the peritoneum. Methods: Exosomes were isolated from peritoneal fluids of patients of gastric cancer with macroscopic (P1) or microscopic (P0CY1) peritoneal metastasis (PM) and comprehensive miRNA expression analysis was carried out. Expressions of candidate miRNAs were then validated in all 58 samples using TaqMan Advanced miRNA Assays. Results: In initial screening, we carried out comprehensive analysis of exosomal miRNA using peritoneal fluids from 11 and 14 patients with or without PM, respectively, and identified 11 dysregulated miRNAs in PM (+) samples. Validation analysis showed that four miRNAs (miR-21-5p, miR-92a-3p, miR-223-3p, and miR-342-3p) were significantly upregulated in 12 PM (+) samples, and their expression levels showed positive correlation with peritoneal cancer index. In contrast, miR-29 family were all downregulated in patients with PM (+) samples. Moreover, in 24 patients with pT4 tumor, miR-29 at gastrectomy tended to be lower in six patients with peritoneal recurrence with significant differences in miR-29b-3p (P = .012). Conclusion: Expression pattern of miRNAs in peritoneal exosomes well reflects the tumor burden in the peritoneal cavity and could be a useful biomarker in the treatment of PM.

Many types of immune cells appear in peritoneal cavity after abdominal surgery. In patients who underwent laparotomy due to gastric cancer, peritoneal lavages were obtained before and after surgical procedure. Cells were recovered from intermediate layer after Ficoll-Hypaque centrifugation and analyzed for phenotypes and functions, especially focused on low density neutrophils (LDN). The number of CD66b (+) LDN with mature phenotype was markedly elevated in postoperative as compared with preoperative lavages. Short term culture of the purified LDN produced many threadlike structures positive for SYTOX, nucleic acid staining, as well as histone and myeloperoxidase, suggesting the NETs formation. Human gastric cancer cells, MKN45, OCUM-1 and NUGC-4, were selectively attached on the NETs, which was totally abolished by the pretreatment of DNAse I. Intraperitoneal (IP) co-transfer of the LDN with MKN45 in nude mice strongly augments the metastasis formation on peritoneum, which was strongly suppressed by the following IP administration of DNAse I. Many NETs-like structures were detected on the surface of human omental tissue resected by gastrectomy. NETs on peritoneal surface can assist the clustering and growth of free tumor cells disseminated in abdomen. Disruption of the NETs by DNAse might be useful to prevent the peritoneal recurrence after abdominal surgery.