Functional precision oncology platform of BRAF(V600E)-mutated colorectal cancer organoids predicts therapy response and reveals RNF43-mediated immunogenicity.

BRAFV600E-mutant colorectal cancer (CRC) represents an aggressive molecular subtype characterized by poor prognosis and resistance to standard therapies. Predictive models to guide treatment selection and overcome drug resistance are urgently needed. We established a biobank of patient-derived organoids (PDOs) and matched xenografts (PDOXs) from BRAFV600E-mutant CRC, capturing inter- and intratumoral heterogeneity. PDOs were characterized by histopathology, whole-exome sequencing, bulk and single-cell RNA sequencing, and drug sensitivity testing to chemotherapies and targeted agents.

Immunogenicity was assessed via PDO-T cell co-culture and humanized xenograft models. PDOs faithfully preserved parental tumor histology, mismatch repair status, mutational landscapes, and copy number alterations (91.6% concordance in driver genes). Single-cell transcriptomics confirmed high fidelity between PDOs and original tumors. Drug sensitivity assays revealed marked interpatient heterogeneity and accurately recapitulated clinical responses to FOLFIRI, FOLFOXIRI, and BRAF/EGFR inhibitor combinations.

Notably, RNF43-mutant PDOs exhibited significantly enhanced sensitivity to encorafenib plus cetuximab. Integrative transcriptomic analysis (TCGA and PDO cohorts) identified that RNF43 mutations are associated with upregulation of E2F, G2M, interferon-α/γ, and inflammatory pathways, along with elevated MHC-I expression. In co-culture with CD8⁺ T cells, RNF43-mutant PDOs triggered stronger T-cell activation, enhanced cytotoxicity, and increased tumor cell apoptosis. In humanized PDOX models, RNF43-mutant tumors showed suppressed growth and heightened CD8⁺ T-cell infiltration compared to wild-type controls.

This functional precision oncology platform integrates multi-omics and immune co-culture to uncover RNF43 mutation as a dual biomarker for targeted therapy sensitivity and tumor immunogenicity in BRAFV600E-mutant CRC.

Our findings provide mechanistic rationale for combining BRAF/EGFR inhibition with immunotherapy to overcome drug resistance and improve outcomes in this aggressive subtype.