Single-cell RNA sequencing of appendiceal adenocarcinoma reveals a low proportion of epithelial cells and a fibroblast enriched tumor microenvironment.

Background: Appendiceal adenocarcinoma (AA) is an understudied gastrointestinal malignancy. Treatment is guided by its proximal counterpart, colorectal cancer (CRC), despite recent studies demonstrating AA's unique mutational landscape and poor response to CRC chemotherapy. In this study, we describe AA on a single-cell level and uncover features highlighting the contrast between AA and CRC; we believe these findings will support AA as a unique disease entity and encourage further disease-specific focus. Materials and methods: Three patients with peritoneal metastases from AA and one from CRC profiled with 5' single-cell RNA sequencing. Results: Traditional k-means clustering analysis of >30 000 cells revealed three canonical compartments and 11 major cell types. AA samples were mostly comprised of stromal cells (56%), while healthy appendix samples had significantly more immune and epithelial cells. Strikingly, fibroblasts were the most abundant cell type in AA with cancer-associated fibroblasts from the mucinous AA tumors showing a distinct profile from goblet cell AA or CRC. Pseudobulk analysis comparing tumor cells from AA with normal appendiceal epithelial cells demonstrated up-regulation of a diverse range of oncogenic pathways including inflammatory, epithelial-mesenchymal transition, and angiogenesis. Conclusions: As the first application of single-cell technology to AA these data provide insight into the intratumor heterogeneity of AA and highlight the important contribution of the tumor microenvironment in this orphan disease. These results also reinforce multiple observations that AA is a unique disease entity from CRC and targeting the tumor microenvironment should be considered as a therapeutic strategy.