Clonal Origin and Lineage Ambiguity in Mixed Neuroendocrine Carcinoma of the Uterine Cervix.

Small-cell neuroendocrine carcinoma (SCNEC) of the cervix is a rare disease characterized by a high incidence of mixed tumors with other types of cancer. The mechanism underlying this mixed phenotype is not well understood. This study established a panel of organoid lines from patients with SCNEC of the cervix and ultimately focused on one line, which retained a mixed tumor phenotype, both in vitro and in vivo. Histologically, both organoids and xenograft tumors showed distinct differentiation into either SCNEC or adenocarcinoma in some regions and ambiguous differentiation in others. Tracking single cells indicated the existence of cells with bipotential differentiation toward SCNEC and adenocarcinomas. Single-cell transcriptional analysis identified three distinct clusters: SCNEC-like, adenocarcinoma-like, and a cluster lacking specific differentiation markers. The expression of neuroendocrine markers was enriched in the SCNEC-like cluster but not exclusively. Human papillomavirus 18 E6 was enriched in the SCNEC-like cluster, which showed higher proliferation and lower levels of the p53 pathway. After treatment with anticancer drugs, the expression of adenocarcinoma markers increased, whereas that of SCNEC decreased. Using a reporter system for keratin 19 expression, changes in the differentiation of each cell were shown to be associated with the shift in differentiation induced by drug treatment. These data suggest that mixed SCNEC/cervical tumors have a clonal origin and are characterized by an ambiguous and flexible differentiation state.

TNF receptor-related factor 3 inactivation promotes the development of intrahepatic cholangiocarcinoma through NF-κB-inducing kinase-mediated hepatocyte transdifferentiation.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is a deadly but poorly understood disease, and its treatment options are very limited. The aim of this study was to identify the molecular drivers of ICC and search for therapeutic targets. APPROACH AND RESULTS: We performed a Sleeping Beauty transposon-based in vivo insertional mutagenesis screen in liver-specific Pten -deficient mice and identified TNF receptor-related factor 3 ( Traf3 ) as the most significantly mutated gene in murine ICCs in a loss-of-function manner. Liver-specific Traf3 deletion caused marked cholangiocyte overgrowth and spontaneous development of ICC in Pten knockout and KrasG12D mutant mice. Hepatocyte-specific, but not cholangiocyte-specific, Traf3 -deficient and Pten -deficient mice recapitulated these phenotypes. Lineage tracing and single-cell RNA sequencing suggested that these ICCs were derived from hepatocytes through transdifferentiation. TRAF3 and PTEN inhibition induced a transdifferentiation-like phenotype of hepatocyte-lineage cells into proliferative cholangiocytes through NF-κB-inducing kinase (NIK) up-regulation in vitro. Intrahepatic NIK levels were elevated in liver-specific Traf3 -deficient and Pten -deficient mice, and NIK inhibition alleviated cholangiocyte overgrowth. In human ICCs, we identified an inverse correlation between TRAF3 and NIK expression, with low TRAF3 or high NIK expression associated with poor prognosis. Finally, we showed that NIK inhibition by a small molecule inhibitor or gene silencing suppressed the growth of multiple human ICC cells in vitro and ICC xenografts in vivo. CONCLUSIONS: TRAF3 inactivation promotes ICC development through NIK-mediated hepatocyte transdifferentiation. The oncogenic TRAF3-NIK axis may be a potential therapeutic target for ICC.

Inhibition of the bone morphogenetic protein pathway suppresses tumor growth through downregulation of epidermal growth factor receptor in MEK/ERK-dependent colorectal cancer.

The bone morphogenetic protein (BMP) pathway promotes differentiation and induces apoptosis in normal colorectal epithelial cells. However, its role in colorectal cancer (CRC) is controversial, where it can act as context-dependent tumor promoter or tumor suppressor. Here we have found that CRC cells reside in a BMP-rich environment based on curation of two publicly available RNA-sequencing databases. Suppression of BMP using a specific BMP inhibitor, LDN193189, suppresses the growth of select CRC organoids. Colorectal cancer organoids treated with LDN193189 showed a decrease in epidermal growth factor receptor, which was mediated by protein degradation induced by leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) expression. Among 18 molecularly characterized CRC organoids, suppression of growth by BMP inhibition correlated with induction of LRIG1 gene expression. Notably, knockdown of LRIG1 in organoids diminished the growth-suppressive effect of LDN193189. Furthermore, in CRC organoids, which are susceptible to growth suppression by LDN193189, simultaneous treatment with LDN193189 and trametinib, an FDA-approved MEK inhibitor, resulted in cooperative growth inhibition both in vitro and in vivo. Taken together, the simultaneous inhibition of BMP and MEK could be a novel treatment option in CRC cases, and evaluating in vitro growth suppression and LRIG1 induction by BMP inhibition using patient-derived organoids could offer functional biomarkers for predicting potential responders to this regimen.

Prohaptoglobin is a possible prognostic biomarker for colorectal cancer.

BACKGROUND AND AIMS: Fucosylated haptoglobin is a novel glycan biomarker for colorectal and other cancers, while the significance of its precursor, prohaptoglobin (proHp), remains to be elucidated. In this study, we investigated whether proHp can be a colorectal cancer (CRC) biomarker and the biological functions of proHp in CRC using 10-7G, a monoclonal antibody recently developed in our laboratory. MATERIALS AND METHODS: Serum proHp level in 74 patients with CRC was semi-quantified by western blotting, and 5-year recurrence-free survival and overall survival were analyzed for groups stratified by proHp status (high vs. low). We also performed immunohistochemical analyses of 17 CRC tissue sections using 10-7G mAb. The biological functions of proHp were evaluated by overexpressing proHp in CRC cell lines. RESULTS: Serum proHp correlated with the clinical stage and poorer prognosis of CRC. In the primary CRC sections, immune cells were stained positive for 10-7G in ∼50% of the cases. Overexpression of proHp in HCT116 human CRC cells induced epithelial-mesenchymal transition-like changes and promoted cell migration in CRC cells. CONCLUSION: We provide evidence for the first time that proHp has potential as a prognostic biomarker for CRC and demonstrated specific biological activities of proHp.

ASURAT: functional annotation-driven unsupervised clustering of single-cell transcriptomes.

MOTIVATION: Single-cell RNA sequencing (scRNA-seq) analysis reveals heterogeneity and dynamic cell transitions. However, conventional gene-based analyses require intensive manual curation to interpret biological implications of computational results. Hence, a theory for efficiently annotating individual cells remains warranted. RESULTS: We present ASURAT, a computational tool for simultaneously performing unsupervised clustering and functional annotation of disease, cell type, biological process and signaling pathway activity for single-cell transcriptomic data, using a correlation graph decomposition for genes in database-derived functional terms. We validated the usability and clustering performance of ASURAT using scRNA-seq datasets for human peripheral blood mononuclear cells, which required fewer manual curations than existing methods. Moreover, we applied ASURAT to scRNA-seq and spatial transcriptome datasets for human small cell lung cancer and pancreatic ductal adenocarcinoma, respectively, identifying previously overlooked subpopulations and differentially expressed genes. ASURAT is a powerful tool for dissecting cell subpopulations and improving biological interpretability of complex and noisy transcriptomic data. AVAILABILITY AND IMPLEMENTATION: ASURAT is published on Bioconductor (https://doi.org/10.18129/B9.bioc.ASURAT). The codes for analyzing data in this article are available at Github (https://github.com/keita-iida/ASURATBI) and figshare (https://doi.org/10.6084/m9.figshare.19200254.v4). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Twin research shows glycan changes are more susceptible to environmental factors than their carrier glycoproteins.

Changes in protein glycosylation are clinically used as biomarkers. In the present study, we employed a twin cohort to investigate the contributions of genetic and environmental factors to glycan modifications of glycoproteins. Mac-2 binding protein (Mac-2 bp), haptoglobin (Hp), and their glycosylated forms are liver fibrosis and cancer biomarkers. Sera from 107 twin pairs without clinical information were used as a training cohort for the Mac-2 bp and Mac-2 bp glycosylation isomer (M2BPGi) assay. As a validation cohort, 22 twin pairs were enrolled in the study. For each twin pair, one twin was diagnosed with liver or pancreatic disease. For the training cohort, the correlation ratios of serum Mac-2 bp and M2BPGi levels in twin sera with random sequences were 0.30 and 0.018, respectively. The correlation ratios between twin pairs in the validation cohort for serum Mac-2 bp and M2BPGi levels were 0.75 and 0.35, respectively. In contrast, correlation ratios of serum Hp and fucosylated haptoglobin (Fuc-Hp) levels between twin sera with liver and pancreatic disease were 0.49 and 0.16, respectively. Although serum protein levels of glycoproteins are susceptible to genetic factors, characteristic glycan changes of these glycoproteins are more susceptible to environmental factors, including liver and pancreatic disease.

Vesicular Integral-Membrane Protein 36 Is Involved in the Selective Secretion of Fucosylated Proteins into Bile Duct-like Structures in HepG2 Cells.

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins in cancer cells. In normal hepatocytes, fucosylated proteins are selectively secreted in the bile duct but not blood. In cases of cancer cells without cellular polarity, this selective secretion system is destroyed. Here, we aimed to identify cargo proteins involved in the selective secretion of fucosylated proteins, such as AFP-L3, into bile duct-like structures in HepG2 hepatoma cells, which have cellular polarity like, in part, normal hepatocytes. α1-6 Fucosyltransferase (FUT8) is a key enzyme to synthesize core fucose and produce AFP-L3. Firstly, we knocked out the FUT8 gene in HepG2 cells and investigated the effects on the secretion of AFP-L3. AFP-L3 accumulated in bile duct-like structures in HepG2 cells, and this phenomenon was diminished by FUT8 knockout, suggesting that HepG2 cells have cargo proteins for AFP-L3. To identify cargo proteins involved in the secretion of fucosylated proteins in HepG2 cells, immunoprecipitation and the proteomic Strep-tag system experiments followed by mass spectrometry analyses were performed. As a result of proteomic analysis, seven kinds of lectin-like molecules were identified, and we selected vesicular integral membrane protein gene VIP36 as a candidate of the cargo protein that interacts with the α1-6 fucosylation (core fucose) on N-glycan according to bibliographical consideration. Expectedly, the knockout of the VIP36 gene in HepG2 cells suppressed the secretion of AFP-L3 and other fucosylated proteins, such as fucosylated alpha-1 antitrypsin, into bile duct-like structures. We propose that VIP36 could be a cargo protein involved in the apical secretion of fucosylated proteins in HepG2 cells.

Polarity switching of ovarian cancer cell clusters via SRC family kinase is involved in the peritoneal dissemination.

Peritoneal dissemination is a predominant pattern of metastasis in patients with advanced ovarian cancer. Despite recent progress in the management strategy, peritoneal dissemination remains a determinant of poor ovarian cancer prognosis. Using various histological types of patient-derived ovarian cancer organoids, the roles of the apicobasal polarity of ovarian cancer cell clusters in peritoneal dissemination were studied. First, it was found that both ovarian cancer tissues and ovarian organoids showed apicobasal polarity, where zonula occludens-1 (ZO-1) and integrin beta 4 (ITGB4) served as markers for apical and basal sides, respectively. The organoids in suspension culture, as a model of cancer cell cluster floating in ascites, showed apical-out/basal-in polarity status, while once embedded in extracellular matrix (ECM), the organoids switched their polarity to apical-in/basal-out. This polarity switch was accompanied by the SRC kinase family (SFK) phosphorylation and was inhibited by SFK inhibitors. SFK inhibitors abrogated the adherence of the organoids onto the ECM-coated plastic surface. When the organoids were seeded on a mesothelial cell layer, they cleared and invaded mesothelial cells. In vivo, dasatinib, an SFK inhibitor, suppressed peritoneal dissemination of ovarian cancer organoids in immunodeficient mice. These results suggest SFK-mediated polarity switching is involved in peritoneal metastasis. Polarity switching would be a potential therapeutic target for suppressing peritoneal dissemination in ovarian cancer.

Aberrant activation of Rho/ROCK signaling in impaired polarity switching of colorectal micropapillary carcinoma.

Micropapillary carcinoma (MPC) is a morphologically distinctive form of carcinoma, composed of small nests of cancer cells surrounded by lacunar spaces. Invasive MPC is associated with poor prognosis. The nests of tumor cells in MPC reportedly exhibit reverse polarity, although the molecular mechanisms underlying MPC patterns are poorly understood. Using the cancer tissue-originated spheroid (CTOS) method, we previously reported polarity switching in colorectal cancer (CRC). When cultured in suspension, the apical membrane promptly switches from the outside surface of the CTOSs to the surface of the lumen inside the CTOSs under extracellular matrix (ECM)-embedded conditions, and vice versa. Here, we investigated two CTOS lines from CRC patient tumors with MPC lesions. Xenograft tumors from the CTOSs exhibited the MPC phenotype. The MPC-CTOSs did not switch polarity in vitro. Time-course analysis of polarity switching using real-time imaging of the apical membrane revealed that local switching was continually propagated in non-MPC-CTOSs, while MPC-CTOSs were unable to complete the process. Integrin ß4 translocated to the outer membrane when embedded in ECM in both MPC and non-MPC-CTOSs. Protein levels, as well as the active form of RhoA, were higher in MPC-CTOSs. The suppression of RhoA activity by GAP overexpression enabled MPC-CTOSs to complete polarity switching both in vitro and in vivo, while overexpression of active RhoA did not affect polarity switching in non-MPC-CTOSs. Pretreatment with a ROCK inhibitor enabled MPC-CTOSs to complete polarity switching both in vitro and in vivo, although delayed treatment after becoming embedded in ECM failed to do so. Thus, the inability to switch polarity might be a cause of MPC, in which the aberrant activation of RhoA plays a critical role. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A smooth muscle-derived, Braf-driven mouse model of gastrointestinal stromal tumor (GIST): evidence for an alternative GIST cell-of-origin.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the gut. GISTs are thought to arise solely from interstitial cells of Cajal (ICC), a KIT-positive population that controls gut motility. Activating gain-of-function mutations in KIT and PDGFRA are the most frequent driver events, and most of these tumors are responsive to the tyrosine kinase inhibitor imatinib. Less common drivers include mutant BRAFV600E and these tumors are resistant to imatinib. A mouse model of GIST was recently reported using Etv1, the master transcriptional regulator of ICC-intramuscular (IM) and ICC-myenteric (MY), to induce mutant Braf expression. ICC hyperplasia was observed in Etv1CreERT2 ;BrafLSL-V600E/+ mice but loss of Trp53 was required for development of GIST. We identified previously expression of the pan-ErbB negative regulator, LRIG1, in two distinct subclasses of ICC [ICC-deep muscular plexus (DMP) in small intestine and ICC-submucosal plexus (SMP) in colon] and that LRIG1 regulated their development from smooth muscle cell progenitors. Using Lrig1CreERT2 to induce BrafV600E , we observed ICC hyperplasia beyond the confines of ICC-DMP and ICC-SMP expression, suggesting smooth muscle cells as the cell-of-origin. To examine this possibility, we selectively activated BrafV600E in smooth muscle cells. Myh11CreERT2 ;BrafLSL-V600E/+ mice developed not only ICC hyperplasia but also GIST and in the absence of Trp53 disruption. In addition to providing a simpler model for mutant Braf GIST, these results provide conclusive evidence for smooth muscle cells as an alternative cell-of-origin for GIST. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Small subset of Wnt-activated cells is an initiator of regrowth in colorectal cancer organoids after irradiation.

Most colorectal cancers (CRCs) are differentiated adenocarcinomas, which maintain expression of both stemness and differentiation markers. This observation suggests that CRC cells could retain a regeneration system of normal cells upon injury. However, the role of stemness in cancer cell regeneration after irradiation is poorly understood. Here, we examined the effect of radiation on growth, stemness, and differentiation in organoids derived from differentiated adenocarcinomas. Following a sublethal dose of irradiation, proliferation and stemness markers, including Wnt target genes, were drastically reduced, but differentiation markers remained. After a static growth phase after high dose of radiation, regrowth foci appeared; these consisted of highly proliferating cells that expressed stem cell markers. Radiosensitivity and the ability to form foci differed among the cancer tissue-originated spheroid (CTOS) lines examined and showed good correlation with in vivo radiation sensitivity. Pre-treating organoids with histone deacetylase inhibitors increased radiation sensitivity; this increase was accompanied by the suppression of Wnt signal-related gene expression. Accordingly, Wnt inhibitors increased organoid radiosensitivity. These results suggested that only a small subset of, but not all, cancer cells with high Wnt activity at the time of irradiation could give rise to foci formation. In conclusion, we established a radiation sensitivity assay using CRC organoids that could provide a novel platform for evaluating the effects of radiosensitizers on differentiated adenocarcinomas in CRC.

Dedifferentiation of neuroendocrine carcinoma of the uterine cervix in hypoxia.

Neuroendocrine carcinoma of small cell type (SCNEC) is a rare pathological subtype in cervical cancer, which has a worse prognosis than other histological cell types. Due to its low incidence and the lack of experimental platforms, the molecular characteristics of SCNEC in the cervix remain largely unknown. Using the cancer tissue-originated spheroid (CTOS) method-an ex vivo 3D culture system that preserves the differentiation status of the original tumors-we established a panel of CTOS lines of SCNEC. We demonstrated that xenograft tumors and CTOSs, respectively, exhibited substantial intra-tumor and intra-CTOS variation in the expression levels of chromogranin A (CHGA), a neuroendocrine tumor marker. Since hypoxia affects differentiation in various tumors and in stem cells, we also investigated how hypoxia affected neuroendocrine differentiation of SCNEC of the uterine cervix. In the CTOS line cerv21, hypoxia suppressed expression of the neuroendocrine markers CHGA and synaptophysin (SYP). Flow cytometry analysis using CD99 (a membrane protein marker of SCNEC) revealed decreased CD99 expression in a subset of cells under hypoxic conditions. These expression changes were attenuated by HIF-1α knockdown, and by a Notch inhibitor, suggesting that these molecules played a role in the regulation of neuroendocrine differentiation. The examined SCNEC markers were suppressed under hypoxia in multiple CTOS lines. Overall, our present results indicated that neuroendocrine differentiation in SCNEC of the uterus is a variable phenotype, and that hypoxia may be one of the factors regulating the differentiation status.

High-throughput screening in colorectal cancer tissue-originated spheroids.

Patient-derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high-throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high-throughput screening of 2427 drugs using the cancer tissue-originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.

Dynamic Change of Polarity in Primary Cultured Spheroids of Human Colorectal Adenocarcinoma and Its Role in Metastasis.

Intestinal epithelial cells possess apical-basal polarity, which governs the exchange of nutrients and waste. Perturbation of cell polarity appears to be a general feature of cancers, although most colorectal cancers are differentiated adenocarcinomas, in which polarity is maintained to some extent. Little is known about the role of dysregulated polarity in cancer. The cancer tissue-originated spheroid method was applied to the preparation and culture of spheroids. Spheroids were cultured in suspension or in type I collagen gel. Polarity was assessed by IHC of apical markers and electron microscopy. Two types of polarity status in spheroids were observed: apical-in, with apical membrane located at cavities inside the spheroids in type I collagen gel; and apical-out, with apical membrane located at the outermost layer of spheroids in suspension. These polarities were highly interchangeable. Inhibitors of Src and dynamin attenuated the polarity switch. In patients, clusters of cancer cells that invaded vessels had both apical-in and apical-out morphologic features, whereas primary and metastatic tumors had apical-in features. In a mouse liver metastasis model, apical-out spheroids injected into the portal vein became apical-in spheroids in the liver within a few days. Inhibitors of Src and dynamin significantly decreased liver metastasis. Polarity switching was observed in spheroids and human cancer. The polarity switch was critical in an experimental liver metastasis model.

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.

LRIG1 Regulates Ontogeny of Smooth Muscle-Derived Subsets of Interstitial Cells of Cajal in Mice.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) control intestinal smooth muscle contraction to regulate gut motility. ICC within the plane of the myenteric plexus (ICC-MY) arise from KIT-positive progenitor cells during mouse embryogenesis. However, little is known about the ontogeny of ICC associated with the deep muscular plexus (ICC-DMP) in the small intestine and ICC associated with the submucosal plexus (ICC-SMP) in the colon. Leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) marks intestinal epithelial stem cells, but the role of LRIG1 in nonepithelial intestinal cells has not been identified. We sought to determine the ontogeny of ICC-DMP and ICC-SMP, and whether LRIG1 has a role in their development. METHODS: Lrig1-null mice (homozygous Lrig1-CreERT2) and wild-type mice were analyzed by immunofluorescence and transit assays. Transit was evaluated by passage of orally administered rhodamine B-conjugated dextran. Lrig1-CreERT2 mice or mice with CreERT2 under control of an inducible smooth muscle promoter (Myh11-CreERT2) were crossed with Rosa26-LSL-YFP mice for lineage tracing analysis. RESULTS: In immunofluorescence assays, ICC-DMP and ICC-SMP were found to express LRIG1. Based on lineage tracing, ICC-DMP and ICC-SMP each arose from LRIG1-positive smooth muscle progenitors. In Lrig1-null mice, there was loss of staining for KIT in DMP and SMP regions, as well as for 2 additional ICC markers (anoctamin-1 and neurokinin 1 receptor). Lrig1-null mice had significant delays in small intestinal transit compared with control mice. CONCLUSIONS: LRIG1 regulates the postnatal development of ICC-DMP and ICC-SMP from smooth muscle progenitors in mice. Slowed small intestinal transit observed in Lrig1-null mice may be due, at least in part, to loss of the ICC-DMP population.

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.

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

BACKGROUND: 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. PATIENTS AND METHODS: Data of consecutive inpatients who underwent colonoscopic polypectomy with antithrombotic therapy at Osaka University Hospital were retrospectively collected and categorized into a HB group or a non-HB group. The incidence and characteristics of PPB were analyzed. RESULTS: A total of 117 patients with 279 lesions were identified, and the HB group included 45 patients. Nine of 10 patients with PPB were in the HB group, and the incidence of PPB was significantly higher in the HB group than in the non-HB group (20.0% vs 1.4%, respectively). PPB onset was later in the HB group than inthe non-HB group (median postoperative day: 4 vs 1, respectively). Five of the nine patients with PPB (55.6%) in the HB group experienced recurrent bleeding. One patient in the HB group required a blood transfusion as a result of massive PPB. All bleeding was eventually controlled endoscopically. Hospitalization was significantly longer in the HB group than in the non-HB group (median hospitalization: 14 vs 4 days, respectively). The univariate analysis showed that the predictors of PPB were warfarin use, HB and pedunculated polyps. CONCLUSIONS: PPB associated with HB is characterized by high incidence, late onset and recurrent bleeding, resulting in long hospitalization.

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.

Serum levels of the N-terminal fragment of connective tissue growth factor is a novel biomarker for chronic pancreatitis.

Chronic inflammation of the pancreas is considered to be one of the causes of pancreatic cancer. However, the diagnosis of chronic pancreatitis (CP) is very difficult in the pancreas, where biopsies are difficult to perform. The prevalence of CP is estimated to be many times more common than in patients with actual symptomatic CP. In recent years, abnormal cleavage of certain proteins has attracted attention as a biomarker for CP other than pancreatic enzymes. Connective tissue growth factor (CTGF) is one of the growth factors involved in tissue repair and other processes and is increased by stimulation of transforming growth factor-ß, suggesting a relationship of CTGF with fibrosis. In this study, we measured the total length of CTGF in blood and N-terminal fragment CTGF in 48 cases of chronic pancreatitis, 64 cases of pancreatic cancer and 45 healthy volunteers (HV). Interestingly, we found that blood N-terminal fragment CTGF level was significantly increased in CP and pancreatic cancer patients. Multiple logistic regression analysis showed serum levels of N-terminal fragment CTGF, CRP and amylase were significant and independent variables for the differential diagnosis of CP from HV. Receiver operating characteristic analysis showed that area under the curve (AUC) value of serum N-terminal fragment CTGF level was 0.933, which can differentiate between CP and HV. Several factors would be involved in the increase in serum N-terminal fragment CTGF level. In conclusion, serum N-terminal fragment CTGF level is a promising new biomarker for CP.