Abstract Intestinal stem cells (ISC) are viewed as the origin of colorectal cancer (CRC), but the early stages of carcinogenesis are unclear. We hypothesize that early, non-transforming mutations reprogram ISCs into a preneoplastic state that increases the risk of transformation. We performed single-cell RNA sequencing (scRNA-seq) on colon epithelial cells from Car1-Cre (CAC) mice, which express Cre specifically in colon epithelial cells. CAC mice were crossed to those with floxed oncogene alleles to generate mice with one (Apc+/-, AC) or two (Apc+/-; KrasG12D/wt, AKC) non-transforming mutations. AC mice had normal crypt morphology and low adenoma incidence, while AKC mice exhibited crypt elongation by 4 weeks and multiple adenomas by 8-10 weeks. Cells from distal colonic crypts (n=7) and from tumors (n=3) representing phenotypes ranging from normal to malignant were profiled using the 10X Genomics Chromium. Processing and clustering with Cell Ranger and Seurat yielded 58,906 cells from crypts and 36,327 from tumors. Initial clustering identified epithelial, immune, endothelial, and cancer groups. The epithelial group was subclustered and visualized by UMAP. Cells from CAC crypts followed the classic differentiation path: Stem, Progenitor, Transit-amplifying, Absorptive epithelial cell, Goblet, Tuft, and Enteroendocrine. Stem cell clusters were classified as Normal (CAC), Abnormal 1 (young AKC), or Abnormal 2 (older AKC). The abnormal ISC states in the crypt structures are distinct from the cancer stem cells found in the cancer group. Trajectory analysis revealed that each stem cell population follows a distinct differentiation sequela, with abnormal ISC giving rise to transcriptionally divergent epithelial lineages. This was validated by integrating scRNA-seq data with spatial transcriptomics data from the same lines, showing that abnormal epithelial populations progressively overtake the normal crypt epithelium while retaining crypt structure. Differential gene expression analysis and Gene Set Enrichment Analysis showed that normal ISC was enriched for classic stemness markers (e.g., Lgr5, Lrig1, Smoc2). Abnormal 1 ISC began to lose these features and instead exhibited increased expression of cell-cycle regulators (e.g., Ccna2, Aurkb) and markers of oxidative stress (e.g., Prdx4, Gsta3, Gpx2). Gene expression profiles in Abnormal 2 ISC showed enrichment of pathways controlling p53 (e.g., Cdkn1a, Smad7), RAS/MAPK (e.g., Map2k1, Epha2), and PI3K/Akt/mTOR signaling (e.g., Akt3, Pik3r3, Mtor), as well as for an inflammatory response (e.g., Fos/Jun, Nfkbiz, Il18). Collectively, these findings show that early, non-transforming mutations push ISC into preneoplastic states marked by increased proliferation and stress response signaling favorable for malignant progression. Citation Format: Yujin Lee, Moray J. Campbell, James C. Fleet. Single-cell mapping of the colon epithelium reveals a pre-malignant stem cell that defines early transformation risk abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5922.
Lee et al. (Fri,) studied this question.