Fig. 1: Upregulation of INCENP and CDCA8 contributes to poor NACT response.

a An experimental design of the present study. Responders (tumor regression grade [TRG] scores of 0–1 or patients who achieved clinical complete or partial response; n = 6) and non-responders (TRG scores of 2–3 or patients who achieved clinical minimal response; n = 8). Created in BioRender. GU, T. (2025) https://BioRender.com/ppcp3bu. b Transcriptomic data from clinical samples (Cohort 1) were subjected to GO enrichment analysis to determine alterations in biological processes, molecular functions, and cellular components. Data are visualized in the provided bubble diagram. c Bubble diagram visualizing the alteration of m⁶A regulators in clinical ESCC sample Cohort 1. d GO enrichment analysis of differentially expressed proteins between shYTHDF3 and Mock cells. The Rich Factor represents the ratio of enriched genes; bubble size denotes gene count, and color indicates -log₁₀P value. Statistical significance was calculated using a two-sided Fisher’s exact test with Benjamini–Hochberg FDR correction. e Rank-based gene set enrichment analysis (GSEA) of signaling pathway alterations and representative protein changes in shYTHDF3 cells. Terms in red represent the primary targets. f Representative immunohistochemical staining images of INCENP, CDCA8, and YTHDF3 in ESCC Cohort 1 tissue samples from the indicated responders and non-responders. Scale bar = 250 μm. g INCENP, CDCA8, and YTHDF3 score levels were measured in tissue derived from ESCC Cohort 1 samples. Responders (TRG1, n = 4) and non-responders (TRG 2-3, n = 8). h Correlation between YTHDF3 protein scores and INCENP or CDCA8 protein scores in Cohort 1. Correlation coefficients (R) and P values were determined using a two-sided Pearson correlation test. Shaded areas represent the 95% confidence interval of the regression line. i Representative radiological images and INCENP, CDCA8 IHC-stained tissues (scale bar = 250 μm) in Cohort 2, ESCC patients with favorable and unfavorable responses to NACT [responders (TRG 0–1, n = 31) and non-responders (TRG 2–3, n = 37)]. Red arrows represent the lesion sites. j Immunohistochemical analysis of INCENP and CDCA8 scores in ESCC tissues from Cohort 2, including responders (TRG 1, n = 10) and non-responders (TRG 2-3, n = 37). k Stacked bar plots showing distributions of clinical outcomes (death, n = 10, progression, n = 10, remission, n = 10) in patients with low and high expression of INCENP and CDCA8. Statistical significance was assessed using a two-sided Chi-square test. Data were presented as means ± SD. P-values were determined by a two-tailed unpaired t-test (g, j). Source data are provided as a Source Data file.