Figure 8. Single-cell RNA-seq analysis of mouse colorectal tumors treated with ATT-I in combination with immune checkpoint blockade therapy.

C57BL/6 mice bearing orthotopic implanted MC38-derived tumors were treated with vehicle control, ATT-I (50 mg/kg, daily), anti–PD-1 (3 times/week, 5 times in total), or ATT-I + anti–PD-1 combo. Tumors were harvested 2 weeks after initial treatment (5 tumor samples were pooled per arm). (A) t-SNE plot of the scRNA-seq data collected from all conditions. Cell types were assessed by the expression levels of known marker genes. (B) Gene expression profiles of functional marker genes in selected immune cell types. Each row and column represents 1 gene and 1 cell type, respectively. (C) Averaged expression levels of T cell activation and cytotoxic marker genes in the CD8⁺ effector T cells from different conditions. The dot size characterizes the proportion of CD8⁺ effector T cells of each condition (y axis) with expression levels (indicated by color intensity) of the selected genes (x axis). The dot color reflects the averaged expression level of each gene in of the CD8⁺ effector T cells of each condition. (D) Distribution of cytotoxicity scores of CD8⁺ effector T cells under each condition. Cytotoxic level of each cell is inferred by the averaged expression level of CD8⁺ T marker genes Prf1, Ifng, Tnf, Pdcd1, Sla2, and Cd8a. The y axis represents the cytotoxicity score. Combo versus anti–PD-1 (P = 1.121 × 10^–6); combo versus ATT-l (P = 0.0024). Statistical analysis was conducted using unpaired 2-tailed t test. (E) Proportion of CD8⁺ effector T cells with significant cytotoxicity genes expressed. The y axis represents the proportion of CD8⁺ effector T cells with (dark blue) and without (red) significant cytotoxicity genes expressed. Combo versus anti–PD-1 (P = 1.006 × 10^–9), combo versus ATT-l (P = 3.816 × 10^–5), combo versus control (P = 1.557 × 10^–8). Statistical analysis was conducted using Fisher’s exact test.