Transcriptome-wide In Vitro Effects of Aspirin on Patient-derived Normal Colon Organoids

Abstract

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

Figure 1:. Differential expression analysis of aspirin treated colon organoids.

A) Volcano plot of DEGs. DEGs with FDR corrected q-values that met significance (red dots) appear above the FDR threshold (horizontal hashed line). Positive log₂fold changes (right of vertical hashed line) indicate higher expression in aspirin treated samples compared to vehicle controls, whereas negative log₂fold changes (left of vertical hashed line) indicate lower expression compared to vehicle controls. Connectors were placed to highlight the most significant 20 DEGs associated with aspirin exposure. B) Plot of five most significantly enriched Gene Ontology terms for each category: Biological Process (BP); Cellular Component (CC) and Molecular Function (MF). Direction corresponds to whether nominally over-expressed (triangle) or under-expressed (circle) DEGs were used for pathway enrichment analysis. Size of each pathway corresponds to the overlap (%) of DEGs identified in aspirin response versus the total number of genes in the pathway annotation.

Figure 2:. Single-cell deconvolution of bulk RNA-seq from aspirin exposed colon organoids.

Nominally significant (P=0.05; light blue) and FDR corrected (q=0.10; dark blue) DEGs were overlaid with known cell markers of colon cell types as identified through scRNA-seq. Positive values indicate the number of genes displaying increased expression, while negative values indicate number of genes with reduced expression in A) aspirin treated organoids or B) regular users of aspirin.

Figure 3:. Differential expression analysis of aspirin-treated colon organoids following adjustment for cell composition.

A) Volcano plot of significant DEGs that were also identified in original analysis (blue) and novel (red). B) Inspection of fold change of nominal DEGs that are positively (orange) or negatively (blue) correlated with Wnt signaling, ranked from left to right in order of significance.

Figure 4:. Bar plot to show significance of modules defined through WGCNA with relation to aspirin treatment status.

Extent of positive correlation to aspirin treatment can be visualized as deepening red hue. For each significant module, the genes with the highest module memberships were annotated. PPI enrichment analysis was performed for each significant module by uploading genes found in each module to STRING. Significance of PPI enrichment can be observed by a deepening blue hue next to the relevant module. Size of circle refers to the number generated when dividing the number of observed edges in the PPI network by the number of expected edges given the size of the network analyzed.