All-trans retinoic acid-driven immune remodeling in esophageal squamous cell carcinoma revealed by single-cell transcriptomics.

Esophageal squamous cell carcinoma (ESCC) remains a highly lethal malignancy with limited durable responses to current therapies. Our previous work showed that all-trans retinoic acid (ATRA) upregulates the deubiquitinase OTUD6B and suppresses ESCC stemness. Here, we integrated in vivo pharmacological treatment, single-cell RNA sequencing, bulk RNA sequencing, immunofluorescence, flow cytometry, and OTUD6B disruption to define how ATRA remodels ESCC tumors. In a syngeneic mEC25 tumor model, ATRA inhibited tumor growth, and the ATRA-plus-anti-PD-1 group showed greater tumor inhibition than the ATRA-alone group. Single-cell profiling of ATRA- and vehicle-treated tumors identified epithelial, stromal, lymphoid, and myeloid compartments and revealed extensive immune remodeling after ATRA treatment. ATRA reduced FOXP3-associated regulatory T-cell features, attenuated M2-like macrophage polarization, and expanded a CD74-high neutrophil state. In epithelial tumor cells, ATRA-regulated genes were enriched in ubiquitination-related protein homeostasis, including protein ubiquitination, ubiquitin-dependent protein catabolism, and proteasome-mediated degradation. These findings prompted transcriptomic analysis of human ESCC cells treated with ATRA or subjected to OTUD6B silencing. ATRA treatment was negatively associated with hallmark epithelial-mesenchymal transition (EMT) enrichment, whereas OTUD6B knockdown was positively associated with EMT enrichment. Importantly, OTUD6B disruption attenuated ATRA-associated tumor suppression and abolished ATRA-induced reductions in CD25+FOXP3+ regulatory cells and F4/80+CD206+ macrophages, as well as the induction of CD74+ neutrophils. These data support a possible contribution of OTUD6B to ATRA responsiveness and provide a rationale for further evaluation of ATRA-based immunotherapy combinations in ESCC.