Shinji Osada

Depletion of CD4+CD25+ regulatory T cells enhances interleukin-2-induced antitumor immunity in a mouse model of colon adenocarcinoma

Interleukin 2 (IL)‐2 induces antitumor immunity and clinical responses in melanoma and renal cell carcinoma. However, IL‐2 also increases the number of CD4+CD25+ regulatory T (Treg) cells that suppress antitumor immune responses. The aim of the present study was to elucidate the effect of depletion of Treg cells on IL‐2‐induced antitumor immunity. IL‐2‐transfected mouse colon adenocarcinoma (MC38/IL‐2) cells were implanted subcutaneously or intrahepatically into male C57BL/6 mice, and tumor growth and the proportion of tumor‐infiltrating lymphocytes with Treg‐cell depletion in response to treatment with anti‐CD25 monoclonal antibody (PC61) were determined. In mice treated with phosphate‐buffered saline, 40–60% of MC38/IL‐2 tumors were rejected. In contrast, all MC38/IL‐2 tumors were rejected in mice treated with PC61. The number of tumor‐infiltrating CD8+ T cells in mice treated with PC61 was approximately twice that in mice treated with PBS. The numbers of tumor‐infiltrating CD4+ and natural killer cells were also increased significantly. To test the antimetastatic effects of IL‐2 treatment in combination with Treg‐cell depletion, human recombinant IL‐2 (rIL‐2) and PC61 were administered to mice implanted with MC38/mock cells in the spleen, and hepatic metastasis was investigated. The average liver weight in mice treated with rIL‐2 plus PC61 was 1.04 ± 0.03 g, less than that in mice treated with rIL‐2 (2.04 ± 0.51 g) or PC61 alone (1.81 ± 0.38 g). We conclude that IL‐2‐induced antitumor immunity is enhanced by Treg‐cell depletion and is due to expansion of the tumor‐infiltrating cytotoxic CD8+ T‐cell population. (Cancer Sci 2007; 98: 416–423)

Hepatocyte growth factor (HGF) mediates the sustained formation of 1,2‐diacylglycerol via phosphatidylcholine—phospholipase C in cultured rat hepatocytes

The addition of hepatocyte growth factor (HGF) to rat hepatocytes in primary culture resulted in the formation of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) and 1,2‐diacylglycerol (DG) by a phosphoinositide‐specific phospholipase C (PI‐PLC). DG showed a biphasic increase; the first phase, corresponding with the peak of Ins(1,4,5)P3 and a second larger and prolonged phase. The HGF stimulates the phosphatidylcholine (PC)‐derived prolonged DG formation by a phospholipase C pathway (PC‐PLC) but not by a phospholipase D pathway. HGF also was found to elicit [Ca2+] oscillations which may be associated with the prolonged DG production from PC via the PC‐PLC phospholipase C pathway.

Intrahepatic cholangiocarcinoma arising 10 years after the excision of congenital extrahepatic biliary dilation

A 52-year-old woman was found to have a liver tumor during treatment for a liver abscess. The tumor was diagnosed as intrahepatic cholangiocarcinoma by closer examinations, including a percutaneous needle biopsy. Ten years previously, she had undergone excision of a choledochal cyst, with reconstruction by Roux-en-Y hepaticojejunostomy, as treatment for Todanis type Ia congenital biliary dilation, which had been confined only to the extrahepatic bile duct. The significant association between congenital biliary dilation and hepatobiliary malignancies is well known. Some patients have been reported to develop biliary cancer long after the excision of the entire extrahepatic bile duct and hepaticoenterostomy. However, in these patients, the development mostly took place in the remnant choledochal cyst, the anastomotic site, or in the dilated intrahepatic bile duct of Todanis type IV-A congenital biliary dilation. The development of intrahepatic cholangiocarcinoma after operation has not been reported previously in a patient with Todanis type I congenital biliary dilation. This case suggests that the entire biliary tree may have a high risk of field cancerization, even in extrahepatic congenital biliary dilation.

The Roles of Surgical Oncologists in the New Era – Minimally Invasive Surgery for Early Gastric Cancer and Adjuvant Surgery for Metastatic Gastric Cancer

In the new era of technical development in surgery, operative devices, molecular targeting and chemotherapeutic agents, surgical oncologists have two main roles in the treatment of gastric cancer. One is to provide patients with minimally invasive surgery, including laparoscopy- or robot-assisted surgery in early gastric cancer patients, and the new concept of surgical intervention toward advanced and metastatic disease. Since recently, laparoscopy-assisted distal gastrectomy has become prevalent in Japan as a surgery which is minimally invasive for the patients and provides them with a good quality of life afterwards. However, the provision of advanced surgical techniques, including lymph node dissection and reconstruction, is more important for patient survival. The second role of surgical oncologists is to evaluate the significant values of the aggressive treatment which we term ‘adjuvant surgery’ for stage IV gastric cancer patients who have successfully responded to initial chemotherapy for curative intent. Stage IV gastric cancer patients are now being informed about the possibility of longer survival with the new chemotherapeutic and surgical strategic approach.

Large solitary fibrous tumor of the retroperitoneum: report of a case.

We report an unusual case of a large solitary fibrous tumor (SFT) in the retroperitoneum. A 53-year-old man was referred to our hospital for surgical treatment of a swelling in the right flank with dull pain. Abdominal computed tomography (CT) and echograms showed a large encapsulated tumor compressing the right kidney and liver. At laparotomy, the tumor was found to be encapsulated but fixed to the capsule of the right kidney within a small area. Therefore, complete removal was achieved. The resected specimen was an encapsulated elastic hard tumor, 14 × 13 × 10 cm in size. Immunohistochemical studies revealed reactivity for CD34 and vimentin, but no staining for keratin, S-100, or Α-smooth muscle actin, confirming a diagnosis of SFT. Although SFT is usually associated with a favorable prognosis, close follow-up is recommended because of the limited information on its long-term behavior.

Cytosolic calcium oscillations induced by hepatocyte growth factor (HGF) in single fura-2-loaded cultured hepatocytes : effects of extracellular calcium and protein kinase C

Hepatocyte growth factor (HGF) induced the periodic fluctuations of cytosolic calcium concentration ([Ca2+]i) in primary cultured rat hepatocytes, which were dependent on extracellular calcium. The HGF-induced [Ca2+]i oscillations were suppressed by the pretreatment with phorbol 12-myristate 13-acetate (PMA). Administration of PMA during oscillations also caused their blockade, but the subsequent addition of protein kinase C (PKC) inhibitor H-7 reversed the inhibitory effects of PMA, thereby resulting in the resumption of the oscillatory responses. Moreover, the prior exposure to H-7 caused apparent increases in [Ca2+]i spike peaks elicited by HGF. These results suggest a negative modulation via PKC in HGF-induced repetitive [Ca2+]i transients. The absence of HGF-induced oscillations after the thapsigargin treatment indicates that the agonist-sensitive intracellular Ca2+ pool plays a crucial role in the [Ca2+] oscillations.

Skewing the Th cell phenotype toward Th1 alters the maturation of tumor-infiltrating mononuclear phagocytes

Mononuclear phagocytes (MPCs) at the tumor site can be divided into subclasses, including monocyte‐lineage myeloid‐derived suppressor cells (MDSCs) and the immunosuppressive tumor‐infiltrating macrophages (TIMs). Cancer growth coincides with the expansion of MDSCs found in the blood, secondary lymphoid organs, and tumor tissue. These MDSCs are thought to mature into macrophages and to promote tumor development by a combination of growth‐enhancing properties and suppression of local antitumor immunoresponses. As little is known about either subset of MPCs, we investigated MPCs infiltrating into murine adenocarcinoma MCA38 tumors. We found that these MPCs displayed immunosuppressive characteristics and a MDSC cell‐surface phenotype. Over 70% of the MPCs were mature (F4/80+Ly6C−) macrophages, and the rest were immature (F480+ Ly6C+) monocytes. MPC maturation was inhibited when the cells infiltrated a tumor variant expressing IL‐2 and soluble TNF type II receptor (sTNFRII). In addition, the IL‐2/sTNFRII MCA38 tumor microenvironment altered the MPC phenotype; these cells did not survive culturing in vitro as a result of Fas‐mediated apoptosis and negligible M‐CSFR expression. Furthermore, CD4+ tumor‐infiltrating lymphocytes (TILs) in wild‐type tumors robustly expressed IL‐13, IFN‐γ, and GM‐CSF, and CD4+ TILs in IL‐2/sTNFRII‐expressing tumors expressed little IL‐13. These data suggest that immunotherapy‐altered Th cell balance in the tumor microenvironment can affect the differentiation and maturation of MPCs in vivo. Furthermore, as neither the designation MDSC nor TIM can sufficiently describe the status of monocytes/macrophages in this tumor microenvironment, we believe these cells are best designated as MPCs.

Fibrosis and Postoperative Fistula of the Pancreas: Correlation with MR Imaging Findings—Preliminary Results

PURPOSE To assess the potential value of magnetic resonance (MR) imaging in evaluating pancreatic fibrosis and predicting the development of postoperative pancreatic fistula. MATERIALS AND METHODS This retrospective study had institutional review board approval, and the requirement for informed consent was waived. MR images obtained in 29 consecutive patients (15 men, 14 women; mean age, 64.9 years; age range, 21-80 years) who underwent pancreatectomy were evaluated. The pancreas-to-muscle signal intensity (SI) ratio on unenhanced T1- and T2-weighted, dynamic contrast material-enhanced, and diffusion-weighted images and the apparent diffusion coefficient (ADC) of the pancreas were measured. MR imaging parameters were correlated with the degrees of pancreatic fibrosis and expression of activated pancreatic stellate cells (PSCs) by using univariate and multivariate regression analyses and receiver operating characteristic curve analysis. The relationships between the development of postoperative pancreatic fistula and the MR imaging measurements were examined by using logistic regression analysis and the Mann-Whitney U test. RESULTS Multiple regression analysis showed that pancreas-to-muscle SI ratios on T1-weighted images and ADC values were independently associated with pancreatic fibrosis (r(2) = 0.66, P < .001) and with activated PSC expression (r(2) = 0.67, P < .001). The mean pancreas-to-muscle SI ratio (± standard deviation) on T1-weighted images was higher (P = .0029) for patients with postoperative pancreatic fistula (1.6 ± 0.2) than for those without (1.2 ± 0.2), and the odds ratio for postoperative pancreatic fistula was 21.3 in patients with an SI ratio of 1.41 and higher. CONCLUSION The pancreas-to-muscle SI ratio on T1-weighted MR images of the pancreas may be a potential biomarker for assessment of pancreatic fibrosis and prediction of postoperative pancreatic fistula.

Contribution of thymidylate synthase to gemcitabine therapy for advanced pancreatic cancer.

Objectives: Thymidylate synthase (TS) inhibitors activate human equilibrative nucleoside transporter 1. We evaluated the contribution of TS expression to determine a treatment method providing an effect from gemcitabine (GEM). Methods: The expression of 5-fluorouracil (5-FU) and GEM metabolic factors (5-FU: TS, dihydropyrimidine dehydrogenase, orotate phosphoribosyltransferase; GEM: human equilibrative nucleoside transporter 1, deoxycytidine kinase, cytidine deaminase, 5&vprime;-nucleotidase) were studied in 7 pancreatic cancer cell lines by Western blotting, and drug resistance was evaluated by 3-[4,5-dimethylthiazol]-2,5-dephenyl tetrazolium bromide assay. The expression of 5-FU factors was observed immunohistochemically in resected pancreatic cancer specimens. Results: Gemcitabine concentrations that inhibited colony formation by 50% correlated with TS protein expression (P = 0.0169). With a 5-FU non-growth-inhibiting dose, GEM concentrations that inhibited colony formation by 50% were significantly reduced by one fourth to one tenth. Knockout of TS expression by small interfering RNA decreased resistance to GEM in the cell lines (P = 0.0019). Immunohistochemically, TS expression related to disease-free survival time of patients treated with GEM (P = 0.0224). A high expression of 5-FU factors was detected: orotate phosphoribosyltransferase: differentiated cases (P = 0.0137), lower T factor (P = 0.0411); dihydropyrimidine dehydrogenase: nerve invasion (P = 0.0188), lymph node recurrence (P = 0.0253); TS, positive N factor (P = 0.0061). Conclusions: The expression of TS provides an alternative source of substrate for DNA synthesis and positively correlates with GEM resistance and shortened patient survival. Abbreviations: GEM - gemcitabine, 5-FU - 5-fluorouracil, TS - thymidylate synthase, DPD - dihydropyrimidine dehydrogenase, OPRT - orotate phosphoribosyltransferase, hENT1 - human equilibrative nucleoside transporter 1

Vascular endothelial growth factor protects hepatoma cells against oxidative stress‐induced cell death

Background:  The aim of the present study was to examine coordination of the vascular endothelial growth factor (VEGF) and VEGF receptor (Flk‐1) system and to study control of VEGF expression by oxidative stress, which is considered a model for chronic liver disease.