大澤 英之

PURPOSE: Although neutrophil extracellular traps (NETs) are present in various tumors, their roles in tumor biology have not been clarified yet. In this study, we examined how NETs affect the pharmacokinetics and effects of doxorubicin (DOX). METHODS: NETs were generated by neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). DOX was added to NETs and their distribution was observed under fluorescein microscopy, and the diffusion of DOX through 3 μM pores from lower to upper chambers was evaluated with a fluorescence-based assay. Ovarian cancer cells, KOC-2S and SKOV3, were embedded in collagen gel droplets and cultured in 3D way and their apoptosis was examined with flow cytometry. RESULTS: DOX was mostly co-localized with NETs. The transfer of DOX to upper chambers increased over time, which was significantly decreased by the presence of neutrophils stimulated with PMA or LPS in the lower chamber. DOX outside of the gel increased the rates of annexin V (+) apoptotic cells, which were significantly reduced by the addition of LPS-stimulated neutrophils in media both in KOC-2S and SKOV3. The reduced diffusion and apoptosis were mostly restored by the destruction of the NETs structure with 1000 u/ml DNAse I. CONCLUSION: NETs efficiently trap and inhibit the diffusion of DOX which may attenuate its ability to induce apoptosis of ovarian cancer cells. Degradation of NETs with DNAse I may augment the response of ovarian cancer to DOX.

Abstract 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.

Peritoneal dissemination is a major metastatic pathway for gastrointestinal and ovarian malignancies. The miR-29b family is downregulated in peritoneal fluids in patients with peritoneal metastases (PM). We examined the effect of miR-29b on mesothelial cells (MC) which play critical a role in the development of PM through mesothelial-mesenchymal transition (MMT). Human peritoneal mesothelial cells (HPMCs) were isolated from surgically resected omental tissue and MMT induced by stimulation with 10 ng/ml TGF-β1. MiR-29b mimics and negative control miR were transfected by lipofection using RNAiMAX and the effects on the MMT evaluated in vitro. HPMC produced substantial amounts of miR-29b which was markedly inhibited by TGF-β1. TGF-β1 stimulation of HPMC induced morphological changes with decreased expression of E-cadherin and calretinin, and increased expression of vimentin and fibronectin. TGF-β1 also enhanced proliferation and migration of HPMC as well as adhesion of tumor cells in a fibronectin dependent manner. However, all events were strongly abrogated by simultaneous transfection of miR-29b. MiR-29b inhibits TGF-β1 induced MMT and replacement of miR-29b in the peritoneal cavity might be effective to prevent development of PM partly through the effects on MC.

Background: The peritoneal cavity contains many site-specific immune cells which constitute a unique immune microenvironment. However, it is unclear how the local immune signature is altered in patients with peritoneal metastases (PM). Methods: Peritoneal lavage fluid or ascites were obtained from 122 patients with various stages of gastric cancer (GC). Cells recovered from peritoneal fluids were immunostained with mAbs for lymphocyte-, macrophage- and tumor cell-specific antigens and the frequencies of leukocyte subsets and antigen expression levels were evaluated with multi-color flowcytometry. Results: The proportions of CD8(+) T cells, CD3(+)CD56(+) NKT-like cells, and CD3(-)CD56(+) NK cells to CD45(+) leukocytes were significantly reduced in patients with PM compared to those without PM. In patients with PM, the rates of CD8 (+) T cells and NKT-like cells correlated inversely with the tumor leukocyte ratio (TLR), the relative frequency of CD326(+) tumor cells to CD45(+) leukocytes. In contrast, the proportion of CD19(+) B cells was significantly increased in patients with PM, and their proportion correlated positively with the TLR and peritoneal carcinomatosis index (PCI) score. In patients with PM, CD14(+) macrophages tended to be increased with enhanced expression of CD14, CD16 and a M2-macrophage marker, CD163. In particular, macrophages in patients with high TLR contained many granules with high side scatter and CD14 expression in their flow profile compared to those without PM. Conclusion: PM are accompanied by a drastic change in phenotypes of lymphocyte and macrophage in the peritoneal cavity, which might be involved in the development and progression of intraperitoneal tumor growth.

Introduction Although new techniques and devices have been introduced, the incidence of postoperative pancreatic fistula (POPF) after distal pancreatectomy remains high. To reduce the risk of POPF, we developed an innovative ligation band and conducted this pilot study to assess the possibility of reducing the incidence of POPF and pancreatic necrosis after distal pancreatectomy. Methods Distal pancreatectomy was performed in three pigs. In two animals, ligation of the pancreas was performed while maintaining arterial blood flow to the stump, and in one animal, the arterial blood flow was occluded. After ligation, the pancreas was sharply divided. Animals were sacrificed seven days later, and the remnant pancreas was assessed histologically. POPF was defined as amylase in ascites > 3x the preoperative serum amylase level. The following equation was used to quantify the extent of necrotic tissue: necrotic tissue residual rate = necrotic tissue area/ cross-sectional area. Results All animals survived, and no POPF developed. For two animals in which arterial blood flow to the stump was maintained, necrotic tissue residual rates at the ligation line were 24% and 31%. At the pancreatic stump, necrotic tissue residual rates were 37% and 50%. In the animal in which arterial blood flow to the stump was occluded, the necrotic tissue residual rate at the ligation line was 83% and that at the pancreatic stump was 78%, both higher than that in animals in which arterial blood flow was maintained. In all animals, there was no injury to pancreatic tissue at the ligation line. Conclusion The pancreas ligation band can potentially prevent POPF after distal pancreatectomy by atraumatic ligation, and the band ligates the pancreatic stump while maintaining arterial blood flow and limiting pancreatic necrosis.

Aim: PD-1/PD-L1 blockade therapy is now widely used for the treatment of advanced malignancies. Although PD-L1 is known to be expressed by various host cells as well as tumor cells, the role of PD-L1 on non-malignant cells and its clinical significance is unknown. We evaluated cell type-specific expression of PD-L1 in colorectal cancer (CRC) specimens using multicolor flow cytometry. Methods: Single cell suspensions were made from 21 surgically resected CRC specimens, and immunostained with various mAbs conjugated with different fluorescent dyes. Tumor cells, stromal cells, and immune cells were identified as CD326(+), CD90(+) and CD45(+) phenotype, respectively. CD11b(+) myeloid cells, CD19(+) B cells and CD4(+) or CD8(+) T cells were also stained in different samples, and their frequencies in the total cell population and the ratio of PD-L1(+) cells to each phenotype were determined. Results: PD-L1 was expressed by all the cell types. The ratio of PD-L1(+) cells to CD326(+) tumor cells was 19.1% ± 14.0%, lower than those for CD90(+) stromal cells (39.6% ± 16.0%) and CD11b(+) myeloid cells (31.9% ± 14.3%). The ratio of PD-L1(+) cells in tumor cells correlated strongly with the ratio in stromal cells, while only weakly with that in myeloid cells. Tumor cells were divided into two populations by CD326 expression levels, and the PD-L1 positive ratios were inversely correlated with the rate of CD326 highly expressing cells as well as mean fluorescein intensity of CD326 in tumor cells, while positively correlated with the frequencies of stromal cells or myeloid cells in CRC. Conclusion: PD-L1 is differentially expressed on various cell types in CRC. PD-L1 on tumor cells may be upregulated together with CD326 downregulation in the process of epithelial mesenchymal transition. Quantification of cell type-specific expression of PD-L1 using multicolor flow cytometry may provide useful information for the immunotherapy of solid tumors.

<title>Abstract</title><sec> <title>Background</title> Intraperitoneal (IP) administration of paclitaxel (PTX) has a great pharmacokinetic advantage to control peritoneal lesions and can be combined with various systemic chemotherapies. In this study, we evaluate the efficacy and tolerability of a combination of IP-PTX and systemic S-1/oxaliplatin (SOX) for induction chemotherapy for patients with peritoneal metastases (PM) from gastric cancer (GC). </sec><sec> <title>Patients and Methods</title> Patients with GC who were diagnosed as macroscopic PM (P1) or positive peritoneal cytology (CY1) by staging laparoscopy between 2016 and 2019 were enrolled. PTX was IP administered at 40 mg/m² on days 1 and 8. Oxaliplatin was IV administered at 100 mg/m² on day 1, and S-1 was administered at 80 mg/m²/day for 14 consecutive days, repeated every 21 days. Survival time and toxicities were retrospectively explored. </sec><sec> <title>Results</title> Forty-four patients received SOX + IP-PTX with a median (range) of 16 (1–48) courses, although oxaliplatin was suspended due to the hematotoxicity or intolerable peripheral neuropathy in many patients. The 1-year overall survival (OS) rate was 79.5% (95% CI 64.4–88.8%) with median survival time of 25.8 months. Gastrectomy was performed in 20 (45%) patients who showed macroscopic shrinkage of PM with a 1-year OS rate of 100% (95% CI 69.5–100%). Grade 2 and 3 histological responses was achieved in four (20%) and one (5%) patients. Grade 3/4 toxicities included neutropenia (11%), leukopenia (39%), and anemia (14%). There were no treatment-related deaths. </sec><sec> <title>Conclusions</title> Combination chemotherapy using SOX + IP-PTX regimen is highly effective and recommended as induction chemotherapy for patients with PM from GC. </sec>