
Immune Remodeling in Barrett’s-Associated Esophageal Adenocarcinoma: Insights from scRNA-seq
Setareh Azarkar 1 ℗, Hossein Safarpour 2 ©, Arezou Khosrojerdi 3
Abstract
Introduction: Barrett’s esophagus, a condition arising from chronic gastroesophageal reflux disease (GERD), is marked by the replacement of normal squamous epithelium with columnar cells and is a key precursor to esophageal adenocarcinoma (EAC). EAC is an aggressive cancer with increasing incidence, and a poor five-year survival rate under 20%. Despite advances in treatment strategies, responses are various among patients. Owing to the pivotal role of immune cells, particularly tumor-infiltrating lymphocytes, in the tumor microenvironment (TME) and determining treatment efficacy and prognosis, we used single-cell RNA sequencing (scRNA-seq) as a powerful tool for profiling cellular heterogeneity to explore immune cell dynamics from healthy tissue through Barrett’s esophagus to EAC, aiming to identify predictive biomarkers and inform targeted therapies. Methods and Materials: To begin, raw single-cell sequencing data from 12 patients with n=64 samples from normal samples to metaplastic, dysplastic and Tumors were obtained from the GEO database using the code GSE251990. Then the Scanpy toolkit was leveraged for data analysis. Initially, we did quality control and normalization, and then cell clustering based on the expression of specific gene markers was performed. We also visualized the immune cell fractions, distinguished rank-genes groups, etc., and for statistical analysis, the Bonferroni formula was used to calculate the adjusted p-value. Results: Finally, we had 72,126 cells from 64 different samples and distinguished more than 20 immune cell subtypes by their marker genes. We found that in the early stages, like normal esophagus and metaplasia, high levels of effector and resident memory T cells were infiltrated, but as the disease progressed to dysplasia and tumor stages, there was a notable increase in regulatory T cells. And a progressive increase was seen in the expression of inhibitory immune checkpoints such as CD274, IDO1, and VSIR, across metaplasia to tumor formation in the tissue microenvironment. We investigated the highly variable expressed genes and their related biological pathways. For example, in the tumor samples, pathways related to response to interferon were highly upregulated, while in metaplastic samples, there was upregulation of NK T cell activation pathways. And finally, we discovered a novel gene named LMNA with higher expression in gastric adenocarcinoma, suggesting a potential role in tumor progression. Conclusion and Discussion: We could elucidate the dynamic changes in immune cell populations through the progression of Barrett’s esophagus to esophageal adenocarcinoma and, in comparison, with normal esophageal tissue using scRNA-seq analysis. We identified different subtypes of immune cells and saw their changes in different disease stages and findings that underscore the immunosuppressive tumor microenvironment in EAC and suggest potential biomarkers and therapeutic targets. Our findings show how single-cell analysis can be used to identify molecular and cellular pathways that might be valuable for immunotherapy in patients with esophageal cancer.
Keywords: Esophageal Adenocarcinoma, Barrett’s Esophagus, Single-Cell RNA-seq, Tumor Microenvironment