For the collection of cSCC samples from patients, analyses were performed with the approval of the Ethical Committee of the First Hospital of Shanxi Medical University. cSCC tissue was collected from 10 patients after surgery and the surgical details of those 10 patients are shown in Table 1. The pathologist confirmed the type of pathology in the sample.

Tumor sections embedded in paraffin were dried at 90 °C for 4 h, dewaxed in xylene, and then rehydrated in graded ethanol solutions. Cooled tissue sections were immersed in 0.3% hydrogen peroxide solution for 15 min to block endogenous peroxidase activity, rinsed with PBS for 5 min, and blocked with 3% BSA solution at room temperature for 30 min. After washing by PBS, the sections were incubated with antibodies (Abcam, Cambridge, MA, USA) for 1 hour in 1% BSA blocking buffer, followed by incubated for 1.5 hours of secondary antibody conjugated with Alexa Fluro 549 (Thermal Fischer) and DAPI. Fluorescence images were obtained using a Zeiss LSM510 confocal microscope, equipped with a 63× immersion objective.

A431(Human cSCC cell) and SCC7 (mouse SCC cell) were obtained from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China). These cells were maintained in a DMEM (Thermo Fisher Scientific, Waltham, MA, USA), which is supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were cultured in a humidified incubator at 37 °C with 5% CO2 (Sanyo Osaka, Japan).

Peripheral blood mononuclear cells from blood donors were isolated by using the Ficoll gradient method, and CD8+T cells were isolated from 1.25 × 107 PBMCs by using the Easy Sep Human CD8+T Cell Isolation Kit.

Extraction of total RNA was performed using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed using the cDNA Synthesis kit (Takara, Japan). The quantity and density of the RNA were verified by a spectrophotometer. QRT-PCR was performed using the SYBR Green Master MIX Kit (Takara, Japan) according to the manufacturer’s instructions. The assays were performed in triplicate and relative gene expression was determined by using the 2-ΔΔCt method.

METTL3 forward: 5’- CATCCGTCTTGCCATCTCTAC-3’

METTL3 reverse: 5’- GACCTCGCTTTACCTCAATCA-3’

METTL14 forward: 5’- GCATCACTGCGAATGAGAAATG-3’

METTL14 reverse: 5’- CAGAACCGCACCAGAGAAATA-3’

RMB15 forward: 5’- GCCCAACTCAAGATCACTCA-3’

RBM15 reverse: 5’- AGCCAGGAGAATGGCATAAC-3’

WTAP forward: 5’- AATGGTAGACCCAGCAATCAA-3’

WTAP reverse: 5’- CGTAAACTTCCAGGCACTCA-3’

VIRMA forward: 5’- GAGGCGGATCCTTTGAGTTT-3’

VIRMA reverse: 5’- AACGACCTGAGAGAGGGATAG-3’

FTO forward: 5’- AGAGCGGGAAGCTAAGAAAC-3’

FTO reverse: 5’- GCCACTGCTGATAGAACTCAT-3’

ALKBH5 forward: 5’- GTGGGTATGCTGCTGATGAA-3’

ALKBH5 reverse: 5’- TCGCGGTGCATCTAATCTTG-3’

To extract total proteins, cell lysates were obtained using RIPA buffer (Beyotime, Shanghai, China) supplemented with phosphatase and protease inhibitors (Yeasen, Shanghai, China). A total of 15 μL protein was injected into a Bis-Tris SDS/PAGE gel and transferred to PVDF membranes. After blocking with 5% BSA, the membranes were incubated with primary antibody overnight at 4 °C. Membranes were then exposed to the secondary antibody for 60 min. The strips were incubated with an ECL kit and analyzed with an imaging system. Densitometric analysis was performed by using Adobe Photoshop CS6.

Purified CD8+T cells were obtained by negative selection. SCC was added to purified CD8+T cells at a 1:10-50 ratio and cultured in 48-well trays (Costar 3548, Cambridge, MA) in RPMI 1640. IL-2 (50 units/mL, Chiron, Emeryville, CA) was added to each well beginning on day 1 and every 2 to 3 days thereafter. SCC cells were harvested on day 7 and assayed for lytic activity in triplicate using CCK-8 assay.

Enzyme-linked immunosorbent assay (ELISA) to detect cytokines was performed with kits (Boster Bio, Wuhan, China).

A total of 103 cells were inoculated into each well of 96-well plates. At each time point (24 h, 48 h, 72 h, and 96 h), 10 μL CCK-8 solution (Yeasen, Shanghai, China) was added to the sextuplicate wells. The wells were incubated for 3 h, and the absorbance of each well was determined at 450 nm.

All of the animal experiments were approved by the Animal Experimentation Ethics Committee of the first Hospital, Shanxi Medical University. Eight-week-old C57BL/6 mice were maintained according to the guidelines of the 3Rs (Replacement, Reduction, and Refinement). A total of 1 × 107 SCC7 cells resuspended in 100 μL of PBS were inoculated subcutaneously in the left flank of the mice. When the tumor volume reaches 50‒100 mm3, anti-PD-1 antibody (200 μg/mouse) or combined HPV vaccine (0.25 μL/mouse) was injected. After the tumor was detected, tumor size was measured every 5 days by a vernier caliper and tumor volume was calculated as volume (cm3) = L × W2 × 0.5 with L and W representing the largest and smallest diameters, respectively.

The m6A RNA Methylation Assay Kit (Abcam, ab185912) was used to evaluate the content of m6A in total RNA.

The poly(A) + RNAs (400 ng) were double-diluted and spotted onto a nylon membrane (GE Healthcare, USA). Briefly, the membranes were then UV crosslinked, blocked, incubated with m6A antibody and horseradish peroxidase-conjugated anti-rabbit IgG, and finally detected with a 3,3’-diaminobenzidine peroxidase substrate kit. The same 400 ng poly(A) + RNAs were spotted on the membrane, stained with 0.02% Methylene blue in 0.3 M sodium acetate for 2 h, and washed with ribonuclease-free water for 5 h.

Statistical analyses were performed using the SPSS software version 19.0 (SPSS, Inc., Chicago, IL) or GraphPad Prism 7.0 (GraphPad Software, USA) as previously described. The values are presented as the mean ± Standard Deviation (SD). For comparisons, Student’s t-test and one-way ANOVA test by Least Significant Difference test were performed, as appropriate; p < 0.05 was considered statistically significant.

The authors selected five cases each of HPV-vaccinated and non-HPV-vaccinated cSCC patients' tumor tissues in order to determine the effect of the HPV vaccine on immune cells in human cSCC tissues, and analyzed the infiltration of CD8, CD4, NK, DC, and Macrophages in cSCC tumors by immunofluorescence. There was a statistical significant difference between the two groups in the five types of immune cells. The expression of CD8, CD4, CD69, CD11c, and CD163 in the tumors of humans in the vaccinated group was significantly increased (Fig. 1A‒B).

Figure 1.

Differences in immune microenvironment in cSCC tissues of HPV vaccinated and unvaccinated patients. (A) Differences in immune microenvironment (CD8, CD4, NK, DC, Macrophage) in cSCC tissues; (B) Image J statistical analysis of the percentage of positive immune cells. **p < 0.01, ***p < 0.001.

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To investigate the effect of the HPV vaccine on CD8+T cell activity, the authors co-cultured CD8+T cells with A431 cells at 10:1, 25:1 and 50:1 ratios and added 2 μg or 10 μg of bivalent HPV vaccine Cervarix (GSK, 0.5 μg/mL). CD8+T cells showed higher cytotoxic activity after adding the HPV vaccine, however, the differences in the killing activity of the HPV vaccine at different concentrations on A431 cells were not statistically significant at a certain potency-target ratio (Fig. 2A). To explore the effect of HPV vaccine on cytokine secretion in the co-culture system, the authors examined IFN-γ, TNF-α, TGF-β, IL-17, IL-12, and IL-33 in the supernatant, and the secretion of IFN-γ, TNF-α, TGF-β, and IL-17 increased significantly after the addition of 2 μg of HPV vaccine, while the secretion increased after the addition of 10 μg of HPV vaccine was IFN-γ, TNF-α, TGF-β, and IL-33 (Fig. 2B).

Figure 2.

Effect of HPV vaccine on CD8+T cell activity in cSCC. (A) CD8+T cell killing assay on A431 cells; (B) secretion of inflammatory factors by HPV vaccine stimulation on CD8+T cells. *p < 0.05, **p < 0.01, ***p < 0.001.

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To further analyze the effect of the HPV vaccine on cSCC at the animal level, the authors hypothesized that the addition of the HPV vaccine would enhance the immunogenicity and antitumor effects of PD-1 treatment in mice. To test this hypothesis, the authors divided 10 mice with cSCC into two groups of 5 mice each and injected PD-1 Abs (10377-M94, SinoBiological) or PD-1Abs+HPV vaccine. The authors did not set up a no-treatment group reflecting the therapeutic effect of PD-1 on cSCC, because it has been concluded that PD-1 monoclonal antibody has a significant inhibitory effect on cSCC in other studies.22–25 To monitor the antitumor response, tumor volume was measured every 5 days, and mice were executed at day 30, tumor tissue was isolated and weight was measured (Fig. 3A). Compared to PD-1 Abs, tumors in the PD-1Abs+HPV vaccine group were reduced in weight (Fig. 3B) and volume (Fig. 3C) at day 30, which demonstrated that stable regression was observed on tumors in all groups. Immunofluorescence analysis of mouse tumor tissues observed a significant increase in intratumoral infiltration of CD8, CD4, NK, DC, and Macrophage in PD-1 monoclonal antibody combined with HPV vaccine mice (Fig. 4A‒B).

Figure 3.

Impact of HPV vaccine combined with PD-1 monoclonal antibody for cSCC. (A) Tumor picture; (B) Volume change; (C) Weight change. ***p < 0.001.

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Figure 4.

Effect of HPV vaccine combined with PD-1 monoclonal antibody on immunofluorescence in mice. (A) Immunofluorescence observation of immune microenvironment differences (CD8, CD4, NK, DC, Macrophage). (B) Image J statistical analysis of the percentage of positive immune cells. ***p < 0.001.

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With the purpose of observing the effect of the HPV vaccine on m6A in tumor tissues, the authors analyzed the m6A content in mouse tumor tissues using a colorimetric method and Dot blot and found that the m6A content in the PD-1Abs+HPV vaccine group was significantly lower than that in the PD-1 Abs group (Fig. 5A‒B). The authors further screened the expression of m6A methylesterase and demethylase by qPCR, and the results were shown in 3H. METTL3 was significantly downregulated in the presence of HPV vaccine, while the differences of METTL14, RBM15, WTAP, VIRMA, FTO, and ALKBH5 were not statistically significant (Fig. 6A). The downregulation of METTL3 expression at the protein level in PD-1Abs+HPV vaccine group was further verified by Western blot (Fig. 6B).

Figure 5.

m6A mRNA and protein expression in tumor tissues. (A) Colorimetric analysis of m6A content in tumors; (B) Dot Blot to verify m6A content in tumors. ***p < 0.001.

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Figure 6.

Selection of targets with aberrant m6A expression. (A) Real-time PCR to screen the expression of m6A-related methylesterase and demethylase in tumor tissues; (B) Western Blot to verify the difference of METTL3 expression. ***p < 0.001.

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To further study the effect of the HPV vaccine on METTL3 expression in cSCC, the authors overexpressed METTL3, qPCR and Western blot were performed to detect the overexpression efficiency at mRNA and protein levels (Fig. 7A‒B). CCK-8 assay revealed that there was no significant difference in cell activity between the six groups within 24 hours of incubation. After 96 hours of culture, cell activity was significantly reduced when co-cultured with CD8+T cells and A431 cells compared with the control group, and further reduced in A431 cell activity after the addition of HPV vaccine, with statistical differences between all groups. After overexpression of METTL3, it still showed the inhibitory effect of CD8+T cells and HPV vaccine on A431 cell activity, and A431 cell activity was significantly higher in the CD8+T cells+HPV vaccine-OE-METTL3 group than in the CD8+T cells+HPV vaccine-OE-NC group (Fig. 8A). Also, the authors examined m6A in each group and found that HPV vaccine significantly reduced the total m6A content in the co-culture system, while METTL3 overexpression significantly abolished this effect. Interestingly, the m6A content in the CD8+T cells+HPV vaccine-OE-NC group was not statistically significantly different from Control -OE-NC, while the m6A content in the CD8+T cells -OE-NC group was elevated compared to Control -OE-NC, although there was no statistical difference (Fig. 8B).

Figure 7.

Identification of METTL3 overexpression. (A‒B) Real-time PCR and Western Blot verified the efficiency of METTL3 overexpression. ***p < 0.001.

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Figure 8.

Mechanisms of HPV-enhanced susceptibility to SCC immunotherapy. (A) CCK8 assay revealed that HPV vaccine significantly promoted the suppressive effect of CD8+T cells on A431 cells, while METTL3 overexpression attenuated the suppressive effect of HPV vaccine treatment on A431 cells by CD8+T cells; (B) m6A assay revealed that HPV vaccine significantly reduced the total m6A content in the high CD8+T cells + A431 cells co-culture system, while METTL3 knockdown significantly abolished this effect. *p < 0.05, **p < 0.01, ***p < 0.001.

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For further evaluation of METTL3 in the HPV vaccine against csCC, xenograft tumor experiments were performed. The authors divided the mouse models of cSCC into 4 groups of 5 mice each and gave PD-1 Abs+OE-NC, PD-1 Abs+HPV vaccine+OE-NC, PD-1 Abs+OE-METTL3, and PD-1 Abs+HPV vaccine+OE-METTL3 treatments respectively. In the combination of anti-PD-1 antibody and HPV vaccine, tumor volume and weight were significantly reduced, and co-treatment with HPV vaccination and anti-mouse PD-1 antibody resulted in complete inhibition of tumor growth, while the inhibitory effect of anti-PD-1 antibody on tumor volume and weight was attenuated after overexpression of mettl3 (Fig. 9A‒C). Histologically, it was found that the PD-1 Abs+HPV vaccine+OE-NC group had significantly more intratumoral infiltrates of CD8, CD4, NK, DC, and Macrophages than the other groups (Fig. 10A‒B).

Figure 9.

Animal-level validation of METTL3 overexpression abolishing HPV vaccine to enhance the sensitivity of cSCC mice treated with PD-1 monoclonal antibody. (A) Tumor picture; (B) volume change; (C) weight change. ***p < 0.001.

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Figure 10.

Effect of overexpression of METTL3 on combination therapy in immunofluorescence. (A) Immunofluorescence observation of immune microenvironment differences (CD8, CD4, NK, DC, Macrophage); (B) Image J statistical analysis of immune cell positive ratio. ***p < 0.001.

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