Biological differences between N-motif–positive and –negative tumor cells. (A) Volcano plot displaying differentially expressed genes between T0 tumor cells with and without N-motifs separately for patients 04 (left) and 07 (right). Genes enriched in N-motif–positive tumor cells are colored red, and those enriched in N-motif–negative cells, blue. Selected genes are highlighted. (B) Heat map exhibiting enriched pathways in N-motif–positive and N-motif–negative T0 tumor cells from patient 04 and 07. P values were calculated using Wilcoxon rank sum tests with false discovery rate: ∗P <  .05; ∗∗P  <  1 × 10e−10; ∗∗∗P  <  1 × 10e−25; ∗∗∗∗P  <  1 × 10e−50. (C) Table showing single-cell complementary DNA–based detected numbers of mutated vs all cells for each reported mutation for the N-motif–positive and N-motif–negative tumor cells for patients 04 and 07 using long-read sequencing. T cells from the same samples served as control. ¹Mutation results in a premature termination codon. ²Mutation results in a frameshift. AA, amino acid; CDS, identification of coding sequences; del, deletion; fs, frameshift; IFN-γ, interferon gamma; TGFβ, transforming growth factor β; TNFA, tumor necrosis factor α; Pt, patient.