Discrete Transcriptional States Define Biphasic Immune Response and Dynamic CMS Transitions in Colorectal Cancer

ABSTRACT Background Sequential alterations in APC , KRAS , TP53 , and SMAD4 have been proposed as a framework for colorectal cancer progression. Human colorectal cancer datasets have not revealed the biological transitions associated with these mutations. When examining a cohort of TCGA-colorectal tumors grouped as AK (APC/KRAS), AKP (APC/KRAS/TP53), and AKPS (APC/KRAS/TP53/SMAD4) , we observed no significant differences in immune-cell composition, four previously defined Consensus Molecular Subtypes (CMS1/2/3/4), or transcriptomic clustering between these genomic groups. Therefore, these canonical alterations do not sufficiently characterize the known properties of metastatic progression in human colorectal cancer. Methods To overcome these limitations, we developed a genetically defined, organoid-based, orthotopic mouse model whereby mouse colon organoids modeling sequential APC , KRAS , TP53 , and SMAD4 alterations were orthotopically injected into the colon. This was followed by RNA-sequence processing, normalization with DESeq2, differential expression, pathway enrichment, and immune/stromal inference. Gene co-expression modules were identified from variance-stabilized mouse expression data, mapped to 1:1 human orthologs, and summarized as eigengenes. A multinomial logistic regression model trained on mouse eigengenes was applied to TCGA-COAD human tumors to assign them to mouse-informed transcriptomic states (AK-like, AKP-like, AKPS-like), which were then used for downstream visualization and comparative analyses. Results Whole-transcriptome analysis revealed discrete transcriptional states and immune-cell differences between the organoid AK/AKP/AKPS groups. Early TP53 loss led to strong activation of immune pathways, accompanied by increased infiltration of NK and T cells. As tumors progressed with SMAD4 loss and metastasis, this immune activity collapsed, giving rise to broad immune suppression. CMS classifications also shifted, with AK tumors resembling epithelial CMS2, AKP tumors displaying immune-rich CMS1 features, and AKPS and metastatic lesions adopting mesenchymal CMS4 characteristics. We then applied a progression-based transcriptomic classifier to 460 human colorectal tumors. This reclassification revealed conserved immune remodeling, CMS transitions, pathway-level differences, and significant differences in patient survival. Conclusion We show that organoid-derived progression profiles reveal hidden evolutionary structure in human colorectal cancer and provide a transcriptional framework for interpreting metastatic potential and clinical outcomes.