2.1 Patient sample

The study incorporated samples collected from OSCC patients undergoing surgical resection at Sun Yat-sen University Cancer Center (Table S1) and The First Affiliated Hospital of Sun Yat-sen University (Table S2). Written informed consent was obtained from all patients before sample collection. Tumour tissues and adjacent normal tissues were obtained immediately after resection and then fixed in neutral-buffered formalin for subsequent processing. The research was carried out with the approval of the Ethics Committee at Sun Yat-sen University.

2.2 Cell culture

KYSE30, KYSE150 and the immortalized oesophageal epithelial cell line SHEE were obtained from the American Type Culture Collection. TE1, TE10 and TE12 were procured from Wuhan Pricella Biotechnology. Cells were maintained in Dulbecco's modified Eagle medium (TransGen Biotech, AQ601) containing 10% foetal bovine serum (FBS, A3160801; GIBCO), 1% penicillin-streptomycin (15140122; GIBCO) at 37°C in a humidified atmosphere with 5% CO₂. Regular mycoplasma detection was conducted throughout the study to ensure the quality of the cell lines.

2.3 Cell transfection

For gene knockdown experiments, short guide RNAs (sgRNAs) targeting PCIF1 were designed and cloned into a lentiviral vector system. The specific sequences of the sgRNAs used for PCIF1 knockdown are available in Table S3. Cells were transfected with sgRNA oligonucleotides targeting MTF2 or a control sgRNA to achieve MTF2 knockdown. The sequences are detailed in Table S3.

The catalytically inactive PCIF1 mutant (amino acid residues 160–163, LFPD mutated to AFPA) was provided by Tianhua Zhou from Zhejiang University School of Medicine, Department of Cell Biology and Gastroenterology. The PCIF1 overexpression plasmid was generated by cloning the full-length open reading frame of the human PCIF1 gene (NM_022104.3) into the pLVX expression vector (GeneCopoeia). Wild-type or mutant PCIF1 plasmids were introduced into cells using Lipofectamine 2000 (11668019; Invitrogen).

2.4 Transgenic mouse model construction

To generate a transgenic mouse model with specific knockout of PCIF1 in oesophageal epithelial cells, we used several mouse strains. K14^CreER mice were sourced from The Jackson Laboratory, whereas Pcif1^flox mice were obtained from Biocytogen Pharmaceuticals and Mtf2^flox mice from Shanghai Model Organisms Center, Inc. For conditional knockout induction, adult mice were administered tamoxifen (T5648; Sigma) intraperitoneally at a dose of 75 mg/kg/day for five consecutive days. Successful gene deletion was confirmed by PCR analysis of oesophageal tissues. To induce OSCC, 6-week-old mice received drinking water containing 50 µg/mL 4-nitroquinoline 1-oxide (4NQO, N8141; Sigma) for a period of 16 weeks. The 4NQO stock solution was prepared at 5 mg/mL in propylene glycol and diluted before administration. For immunotherapy experiments, an anti-PD1 monoclonal antibody (BE0146; BioXcell) was administered intraperitoneally at a dose of 200 µg per mouse every 2 days, starting at Week 22 and continuing until Week 26. Control group mice were administered an equivalent volume of IgG isotype control antibody. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.

2.5 Nude mice xenograft tumourigenesis

Male BALB/c nude mice (6 weeks old) were injected subcutaneously with 1 × 10⁶ KYSE150 OSCC cells stably expressing either PCIF1 sgRNA or control sgRNA. Tumour dimensions were recorded every 3 days using calipers, and volumes were calculated with the formula (length × width²)/2. Mice were sacrificed on Day 30, and the tumours were collected for subsequent examination.

2.6 Western blotting

OSCC cell proteins were extracted using RIPA buffer (P0013B; Beyotime) with added protease inhibitors. The bicinchoninic acid (BCA, 23227; ThermoFisher) assay was used to measure protein concentrations. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in equal amounts and transferred to polyvinylidene fluoride (PVDF) membranes. These membranes were incubated with primary antibodies against PCIF1 (ASE-39532; SAB), MTF2 (16208-1; Proteintech), GOLIM4 (31083-1; Proteintech) and GAPDH (10494-1; Proteintech), followed by HRP-conjugated secondary antibodies (SA00001; Proteintech). Protein detection was carried out using an enhanced chemiluminescence system.

2.7 Quantitative real-time PCR (qPCR)

Total RNA extraction from OSCC cells was performed using TRIzol reagent (15596018; ThermoFisher) per the manufacturer's protocol. RNA concentration and purity were measured using a NanoDrop spectrophotometer. For reverse transcription, 1 µg of RNA was converted to cDNA using a reverse transcription kit (R323-01; Vazyme). qPCR analysis, using SYBR Green Master Mix (Q711-02; Vazyme) on a real-time PCR system, quantified PCIF1 and MTF2 expression, with GAPDH as the internal control. Primer details are provided in Table S3.

2.8 Polysome profiling

KYSE150 cell lines with or without PCIF1 knockout were utilized for polysome profiling. Cells were pre-treated with cycloheximide (HY-12320; MCE; 100 µg/mL) to stabilize ribosome–mRNA complexes, followed by lysis in polysome lysis buffer. A 10%–50% sucrose gradient was used to separate the cell lysates by ultracentrifugation at 36 000 rpm for 2 h. RNA was extracted from the collected polysome-associated fractions. qPCR was used to assess MTF2 mRNA levels, comparing the PCIF1 control group with the PCIF1 knockout group.

2.9 MTF2 5′UTR luciferase reporter assay

To explore the role of PCIF1 in regulating MTF2 translation, the 5′UTR WT (where WT is wild type) and 5′UTR mut (mutant) of MTF2 was cloned into a luciferase reporter construct. The 5′UTR WT construct contains the full wild-type sequence of MTF2, whereas the 5′UTR mut construct includes mutations at the key regulatory sites. To assess luciferase activity, OSCC cells were co-transfected with 5′UTR WT or 5′UTR mut reporter constructs along with a control plasmid, a PCIF1 overexpression plasmid or a catalytically inactive PCIF1 mutant plasmid. Forty-eight hours post-transfection, luciferase activity was assessed using a dual-luciferase reporter assay, with Renilla luciferase serving as the normalization control.

2.10 Actinomycin D assay

To evaluate the stability of MTF2 mRNA, OSCC cells were exposed to actinomycin D (A432787; Aladdin) to inhibit new RNA synthesis. Cells were harvested at multiple time points, typically at 0-, 6- and 12-h post-treatment, to monitor the decay of MTF2 mRNA. MTF2 mRNA levels were assessed at different time points through qPCR.

2.11 m6Am sequencing

Total RNA isolation from OSCC cells was performed with TRIzol reagent as per the manufacturer's guidelines. The extracted RNA (∼10 µg) was fragmented to obtain ∼100–200 nt fragments using RNA fragmentation buffer (E6150S; NEB) at 70°C for 7.5 min. The fragmented RNAs were then subjected to m7G immunoprecipitation using anti-m7G antibodies (RN017 M; Medical & Biological Laboratories) conjugated to pierce protein A/G magnetic beads at 4°C for 3 h. The m7G-enriched RNA was eluted, followed by a second round of immunoprecipitation with anti-m6A antibodies (202003; Synaptic Systems) under similar conditions to enrich for m6Am-containing fragments. After stringent washing, the antibody-bound RNA was extracted using TRIzol reagent and ethanol-precipitated overnight. Library preparation was carried out with the NEBNext Ultra II Directional RNA Library Prep Kit using the enriched RNA, and sequencing was conducted on the Illumina HiSeq4000 platform.

2.12 m6Am peak calling

The sequenced reads obtained from m6Am-Seq were first trimmed for quality and adapters using Trim Galore. Cleaned reads were aligned to the hg38 version of the human genome with STAR. Aligned BAM files were used to identify m6Am peaks using exomePeak2. A false discovery rate threshold (FDR < .05) was used to filter peak calling results and ensure statistical significance.

2.13 Motif discovery

To identify potential motifs associated with m6Am peaks, the sequences surrounding the called peaks were extracted using BEDTools. Motif discovery was performed using HOMER, which was set to identify enriched motifs within a ±50 nt window around the m6Am sites.

2.14 Colony formation assay

In six-well plates, 500 OSCC cells per well were cultured under standard conditions for 14 days to facilitate colony formation. The colonies were then fixed with methanol, stained with crystal violet (.1%) and counted manually.

2.15 Transwell migration and invasion assay

Transwell chambers with an 8.0 µm pore size (Corning) were used to assess cell migration and invasion. For migration, 2 × 10⁴ cells in serum-free medium were added to the upper chamber, with the lower chamber containing medium with 20% FBS. For invasion, matrigel-coated inserts were used to mimic the extracellular matrix. After 24 h, migrated or invaded cells on the lower surface of the membrane were fixed, stained with crystal violet and microscopically counted.

2.16 Cell apoptosis detection

To assess apoptosis, OSCC cells were harvested, rinsed in Phosphate buffer saline (PBS) and subjected to staining with an Annexin V-FITC/PI Apoptosis Detection Kit (70-AP107; MultiSciences). Following a 15-min incubation in the dark at room temperature, the cells underwent analysis on a flow cytometer. Annexin V-positive cells indicate apoptosis, whereas propidium iodide staining indicates cell membrane permeability.

2.17 Immunohistochemistry (IHC) staining

Sections from patient-derived samples and xenograft tumours were prepared for IHC staining. Deparaffinized and rehydrated sections were treated with sodium citrate buffer (pH 6.0) at high pressure for 15 min to retrieve antigens. To inhibit endogenous peroxidase activity, 3% hydrogen peroxide was applied for 10 min at room temperature. Then sections were blocked with 5% bovine serum albumin for 30 min. Primary antibodies specific to PCIF1 (NBP2-13740; Novus), Ki67 (NB500-170; Novus) and MTF2 (16208-1; Proteintech) were applied and incubated overnight at 4°C. Slides were washed and treated with biotinylated secondary antibodies at room temperature for 30 min. Visualization was performed with a streptavidin–HRP complex and DAB chromogen, followed by haematoxylin counterstaining, dehydration, clearing and mounting. Microscopic evaluation was performed to quantify staining intensity and localization.

2.18 Haematoxylin and eosin (H&E) staining

Tissue sections from formalin-fixed, paraffin-embedded patient specimens and xenograft tumours were utilized for H&E staining. Deparaffinization with xylene, rehydration through graded ethanol and rinsing in distilled water were performed before staining with haematoxylin for 5 min. Sections were washed, differentiated with acid alcohol, blued with alkaline water and counterstained for 2 min with eosin. After dehydration in ethanol, clearing with xylene and mounting, sections were observed with a light microscope for histological analysis.

2.19 RNA immunoprecipitation (RIP)

For assessing the interaction between PCIF1 and MTF2 mRNA, OSCC cells were lysed in RIP lysis buffer containing protease and RNase inhibitors. The lysate was incubated overnight at 4°C with PCIF1 antibody (98085S; Cell Signaling Technology) conjugated to Protein A/G magnetic beads. RNA from the immunoprecipitated complexes was extracted using TRIzol reagent following washing. qPCR was performed to measure MTF2, GAPDH and GOLIM4 mRNA levels. IgG served as a negative control, and input RNA was used for normalization.

2.20 m6A-RIP qPCR

RNA was isolated using TRIzol Reagent (15596018; ThermoFisher), followed by mRNA purification with the magnetic mRNA isolation kit (S1550S; NEB). For m6A immunoprecipitation, 2 µg of m6A antibody (202003; Synaptic Systems) was coupled to protein A/G magnetic beads (88803; ThermoFisher) in IP buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4,. 1% NP-40) and incubated at 4°C for 2 h. Ten percent of the mRNA was reserved as input, and the remaining portion (∼2 µg) was incubated with the antibody-bead complex in 500 µL IP buffer containing 200 U RNase inhibitor (EO0381; ThermoFisher) at 4°C for 2 h. The enriched m6A RNA was eluted twice with 100 µL elution buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4,. 1% NP-40, 200 U RNase inhibitor) and recovered by ethanol precipitation. Both input and m6A-enriched RNA were subjected to qRT-PCR analysis.

2.21 Flow cytometry analysis

Tumour tissues were collected from mice and processed into single-cell suspensions by enzymatic digestion with collagenase IV (07909; Stemcell) and DNase I (E127-01B; NovoProtein), followed by filtration through a 70-µm cell strainer. The resulting single-cell suspensions were stained with anti-mouse CD3 APC (20–0032; Tonbo; 1:200), anti-mouse CD8a FITC (35–0081;Tonbo; 1:200), anti-Fpr1 (NLS1878; Novus) and anti-GZMB PE (12-8898-82;Invitrogen; 1:200). Stained cells were analyzed using a flow cytometer, and data were processed with CytExpert software. CD8⁺ T cells were gated from CD3⁺ populations, and the expression of FPR1 and GZMB within CD8⁺ T cells was quantified.

2.22 Statistical analysis

Unless stated otherwise, experiments were carried out in triplicate, and data were presented as mean ± standard deviation (SD). GraphPad Prism 9 was used for statistical analyses. The unpaired, two-tailed Student's t-test was used to compare two groups, whereas one-way analysis of variance followed by Tukey's multiple comparison test was applied for comparisons across multiple groups. Survival analyses were conducted using Kaplan–Meier curves and differences in survival rates were determined by log-rank tests.

3.1 Comprehensive analysis of PCIF1 expression and its prognostic significance in OSCC

To elucidate the clinical significance of PCIF1 in OSCC, we first analyzed its expression across various cancer types using data from the TCGA database (Figure 1A). PCIF1 was found to be significantly upregulated in several cancers, with particularly high expression observed in oesophageal cancer (Figure S1A), suggesting its potential role in tumourigenesis.

Next, we examined PCIF1 protein expression in two independent patient cohorts (SYSUCC and FAH-SYSU) using IHC. In the SYSUCC cohort, tumour tissues exhibited markedly stronger PCIF1 staining compared to adjacent normal tissues (Figure 1B). Quantitative analysis further confirmed significantly higher IHC scores in tumour samples (Figure 1C). Consistent results were observed in the FAH-SYSU cohort, with elevated PCIF1 staining in tumour tissues and higher IHC scores compared to normal tissues (Figure 1E,F). Besides, in the SYSUCC cohort, the elevated PCIF1 expression was associated with higher tumour grades and more advanced clinical stages (Figure S1B,C). Similarly, the FAH-SYSU cohort demonstrated consistent findings, with higher PCIF1 levels correlating with aggressive tumour characteristics, such as advanced grade, reinforcing the role of PCIF1 in promoting OSCC progression (Figure S1E,F). In addition to its association with aggressive clinical features, PCIF1 was also linked to treatment response and recurrence involvement. Specifically, higher PCIF1 expression was observed in patients who experienced tumour recurrence and poor response to radiotherapy, as shown by increased IHC scores (Figure S1D,G).

Kaplan–Meier survival analysis consistently demonstrated that high PCIF1 expression was associated with significantly poorer overall survival in both cohorts (Figure 1D,G). Western blot analysis of PCIF1 protein levels in cell lines supported these findings. OSCC cell lines (TE1, TE9, TE12, K30 and K150) demonstrated markedly higher PCIF1 expression compared to the immortalized oesophageal epithelial cell line (SHEE) (Figure 1H). Multivariate analysis identified PCIF1 as an independent prognostic factor, further supporting its potential as a key biomarker in OSCC progression and prognosis.

3.2 PCIF1 regulates migration, invasion and proliferation in OSCC

To investigate the role of PCIF1 in OSCC, we selected KYSE30 and KYSE150 cell lines due to their notably high PCIF1 expression levels, as observed in Figure 1H. Functional assays were performed following the knockdown of PCIF1 using two specific sgRNAs (sg1 and sg2), which effectively reduced PCIF1 protein levels (Figure 2A). PCIF1 knockdown significantly reduced cell proliferation, as demonstrated by decreased optical density values in CCK-8 assays (Figure 2B), and colony formation assays showed fewer colonies, indicating impaired proliferative capacity (Figure 2C). Migration and invasion assays revealed that PCIF1 silencing also significantly inhibited the motility of OSCC cells, suggesting a key role in promoting metastasis (Figure 2DE). Additionally, PCIF1 knockdown led to increased apoptosis, highlighting its importance in cell survival (Figure 2F). In vivo experiments further supported the tumour-promoting role of PCIF1. PCIF1-knockdown KYSE150 cells were injected subcutaneously into nude mice, resulting in a marked reduction in tumour volume and weight, confirming that PCIF1 promotes tumour growth (Figure 2G–I). Immunohistochemical analysis of xenograft tumours demonstrated significantly lower expression of PCIF1 and Ki67 in the PCIF1-knockdown group, indicating reduced cell proliferation (Figure 2J–L).

To further elucidate the role of PCIF1's enzymatic activity in OSCC progression, we introduced both wild-type PCIF1 (OE) and a catalytically inactive mutant (OE-mut) into KYSE30 cells (Figure S2A). Overexpression of wild-type PCIF1 significantly increased cell proliferation, as shown by higher optical density values in CCK-8 assays (Figure S2B), along with enhanced colony formation (Figure S2C). Overexpression of PCIF1 also led to increased cell migration and invasion, whereas the catalytically inactive mutant showed diminished effects, indicating that PCIF1's enzymatic activity is critical for these processes (Figure S2DE). Apoptosis assays demonstrated reduced cell death in PCIF1-overexpressing cells, further supporting its role in promoting OSCC progression by enhancing cell survival (Figure S2F).

Overall, these findings imply that PCIF1's enzymatic activity may play a crucial role in the aggressive features of OSCC, highlighting its potential as a target for therapeutic strategies that aim to disrupt m6Am methylation in cancer cells.

3.3 m6Am sequencing identifies MTF2 as a potential downstream target of PCIF1

To further investigate the downstream effects of PCIF1-mediated m6Am modification in OSCC, we performed m6Am sequencing in PCIF1-knockdown and control KYSE150 cell lines. The results showed a global reduction in m6Am-modified transcripts upon PCIF1 knockdown, with a pronounced decrease around the 5′ UTR and CDS regions, suggesting that PCIF1 specifically targets these regions for m6Am modification (Figure 3A,B). Motif analysis revealed the presence of a GCTCA motif near the m6Am sites, potentially recognized by PCIF1 (Figure 3C). Among the top 10 differentially methylated genes identified from m6Am sequencing, MTF2 and GOLIM4 emerged as potential candidates (Figure 3D). To validate these findings, we incorporated protein expression data from the DepMap portal for OSCC cell lines. A negative correlation was observed between PCIF1 expression and MTF2 protein levels (R = −.52, p < .01), whereas GOLIM4 protein levels showed no significant association (R = −.09, p = .62) (Figure 3E,F). These results suggest that PCIF1 specifically regulates MTF2 expression through m6Am modification. Western blot analysis also confirmed that PCIF1 knockdown led to a marked increase in MTF2 protein levels, whereas GOLIM4 levels remained largely unchanged (Figure 3G). RIP assays further demonstrated a direct interaction between PCIF1 and MTF2 mRNA, supporting the hypothesis that PCIF1 directly modulates MTF2 translation via m6Am modification (Figure 3H–J). Bioinformatics analysis confirmed that MTF2 mRNA contains a high-confidence, PCIF1-dependent m6Am site (Figure 3K).

m6A-RIP followed by qPCR further demonstrated a significant reduction in m6Am modification on MTF2 mRNA in PCIF1-knockdown cells, confirming the loss of m6Am methylation on MTF2 transcripts (Figure 3L). Together, these findings establish MTF2 as a key downstream target of PCIF1, mediated by m6Am modification, and highlight its potential role in OSCC progression.

3.4 MTF2 as a translational target of PCIF1 via m6Am modification in OSCC

Building on our previous findings, we sought to clarify the mechanism by which PCIF1 regulates MTF2 expression. Despite targeting PCIF1, there was no significant change in MTF2 mRNA levels across different cell lines (Figure 4A,B). Similarly, MTF2 mRNA stability remained unchanged, suggesting that PCIF1's regulatory effects are likely post-transcriptional (Figure 4C,D). To further investigate this mechanism, we hypothesized that PCIF1-mediated m6Am modification may affect MTF2 mRNA's interaction with translation initiation factors or ribosomal machinery. To further explore the translational regulation, we performed polysome profiling. The results revealed that MTF2 mRNA was significantly enriched in polysome fractions upon PCIF1 knockdown, indicating enhanced translation (Figure 4E). Subsequent qPCR analysis of MTF2 mRNA in the extracted polysome fractions further confirmed enhanced MTF2 translation following PCIF1 knockdown (Figure 4F). We further analyzed MTF2 protein expression in different PCIF1-modified cell lines. In line with the translation assay results, PCIF1 knockout led to a marked increase in MTF2 protein levels, whereas overexpression of wild-type PCIF1 decreased MTF2 expression (Figure S3A–C). Importantly, overexpression of catalytically inactive PCIF1 failed to affect MTF2 protein levels, underscoring the necessity of PCIF1's methyltransferase activity in regulating MTF2 translation (Figure S3A–C).

We next employed a 5′ UTR luciferase reporter assay to dissect the mechanism of PCIF1-mediated translational control. In PCIF1-knockout cells, luciferase activity was significantly higher when the wild-type MTF2 5′ UTR construct was transfected, while the mutant construct showed no response, confirming that PCIF1 specifically regulates MTF2 translation via its 5′UTR (Figure 4G,H). These results collectively confirm that PCIF1 negatively regulates MTF2 translation via m6Am modification in OSCC, implicating the PCIF1-m6Am-MTF2 axis as a potential pathway driving tumour progression.

3.5 Functional role and clinical implications of MTF2 in OSCC

To further validate the role of MTF2 as a downstream target of PCIF1 and its clinical relevance in OSCC, rescue experiments were performed in KYSE30 and KYSE150. Co-knockdown of PCIF1 and MTF2 reversed the suppressive effects of PCIF1 knockdown on cell proliferation, migration and invasion, restoring these tumorigenic capabilities to levels similar to the control group (Figure 5B–E). This indicates that PCIF1 may exert its oncogenic functions partly through MTF2 downregulation.

Subsequent immunohistochemical analysis of OSCC patient samples revealed a consistent pattern of low MTF2 expression correlating with worse clinical outcomes, mirroring the trends observed for PCIF1 (Figure 5F). In both cohorts, reduced MTF2 levels were associated with higher tumour grade, advanced stage, recurrence and poor treatment response (Figure S4A–F). Moreover, Kaplan–Meier survival analysis showed that patients with low MTF2 expression had significantly lower overall survival compared to those with high MTF2 expression, confirming its prognostic value (Figure 5H,J). These results collectively underscore MTF2's role as a tumour suppressor in OSCC and suggest that PCIF1-mediated suppression of MTF2 contributes to tumour progression and adverse clinical features.

3.6 PCIF1-MTF2 axis drives OSCC tumourigenesis in transgenic mouse models

To investigate the in vivo role of the PCIF1-MTF2 axis in OSCC tumourigenesis, we generated transgenic mice models with conditional knockouts of PCIF1, MTF2 or both genes in oesophageal epithelial cells. These transgenic models were created by breeding PCIF1-floxed and MTF2-floxed mice with K14-CreERT2 mice, ensuring specific gene deletions in oesophageal tissues. Following tamoxifen induction, these mice were treated with 4-nitroquinoline 1-oxide (4NQO) for 16 weeks to induce OSCC (Figure 6A). Histological analysis at Week 26 revealed that PCIF1 knockout significantly reduced tumour lesion size and incidence, suggesting its critical role in tumour initiation and growth suppression (Figure 6B,C). In contrast, MTF2 knockout led to increased tumour size, whereas the combined knockout of both PCIF1 and MTF2 further exacerbated tumour formation, demonstrating a compensatory oncogenic effect in the absence of MTF2 (Figure 6D–F). H&E staining confirmed varying levels of tumour invasion across different genotypes, with double-knockout mice exhibiting more aggressive tumour phenotypes compared to PCIF1 knockout alone (Figure 6D–F). IHC analyses showed that PCIF1 deletion was associated with reduced expression of Ki67, a proliferation marker, whereas MTF2 deletion increased Ki67 expression, highlighting MTF2's tumour-suppressive role (Figure 6K,L). The expression of PCIF1 and MTF2 in tumour tissues was consistent with these findings: PCIF1 loss decreased PCIF1⁺ cell populations, whereas MTF2 loss increased MTF2⁺ cells (Figure 6G–J). These results reinforce the pivotal role of the PCIF1-MTF2 axis in modulating OSCC progression and underscore MTF2's role as a tumour suppressor, potentially counteracting PCIF1's oncogenic effects.

3.7 PCIF1 knockout potentiates anti-PD1 immunotherapy in OSCC

To investigate whether targeting PCIF1 could enhance the efficacy of anti-PD1 immunotherapy in OSCC, we conducted a comparative analysis of tumour progression in PCIF1-knockout mice treated with either anti-PD1 antibody or IgG control (Figure 7A). In comparison to anti-PD1 monotherapy, the combination of PCIF1 knockout with anti-PD1 treatment significantly reduced tumour burden (Figure 7B), as evidenced by decreased lesion area and a reduced number of tumour lesions (Figure 7C and E). Histopathological evaluation revealed that tumours in the combination group were of lower histological grade and exhibited less invasive characteristics, suggesting that PCIF1 deficiency enhances the tumour-suppressive effects of PD1 blockade (Figure 7D,F). Mechanistically, immunohistochemical analysis demonstrated that PCIF1 knockout combined with anti-PD1 treatment not only reduced the proliferation marker Ki67 but also led to a pronounced upregulation of the tumour suppressor MTF2 (Figure 7G–L). Specifically, while anti-PD1 therapy alone modestly decreased Ki67 levels, the combination with PCIF1 knockout further suppressed cellular proliferation, implying that the absence of PCIF1 sensitizes tumours to immunotherapy. Moreover, the increase in MTF2 expression observed in the PCIF1 knockout group suggests a potential mechanism by which PCIF1 inhibition augments the therapeutic impact of PD1 blockade, possibly by restoring MTF2-mediated suppression of oncogenic pathways (Figure 7I,J).

Interestingly, flow cytometric analysis of CD8⁺ T cells showed no significant differences in effector molecule expression, such as granzyme B (GZMB) and perforin (PRF1), between the PCIF1 knockout group and the IgG control group (Figure 7M–P). This suggests that PCIF1's tumour-suppressive effects are independent of direct immune modulation. Instead, PCIF1 knockout appears to enhance anti-PD1 efficacy by reducing tumour aggressiveness rather than altering the immune microenvironment, suggesting a novel mechanism for sensitizing tumours to immunotherapy. Together, these findings demonstrate that PCIF1 depletion and PD1 inhibition act through complementary but mechanistically distinct pathways to suppress tumour growth. This highlights the potential of dual targeting PCIF1 and PD1 as a novel therapeutic strategy for OSCC, leveraging their synergistic effects to maximize anti-tumour efficacy.