Differential 5-FU responses of crypt-derived organoids embedded in collagen versus Matrigel, and their transcriptomic comparison with villus-derived organoids

We compared the effects of 5-fluorouracil (5-FU) on gene expression profiles of mouse crypt-derived intestinal organoids embedded in either Matrigel (MG) or collagen type I (COL). Distinct sets of genes were upregulated in COL-embedded organoids following 5-FU treatment, suggesting that collagen-based ECM uniquely modulates inflammatory and DNA-damage responses. We further compared COL-embedded crypt-derived organoids with COL-embedded villus-derived organoids (V-organoids) and found that their transcriptional profiles were highly similar. These results indicate that collagen embedding promotes the emergence of revival stem cell–like signatures regardless of the cellular origin of the organoids. Overall design: To evaluate how ECM environments influence responses to chemotherapeutic stress, organoids in each ECM condition (MG and COL) were either left untreated (Control) or treated with 5-fluorouracil (5-FU). This resulted in a 2 × 2 factorial design consisting of four experimental groups: MG_Control, MG_5FU, COL_Control, and COL_5FU, with three biological replicates per condition (n = 3). We established villus-derived intestinal organoids (V-organoids) from mouse small intestine and compared them with crypt-derived organoids cultured under two distinct extracellular matrix (ECM) conditions: Matrigel (MG) and collagen type I (COL-cWRNE).