Abstract 6602: Spatial transcriptomic profiling of the mouse distal colon during early carcinogenesis reveals transitional cell states that precede cancer

Abstract Early onset colorectal cancer (EOCRC) is increasing yet the mechanism for the accelerated shift to a younger age is unknown. We hypothesize that sporadic gene mutations that are necessary for colon carcinogenesis but not sufficient to cause cancer, create a preneoplastic cell state for increased risk of transformation. To identify transitional cell phenotypes, we used Spatial Transcriptomics (ST) on distal colon samples from Car1-Cre (CAC) mice with Cre expression limited to ∼6% of the distal colon/rectum epithelium, where EOCRC is more prevalent. CAC;ROSAmT/mG (CR) mice (where eGFP marks cells expressing Cre) were crossed to those with floxed oncogene alleles to generate mice with one (Apc+/-, ACR) or two (Apc+/-;KrasG12D/wt, AKCR) non-transforming mutations. ACR mice had normal crypt morphology and low adenoma incidence (<5% of mice), while 4-week-old AKCR mice exhibited crypt elongation, and 8-10 wk-old AKCR developed 3-8 advanced adenomas/mouse. Distal colon Swiss rolls (n=9) and tumors (n=3) with phenotypes ranging from normal to malignant were collected and processed using the 10X Visium Spatial Gene Expression System. Sequencing data was processed, and sample data were pooled for analysis using Seurat. Spatial counts were normalized, dimensionality was reduced with PCA, then clustered and visualized as UMAP projections. Clusters were annotated to known cell types, and differential gene expression (DEG) analysis was followed by functional annotation using GO and Pathway terms. In normal tissue, clusters for the crypt base, middle, and top were identified. These spatially restricted clusters were replaced in early AKCR crypts and again in late AKCR crypts. The clusters from late AKCR crypts were distinct from those in ACR tumors, which were different from clusters in AKCR tumors. This indicates the existence of transition states from normal to “not normal, not cancer” to early adenoma to late adenoma. Normal epithelial clusters were enriched for genes controlling typical colonic functions, e.g. metabolism, and transmembrane transport pathways. AKCR crypt clusters lost these functions and gained others, e.g. immune and antimicrobial genes normally associated with Paneth cells (e.g., Defa22, Defa24, Defa30), gene markers associated with CRC tumors (e.g. Egfr, Hamp, Plet1, Wfdc18), genes for xenobiotic inactivation (Maoa, various Cyp, Gst, Ugt, and Sult genes). Compared to late AKCR crypts, AKCR tumor clusters lost even more colonic functions (e.g. downregulation of differentiation marker Krt20 and xenobiotic metabolism markers) and expressed genes related to mitosis, DNA replication, and DNA repair, highlighting a transition to uncontrolled proliferation and genomic instability. Our findings delineate key early molecular transitions in epithelial cells during colon carcinogenesis that may increase the risk of transformation. Citation Format: Yujin Lee, James C. Fleet. Spatial transcriptomic profiling of the mouse distal colon during early carcinogenesis reveals transitional cell states that precede cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6602.