Transcriptomic benchmarking positions iPSC-Derived intestinal organoids as immature, non-malignant epithelial references for colorectal cancer tissue

Primary colorectal cancer (CRC) biopsies and intestinal organoids are frequently compared to define tumor-associated epithelial alterations; however, these systems differ in malignant status, tissue composition, developmental state, and epithelial maturation. In this study, transcriptomic profiling of CRC biopsies, matched normal intestinal tissue, and iPSC-derived intestinal organoids (iIOs) was performed to define the biological axes separating primary tissue from organoid epithelial states. Transcriptome-wide analysis revealed strong separation between CRC biopsies and iIOs, with iIOs diverging from primary tissue more extensively than tumors differed from normal mucosa. Pathway enrichment and cell deconvolution showed that biopsies were dominated by immune, inflammatory, and stromal programs reflecting tissue complexity, whereas iIOs were enriched for proliferative, biosynthetic, and metabolic programs. Projection onto a malignancy axis placed iIOs at a neutral position, indicating that their divergence stems from developmental state rather than malignant transformation. Compared to normal mucosa, organoids reproducibly lost adult tissue-associated immune/stromal programs while gaining proliferative, fetal-like epithelial programs. Gut Cell Atlas scoring, crypt–surface positioning, and lineage deconvolution showed that iIOs retain intestinal epithelial identity but occupy a developmentally immature, crypt/progenitor-biased state dominated by transit-amplifying programs with limited differentiated lineage diversity. Metabolic module analysis and anabolism-proliferation correlation showed that elevated metabolic activity in iIOs, including biosynthetic and nucleotide synthesis programs. These reflect proliferation-coupled anabolism rather than malignancy-like metabolic reprogramming; the glycolysis-OXPHOS balance did not differ significantly between groups. Consequently, iIOs represent metabolically active, fetal-like, crypt/progenitor epithelial reference models rather than direct transcriptomic surrogates of adult intestinal mucosa or primary CRC tissue. These results clarify the utility and limitations of iIOs as epithelial reference models for colorectal cancer research.