Biological characteristics of intestinal organoids versus in vivo intestinal crypts and mechanisms underlying their differential responses to nobiletin

Abstract
Objective
Intestinal organoids serve as important tools for drug screening, but the extent to which they recapitulate in vivo tissue microenvironments and drug responses remains inadequately evaluated. This study aims to verify systematic differences between intestinal organoids and in vivo intestinal crypts in fundamental biological characteristics and responses to nobiletin (NOB) through parallel comparison.

Methods
A standard mouse small intestinal organoid culture system was established, with synchronous isolation of homologous mouse small intestinal crypt tissues. Whole-transcriptome sequencing was employed to analyze gene expression profiles, flow cytometry was used to analyze cell subpopulation proportions, and Western blotting was performed to detect key protein expression levels. In vivo crypts and ex vivo organoids were treated with different concentrations of NOB: in vivo administration via gavage at 200 mg/kg for 12 h; organoids cultured for 48 h followed by 100 μmol/L NOB treatment for 12 h. GSEA, GO, and KEGG enrichment analyses and morphological observations were conducted on crypt cells from both groups to comprehensively evaluate the regulatory effects of NOB on cell cycle, metabolic flux, and signaling pathways.

Results
Intestinal organoids effectively recapitulated core biological characteristics of in vivo crypts, including active proliferation of intestinal stem cells, multi-lineage differentiation potential, and CD24-marked cell clustering patterns. However, organoids exhibited specific metabolic reprogramming and immune-silent phenotypes, with differential glycosylation modification of the stem cell marker OLFM4. NOB treatment further amplified model-dependent differences: in in vivo crypts, NOB primarily downregulated the genes related to immune defense and oxidative stress, without significant effects on crypt morphology or OLFM4+ cell numbers; in organoids, NOB significantly suppressed core anabolic pathways including DNA replication, cell cycle, and amino acid and nucleotide synthesis, downregulated the expression of stem cell markers, and markedly reduced organoid budding number (P<0.001).

Conclusion
Intestinal organoids and in vivo tissues share commonalities while exhibiting differences at both baseline states and drug response levels. Organoids can recapitulate intestinal epithelial self-renewal processes but differ from in vivo tissues in metabolic programming and immune response. NOB mainly modulates immune microenvironment-related pathways in the in vivo context, whereas it directly targets epithelial cell-autonomous proliferation and metabolic programs in organoids.