Abstract 2359: Age-dependent transcriptional changes in colonic fibroblasts revealed through single-cell RNA sequencing

Abstract Introduction: Fibroblasts are key enhancers of colorectal cancer (CRC) development. Cancer-associated fibroblasts (CAF)s promote oncogenesis via extracellular matrix remodeling and the secretion of inflammatory cytokines. CAFs likely arise from normal tissue-resident fibroblast populations that are altered with aging/senescence and environmental exposures. Thus, fibroblasts may mediate CRC development by amplifying etiologic exposures or age-related mucosal changes. This study examines age-related transcriptional changes in fibroblasts from intestinal biopsies to assess the effects of aging. Methods: Using 10X Genomics Chromium single-cell RNA sequencing, we profiled cells derived from rectosigmoid biopsies of 48 individuals with a history of colorectal adenoma (Young: 24, aged <55; Older: 24, aged >65). Cellranger, Cellbender, and Seurat were used to derive gene counts, cell types, and clusters. Analyses focused on the stromal cell compartment. Milo and Nebula identified age-related differences in cell abundance and differentially expressed genes (DEGs). Results: Among 61, 605 stromal cells, 39, 074 fibroblasts (ADAMDEC1high) were subtyped as crypt top fibroblasts (CTFs; PDGFRAhigh), colon crypt fibroblasts type-1 (cCF1, PDGFRAlow/GREM1-), colon crypt fibroblasts type-2 (cCF2, GREM1+/RSPO3+), and fibroblastic reticular-like cells (FRLCs, CCL19+). We did not observe changes in the abundance of fibroblasts between the age groups. However, we found 181 ccF1, 68 ccF2, and 44 CTF specific age-associated DEGs. FRLCs had one significant DEG: PLAU (LogFC = 1.96; FDR = 0.021), a factor that converts fibroblasts into CAFs. In cCF1, GDF15 (LogFC = 1.16; FDR = 5.73 x 10-5), a marker of senescence and CRC risk, was the most upregulated age-associated gene. Gene set enrichment analysis revealed several inflammaging pathways were upregulated in aged fibroblasts, including interleukin-2, interleukin-6, tumor necrosis factor-α, and interferon-γ pathways. Additionally, KRAS signaling (all fibroblasts), p53 (cCF1), and epithelial-mesenchymal transition (cCF2) were upregulated with aging, while Myc targets (all fibroblasts) were downregulated. Conclusions: Fibroblasts in individuals at CRC risk acquire a senescent phenotype with aging, promoting a chronic inflammatory microenvironment. These changes may alter colon crypt regeneration and increase susceptibility to CRC risk factors. Further studies may clarify fibroblast roles in early- and typical-onset CRC. Citation Format: Jonathan M. Downie, Ryan J. Musich, Saleh Khawaled, Alexander Caraballo, Kylor Lachut, Shijie He, Marina Magicheva-Gupta, Ingrid Crumpton, Liza Hote, Karen Kirunda, Perry Labelle, Omer H. Yilmaz, Thaddeus Stappenbeck, Andrew T. Chan, David A. Drew. Age-dependent transcriptional changes in colonic fibroblasts revealed through single-cell RNA sequencing [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 2359.