Hidemitsu Sugai

CCL17 and CCL22 chemokines within tumor microenvironment are related to accumulation of Foxp3+ regulatory T cells in gastric cancer

It has been reported that an increased population of regulatory T cells (Tregs) is one of the reasons for impaired anti‐tumor immunity. Recently, Foxp3 has been reported as a reliable marker of Tregs. The authors investigated the frequency of Foxp3+ Tregs within CD4+ cells in TILs, regional lymph nodes and PBLs of gastric cancer patients (n = 45). Furthermore, to elucidate the mechanisms behind Treg accumulation within tumors, they evaluated the relationship between CCL17 or CCL22 expression and the frequency of Foxp3+ Tregs in gastric cancer. CD4+CD25+Foxp3+ Tregs as a percentage of CD4+ cells were counted by flow cytometry and evaluated by immunohistochemistry. Moreover, an in vitro migration assay using Tregs derived from gastric cancers was performed in the presence of CCL17 or CCL22. As a result, the frequency of Foxp3+ Tregs in TILs was significantly higher than that in normal gastric mucosa (12.4% ± 7.5% vs. 4.1% ± 5.3%, p < 0.01). Importantly, the increase in Tregs in TILs occurred to the same extent in early and advanced disease. Furthermore, the frequency of CCL17+ or CCL22+ cells among CD14+ cells within tumors was significantly higher than that of normal gastric mucosa, and there was a significant correlation between the frequency of CCL17+ or CCL22+ cells and Foxp3+ Tregs in TILs. In addition, the in vitro migration assay indicated that Tregs were significantly induced to migrate by CCL17 or CCL22. In conclusion, CCL17 and CCL22 within the tumor are related to the increased population of Foxp3+ Tregs, with such an observation occurring in early gastric cancer.

Vascular endothelial growth factor inhibits maturation of dendritic cells induced by lipopolysaccharide, but not by proinflammatory cytokines

Purpose: Dendritic cells (DCs) play an important role in the host’s immunosurveillance against cancer. It has been shown that the function of DCs is impaired and their population decreased in a cancer-bearing host. In the present study, we investigated the mechanism of down-regulation of DCs in a cancer-bearing host. Methods: We evaluated the relationship between DC infiltration and production of vascular endothelial growth factor (VEGF) in carcinoma tissue by immunohistochemistry. Furthermore, functional and phenotypical alterations of DCs were evaluated when monocyte-derived, mature DCs were treated with VEGF in vitro. Monocyte-derived DCs were generated in a culture of monocyte with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor, and the maturation of DCs was induced by either lipopolysaccharide (LPS) or a proinflammatory cytokine cocktail: tumor-necrosis factor α, prostaglandin E2, IL-6, and IL-1β. Results: A significant inverse correlation was found between the density of DCs and the quantity of VEGF production in gastric carcinoma tissue (r=−0.39, p<0.05). In LPS-induced maturation, the ability of mature DCs to stimulate allogenic T cells and produce IL-12 (p70 heterodimer) was suppressed by the addition of VEGF in a dose-dependent manner. A lesser expression of costimulatory molecules (CD80 and CD86) was seen in DCs treated with exogenous VEGF than in DCs not treated with VEGF. The population of dead DCs (early and late apoptosis) treated with VEGF increased more than that without VEGF treatment, using the annexin V and propidium iodide evaluation in DCs matured by LPS. In contrast, in DCs matured by the proinflammatory cytokine cocktail, the down-regulation of costimulatory molecules and induction of DC apoptosis was not seen. Conclusions: These findings show that the inhibition of DC maturation by VEGF differs depending on the maturation status of the DCs.

Improved quality of life with jejunal pouch reconstruction after total gastrectomy

BACKGROUND There is increasing evidence that the effect of jejunal pouch reconstruction is satisfactory for reservoir function in several randomized control studies. However, these studies were performed in patients with advanced gastric cancer, where significant numbers of the patients died of disease recurrence. In order to exclude the influence of disease recurrence, we performed jejunal pouch reconstruction after total gastrectomy in patients with early gastric cancer in a randomized controlled study and investigated whether or not an improved quality of life (QOL) was observed with jejunal pouch reconstruction. METHODS Fifty consecutive patients receiving total gastrectomy for early gastric cancer were prospectively divided into the Roux-en-Y reconstruction group without pouch (RY group) or the jejunal pouch reconstruction group (pouch group). Body weight, eating capacity, QOL assessment by gastrointestinal symptom rating scale (GSRS), nutritional parameters, endoscopical examination, 24-hour pH monitoring and Bilitec monitoring were evaluated at 3, 12, and 48 months after surgery. RESULTS Jejunal pouch reconstruction provided the better QOL than Roux-en-Y reconstruction without pouch both at short-term and long-term periods in a randomized control study. Moreover, as a new finding, pouch reconstruction provided less bile reflux into the esophagus compared with Roux-en-Y reconstruction. CONCLUSIONS Jejunal pouch reconstruction provided improvement of QOL in patients receiving total gastrectomy.

Evaluation of VEGF and VEGF-C expression in gastric cancer cells producing α-fetoprotein

Background. Gastric cancers producing α-fetoprotein (AFP) are known to have a poor prognosis and to show a high incidence of liver metastasis. Vascular endothelial growth factor (VEGF) and its isoform VEGF-C are reported to be associated with tumor progression through an angiogenic or lymphangiogenic function. In the present study, to clarify the cellular characteristics of AFP-producing gastric cancers, the expression of VEGF and that of VEGF-C in AFP-producing gastric cancer was compared with their expression in gastric cancers that do not produce AFP. Methods. Twenty-six patients with AFP-producing gastric cancers [AFP(+)] and 26 patients with stage-matched gastric cancers that did not produce AFP [AFP(−)] were evaluated for VEGF and VEGF-C expression and vessel density, using immunohistochemical analysis. Results. The survival rate of the AFP(+) group was significantly worse than that of the stage-matched AFP(−) group (P < 0.05). The frequency of VEGF-C expression was significantly higher in the AFP(+) group than in the AFP(−) group (P < 0.01). There was no significant difference in VEGF expression between the AFP(+) and AFP(+) groups. The microvessel density was higher in the AFP(+) group than in the AFP(−) group (P < 0.05). Conclusions. A higher expression of VEGF-C might be one explanation for the poorer prognosis of AFP-producing gastric cancers.

Frequencies of HER-2/neu overexpression relating to HLA haplotype in patients with gastric cancer.

We have identified that HER‐2/neu‐derived peptides are naturally processed as tumor rejection antigens recognized by tumor‐specific, HLA‐A2‐restricted cytotoxic T lymphocytes in gastric cancer. To evaluate candidates for immunotherapy using HER‐2/neu‐derived, HLA‐A2‐restricted peptides, we examined the frequency of HLA‐A2 relating to HER‐2/neu overexpression or the infiltrating grade of tumor‐infiltrating lymphocytes (TILs) in Japanese patients with gastric cancer. HER‐2/neu‐overexpressing tumors detected by immunohistochemistry amounted to 19% of primary gastric cancers and HLA‐A2‐positive patients with gastric cancer were 31% of primary gastric‐cancer cases. Finally, gastric‐cancer patients with both HLA‐A2‐positive and HER‐2/neu‐overexpressing tumors amounted to 6.6% of these cases. There was no significant difference in the infiltrating grade of TILs between gastric cancers overexpressing HER‐2/neu and those that did not. The candidate for HER‐2/neu‐based immunotherapy with HLA‐A2‐restricted peptides represent a very limited population of Japanese patients.

Macrophages in tumor‐draining lymph node with different characteristics induce T‐cell apoptosis in patients with advanced stage‐gastric cancer

A hosts immune‐defense system is suppressive by many factors in patients with cancer. We have previously shown one possible mechanism behind the T‐cell dysfunction, whereby H2O2 secreted from macrophages in tumor‐draining lymph node (MTDL) induced T‐cell dysfunction with down‐regulation of TCR ζ molecules. In the present study, we analyzed how MTDL affect T cells, with a particular focus on T‐cell apoptosis, by co‐culturing MTDL with autologous peripheral blood T cells in gastric cancer. Moreover, we characterized the MTDL according to surface marker, oxygen‐burst capacity and intracellular cytokine status. T‐cell apoptosis was significantly induced in comparison to T‐cell alone control in patients with advanced disease, concomitant to the elevated caspase activity and following impaired T‐cell function. In patients with early disease, no significant difference was seen in the proportions of T cells that underwent apoptosis between T cells plus MTDL and T cells alone. Moreover, the addition of a selective scavenger of H2O2, catalase inhibited the apoptosis of T cells co‐cultured with MTDL in patients with advanced disease. In the characterization of MTDL, the production of H2O2 in MTDL from advanced disease was significantly higher than that in early disease. The amounts of intracellular IL‐10 and IL‐12 in MTDL in advanced disease were significantly higher than those in early disease. These results indicated that MTDL induced apoptosis of autologous T cells and this T‐cell dysfunction was mediated by H2O2 derived from MTDL. Furthermore, the characteristics of MTDL including the capacity of oxygen‐burst and intracellular cytokine production were different depending on the disease progression.

A Newly Identified MAGE-3-Derived, HLA-A24-Restricted Peptide Is Naturally Processed and Presented as a CTL Epitope on MAGE-3-Expressing Gastrointestinal Cancer Cells

Purpose: In order to broaden the possibility for anti-MAGE-3 immune targeting, it is important to identify HLA-A24-restricted epitopes derived from MAGE-3, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we defined a new MAGE-3 derived, HLA-A24-binding peptide presented as a CTL epitope on gastrointestinal cancer cells. Materials and Methods: A panel of MAGE-3-derived peptides (9mer and 10mer) with the HLA-A24-binding motif was selected, and identification of MAGE-3-derived, HLA-A24-restricted CTL epitopes was performed by a reverse immunology approach. To induce MAGE-3-peptide specific CTLs, PBMCs were repeatedly stimulated with monocyte-derived, mature DCs pulsed with the peptides. Subsequent peptide-induced T cells were tested for their specificities by ELISPOT, tetramer and cytotoxic assay. CTL clones were then obtained from the CTL line by limiting dilution. Results: The peptide-inducing CTLs revealed that MAGE-3(113)-peptide was reacted as a CTL epitope in a HLA-A24-restricted fashion, confirmed by ELISPOT and cytotoxic assays. In addition, the MAGE-3(113)-specific CTL clones, confirmed by tetramer assay, showed that the MAGE-3(113) epitope is naturally processed and presented as the CTL epitope on MAGE-3-expressing gastrointestinal cancer cells by evaluating the cold target inhibition assays. Conclusion: The newly identified MAGE-3(113)-peptide epitope is naturally processed and presented as the CTL epitope on MAGE-3-expressing gastrointestinal cancer cells, indicating that anti-MAGE-3 immune targeting with the MAGE-3(113) peptide is a promising approach for treatment.

Protein-Bound Polysaccharide K Partially Prevents Apoptosis of Circulating T Cells Induced by Anti-Cancer Drug S-1 in Patients with Gastric Cancer

Background and Methods:It has been shown that T-cell dysfunction, including apoptosis of peripheral blood T cells, commonly occurs in patients receiving chemotherapy. In order to evaluate whether concomitant administration of the oral biological response modifier protein-bound polysaccharide K (PSK) could induce anti-apoptotic effects in patients treated with the anti-cancer drug, S-1, peripheral blood T cells were analyzed for induction of apoptosis, caspase-3 activities and expression of proapoptotic protein Bax and anti-apoptotic protein Bcl-2 in patients with curatively resected stage III gastric cancer, who were randomly assigned to postoperative adjuvant therapy with S-1 alone (n = 10) or S-1 combined with PSK (n = 10). Results: T-cell apoptosis 5 weeks after adjuvant therapy was significantly higher in the S-1 group (24.1 ± 5.0%) than in the S-1 + PSK group (19.1 ± 3.9%). S-1 induced T-cell apoptosis and concomitantly elevated caspase-3 activities and Bax expression in peripheral blood T cells. In addition, PSK partially prevented the T-cell apoptosis induced by S-1. Conclusion:PSK could partially prevent the T-cell apoptosis induced by S-1.

Trypsin activity and bile acid concentrations in the esophagus after distal gastrectomy.

The pathogenesis of reflux esophagitis is not well understood and remains controversial. Distal gastrectomy serves as a model to assess the role of duodenal reflux with low gastric acidity in the development of reflux esophagitis. We investigated the relationship between the severity of esophagitis and gastroduodenal juice reflux, with particular focus on trypsin and bile acids after distal gastrectomy reconstructed with Billroth I anastomosis. Twenty-eight patients with gastroesophageal reflux disease after distal gastrectomy were enrolled. Esophageal and duodenal contents were aspirated under endoscopical examination, and their trypsin activity and bile acid concentrations were measured. The grade of reflux esophagitis was assessed by endoscopy and the symptoms were scored. Moreover, the grade of infiltration of inflammatory cells and the expression of COX-2 mRNA in the esophageal epithelium were evaluated. Patients with severe esophagitis had a higher amount of trypsin activity and bile acid concentrations in the esophagus, but not in the duodenum, compared to patients with mild esophagitis (P < 0.05). There was a strong positive correlation between the trypsin activity and the bile acid concentrations in the esophagus (r = 0.743, P = 0.0001). Moreover, the COX-2 mRNA expression and the grade of infiltrating inflammatory cells in the esophageal mucosa significantly correlated with the trypsin activity and bile acid concentrations in the esophagus. Thus, duodenogastroesophageal reflux with low gastric acidity is one of the pathogeneses in the development of reflux esophagitis from the present clinical study with patients after distal gastrectomy reconstructed with Billroth I anastomosis.

c-Met Expression in a Gastric Cancer Cell Line Producing α-Fetoprotein

PurposeWe previously reported a higher frequency of c-Met protein expression and high proliferative status in gastric cancers producing α-fetoprotein (AFP) than in those not producing AFP.MethodsTo investigate this further, we established an AFP-producing gastric cancer cell line, designated as AME-1, from the primary tumor of a patient with AFP-producing gastric cancer.Resultsα-Fetoprotein production in the AME-1 cell line was confirmed at the protein level by immunocytochemistry and at the mRNA level by reverse transcription–polymerase chain reaction (RT-PCR). A high amount of AFP was also detected in the supernatant of cultured AME-1. The AME-1 cell line expressed c-Met both at the mRNA and protein levels. A semiquantitative RT-PCR method indicated that AME-1 had a higher amount of c-Met mRNA than well-known gastric cancer cell lines (MKN-28, MKN-45) with strong c-Met expression. Hepatocyte growth factor (HGF), a ligand for the c-Met receptor, was not detected in the mRNA or protein of AME-1. The AME-1 cell line showed a proliferative response to exogenous HGF treatment in a dose-dependent manner, but the activity of migration was not stimulated by exogenous HGF.ConclusionsThe AME-1 cell line may be a useful model for elucidating aggressive behavior, especially related to regulation of the c-Met/HGF system, in AFP-producing gastric cancers.