Supplementary Material for: Network Analysis of Gut Microbiota Including <b><i>Fusobacterium</i></b> and Oral Origin Bacteria and Their Distribution on Tumor Surface, Normal Mucosa, and in Feces in Patients with Colorectal Cancer

Introduction: Fusobacterium and several bacteria are reported to be associated with colorectal cancer (CRC). However, their relationship and whether they cause CRC or are just adapted to the cancerous environment is not known. We approached this subject by investigating the correlation and distribution of the bacteria throughout the colon in patients with CRC and elucidated the relationship between microbiota and CRC. Methods: Twenty-five patients with CRC who underwent colonoscopy for endoscopic submucosal dissection or surgery were prospectively enrolled. Fecal samples were taken before bowel preparation, and mucosal samples were collected from three sites (tumor surface, tumor-adjacent mucosa, and cecum) during colonoscopy using a cytology brush. The microbiota was identified and analyzed by sequencing of the 16S rRNA gene of the V3–V4 region. We evaluated the correlation between the bacteria based on network analysis and the distribution of Fusobacterium in the colon. Results: A network consisting of many bacteria was found in all sites; especially, oral origin bacteria including Fusobacterium formed a positively correlated network on tumor surface. Streptococcus showed a significantly higher relative abundance on tumor surface than in feces. The relative abundance of Fusobacterium had significant positive correlations between tumor surface and feces, tumor-adjacent mucosa, and cecum. Conclusion: In patients with CRC, many bacteria were correlated with each other, and Fusobacterium and oral origin bacteria formed a positively correlated network on tumor surface. Fusobacterium was equally distributed on tumor surface and throughout the lumen and mucus in the colon. In the colon where Fusobacterium is widely distributed, Fusobacterium would adhere to the tumor surface and be correlated with oral origin bacteria to make a microenvironment that is favorable for CRC

Supplementary Material for: Reduced DEFA5 expression and STAT3 activation underlie the submucosal invasion of early gastric cancers

Introduction Submucosal invasion is a core hallmark of early gastric cancer (EGC) with poor prognosis. However, the molecular mechanism of the progression from intramucosal gastric cancer (IMGC) to early submucosal-invasive gastric cancer (SMGC) is not fully understood. The objective of this study was to identify genes and pathways involved in the submucosal invasion in EGC using comprehensive gene expression analysis. Methods Gene expression profiling was performed for eight cases of IMGC and eight cases of early SMGC with submucosal invasion ≥ 500 μm. To validate the findings of gene expression analysis and to examine the gene expression pattern in tissues, immunohistochemical (IHC) staining was performed for 50 cases of IMGC and SMGC each. Results Gene expression analysis demonstrated that the expression levels of small intestine-specific genes were significantly decreased in SMGC. Among them, defensin alpha 5 (DEFA5) was the most downregulated gene in SMGC, which was further validated in SMGC tissues by IHC staining. Gene set enrichment analysis showed a strong association between SMGC, the JAK-STAT signaling pathway, and the upregulation of STAT3-activating cytokines. The expression of phosphorylated STAT3 was significant in the nucleus of tumor cells in SMGC tissues but not in areas expressing DEFA5. Conclusion The results of this study strongly suggest that the downregulation of DEFA5 and the activation of STAT3 play a significant role in the submucosal invasion of EGC

Supplementary Material for: Gastroesophageal Reflux Disease-Related Disorders of Systemic Sclerosis Based on the Analysis of 66 Patients

<b><i>Background/Aims:</i></b> Gastroesophageal reflux disease (GERD)-related disorders of systemic sclerosis (SSc) patients have not been adequately investigated. <b><i>Methods:</i></b> Sixty-six SSc patients (5 males and 61 females; 56.6 ± 14.6 years old) who underwent esophagogastroduodenoscopy were analyzed on the basis of 16 background factors. They were additionally compared with 116 matched non-SSc subjects controlling age, sex, and use of proton pump inhibitors (PPIs). <b><i>Results:</i></b> The mean disease duration of 66 patients was 5.1 ± 8.1 years, and their breakdown was as follows: 53 (80.3%) with GERD, 38 (57.6%) with GERD-related symptoms, and 20 (30.3%) with reflux esophagitis (RE; LA-A: 10, LA-B: 5, LA-C: 4, LA-D: 1). Use of PPI (<i>p</i> = 0.0455), complication of interstitial lung disease (<i>p</i> = 0.0242), and history of cyclophosphamide therapy (<i>p</i> = 0.0184) denoted significant association with GERD-related symptoms. Older age (<i>p</i> = 0.0211) was significantly associated with RE. None of GERD-related disorders showed any difference between 37 diffuse cutaneous SSc and 29 limited cutaneous SSc patients. The matched analysis indicated that SSc patients had higher prevalence of GERD (<i>p </i>< 0.0001), GERD-related symptoms (<i>p</i> = 0.0034), and RE (<i>p </i>= 0.0002). <b><i>Conclusion:</i></b> SSc patients tend to have worse GERD symptoms and severer RE. However, most SSc-associated factors did not show significant association with GERD-related disorders, indicating the difficulty in predicting GERD-related disorders among SSc patients