Fig 2: Functional role of PCIF1 in oesophageal squamous cell carcinoma (OSCC) cell proliferation, migration and invasion. (A) Western blot analysis of PCIF1 expression in different OSCC cell lines, confirming successful knockdown by sgRNAs (sg1, sg2) in KYSE30 and KYSE150 cells. (B) Cell proliferation assays showing reduced proliferation in PCIF1 knockdown cells compared to control (vector). Optical density (OD) values at 450 nm were measured at different time points. *p < .05, **p < .01 by one‐way analysis of variance (ANOVA), Dunnett's test. (C) Colony formation assays in PCIF1 knockdown cells, showing decreased colony formation relative to controls. *p < .05, **p < .01 by one‐way ANOVA with Tukey's multiple comparison test. (D) Migration assays in PCIF1 knockdown cells, showing decreased migration relative to controls. **p < .01 by one‐way ANOVA with Tukey's multiple comparison test. (E) Invasion assays demonstrating reduced invasive capabilities in PCIF1‐knockdown cells. *p < .05 by one‐way ANOVA with Tukey's multiple comparison test. (F) Increased apoptosis rates in PCIF1‐knockdown cells compared to control group. **p < .01 by one‐way ANOVA with Tukey's multiple comparison test. (G) Representative images of xenograft tumours in nude mice injected with PCIF1‐knockdown OSCC cells compared to vector controls. Scale bar, 1 cm. (H) Tumour growth curves depicting a significantly slower growth rate in mice injected with PCIF1‐knockdown cells compared to the control group. Tumour volume was measured at regular intervals. ***p < .001 by one‐way ANOVA, Dunnett's test. (I) Comparison of final tumour weights between PCIF1‐knockdown and control groups. ***p < .001, Student's t‐test. (J) Representative images of Haematoxylin and eosin (H&E), PCIF1 and Ki67 staining in xenograft tumours from PCIF1‐knockdown and control groups. Scale bar, 100 µm. (K) Quantification of Ki67+cells in PCIF1‐knockdown versus control tumours. ***p < .001, Student's t‐test. (L) Quantification of PCIF1+ cells levels in xenograft tumours, demonstrating successful knockdown with significantly lower PCIF1 levels compared to controls. ***p < .001, Student's t‐test.

Fig 3: PCIF1 expression and clinical correlation in oesophageal squamous cell carcinoma (OSCC) patient samples. (A) Boxplot showing PCIF1 expression levels across TCGA cancer types, with significantly higher expression in Esophageal adenocarcinoma (ESCA) compared to normal tissues. p > .05, *p < .05, **p < .01, ***p < .001, Wilcoxon test. (B) Representative images of PCIF1 immunohistochemistry (IHC) staining in normal and OSCC tumour tissues from Sun Yat‐sen University Cancer Center (SYSUCC) patients' cohort. Insets show higher magnification views. Scale bar, 100 µm. (C) Quantification of PCIF1 IHC scores in normal (n = 53) versus tumour (n = 297) tissues in SYSUCC cohort, showing significantly higher PCIF1 expression in tumour samples. ***p < .001, Student's t‐test. (D) Kaplan–Meier survival analysis in SYSUCC cohort, demonstrating poorer survival for patients with high PCIF1 expression compared to low expression. p < .0001, log‐rank test. (E) Representative images of PCIF1 IHC staining in normal and OSCC tumour tissues from The First Affiliated Hospital Sun Yat‐sen University (FAH‐SYSU) patients' cohort. Insets show higher magnification views. Scale bar, 100 µm. (F) Quantification of PCIF1 IHC scores in normal (n = 130) versus tumour (n = 130) tissues in FAH‐SYSU cohort, showing significantly higher PCIF1 expression in tumour samples. ***p < .001, Student's t‐test. (G) Kaplan–Meier survival curves for FAH‐SYSU cohort. PCIF1 expression is higher in tumours, and high expression correlates with worse prognosis. p < .0001, log‐rank test. (H) Western blot analysis of PCIF1 protein levels in normal oesophageal epithelial cells (Simian Virus 40‐Immortalized Human Oesophageal Epithelial Cell Line [SHOE]) and OSCC cell lines (Oesophageal Squamous Cell Carcinoma Cell Line TE1, TE10, TE12 and Kyoto Oesophageal Squamous Cell Carcinoma Cell Line 30, 150), showing higher PCIF1 expression in OSCC cell lines.

Fig 4: MTF2 as a direct target of PCIF1‐mediated m6Am modification in oesophageal squamous cell carcinoma (OSCC) cells. (A) Western blot analysis of MTF2 protein levels in PCIF1‐knockdown OSCC cells. (B) Quantitative real‐time PCR (qPCR) analysis of MTF2 mRNA levels in PCIF1‐knockdown OSCC cells, showing no significant change in MTF2 transcription compared to control cells. (C) mRNA stability assay of MTF2 in PCIF1‐knockdown KYSE30 cells, indicating increased mRNA stability over time, as measured by qPCR following actinomycin D treatment. (D) mRNA stability assay of MTF2 in PCIF1‐knockdown KYSE150 cells, indicating increased mRNA stability over time, as measured by qPCR following actinomycin D treatment. (E) Polysome profiling of PCIF1‐knockdown cells, showing enhanced MTF2 mRNA association with polysomes, indicating increased translation. (F) qPCR analysis of polysome fractions, confirming increased MTF2 mRNA abundance in translationally active fractions. (G) Schematic representation of mutant (mut) constructs of the MTF2 5′UTR used in the luciferase reporter assay. (H) Dual‐luciferase assay results in control (NC), PCIF1 overexpression (PCIF1 OE) and PCIF1 catalytic inactive mutant (PCIF1 OEmut) OSCC cells, transfected with either wild‐type (WT) or mut MTF2 5′UTR constructs.

Fig 5: PCIF1 knockout enhances the efficacy of anti‐PD1 therapy in oesophageal squamous cell carcinoma (OSCC). (A) Schematic of the experimental design involving the generation of PCIF1 knockout in OSCC mouse models, followed by treatment with anti‐PD1 antibody or IgG control. Mice were administered 4NQO in drinking water to induce tumour formation. Anti‐PD1 or IgG treatment was administered starting from Week 22, and mice were sacrificed at Week 26 for analysis. (B) Representative images of oesophageal tumours from different treatment groups: IgG control, Anti‐PD1 treated and combined anti‐PD1 + PCIF1 knockout. Scale bar, 2 mm. (C) Quantification of lesion area, showing a reduction in tumour size in the PCIF1 knockout group and in the combined anti‐PD1 + PCIF1 knockout group compared to controls. **p < .01, ***p < .001 by one‐way analysis of variance (ANOVA) with Tukey's multiple comparison test. (D) Haematoxylin and eosin (H&E) staining of oesophageal tissue sections from each treatment group. Scale bar, 200 µm. (E) Quantification of the number of lesions, indicating a significant reduction in the combined treatment group. *p < .05 by one‐way ANOVA with Tukey's multiple comparison test. (F) Tumour invasion grade analysis, showing decreased aggressiveness in the anti‐PD1 + PCIF1 knockout group. *p < .05 by Pearson chi‐square test. (G and H) Immunohistochemistry (IHC) staining (G) and quantification (H) of PCIF1‐positive cells across the treatment groups, showing a marked reduction in PCIF1 expression in the knockout group. Scale bar, 100 µm. ***p < .001 by one‐way ANOVA with Tukey's multiple comparison test. (I and J) IHC staining (I) and quantification (J) of MTF2‐positive cells, with increased MTF2 expression in the Anti‐PD1 + PCIF1 knockout group. Scale bar, 100 µm. ***p < .001 by one‐way ANOVA with Tukey's multiple comparison test. (K and L) IHC staining (K) for Ki67 with quantification (L) showing decreased Ki67‐positive cells in the anti‐PD1 and PCIF1 knockout groups, particularly in the combined treatment group, indicating reduced tumour proliferation. Scale bar, 100 µm. ***p < .001 by one‐way ANOVA with Tukey's multiple comparison test. (M–P) Flow cytometry analysis showing no significant changes in CD8+ T cell function across the treatment groups, including FPR1+ and GZMB+ CD8+ T cells, suggesting that PCIF1 knockout and anti‐PD1 treatment operate via distinct mechanisms. p > .05, ***p < .001 by Student's t‐test.