Modeling colorectal cancer initiation using human iPSC-derived intestinal organoids
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Abstract
OBJECTIVE: Human induced pluripotent stem cell (hiPSC)-derived human intestinal organoids (HIOs) are increasingly utilized as in-vitro models for studying colorectal cancer (CRC). Prevalent oncogenic drivers in CRC include mutations in EGFR (L858R), KRAS (G12D) and CTNNB1 (S33C) which are known to contribute to neoplastic growth in epithelial tissue. However, Investigating the early molecular events associated with CRC continues to be an important topic of study. We generated inducible oncogenic mesenchyme-free hiPSC-derived HIOs to study the early molecular events associated with three oncogenes employing bulk-RNA sequencing to examine genetic expression and pathway commonalities compared to existing primary literature in the field of CRC.
METHODS: A CDX2-GFP reporter hiPSC line was transfected independently with three constructs each containing a doxycycline (Dox) -inducible promoter driving expression of a mutant EGFR, KRAS, or CTNNB1 followed by the BFP reporter knocked-in into the AAVS1 safe harbor locus. Correct knock-in was validated before differentiation. HiPSCs were subsequently cultured and differentiated for 15 days to achieve CDX2-GFP+ progenitor differentiation, followed by HIO maturation and were induced for three weeks, using DMSO as a control, before harvesting for RT-qPCR and bulk-RNA sequencing. Principle component analysis and Heatmaps were generated from bulk-RNA sequencing for enrichment analysis.
RESULTS: Gel electrophoresis revealed biallelic integration of mutant EGFR and KRAS whereas CTNNB1 exhibited monoallelic integration, potentially impacting the levels of induction and expression. Upon induction, we used RT-qPCR to demonstrate expression with statistical significant of oncogenic EGFR and KRAS in both hiPSC and HIOs, and expression without statistical significant expression for CTNNB1 in either state, suggesting potential silencing affecting significant expression in the AAVS1 locus. Bulk-RNA sequencing enrichment analysis identified key pathways, including EGFR-STAT2, interferon alpha/beta signaling activation, and anti-apoptotic signaling upon EGFR induction; KRAS-CDX2 regulation and epithelial homeostasis dysregulation upon KRAS induction; and CTNNB1 to KRAS regulation upon CTNNB1 induction. Some of these pathways have been previously described as having a role in CRC development.
CONCLUSION: We have established a novel model for studying the early molecular events associated with oncogene activation in CRC, by utilizing three inducible oncogene hiPSC engineered lines and their intestinal organoid progeny. Using this platform we confirmed robust induction of the oncogenes and specific transcriptional changes associated with oncogene expression, as determined by RNA-Sequencing. Validation of these pathways and their functional impact on intestinal transformation is warranted. These studies underlined several potential mechanisms of early tumorigenesis and open novel opportunities for therapeutic intervention.
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2025