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

Background & Aims: Approximately one-third of colorectal cancers develop from serrated lesions (SLs), including hyperplastic 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 immune 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 epithelium showed Paneth cell metaplasia with aberrant lysozyme expression. As for immune contexture, enhanced cytotoxic activity 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.