Figure 7. Functional validation of the tumor cell-differentiation status and the effects on cytotoxic immunity.

(A) IF images of Apc-driven colonic tumor and Braf-driven proximal colon villiform metaplasia (white arrows).

(B) Quantification of CD8-positive pixels from IF. Red line denotes the mean level detected in adjacent normal colon in Braf mice. Error bars represent SEM from n = 3 animals per group.

(C) IF images of CD8⁺ T cells in tumor, villiform metaplasia (white arrows), and control colon. Dotted line demarcates border between villus and crypt compartments.

(D and E) H&E (D) and β-catenin IHC (E) of colonic tissues and tumors of tamoxifen-induced Lrig1 or Mist1 tumor mice 28 days after DSS.

(F–H) UMAP of epithelial scRNA-seq data generated from mouse colonic tissues and tumors, with overlays indicating (F) cell type, (G) gene overlays, and (H) biological replicates.

(I) Heatmap of genes defining human metaplastic and cell signatures in specified epithelial populations from mouse scRNA-seq.

(J and K) Combined UMAP of immune cell scRNA-seq data from mouse colonic tissues and tumors, with overlays indicating (J) conditions and (K) cell type.

(L and M) Quantification of (L) general immune cell types and (M) specific lymphocyte populations from Lrig1 (left) and Mist1 (right) scRNA-seq data.

(N) UMAP overlays of genes related to immunosuppression or cytotoxicity in myeloid and lymphoid cells.

(O) MxIF images of T cells in tumors.

(P) Image quantification of T cells. Each dot represents a field of view. Error bars represent SEM from n = 3 animals per group.

(Q) CytoTRACE score for TSCs from scRNA-seq.

(R) Organoid formation efficiency of single cells isolated from tumors and control colons. Each dot represents data from a well with representative images shown in insets. Error bars represent SEM from n = 4 animals per tumor, 2 for control.

(S) SCI, II, and III metagene signatures for TSCs from scRNA-seq.

(T) Heatmap of individual antigen-presentation genes at single-cell level.

(U) MHCII metagene signature expression for TSCs.

(V) Quantification of DQ-OVA⁺/I-AI-E⁺ epithelial tumoroid cells from flow plots. Error bars represent SEM from n = 6 animals per condition.

(W) Percentage of proliferating T cells determined by CellTrace Violet assay when co-cultured with organoids derived from colonic tumors or normal tissues (+DSS) +/— 50 mg/mL OVA peptide. Error bars represent SEM of organoids from n = 5 mice for tumors and two for normal.

*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S7 and Table S5.