Single Cell RNA sequencing (DropSeq) of esophageal adenocarcinoma and matched normal samples

Cancer grows within the complex ecosystem of the tumor microenvironment (TME) which can define responses to treatment. Esophageal adenocarcinoma (EAC) is typically resistant to cytotoxic therapies and immunotherapies have gained little traction against this disease. We used single cell RNA sequencing to identify the key constituents of the TME in EAC in a cohort of 22 patients. We found differences within and between individual patients in cancer cells and also in the stromal and immune cell populations. We found a subtype of cancer-associated fibroblasts, the myofibroblastic CAFs (myoCAFs), acting as TME interaction hubs to drive the hallmarks of cancer. Finally, we demonstrated the efficacy of repurposed PDE5 inhibitors in targeting myoCAFs in near-patient models of the disease. Overall design: EAC tumor and normal tissue samples were obtained from patients from a single centre. To isolate single cells for droplet-based sequencing (Drop-seq) tumor material obtained by 8mm biopsy punch from the epicenter of the tumor on the luminal surface (or at the proximal esophageal resection margin >5cm from tumor to obtain normal tissue), was enzymatically dissociated using a protocol optimized to recover all of the broad cell categories in the TME. Following the Drop-seq procedure, approximately 1,000 single-cell transcriptomes per sample were selected for sequencing using Illumina NextSeq 500. In total, after filtering for feature cut-offs (number of genes, % mitochondrial content, dissociation induced artefacts. STAR Methods), we retained 16,328 cells and 28,620 genes for downstream analysis. In total 10,250 cells were from tumors and 6,078 cells were from normal esophagus.