Masahiko Tsujii

Retaining cell–cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer

Primary culture of the cancer cells from patients’ tumors can provide crucial information of individual tumors, yet the technology has not been optimized until now. We developed an innovative culture method for primary colorectal cancer cells, based on the principle that cell–cell contact of cancer cells was maintained throughout the process. When tumor tissue was dissociated into cell clusters, in which cell–cell contact was retained, they rapidly formed spheroids that we termed cancer tissue-originated spheroids (CTOSs). CTOSs of colorectal cancer consisted of highly purified and viable cancer cells, and they were prepared with high efficiency. In immunodeficient mice, CTOSs formed xenograft tumors that retained the features of the parental tumors. Moreover, CTOSs were able to be cultured and expanded in vitro using a 3D culture system and stem cell culture medium. This method allowed evaluation of chemosensitivity and signal pathway activation in cancer cells from individual patients. Easy preparation and culture of pure primary cancer cells provides an innovative platform for studying cancer biology and developing personalized medicine.

Scheduled endoscopic surveillance controls secondary cancer after curative endoscopic resection for early gastric cancer: a multicentre retrospective cohort study by Osaka University ESD study group

Background After endoscopic submucosal dissection (ESD) of early gastric cancer (EGC), patients are at high risk for synchronous or metachronous multiple gastric cancers. Objective To elucidate the time at which multiple cancers develop and to determine whether scheduled endoscopic surveillance might control their development. Design A multicentre retrospective cohort study from 12 hospitals was conducted. Patients with EGC who underwent ESD with en bloc margin-negative curative resection were included. Synchronous cancer was classified as concomitant cancer or missed cancer. The cumulative incidence of metachronous cancers and overall survival rate were calculated using the Kaplan–Meier method. Results From April 1999 to December 2010, 1258 patients met the inclusion criteria. Synchronous or metachronous multiple cancers were detected in 175 patients (13.9%) during a mean of 26.8 months. Among the 110 synchronous cancers, 21 were missed at the time of the initial ESD. Many of the missed lesions existed in the upper third of the stomach and the miss rate was associated with the endoscopists inexperience (<500 oesophagogastroduodenoscopy cases). The cumulative incidence of metachronous cancers increased linearly and the mean annual incidence rate was 3.5%. The incidence rate did not differ between patients with or without Helicobacter pylori eradication. Four lesions (0.32%) were detected as massively invading cancers during the follow-up. Conclusions Nineteen per cent of synchronous cancers were not detected until the initial ESD. The incidence rate of metachronous cancer after ESD was constant. Scheduled endoscopic surveillance showed that almost all recurrent lesions were treatable by endoscopic resection.

Topical Implantation of Mesenchymal Stem Cells Has Beneficial Effects on Healing of Experimental Colitis in Rats

Mesenchymal stem cells (MSCs) are attractive cell sources in regenerative medicine. We examined the effects of topical MSCs implantation on an experimental model of inflammatory bowel disease. Putative MSCs, isolated from bone marrow aspirates of male rats by dish adherence and expanded in vitro, were characterized by flow cytometry, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and differentiation assays. Experimental colitis was induced by intraluminal instillation of 2,4,6-trinitrobonzene sulfonic acid (TNBS) in the colons of male rats. The putative MSCs and unselected fresh bone marrow cells were injected into the colonic submucosa surrounding the area exposed to TNBS. The healing process of the injury was examined macroscopically and immunohistologically. Multipotent MSCs positive for CD29 and CD90, and negative for CD31 and CD34, were implanted into colon tissue surrounding the lesion; a majority of the engrafted cells were positive for vimentin. The implantation significantly accelerated healing of the damaged mucosa compared with vehicle-injected controls. The MSCs expressed vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β1 in vitro and after the implantation. In conclusion, we found that MSCs were successfully topically implanted in the colon and that they were associated with accelerated healing of TNBS-induced colitis. The beneficial effects of the MSCs might be mediated, at least in part, by their ability to differentiate into colonic interstitial cells and by their ability to provide VEGF and TGF-β1 to the injured area.

Oral Glucose Tolerance Test Predicts Prognosis of Patients with Liver Cirrhosis

OBJECTIVE:The aim of this study was to evaluate whether oral glucose tolerance test (OGTT) was useful in evaluating the prognosis of patients with liver cirrhosis.METHODS:Fifty-six patients with liver cirrhosis were enrolled in a prospective cohort study. In all cases, glucose tolerance was diagnosed by a 75-g OGTT according to World Health Organization (WHO) criteria. The relationship of clinical variables to the cirrhosis-related prognosis was investigated using univariate and multivariate regression models.RESULTS:Diabetes mellitus (DM) was diagnosed in 21 subjects (38%), impaired glucose tolerance (IGT) in 13 subjects (23%), and normal glucose tolerance (NGT) in 22 subjects (39%) using OGTT. The cumulative survival rates of patients with liver cirrhosis and NGT were 94.7% at 5 yr; liver cirrhosis and IGT, 68.8% at 5 yr; liver cirrhosis and DM, 56.6% at 5 yr. The survival rates of patients with liver cirrhosis and DM significantly differed from those with NGT. Univariate analysis demonstrated that serum albumin, total bilirubin, prothrombin activity, Child-Pugh scores, and glucose intolerance were highly significant prognostic factors. Multiple regression analysis yielded albumin and DM as the most powerful independent negative predictors of survival.CONCLUSIONS:OGTT appears to be useful for evaluating the prognosis of cirrhotic patients.

SHORT-TERM OUTCOMES OF ENDOSCOPIC SUBMUCOSAL DISSECTION (ESD) FOR EARLY GASTRIC NEOPLASM: MULTICENTER SURVEY BY OSAKA UNIVERSITY ESD STUDY GROUP

Background:  Endoscopic submucosal dissection (ESD) was developed for en bloc removal of large and flat gastrointestinal tract neoplasms. In Japan, ESD is performed under conscious sedation. The risks for sedation‐related complications of ESD, such as postoperative pneumonia, have not been evaluated. The aim of this study was to evaluate the incidence of postoperative pneumonia after ESD in a multicenter survey.

CagA mediates epigenetic regulation to attenuate let-7 expression in Helicobacter pylori-related carcinogenesis

Objective MicroRNAs (miRNAs) act as tumour suppressor genes or oncogenes in the regulation of multiple carcinogenic processes. Aberrant miRNA expression is reported in Helicobacter pylori (H pylori)-related gastritis and gastric cancer. The cytotoxin-associated gene A (CagA) of H pylori has a pathophysiologically important role in gastric carcinogenesis. A study was undertaken to evaluate the effect of CagA on miRNA expression and its regulatory mechanism. Methods The effect of CagA on miRNA expression was assessed by comprehensive miRNA microarray. The mechanisms of the in vitro and in vivo effects of CagA on histone modification and DNA methylation and the involvement of CagA-dysregulated signal transduction on let-7, an important representative miRNA in gastric carcinogenesis, were investigated. Results In in vitro experiments, CagA significantly attenuated let-7 expression leading to Ras pathway activation. CagA enhanced c-myc, DNA methyltransferase 3B (DNMT3B) and Enhancer of Zeste homologue 2 (EZH2) expression and attenuated miR-26a and miR-101 expression, which resulted in the attenuation of let-7 expression by histone and DNA methylation. Experiments performed in CagA transgenic mice revealed that c-myc, EZH2 and DNMT3B expression were enhanced and let-7 expression was attenuated to induce Ras oncoprotein expression in the stomach, with no associated inflammation. Conclusions H pylori CagA induces aberrant epigenetic silencing of let-7 expression, leading to Ras upregulation.

Helicobacter bilis infection in biliary tract cancer

Background:  Biliary tract cancer is a highly fatal disease with poor prognosis, but the aetiology is poorly understood.

Involvement of bone marrow-derived cells in healing of experimental colitis in rats

Bone marrow is reported to contain hematopoietic stem cells and other adult somatic stem cells that have phenotypes of cells composing tissues other than bone marrow. To explore the implication of bone marrow‐derived cells in the treatment of inflammatory bowel diseases, experimental colitis was induced in wild‐type rats after transplantation of bone marrow from transgenic rats expressing green fluorescence protein (GFP). Chronic colitis was induced 21 days later using 30 mg 2,4,6‐trinitrobenzenesulfonic acid (TNBS). Control rats received saline. At 28, 56, and 224 days after TNBS administration, rats were euthanized, and tissues were removed and processed for paraffin‐embedded sections. Cells derived from bone marrow were identified by immunohistochemistry using anti‐GFP antibody. To identify the phenotypes of the cells expressing GFP, we conducted serial‐section analysis and double‐staining analysis using antibodies against cytokeratin (epithelial cells) or vimentin (interstitial cells). In the present study, GFP‐positive, bone marrow‐derived cells occupied 37.6% and 4.25% of the colonic epithelium at 28 days and 56 days after the induction of TNBS‐colitis, respectively. Also, significant amounts of mucosal and submucosal interstitial cells were derived from the bone marrow. These findings showed that a large amount of bone marrow‐derived cells were involved in regeneration of the colon after experimental colitis in rats.

Caldesmon suppresses cancer cell invasion by regulating podosome/invadopodium formation

The podosome and invadopodium are dynamic cell‐adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin‐binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmons involvement in podosome/invadopodium‐mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.

Gastroesophageal reflux disease related to diabetes: Analysis of 241 cases with type 2 diabetes mellitus

Background and Aim:  We examined the incidence of symptomatic gastroesophageal reflux disease (GERD) in patients with type 2 diabetes mellitus (DM).