The histological changes in the gastric epithelium are crucial in the progression from premalignant to neoplastic lesions. TLR4, TLR5 and TLR9 have been localized in the gastric epithelium and studied separately using conventional histological techniques without a focus on the protein or cell interactions within the microenvironment. Therefore, we developed a multiplex immunohistochemistry/immunofluorescence (mIHC/IF) technology for the simultaneous detection of TLR4, TLR5 and TLR9 on a single tissue section of human gastric biopsies from 10 paired cases collected in two independent visits, and its correlation with the OLGA/OLGIM scoring and H. pylori status after eradication. The results confirmed that mIHC/IF is useful for simultaneously interrogating six biomarkers and demonstrated that TLR4 and TLR9 are significantly associated with H. pylori infection. However, only TLR9 is positively related to the presence of intestinal metaplasia. TLR5 was mainly present in goblet cells (TFF3+) but did not show any significant association with H. pylori or the presence of intestinal metaplasia. Our results suggest that a more comprehensive strategy to interrogate the tissue microenvironment in premalignant lesions may improve the interpretation of the earned risk of gastric cancer in patients with chronic gastritis and evidence of failure in H. pylori eradication.

Chemoresistance is one of the major barriers to effective treatment in gastric cancer (GC), contributing to disease progression and poor patient survival. SRY-box transcription factor 5 (SOX5) has been associated with aggressive cancer phenotypes in several malignancies; however, its relationship with chemoresistance in GC has yet to be fully characterized. This study investigated whether SOX5 expression is associated with molecular features linked to aggressive GC phenotypes, including multi-drug resistance proteins, cancer stem cell (CSC) antigens, epithelial–mesenchymal transition (EMT) markers, and proteins involved in drug efflux and DNA repair. SOX5 expression was evaluated in AGS and MKN-45 GC cell lines using Western blotting, real-time quantitative PCR, and immunofluorescence. Markers associated with EMT (Snail1, ZEB1, and E-cadherin), CSC phenotypes (CD44 and CD133), DNA repair (XRCC1 and ERCC2), and drug efflux (ABCB1) were analyzed. Cellular sensitivity to 5-fluorouracil (5-FU) and oxaliplatin (Ox) was assessed using an MTT-based viability assay. In addition, publicly available GC datasets were analyzed to evaluate the association between SOX5 expression and overall survival. AGS cells exhibited significantly higher SOX5 expression at both the mRNA and protein levels compared with MKN-45 cells. High SOX5 expression was associated with increased expression of the transcription factors Snail1 and ZEB1, which are associated with EMT, leading to decreased E-cadherin expression. Furthermore, SOX5 expression was associated with increased expression of CD133, the DNA repair genes XRCC1 and ERCC2, and ABCB1 transcripts. Consistent with these molecular findings, AGS cells exhibited greater viability following exposure to 5-FU and Ox, either alone or in combination, suggesting reduced sensitivity to these agents. Furthermore, high SOX5 expression was associated with poorer overall survival across independent GC patient cohorts. Overall, these findings suggest SOX5 as a potential biomarker associated with an aggressive molecular phenotype characterized by EMT activation, enhanced DNA repair capacity, stemness-associated features, and reduced sensitivity to FOLFOX-based chemotherapy. Future mechanistic studies are warranted to establish whether SOX5 directly drives these processes.