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Additional file 1. Supplementary Table 1. Characteristics of patients.

Additional file 15 Supplementary Fig. 14. (A) Screening of synthetic lethality target of FBXW7 mutation using the DepMap dataset. (B, C) Sensitivity of the FH535 molecule in colorectal cancer cell lines with FBXW7 mutation (Wilcoxon rank sum test).

Additional file 3 Supplementary Fig. 2. (A) Spearman’s correlation of variant allele fractions (VAFs) between organoid RNA and organoid DNA. (B) Characteristics of variants detected in organoid DNA and not in tissue RNA; the variants detected in organoids only were associated with low read depth in primary tissue sequences or subclonal events based on organoid sequencing (Wilcoxon rank-sum test). (C) Spearman’s correlation of VAFs between organoid DNA and tissue RNA. (D) Spearman’s correlation of VAF in each gene between organoid DNA and tissue RNA.

Additional file 10 Supplementary Fig. 9. (A) Classification of the TIM using primary cancer tissue. (B) Immune cell types from CIBERSORT based on the TIM class (Wilcoxon rank-sum test). (C) Immune cell types using MCP score based on the TIM class (Kruskal Wallis test). (D) Validation of the exhausted TIM class based on CD8+ T cell exhaustion score using GSVA analysis (Wilcoxon rank-sum test). (E) Significant correlation of HLA-I expression in CCOs versus primary tumor tissues (Spearman correlation test). (F) Fraction of copy number variation (CNV) based on the TIM class (Kruskal Wallis test). (G) Fusion number based on the TIM class (Kruskal Wallis test). TIM, tumor immune microenvironment; CCO, colorectal cancer organoid.

Additional file 13 Supplementary Fig. 12. (A) Enrichment of KRAS (biallelic) mutations in tumors with decreased CD8+ T cells (10,000 randomly permutated p values and one-sided Wilcoxon rank sum test). (B) Increased frequency of KRAS biallelic mutation in non-immunogenic (immuno-desert TIM) tumors (Fisher’s exact test). (C, D) KRAS mutation status and TIM. (E) Characteristics of gene expression and infiltrating immune cell types based on the KRAS mutation status (Spearman’s correlation test). TIM, tumor immune microenvironment.

Additional file 6 Supplementary Fig. 5. (A) Ki-67 immunohistochemistry in the primary tumor tissues and its interpretation by a pathologist and by image analysis. (B) Significant correlation of the Ki-67 proliferation index between the pathologist’s result and image analysis. (C) No correlations in Ki-67 mRNA expression between bulk primary tissues including tumor microenvironment and cancer cells and CCOs. (D) Comparison of our molecular subgrouping and CMS classification in CCOs (Chi-squared test). CCO, colorectal cancer organoid.

Additional file 11 Supplementary Fig. 10. No significant differential gene expression in organoids based on the tumor immune microenvironment (TIM) class of primary tissues (FDR q > 0.25 by one-way ANOVA test).

Additional file 12 Supplementary Fig. 11. (A) Pathway analysis using tissue RNA sequence data based on the tumor immune microenvironment (TIM) class. (B, C) Significance of Wnt/β-catenin signaling pathway in the non-immunogenic group using GSVA analysis (Wilcoxon rank-sum test). (D) APC mutation status based on the TIM class. (E) Significance of biallelic APC mutation and TIM class (Fisher’s exact test). (F) Expression of target genes belonging to the Wnt/beta-catenin pathway (Wilcoxon rank-sum test).

Additional file 2 Supplementary Fig. 1. (A) Histology and immunohistochemistry of the established CCOs and the corresponding primary tumors. (B) Distribution of variant allele fraction (VAF) of detected variants in the MSI-high group and the MSI-low/MSS group. CCO, colorectal cancer organoid; MSI, microsatellite instability; MSS, microsatellite stable; H&E, hematoxylin and eosin.

Additional file 16 Supplementary Fig. 15. (A) FH535 induced cell death and destroyed the organoid structure in the two CCOs with FBXW7 mutation (AMC-17CT-019 and AMC-17CT-048). (B) Sensitivity of the FH535 molecule in CCOs with FBXW7 mutation (Wilcoxon-rank sum test). (C) Sensitivities of various WNT signaling drugs targets showing non-significant responses to drugs targeting WNT pathways in colorectal cancer cells with FBXW7 mutation. (D) CCOs with FBXW7 mutation also showed non-significant responses to ICG-001, which also targets the WNT/β-catenin pathway, suggesting that FH535 has a different mechanism action in colorectal cancers harboring the FBXW7 mutation. CCO, colorectal cancer organoid.

Additional file 5 Supplementary Fig. 4. (A) Spearman’s correlation coefficient matrix of CCOs versus primary tumor tissues. (B) Total immune score and stromal score of CCOs versus primary tumor tissues. (C) Genes that are differentially expressed between CCOs and primary tumor tissues. (D) Spearman correlation of the expression of differentiation or stem cell markers in CCOs versus primary tumor tissues. CCO, colorectal cancer organoid; TIM, tumor immune microenvironment.

Additional file 4 Supplementary Fig. 3. (A) Profiles of copy number variations with microsatellite instability (MSI) information in the 87 CCOs. (B) Circos plot of the detected fusions in the 87 CCOs. (C) HLA class I molecular typing and its frequency in 87 CCOs. (D) Frequency of the HLA supertype in 87 CCOs. CCO, colorectal cancer organoid.

Additional file 14 Supplementary Fig. 13. (A) Frequency of TP53 gain-of-function (GOF) mutation based on the tumor immune microenvironment (TIM) class, showing a high frequency of TP53 GOF mutations in the Desert group (p = 0.033, Fisher’s exact test). (B) Association between TP53 GOF mutations and decreased CD8+ T cell (10,000 random permutation test and Spearman’s correlation test). (C) Tendency for the exclusive occurrence of TP53 and KRAS mutation. (D) Enriched unfolded protein response pathway (GSVA score) in the immune-exhausted group (Wilcoxon rank-sum test).

Additional file 8 Supplementary Fig. 7. (A) Prognostic impact of gene expression in HLA class II in CCOs. (B) Patient survival based on HLA class II expression level using immunohistochemistry (IHC) in cancer cells from primary tissue (log-rank test).