Oral Presentations

Background: Caco-2 cell line is the gold standard in vitro model for studying xenobiotic intestinal absorption and metabolism. Nevertheless, it does not fully replicate normal intestinal epithelium and shows cellular heterogeneity. To overcome this, Caco-2/TC-7 was isolated, but both lines are tumour-derived. The porcine IPEC-J2 line represents a non-tumorigenic alternative. Hypothesis: We hypothesised that these cell lines exhibit distinct transcriptomic profiles reflecting differential responses to external stressors, including xenobiotics. Methods: Cells were differentiated for 21 days. Total RNA was extracted and sequenced (Illumina NovaSeqX, PE150). Reads were processed using nf-core/rnaseq pipeline and quantified with Salmon. For IPEC-J2 vs Caco-2, one-to-one orthologs were identified. Differential expression analysis for IPEC-J2 vs Caco-2 and TC-7 vs Caco-2 was performed with DESeq2 (padj < 0.01; |log2FC| > 1). Functional enrichment was conducted using clusterProfiler (GO Biological Process, q-value < 0.01). Results: Intra-species analysis identified 2,667 differentially expressed genes (DEGs). TC-7 showed upregulation of genes involved in xenobiotic biotransformation (e.g., CYP3A4/5, UGT1A1) and lipid metabolism (e.g., CYP4F12), and downregulation of cholesterol- (e.g., INSIG1) and cytoskeletal- related genes (e.g., VIM). Cross-species analysis identified 7,175 DEGs. IPEC-J2 showed higher expression of extracellular matrix (ECM) and epithelial organization genes (e.g., CCN3, MUC1), whereas Caco-2 exhibited upregulation of lipid/nutrient transport (e.g., APOA4, SLC23A1) and detoxification- related genes (e.g., GPX1/4). Conclusion: Distinct transcriptional pathways related to epithelial organization and metabolic specialization were identified across models. Compared with Caco-2, TC-7 showed upregulation of genes involved in xenobiotic and lipid metabolism, supporting its use in biotransformation studies. Compared to Caco-2, IPEC-J2 showed higher expression of ECM and structural genes, suggesting a more dynamic epithelial organization; compared to IPEC-J2, Caco-2 showed upregulation of detoxification-related transcripts, consistent with greater metabolic capacity. These findings underscore the importance of selecting the most appropriate in vitro model based on specific research goals. Acknowledgements: This study received funding from PRIN PNRR 2022 (2022MYJX94).