Mechanisms underlying aspirin chemoprevention of transit-amplifying cells following aspirin treatment (P ¼ colorectal cancer remain unclear. Prior studies have been 0.01). Following deconvolution, DEGs included novel limited because of the inability of preclinical models to putative targets for aspirin such as TRABD2A (q ¼ recapitulate human normal colon epithelium or cellular 0.055), a negative regulator of Wnt signaling. Weighted heterogeneity present in mucosal biopsies. To overcome gene co-expression network analysis identified 12 signifsome of these obstacles, we performed in vitro aspirin icant modules, including two that contained hubs for treatment of colon organoids derived from normal mucoEGFR and PTGES2, the latter being previously implicated sal biopsies to reveal transcriptional networks relevant to in aspirin chemoprevention. In summary, aspirin treat-aspirin chemoprevention. Colon organoids derived from ment of patient-derived colon organoids using physiolog-38 healthy individuals undergoing endoscopy were treated ically relevant doses resulted in transcriptome-wide with 50 mmol/L aspirin or vehicle control for 72 hours and changes that reveal altered cell composition and improved subjected to bulk RNA sequencing. Paired regression understanding of transcriptional pathways, providing analysis using DESeq2 identified differentially expressed novel insight into its chemopreventive properties. genes (DEG) associated with aspirin treatment. Cellular composition was determined using CIBERSORTx. Aspirin Prevention Relevance: Numerous studies have highlight-treatment was associated with 1,154 significant (q < 0.10) ed a role for aspirin in colorectal cancer chemoprevention, DEGs prior to deconvolution. We provide replication of though the mechanisms driving this association remain these findings in an independent population-based RNA-unclear. We addressed this by showing that aspirin treat-sequencing dataset of mucosal biopsies (BarcUVa-Seq), ment of normal colon organoids diminished the transit-where a significant enrichment for overlap of DEGs was amplifying cell population, inhibited prostaglandin syntheobserved (P < 2.2E–16). Single-cell deconvolution revealed sis, and dysregulated expression of novel genes implicated in changes in cell composition, including a decrease in colon tumorigenesis.