Comparative single-cell analysis of esophageal cancer subtypes reveals cellular distinctions explaining varied immunotherapy responses

Esophageal cancer comprises two main subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Previous studies revealed their distinct genomic characteristics, sharing similarities with head and neck squamous cell carcinoma (HNSCC) and gastric adenocarcinoma (GAC) for ESCC and EAC, respectively. This study compares ESCC and EAC at single-cell resolution to identify factors explaining differences in immunotherapy response rates. We performed comparative single-cell transcriptome analysis for four cancer types near the esophagus using data from 35 patients with ESCC, EAC, HNSCC, or GAC. Malignant epithelial cells showed distinct separations between subtypes based on histological origins. We identified enrichment of CXCL13+ CD8+ T cells and CXCL9+CXCL10+ TAMs in ESCC and HNSCC, promoting a hot-tumor environment through interferon-? pathways. Conversely, hypoxia and cold-tumor related populations, such as heat-shock protein high CD8+ T cells and MARCO+ TAMs, were enriched in EAC and GAC. These immune subsets' expression signatures predicted immunotherapy responses across diverse cancer types. Overall design: Libraries were prepared using the Chromium controller following the protocol outlined in the 10x Chromium Next GEM Single Cell 5-v2 Cell Surface Protein User Guide (CG000330). Initially, cell suspensions were diluted with nuclease-free water to achieve a targeted cell count of 10,000. These suspensions were then mixed with master mix and loaded with Single Cell 5' Gel Beads and Partitioning Oil into a Next GEM Chip K. Within the droplets, RNA transcripts from single cells were uniquely barcoded and reverse-transcribed. The purified libraries were quantified using qPCR following the qPCR Quantification Protocol Guide (KAPA) and qualified using the Agilent Technologies 4200 TapeStation. Finally, the libraries were sequenced on the HiSeq platform (Illumina) according to the specified read length in the user guide.