Abstract B39: Understanding the association of gut microbiota and tumor microenvironment in gastric and esophageal cancer
Authors: Thakkar Prashant V. , Zhang Chao , Sharma Prateek , Vennelaganti Sreekar , Betel Doron , Shah Manish A. 
Published: February 2019

Gastric and esophageal cancers remain the second and the sixth leading cause of cancer related deaths respectively. Dysbiosis of the gut microbiome remains a strong risk factor for gastrointestinal cancer, likely related to altered mucosal immunity and a pro-inflammatory immune microenvironment. However, the molecular interactions between host human cells and the microbiome are poorly understood as it pertains to gastric and esophageal cancer. Our main goal is to understand how alteration of regional mucosal microbiome alters the tumor microenvironment, thus creating conditions conducive to tumorigenesis. Understanding these changes at the molecular level will help contribute to our knowledge of the disease pathophysiology.

Using a novel customized computational pipeline to identify and characterize bacteria from low microbial content endoscopic samples (Zhang et al., Genome Biol 2015), we used low-pass (10-30x) whole genome sequencing (WGS) to examine the microbiome composition of 15 gastric cancer samples along with their adjacent non-cancer mucosal tissue (n=30). We found a higher overall bacterial content in tumor samples compared to that of their corresponding matching normal tissues. Furthermore, we observed a dysbiosis in samples positive for Helicobacter pylori, consistent with previous findings that H. pylori alters composition of the gastric microbiome. Although a wide variety of other microbes were detected, we found specific enrichment of organisms like Veilonella Parvula (12/15) and Prevotella melaninogenica (10/15) in tumor tissues, both of which have been implicated in tumorigenicity.

A subset of these samples (10 gastric cancer and 12 adjacent normal) underwent parallel RNAseq analysis and using a gene expression signature panel of 176 genes compiled from several published series, we examined immune cell types, specifically B cells, CD8+ T cells, Treg cells, CD4+ Th1, Th2 and Th17 helper cells and macrophages, to determine an association with tumor versus the normal mucosa. We found an overall higher immune response, and more specifically a higher association of Th1 and Th2 helper cells as well as macrophages, with tumors as compared to normal mucosa. We further corroborated these findings using a single cell RNA sequencing approach on three tumor and matching normal samples. Using ELISA, we detected a significantly increased expression of pro-inflammatory cytokines such as TNFα, IL-8, GRO, MCP-1 and IL-1a in tumor samples than in normal mucosa. Increased expression of TNF-α and IL-8 has been directly correlated to tumor invasion and metastasis, and tumor vascularity respectively. Additionally, MCP-1 has been shown to be important in recruitment of macrophages and is secreted by gastric epithelial cells in response to pro-inflammatory cytokines upon H. pylori infection.

We used a similar approach, of performing WGS and RNAseq analysis in parallel, on 9 normal squamous cell carcinoma, 8 non-dysplastic Barrett’s esophagus and 6 esophageal adenocarcinoma samples (n = 23 samples). WGS analysis revealed a higher microbial diversity in normal squamous cell carcinomas compared to the non-dysplastic Barrett’s esophagus with high abundance of Veilonella Parvula, Prevotella melaninogenica and Streptococcus parasanguinis. Unsupervised clustering analysis of the entire data set revealed high abundance of Fusobacterium nucleatum in adenocarcinoma samples, which has been associated with a shorter survival in Esophageal cancer. Unsupervised clustering analysis of RNAseq results revealed a high enrichment of CD4+ Th1 and Th2 helper cell as well as CD8+ T cell associated immune response in some adenocarcinoma and non-dysplastic Barrett’s esophageal samples.

Together, our data are derived from endoscopic biopsy samples from patients, and suggest that both gastric and esophageal cancers display distinct microbial patterns associated with chronic inflammation and a tumor-promoting pro-inflammatory microenvironment.

Citation Format: Prashant V. Thakkar, Chao Zhang, Prateek Sharma, Sreekar Vennelaganti, Doron Betel, Manish A. Shah. Understanding the association of gut microbiota and tumor microenvironment in gastric and esophageal cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B39.