Single-cell Transcriptomics Reveals Early Molecular and Immune Alterations Underlying the Serrated Neoplasia Pathway Toward Colorectal Cancer

BACKGROUND & AIMS: Approximately one-third of colorectal cancers develop from serrated lesions (SLs), including hyper -plastic polyp (HP), sessile serrated lesion (SSL), traditional serrated adenoma (TSA), and SSL with dysplasia (SSLD) through the serrated neoplasia pathway, which progresses faster than the conventional adenoma-carcinoma pathway. We sought to depict the currently unclarified molecular and im-mune alterations by the single-cell landscape in SLs. METHODS: We performed single-cell RNA sequencing of 16 SLs (including 4 proximal HPs, 5 SSLs, 2 SSLDs, and 5 TSAs) vs 3 normal colonic tissues. RESULTS: A total of 60,568 high-quality cells were obtained. Two distinct epithelial clusters with redox imbalance in SLs were observed, along with upregulation of tumor-promoting SerpinB6 that regulated ROS level. Epithelial clusters of SSL and TSA showed distinct molecular features: SSL-specific epithelium manifested overexpressed proliferative markers with Notch pathway activation, whereas TSA-specific epithe-lium showed Paneth cell metaplasia with aberrant lysozyme expression. As for immune contexture, enhanced cytotoxic ac-tivity of CD8(+) T cells was observed in SLs; it was mainly attributable to increased proportion of CD103(+)CD8(+) tissue -resident memory T cells, which might be regulated by retinoic acid metabolism. Microenvironment of SLs was generally immune-activated, whereas some immunosuppressive cells (regulatory T cells, anti-inflammatory macrophages, MDK(+)IgA(+) plasma cells, MMP11-secreting PDGFRA(+) fibroblasts) also emerged at early stage and further accumulated in SSLD. CONCLUSION: Epithelial, immune, and stromal components in the serrated pathway undergo fundamental alterations. Future molecular subtypes of SLs and potential immune therapy might be developed.