Organ Dependent Diversity in Gastrointestinal Fibroblasts.

<p>(A) Unsupervised clustering within submucosal fibroblasts (SMFs) or subperitoneal fibroblasts (SPFs) samples. In both analyses, samples tended to be separated with their organs. The bar indicates each gastrointestinal organ: esophagus (brown), stomach (green), duodenum (light blue), ileum (gray), and colon (orange). (B) Organ signature genes in SMFs and SPFs. The probe sets that express specifically in each organ were selected based on <i>P</i> < 0.05, fold change > 2.0. The 433 probe sets were selected as SMFs organ signature genes, and 526 probe sets were selected as SPFs organ signature genes; of these, 87 probe sets expressed both SMFs and SPFs, defined as common organ signature genes. (C) Hierarchical clustering based on 87 common organ signature genes. The samples were clustered with their organs, but not with their anatomical site. (D) A schematic image of the expression pattern of homeotic genes in GIFs. The regional expression of homeotic genes depending on the anterior-posterior axis of the gastrointestinal tract was observed.</p

Gastrointestinal Fibroblasts Have Specialized, Diverse Transcriptional Phenotypes: A Comprehensive Gene Expression Analysis of Human Fibroblasts

<div><p>Background</p><p>Fibroblasts are the principal stromal cells that exist in whole organs and play vital roles in many biological processes. Although the functional diversity of fibroblasts has been estimated, a comprehensive analysis of fibroblasts from the whole body has not been performed and their transcriptional diversity has not been sufficiently explored. The aim of this study was to elucidate the transcriptional diversity of human fibroblasts within the whole body.</p><p>Methods</p><p>Global gene expression analysis was performed on 63 human primary fibroblasts from 13 organs. Of these, 32 fibroblasts from gastrointestinal organs (gastrointestinal fibroblasts: GIFs) were obtained from a pair of 2 anatomical sites: the submucosal layer (submucosal fibroblasts: SMFs) and the subperitoneal layer (subperitoneal fibroblasts: SPFs). Using hierarchical clustering analysis, we elucidated identifiable subgroups of fibroblasts and analyzed the transcriptional character of each subgroup.</p><p>Results</p><p>In unsupervised clustering, 2 major clusters that separate GIFs and non-GIFs were observed. Organ- and anatomical site-dependent clusters within GIFs were also observed. The signature genes that discriminated GIFs from non-GIFs, SMFs from SPFs, and the fibroblasts of one organ from another organ consisted of genes associated with transcriptional regulation, signaling ligands, and extracellular matrix remodeling.</p><p>Conclusions</p><p>GIFs are characteristic fibroblasts with specific gene expressions from transcriptional regulation, signaling ligands, and extracellular matrix remodeling related genes. In addition, the anatomical site- and organ-dependent diversity of GIFs was also discovered. These features of GIFs contribute to their specific physiological function and homeostatic maintenance, and create a functional diversity of the gastrointestinal tract.</p></div

Anatomical Site Dependent Diversity in Gastrointestinal Fibroblasts.

<p>(A) Unsupervised clustering in the colon, ileum, stomach, duodenum, and esophagus derived fibroblasts samples. Submucosal fibroblasts (SMFs) and subperitoneal fibroblasts (SPFs) from the colon, ileum, and stomach showed different gene expression profiles, whereas those from the duodenum and esophagus were not separated into different clusters. The red bar indicates SMFs samples and the blue bar indicates SPFs samples. The color scale of gene expression is same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129241#pone.0129241.g001" target="_blank">Fig 1A</a>. (B) Supervised clustering between SMFs and SPFs samples in the colon, ileum, and stomach. A total of 498 probe sets were selected based on <i>P</i> < 0.05 and fold change > 2.0 (<i>t</i>-test unpaired). The top significant genes are shown on the right. (C) Distinct expression of the genes related with transcriptional regulation, signal ligands, and extracellular matrix remodeling in SMFs and SPFs.</p

Diversity of Gene Expression in Human Gastrointestinal Fibroblasts.

<p>(A) Diversity of gene expression of 63 primary human fibroblasts. Each column is colored with the fibroblast origin; dermal fibroblasts (DeFs), mammary fibroblasts from Japanese (J_MaFs), mammary fibroblasts from Caucasians (C_MaFs) are colored brown; prostate fibroblasts (PrFs) and uterus fibroblasts (UtFs) are colored light blue; gallbladder fibroblasts (GaFs), hepatic stellate cells (HSCs), and liver fibroblasts (LiFs) are colored gray; lung fibroblasts (LuFs) are colored green; submucosal fibroblasts from the gastrointestinal tract (SMFs) are colored red; subperitoneal fibroblasts from the gastrointestinal tract (SPFs) are colored blue; and vascular adventitial fibroblasts (VAFs) are colored pink. The color scale of the gene expression range is +5 to -5 logs on log base 2. (B) Expansion view of the dendrogram seen in (A). The first bifurcation of the dendrogram separated gastrointestinal fibroblasts (GIFs) and non-GIFs significantly (<i>P</i> < 0.001). Furthermore, clustering depending on anatomical site and gastrointestinal organ was also observed.</p

Anatomical Site and Organ Signature Genes Discriminate the Topological Diversity of Gastrointestinal Fibroblasts.

<p>(A) Hierarchical clustering of the stomach, ileum, and colon fibroblasts, based on 585 probe sets that consisted of 498 anatomical site signature genes and 87 common organ signature genes, as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129241#pone.0129241.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129241#pone.0129241.g004" target="_blank">4</a>. The bar indicates the positional information of the samples: upper bar indicates submucosal fibroblasts (red) or subperitoneal fibroblasts (blue), and the lower bar indicates stomach (gray), ileum (green), or colon (orange). (B-E) The validation study of the anatomical site and organ signature genes in independent fibroblasts samples. The mRNA expression of SPFs signature: <i>MSX1</i> (B), SMFs signature: <i>PITX1</i> (C), colon fibroblasts signature: <i>HOXA10</i> (D), and stomach fibroblasts signature: <i>HOXB8</i> (E) were calculated (<i>n</i> = 3).</p

The Transcriptional Differences between Gastrointestinal Fibroblasts and Non–Gastrointestinal Fibroblasts.

<p>(A) Supervised clustering using the significant different expressed probe sets between gastrointestinal fibroblasts (GIFs) and non-GIFs. The 995 significant probe sets were selected based on <i>P</i> < 0.05 and fold change > 2.0 (one-way ANOVA). The red bars indicate a GIF sample, and blue bars indicate a non-GIF sample. The top significant genes are shown on the right. The color scale of gene expression is the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129241#pone.0129241.g001" target="_blank">Fig 1A</a>. (B) Distinct expression of the genes related with transcriptional regulation, signal ligands, and extracellular matrix remodeling in GIFs and Non-GIFs.</p

The Expression of Anatomical Site and Organ Signature Genes in Human Tissue.

<p>(A-D) The mRNA expression of anatomical and organ signature genes in human mesenchymal tissue (<i>n</i> = 3). SPFs signature: <i>MSX1</i> (A), SMFs signature: <i>PITX1</i> (B), colon fibroblasts signature: <i>HOXA10</i> (C), stomach fibroblasts signature: <i>HOXB8</i> (D). (E) Immunofluorescence staining of <i>MSX1</i> in human colonic tissue. Arrow heads show vimentin-positive, spindle-shaped fibroblastic cells. (F) Semi-quantitative value of nuclear <i>MSX1</i> expression in human colonic and gastric tissue (<i>n</i> = 3).</p

MicroRNA Markers for the Diagnosis of Pancreatic and Biliary-Tract Cancers

<div><p>It is difficult to detect pancreatic cancer or biliary-tract cancer at an early stage using current diagnostic technology. Utilizing microRNA (miRNA) markers that are stably present in peripheral blood, we aimed to identify pancreatic and biliary-tract cancers in patients. With “3D-Gene”, a highly sensitive microarray, we examined comprehensive miRNA expression profiles in 571 serum samples obtained from healthy patients, patients with pancreatic, biliary-tract, or other digestive cancers, and patients with non-malignant abnormalities in the pancreas or biliary tract. The samples were randomly divided into training and test cohorts, and candidate miRNA markers were independently evaluated. We found 81 miRNAs for pancreatic cancer and 66 miRNAs for biliary-tract cancer that showed statistically different expression compared with healthy controls. Among those markers, 55 miRNAs were common in both the pancreatic and biliary-tract cancer samples. The previously reported miR-125a-3p was one of the common markers; however, it was also expressed in other types of digestive-tract cancers, suggesting that it is not specific to cancer types. In order to discriminate the pancreato-biliary cancers from all other clinical conditions including the healthy controls, non-malignant abnormalities, and other types of cancers, we developed a diagnostic index using expression profiles of the 10 most significant miRNAs. A combination of eight miRNAs (miR-6075, miR-4294, miR-6880-5p, miR-6799-5p, miR-125a-3p, miR-4530, miR-6836-3p, and miR-4476) achieved a sensitivity, specificity, accuracy and AUC of 80.3%, 97.6%, 91.6% and 0.953, respectively. In contrast, CA19-9 and CEA gave sensitivities of 65.6% and 40.0%, specificities of 92.9% and 88.6%, and accuracies of 82.1% and 71.8%, respectively, in the same test cohort. This diagnostic index identified 18/21 operable pancreatic cancers and 38/48 operable biliary-tract cancers in the entire cohort. Our results suggest that the assessment of these miRNA markers is clinically valuable to identify patients with pancreato-biliary cancers who could benefit from surgical intervention.</p></div

Plots of p-values in the analysis of pancreatic cancer (P can) (A) or biliary-tract cancer (B can) (B) with healthy control (HC) in z-axis, pancreato-biliary cancer (PB can) with non-malignant abnormalities (NMA) in y-axis, or pancreato-biliary cancer (PC can) with other types of cancer (OTC) in x-axis.

<p>The absolute values of the exponents of p-values on a log₁₀ scale were plotted; therefore, miRNAs that were located in further outside are more statistically significant. The ten miRNA used in the diagnostic indices were specified.</p

ROC analysis of a combination of four miRNAs (miR-6075, miR-6799-5p, miR-125a-3p, and miR-6836-3p) in a solid line, CEA in a dotted line, and CA19-9 in a slashed line.

<p>The AUC values were 0.949, 0.682 and 0.845 for a combination of the four miRNAs, CEA, and CA19-9, respectively. The analysis was performed in the test cohort.</p