The gatekeeper to gastric cancer; gastric microbiota invade the lamina propria in Helicobacter pylori-associated gastric carcinogenesis

Stomach cancer is the fourth leading cause of cancer-related deaths worldwide. Helicobacter pylori is the main risk factor for gastric adenocarcinoma (GAC), yet the mechanism underpinning this association remains uncharacterised. Gastric intestinal metaplasia (GIM) represents the pre-cancerous stage and follows H. pylori- associated chronic gastritis (CG). Sequencing studies have revealed fewer H. pylori and more non- H. pylori bacteria in GAC. However, the spatial organisation of the gastric microbiota in health and disease is unknown. Here, we have combined RNA in situ hybridisation and immunohistochemistry to detect H. pylori , non- H. pylori bacteria and host cell markers (E-cadherin, Mucins 5AC and 2) from patients with CG (n=9), GIM (n=12), GAC and normal tissue adjacent to tumours (NATs) (n=3). Quantitative analysis of whole slide scans revealed significant correlations of H. pylori and other bacteria in CG and GIM samples. In contrast to sequencing studies, significantly fewer non- H. pylori bacteria were detected in H. pylori- negative patients. Importantly, whilst H. pylori exclusively colonised the gastric glands, non- H. pylori bacteria invaded the lamina propria in 3/4 CG and 5/6 GIM H. pylori -positive patients. Bacterial invasion was observed in 3/3 GAC samples and at higher levels than matched NATs. We propose that H. pylori ‘holds the keys’ to disrupt the gastric epithelial barrier, facilitating the opportunistic invasion of non- H. pylori bacteria to the lamina propria. Bacterial invasion could be a significant driver of inflammation in H. pylori- associated carcinogenesis. This proposed mechanism would both explain the synergistic roles of H. pylori and other bacteria and redirect attempts to prevent, diagnose and treat GAC. ### Competing Interest Statement The authors have declared no competing interest.