Jumpei Kondo

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.

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.

Spheroid Culture of Primary Lung Cancer Cells with Neuregulin 1/HER3 Pathway Activation

Introduction: Primary culture of cancer cells is expected to be useful for investigating the biology of cancer and predicting chemosensitivity for individual patients, yet has been hampered by technical difficulties. We recently developed the cancer tissue–originated spheroid (CTOS) method for the primary culture of colorectal cancer cells. In the present study, we applied this system to the primary culture of non–small-cell lung cancer. Methods: We used 125 surgical specimens and 18 pleural effusions for CTOS preparation. Partially digested tumor fragments were cultured in a medium for embryonic stem cells. CTOSs were subjected to sensitivity assay and signal transduction assay for the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib. We also investigated the effects of growth factors in culturing lung cancer CTOS. Results: The success rate of CTOS preparation from surgical specimens was 80.0%. The CTOS method was also suitable for culturing tumor spheroids from pleural effusions. CTOSs from lung cancer consisted mostly of pure cancer cells. CTOSs and CTOS-derived xenografts retained the characteristics of the original tumors. In vitro assay results showed that EGFR mutation status and expression levels corresponded with erlotinib sensitivity, confirming previous clinical findings. Furthermore, we found that neuregulin 1, a ligand of HER3, potently induced CTOS growth. Conclusions: The CTOS method enables us to obtain primary lung tumor cells of high viability and purity. CTOS could be a new platform for studying lung cancer biology.

Clinical features of post-polypectomy bleeding associated with heparin bridge therapy

 Heparin is given to patients undergoing colonoscopic polypectomy at high risk for thromboembolism. Little is known, however, about how heparin bridge therapy (HB) affects post‐polypectomy bleeding (PPB). The present study aimed to identify the clinical features of PPB associated with HB.

Functional analysis of agalactosyl IgG in inflammatory bowel disease patients

Background: Agalactosyl immunoglobulin (Ig) G is increased in inflammatory bowel disease (IBD) similarly to rheumatoid arthritis (RA). The lectin complement pathway is shown to be activated through association of agalactosyl IgG with mannan‐binding lectin (MBL) in RA. Functional changes of IgG agalactosylation in IBD, however, have not yet been clarified. Methods: The ratio of the agalactosyl/non‐agalactosyl fraction in fucosylated IgG oligosaccharides (G0F/G2F) and serum MBL levels were analyzed in 59 patients with Crohns disease (CD), 64 ulcerative colitis (UC), and 39 healthy volunteers (HV). The MBL levels associated with serum IgG were analyzed by enzyme‐linked immunosorbent assay. MBL expression in the intestinal mucosa was analyzed by immunohistochemistry. Phagocytosis of sheep red blood cells (SRBC) reacted with either an agalactosyl or non‐agalactosyl SRBC‐specific IgG antibody was determined by flow cytometry. Results: The serum MBL levels were not significantly different among CD, UC, or HV. In patients with CD, the serum MBL levels were negatively correlated with the Crohns Disease Activity Index (CDAI). The levels of MBL associated with agalactosyl IgG were not different from those associated with non‐agalactosyl IgG. Immunoreactivity to MBL was less in the inflamed mucosa compared with the noninflamed mucosa. Phagocytic activity of SRBC was significantly higher in the presence of agalactosyl IgG compared to non‐agalactosyl IgG. Conclusions: Agalactosyl IgG oligosaccharides enhanced antibody‐dependent phagocytosis in vitro but did not activate the lectin complement pathway. Oligosaccharide alterations of IgG are not only a marker of IBD but also functionally modulate the immune function of IBD. (Inflamm Bowel Dis 2010;)

Roles of double-balloon endoscopy in the diagnosis and treatment of Crohn’s disease: a multicenter experience

BackgroundDouble-balloon endoscopy (DBE) examinations are not yet widely accepted as routine procedures for examining the small bowel of patients with Crohn’s disease (CD).AimTo evaluate the feasibility and usefulness of DBE for CD in tertiary-care hospitals.MethodsBetween July 2004 and September 2008, 1444 DBE procedures were performed for 704 patients in 6 tertiary-care hospitals. Patient profile, indication, diagnosis and treatment of DBE were evaluated using a multicenter database.ResultsDBE examinations were most frequently performed in 75 patients with CD, corresponding to 10.5% of all the patients examined by DBE. Fifty patients were diagnosed with CD before DBE, while DBE was performed for the diagnosis of 25 new CD patients. Small bowel lesions were often detected even when the terminal ileum was not involved. In the 75 patients, 21 patients were asymptomatic at the time of DBE examinations. Active inflammatory lesions were detected in 51.2% of the CD patients, and were even detected in 33.3% of the asymptomatic CD patients. The treatment was altered in 53.3% of the CD patients after the DBE evaluation. No severe complications were experienced.ConclusionsDBE procedures can be safely performed in patients with CD and should be considered for the precise evaluation of and to determine the treatment strategy for CD.

Evaluation of Endoscopic Ultrasound Image Quality Is Necessary in Endosonographic Assessment of Early Gastric Cancer Invasion Depth

We evaluated whether endoscopic ultrasonography (EUS) image quality affects the accuracy of diagnosing the vertical invasion depth of early gastric cancer (EGC). A total of 75 lesions in 75 patients suspected of having EGC were enrolled. All patients underwent EUS examination. Findings of EUS were compared with histopathologic results. We evaluated the effect of the following clinicopathologic factors: location, diameter, surface pattern, concomitant ulceration, histology type, and EUS image quality score. EUS image quality was scored based on detection repeatability, appropriate probe placement, and clarity of the five gastric wall layers including the lesion. Sixty-three lesions (84%) were pathologically mucosal and 12 lesions (16%) were submucosal cancer. Overall accuracy was 82.7%. Significantly more lesions in the upper and middle portions of the stomach were incorrectly diagnosed than in the lower portion (P = 0.0019). Lesion diameter was significantly larger among incorrectly diagnosed lesions (P = 0.0257). Low-quality images were significantly more often associated with incorrectly diagnosed lesions than with correctly diagnosed lesions (P = 0.0001). Multivariate analysis revealed that EUS image quality was associated with EUS staging accuracy (odds ratio, 21.8; 95% confidence interval, 4.5–137.6). Low-quality EUS images led to an incorrect diagnosis of invasion depth of EGC, independent of tumor location or size.

Upregulation of GRAIL is associated with remission of ulcerative colitis

Abrogating tolerance against unidentified antigens is a critical step in the pathogenesis of ulcerative colitis (UC). T cell anergy, one of the main mechanisms of tolerance, has been shown to be induced by E3 ubiquitin ligases, such as gene related to anergy in lymphocytes (GRAIL), Itch, and c-Cbl in mice. However, it is not well known whether these E3 ligases play roles in human diseases. The pathophysiological role of the E3 ligases in patients with UC was investigated. At first, the expression of GRAIL, Itch, and c-Cbl in human anergic T cells was analyzed by quantitative RT-PCR and Western immunoblotting. Next, the mRNA expression of the E3 ligases was analyzed in peripheral CD4+ T cells of 20 patients with UC and 10 healthy volunteers (HV). mRNA expression was analyzed in patients with active UC before and after treatment with prednisolone and leukocytapheresis. Anergic human CD4+ T cells expressed significantly higher levels of GRAIL, Itch, and c-Cbl than nonanergic cells. GRAIL expression was significantly higher in patients with UC in remission than in patients with active disease and in HV (P < 0.01). The level of GRAIL expression was also significantly increased in patients with active disease whose clinical activity index scores improved after treatment (P < 0.05). There were no significant differences in Itch and c-Cbl expression among patients with active UC, patients with UC in remission, and HV. These data suggest that GRAIL plays an important role in maintaining remission in patients with UC.

The effectiveness of an anti-human IL-6 receptor monoclonal antibody combined with chemotherapy to target colon cancer stem-like cells

Recent studies have demonstrated that cancer stem cells (CSCs) can initiate and sustain tumor growth and exhibit resistance to clinical cytotoxic therapies. Therefore, CSCs represent the main target of anticancer therapy. Interleukin-6 (IL-6) promotes cellular proliferation and drug resistance in colorectal cancer, and its serum levels correlate with patient survival. Therefore, IL-6 and its downstream signaling molecule the signal transducer and activator of transcription-3 (STAT3) represent potential molecular targets. In the present study, we investigated the effects of IL-6 and its downstream signaling components on stem cell biology, particularly the chemoresistance of CSCs, to explore potential molecular targets for cancer therapy. The colon cancer cell line WiDr was cultured in serum-free, non-adherent, and three-dimensional spheroid-forming conditions to enrich the stem cell-like population. Spheroid-forming cells slowly proliferated and expressed high levels of Oct-4, Klf4, Bmi-1, Lgr5, IL-6, and Notch 3 compared with adherent cells. Treatment with an anti-human IL-6 receptor monoclonal antibody reduced spheroid formation, stem cell-related gene expression, and 5-fluorouracil (5-FU) resistance. In addition, IL-6 treatment enhanced the levels of p-STAT3 (Tyr705), the expression of Oct-4, Klf4, Lgr5, and Notch 3, and chemoresistance to 5-FU. siRNA targeting Notch 3 suppressed spheroid formation, Oct-4 and Lgr5 expression, and 5-FU chemoresistance, whereas STAT3 inhibition enhanced Oct-4, Klf4, Lgr5, and Notch 3 expression and 5-FU chemoresistance along with reduced spheroid growth. Taken together, these results indicate that IL-6 functions in dichotomous pathways involving Notch 3 induction and STAT3 activation. The former pathway is involved in cancer stem-like cell biology and enhanced chemoresistance, and the latter pathway leads to accelerated proliferation and reduced chemoresistance. Thus, an anti-human IL-6 receptor monoclonal antibody or Notch 3 inhibition may be superior to STAT3 inhibition for CSC-targeting therapies concomitant with anticancer drugs.

p53 functional deficiency in human colon cancer cells promotes fibroblast-mediated angiogenesis and tumor growth

Cancer-associated fibroblasts (CAFs) create a microenvironment that contributes to tumor growth; however, the mechanism by which fibroblasts are phenotypically altered to CAFs remains unclear. Loss or mutation of the tumor suppressor p53 plays a crucial role in cancer progression. Herein, we analyzed how the p53 status of cancer cells affects fibroblasts by investigating the in vivo and in vitro effects of loss of p53 function in cancer cells on phenotypic changes in fibroblasts and subsequent tumor progression in human colon cancer cell lines containing wild-type p53 and in cells with a p53 functional deficiency. The growth of p53-deficient tumors was significantly enhanced in the presence of fibroblasts compared with that of p53-wild-type tumors or p53-deficient tumors without fibroblasts. p53-deficient cancer cells produced reactive oxygen species, which activated fibroblasts to mediate angiogenesis by secreting vascular endothelial growth factor (VEGF) both in vivo and in vitro Activated fibroblasts significantly contributed to tumor progression. Deletion of fibroblast-derived VEGF or treatment with N-acetylcysteine suppressed the growth of p53-deficient xenograft tumors. The growth effect of blocking VEGF secreted from cancer cells was equivalent regardless of p53 functional status. Human colon cancer tissues also showed a significant positive correlation between p53 cancer cell staining activated fibroblasts and microvessel density. These results reveal that fibroblasts were altered by exposure to p53-deficient epithelial cancer cells and contributed to tumor progression by promoting neovascularization. Thus, p53 acts as a modulator of the tumor microenvironment.