Patients and tissue samples
A total of one hundred ninety-seven CC patients who received therapy at Sun Yat-Sen University Cancer Center in Guangzhou, China, and who accepted medical follow-up that continued until 2019 were included. Paraffin tumour specimens from 197 CC patients were collected at Sun Yat-Sen University Cancer Center between 2008 and 2010. In this retrospective study, none of the patients received antitumour treatment before tumour tissue was obtained, and all 197 patients were histologically confirmed as having primary CC. As shown in Table 1, according to the WHO and the International Federation of Gynecology and Obstetrics classification criteria, 127 (64.5%) patients had stage I disease, and 70 (35.5%) had stage II-IV disease; 26 (13.2%) died. Forty-five (22.8%) patients received surgery alone, 41 (20.8%) patients received radiation therapy alone, 27 (13.7%) patients received surgery + radiation therapy, and 84 (42.6%) patients received surgery + radiation therapy + chemotherapy. The tumour specimens and clinical information were provided by the Pathology Department of Sun Yat-Sen University Cancer Center. The study was approved by the Research Ethics Committee of the Sun Yat-sen University Cancer Center, and written informed consent was obtained from all 197 patients.
The HeLa cervical cancer cell line is maintained in our laboratory. HeLa cells were cultured RPMI 1640 (Gibco, Grand Island, NY, USA) containing 10% foetal bovine serum (FBS; EXCELL BIO, Florida, USA).
Antibodies and Reagents
The antibodies and reagents used in this study were as follows. For immunoblotting, immunohistochemistry (IHC) and immunofluorescence staining, rabbit anti-METTL3 antibody (ab195352; Abcam, Cambridge, UK), rabbit anti-CD33 antibody (ab199432; Abcam, Cambridge, UK), rabbit mAb IgG control (ab172730; Abcam, Cambridge, UK), and rabbit anti-GAPDH antibody (10494; Proteintech, Wuhan, China)) were used. Rabbit anti-human IgG (H+L) secondary antibody, HRP (PV-6001-6.0; ZSGB Bio, Beijing, China), a DAB Horseradish Peroxidase Colour Development Kit (ZLI-9017, ZSGB Bio, Beijing, China), and RIPA cell lysis buffer (CW2333S; CWBIO, Beijing, China) were also applied. Milk (1706404, Bio-Rad, Hercules, USA) and West dura extended duration substrate (34075, Thermo Scientific, Carlsbad, USA), haematoxylin (DH0001; Solarbio, Beijing, China) and DAPI (GB1012, Servicebio, Wuhan, China); Lipofectamine™ LTX Reagent with PLUS™ Reagent (15338100; Invitrogen, Carlsbad, USA) were used for the siRNA assay. For CD33+ cell isolation, human CD33-antibody-linked magnetic beads (23227, Invitrogen, Carlsbad, USA) were applied. Antibodies for flow cytometry were as follows: PerCP-Cyanine5.5 anti-human CD33 antibody (45-0338-42; eBioscience, San Diego, USA), APC anti-human HLA-DR antibody (17-9956-42; eBioscience, San Diego, USA), and PE anti-human CD11b antibody (12-0118-42; eBioscience, San Diego, USA). Fixable Viability Stain 700 (564997, BD, San Jose, USA) was used to distinguish the dead cells.
Immunohistochemistry (IHC) and immunofluorescence staining
Paraffin-embedded tissues were continuously sectioned at a thickness of 4 μm, and the immunohistochemistry kit was used according to the manufacturer’s instructions. In brief, tissue sections were deparaffinized by xylene, rehydrated in graded alcohols and immersed in EDTA (PH 8.0). Microwave (95°C 12 min) was applied for antigen retrieval, and samples were cooled to room temperature. The endogenous enzyme block reagent was used to block the activities of endogenous peroxidase. The goat serum was applied to block nonspecific binding sites at room temperature for 30 min. Primary antibodies, including rabbit anti-METTL3 antibody (1:400), rabbit anti-CD33 antibody (1:200), and rabbit mAb IgG control (1:200) were incubated at 37°C for 1 h and developed with peroxidase. After staining by haematoxylin, images were taken under a microscope (NIKON ECLIPSE 80i). The expression of METTL3 on CD33+ cells was measured by immunofluorescence staining; DAPI was used to stain the nuclei. The images were taken with a fluorescence microscope (NIKON ECLIPSE C1).
The METTL3 expression level was scored in tumour cells in five to ten separate ×400 high-power fields (HPFs). We scored METTL3 expression in the tumour cells of each specimen using a semiquantitative immunoreactivity scoring system, which ranged from zero to twelve and was equal to multiplication of the intensity of immunohistochemical staining (zero: no staining; one: weak staining; two: moderate staining; and three: strong staining) and the percentage of positive tumour cells (one: less than 25%; two: 25–50%; three: 50–75%; and four: more than 75%). The expression of CD33 was determined by counting CD33-positive cells from five to ten separate ×400 HPFs from the same patient. METTL3 expression level in tumour-infiltrating cells (TILs) was evaluated based on the mean percentage from five to ten separate ×400 HPFs from the same patient. These METTL3- and CD33-positive scores were determined separately by two pathologists. An isotype control IgG antibody was included.
Knockdown of METTL3 by siRNA
To knock down METTL3 in HeLa cells or CD33+ cells, we generated METTL3-specific siRNA (siMETTL3) with the help of RiboBio; a control-siRNA vector was also generated. The siRNAs were transiently transfected into HeLa or CD33+ cells by Lipofectamine™ LTX Reagent with PLUS™ Reagent according to the manufacturer's instructions. After 48 h, the cells were harvested for immunoblotting and MDSC induction.
The sequences of siMETTL3 siRNAs were as follows:
siMETTL3_001 5'- CAAGTATGTTCACTATGAA -3';
siMETTL3_002 5'- GACTGCTCTTTCCTTAATA -3'; and
siMETTL3_003 5'- GGACTCGACTACAGTAGCT -3’.
MDSCs induction in vitro and fluorescence-activated cell sort (FACS) staining
Peripheral blood mononuclear cells (PBMCs) were derived from the peripheral blood of healthy donors by gradient centrifugation separation. The CD33+ cells were sorted by human CD33-antibody-linked magnetic beads. After isolation, the 1×10^6 CD33+ cells were seeded in a 24-well plate (outer well) and co-cultured with HeLa cells (inner well, at 1:2 ratio) with or without METTL3 knockdown in a Transwell system (3421, Corning, New York, NY, USA) for 48 h. The cells were harvested for FACS staining and detected by cytometry CytExpert software (Beckman Coulter, San Jose, USA) or immunoblotting. Cells were pipetted into single-cell suspensions and incubated with corresponding fluorescence-labelled antibodies according to the manufacturer’s instructions. The flow cytometer used was a cytoFLEX (Beckman), and all data were analysed by the original analysis software provided with the flow cytometer (CytExpert). The CD33+CD11b+HLA-DR- cells were defined as peripheral MDSCs in this study.
The harvested cells were lysed with pre-cooled RIPA buffer, and the proteins were quantified by a BCA protein assay kit (23227, Invitrogen, Carlsbad, USA) and then separated using a 10% SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membrane (IPVH00010; Millipore, Massachusetts, USA). The membrane was blocked with 5% milk and incubated with the corresponding primary antibodies at 4°C overnight. Next, the membrane was incubated with HRP-coupled secondary antibodies at room temperature and detected using a West dura extended duration substrate.
SPSS 19.0 software (SPSS Inc., Chicago, USA) was used to analyse all the data, and GraphPad Prism 7 software (La Jolla, USA) was used to obtain the curves. The median values were used as cut-off values to divide the patients into two groups (high level and low level). We used Pearson’s chi-square test or Spearman's chi-square test to analyse the relationships between immunohistochemical variants in different cell populations and patients’ clinical parameters. The relationships among the expression of METTL3 in tumour cells, METTL3 in tumour-infiltrating immune cells and CD33 in tumour-infiltrating immune cells were determined using Pearson's or Spearman’s correlation coefficient and linear regression analyses. Cut-off selection was based on X-tile (Version 3.6.1, New Haven, USA). Then, we evaluated prognostic factors in univariate and multivariate analyses using the Cox proportional hazards model. In our research, *P < 0.05 was considered significant. The authenticity of this article has been validated by uploading the key raw data onto the Research Data Deposit (RDD) public platform (www.researchdata.org.cn), with the RDD approval number RDDB2020000943.