Connecting METTL3 and intratumoural CD33+ MDSCs in predicting clinical outcome in cervical cancer

Background Methyltransferase-like 3 (METTL3) is a member of the m6A methyltransferase family and acts as an oncogene in cancers. Recent studies suggest that host innate immunity is regulated by the enzymes controlling m6A epitranscriptomic changes. Here, we aim to explore the associations between the levels of METTL3 and CD33+ myeloid-derived suppressor cells (MDSCs) in tumour tissues and the survival of patients with cervical cancer (CC). Methods Specimens of paraffin embedded tumour from 197 CC patients were collected. The expression levels of METTL3 and CD33 were measured by immunohistochemical (IHC) staining. The clinical associations of the IHC variants were analysed by Pearson’s or Spearman’s chi-square tests. Overall survival (OS) and disease-free survival (DFS) were estimated by the Kaplan–Meier method and log-rank test. Hazard ratios (HRs) and independent significance were obtained via Cox proportional hazards models for multivariate analyses. METTL3 in CD33+ cells or CC-derived cells was knocked down by METTL3-specific siRNA, and MDSC induction in vitro was performed in a co-culture system in the presence of METTL3-siRNA and METTL3-knockdown-CC-derived cells compared with that of the corresponding controls. Results We found that tumour tissues displayed increased levels of METTL3 and CD33+ MDSCs compared with tumour-adjacent tissues from the same CC patients. Importantly, METTL3 expression was positively related to the density of CD33+ cells in tumour tissues (P = 0.011). We further found that the direct CD33+CD11b+HLA-DR− MDSC induction and tumour-derived MDSC induction in vitro were decreased in the absence of METTL3. The level of METTL3 in tumour microenvironments was significantly related to advanced tumour stage. The levels of METTL3 and CD33+ MDSCs in tumour tissues were notably associated with reduced DFS or OS. Cox model analysis revealed that the level of METTL3 in tumour cells was an independent factor for patient survival, specifically for DFS (HR = 3.157, P = 0.022) and OS (HR = 3.271, P = 0.012), while the CD33+ MDSC number was an independent predictor for DFS (HR: 3.958, P = 0.031). Interestingly, in patients with advanced-disease stages (II–IV), METTL3 in tumour cells was an independent factor for DFS (HR = 6.725, P = 0.010) and OS (HR = 5.140, P = 0.021), while CD33+ MDSC density was an independent factor for OS (HR = 8.802, P = 0.037). Conclusion Our findings suggest that CD33+ MDSC expansion is linked to high levels of METTL3 and that METTL3 and CD33+ MDSCs are independent prognostic factors in CC.

addition to traditional prognostic factors, including age, WHO grade, TNM stage and clinical status, some of the molecular markers could be new predictors of CC prognosis (6,9,10). However, there are no con rmed molecular markers for tumour progression or prognosis in CC patients. The related viral proteins E6 and E7 have been the focal points of research for the past several years (11,12). In other words, easily detectable and meaningful molecular markers need to be con rmed.
CD33-positive cells are usually de ned as myeloid-derived suppressor cells (MDSCs) with suppressive in uence on human tumour tissues (25,26). MDSCs in the tumour environment were con rmed to be an independent indicator of poor prognosis in patients with many solid tumours (25,27,28). In our previous studies, the MDSC proportion was expanded in the tumour microenvironment and showed extensive negative regulatory function for antitumour immunity in malignancies (29)(30)(31). Recent studies have indicated that the differentiation of myeloid cells is regulated by m 6 A methyltransferases, including METTL3 (22,32,33). We hypothesized that MDSC expansion may be linked to the level of METTL3 in the microenvironment of CC.
In the present study, we detected the levels of METTL3 and CD33 + MDSCs in tumour specimens from 197 CC patients by immunohistochemical (IHC) staining. We observed increased levels of METTL3 and CD33 + MDSCs in tumour tissues and positive associations between the levels of METTL3 and CD33 + MDSCs. The high levels of METTL3 and CD33 + MDSCs in the CC tumor microenvironment were signi cantly associated with poor disease-free survival (DFS) and overall survival (OS) in CC patients.
Importantly, METTL3 and CD33 + MDSCs were independent prognostic predictors for CC patients. These ndings suggest that METTL3 and MDSCs contribute to the development of disease and that METTL3 may respond to MDSC expansion in tumour microenvironments in CC.

Methods
Patients and tissue samples The METTL3 expression level was scored in tumour cells in ve to ten separate ×400 high-power elds (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 ve to ten separate ×400 HPFs from the same patient. METTL3 expression level in tumour-in ltrating cells (TILs) was evaluated based on the mean percentage from ve 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-speci c 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: , and all data were analysed by the original analysis software provided with the ow cytometer (CytExpert). The CD33 + CD11b + HLA-DRcells were de ned as peripheral MDSCs in this study.

Immunoblotting
The harvested cells were lysed with pre-cooled RIPA buffer, and the proteins were quanti ed by a BCA protein assay kit (23227, Invitrogen, Carlsbad, USA) and then separated using a 10% SDS-PAGE. Proteins were transferred onto polyvinylidene di uoride 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.

Results
The level of METTL3 is positively linked to the number of CD33 + MDSCs and contributes to tumour development In the present study, the levels of METTL3 and CD33 + MDSCs were examined in tumour specimens from 197 patients with CC by IHC. METTL3 was located in the nuclei of tumour cells and tumour-in ltrating immune cells, while CD33 + cells were scattered mainly in the tumour stroma ( Figure 1 A and 1B); isotype IgG was used as a control ( Figure 1C). Importantly, we found that CD33 and METTL3 co-localized in some tumour-in ltrated immune cells ( Figure 1D). We further demonstrated that the percentage of CD33 + CD11b + HLA-DRperipheral MDSCs was increased in CC patients compared with healthy donors, as was the percentage of tumour-derived CD33 + CD11b + HLA-DR -MDSCs compared with that of tumouradjacent tissues ( Figure 1E and 1F, n = 3). Consistent with the increase in the MDSC population in CC patients, the level of METTL3 was increased in the peripheral and tumour-in ltrating immune cells compared with the corresponding controls ( Figure 1G and 1H). Among the 197 patients with CC, the median survival time was 96 months (range: 0 to 120 months), and the 10-year DFS and 10-year OS rates were 88.83% and 86.80%, respectively (Figure S1A and S1B). Table 2 shows the results of the relationships between clinicopathological features and immunohistochemical variants in different cell types in the tumour microenvironment. High METTL3 expression in the tumour and in tumour-in ltrating immune cells was linked to tumour stage (P = 0.040 and 0.020, respectively).
In addition, we analysed the relationship between METTL3 expression in tumour cells and in tumour- To further investigate the role of METTL3 in the regulation of MDSC expansion, we knocked down METL3 expression in CD33 + cells or HeLa cells. We found that CD33 + CD11b + HLA-DR -MDSCs and tumour-derived MDSCs were decreased when METTL3 was knocked down in CD33 + cells or HeLa cells ( Figure 2D -2G).
High METTL3 levels and CD33 + MDSC density are associated with poor outcomes To evaluate the expression of METTL3 and CD33 as predictors for the prognosis of the 197 patients, Kaplan-Meier survival curves were used for analysis. The high level of METTL3 in tumour cells was signi cantly correlated with decreased DFS (P < 0.001, Figure 3A) and OS (P < 0.001, Figure 3B) in CC patients. Accordingly, a high level of METTL3 in tumour-in ltrating immune cells was negatively correlated with DFS (P = 0.002, Figure 3C) and OS (P < 0.001, Figure 3D) in CC patients. The high density of CD33 + MDSCs was obviously correlated with decreased DFS (P < 0.001, Figure 3E) and OS (P < 0.001, Figure 3F) in CC patients.
METTL3 and CD33 + MDSCs are independent factors for patient prognosis As shown in Table 3, univariate analysis showed that in addition to lymph node involvement and clinical stage, high levels of METTL3 in tumour cells (HR: 4.244, P = 0.002) and in tumour-in ltrating immune cells (HR: 4.857, P = 0.004) and a high density of CD33 + MDSCs (HR: 6.579, P = 0.002) were noticeably correlated with reduced DFS. In addition, we found that high levels of METTL3 in tumour cells (HR: 5.502, P = 0.001) and in tumour-in ltrating immune cells (HR: 6.021, P = 0.001) and a high density of CD33 + MDSCs (HR: 5.755, P = 0.001) were associated with decreased OS. Moreover, clinicopathological parameters such as clinical stage (HR: 3.511, P = 0.005) and nodal status (HR: 2.798, P = 0.032) also had prognostic value with decreased DFS, and clinical stage (HR: 3.820, P = 0.001) was related to decreased OS. In addition, other clinical characteristics, such as age and tumour status, were not clearly related to DFS and OS (Table 3). When we performed multivariate Cox proportional hazards regression analysis in Table 4, we included all the signi cant univariate variables. For all 197 patients, in addition to clinical stage (HR: 3.827, P = 0.003), N status (HR: 3.219, P = 0.021) was an independent factor for DFS, clinical stage (HR: 4.248, P < 0.001) was an independent factor for OS, and METTL3 levels in tumour cells (HR: 3.157, P = 0.022) and in tumour-in ltrating immune cells (HR: 3.368, P=0.036) and CD33 + MDSCs (HR: 3.958, P = 0.031) were independent factors for both DFS and OS (Table 4).  Table 4).
The combination of METTL3 levels and CD33 + MDSCs was associated with the survival of patients with CC Finally, considering that METTL3 levels were positively correlated with high CD33 + MDSC in ltration, we calculated the signi cance of the combination of these two biomarkers for the survival of CC patients. All 197 patients were divided into three groups. Patients with low levels of both METTL3 in tumourin ltrating immune cells and CD33 + MDSCs were included in the combined low expression group, those with high levels of only one of the two biomarkers were included in the combined medium expression group, and those with high levels of both were included in the combined high expression group. The high combination of METTL3 and intratumoural CD33 + MDSCs was associated with reduced DFS (P < 0.001, Figure 5A) and OS (P < 0.001, Figure 5B). In the patients (127) with early-stage disease, the high combination of METTL3 and CD33 + MDSCs was not related to DFS (P = 0.063, Figure 5C) but was clearly negatively related to OS (P = 0.037, Figure 5D). In the patients (70) with advanced-stage disease, the combination of high METTL3 levels and CD33 + MDSCs was clearly related to unfavourable DFS (P < 0.001, Figure 5E) and OS (P < 0.001, Figure 5F).

Discussion
The development of tumour cells depends on the tumour microenvironment, which includes tumour cells, various other cells and extracellular components (7). The immunosuppressive cells in the tumour microenvironment, such as Tregs and MDSCs, not only affect each other, but their changes in number and types will affect tumour development (34,35). METTL3 is one of the 'writers', and its role is to catalyse the m 6 A methylation of mRNA (and other nuclear RNAs); after the methylation of m 6 A, RNAs will nucleate and transport to the cytoplasm faster and then produce more proteins for function and proliferation.
Some studies have shown that METTL3 expression can promote tumour cell proliferation, leading to poor patient prognosis. The tumour-in ltrated MDSC population usually induces antitumour immunity tolerance by inhibiting the proliferation and function of T cells, such as hindering antigen presentation by antigen-presenting cells (36). Increased METTL3 levels and CD33 + MDSCs have been found in tumour microenvironments and lead to a poor prognosis (37)(38)(39)(40). In this study, we focused on the distribution of

Consent for publication
The authors declare no con icts of interest. All authors read and approved the nal manuscript.

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The authenticity of this article has been validated by uploading the key raw data to the Research Data Deposit (RDD) public platform (www. researchdata.org.cn).

Competing interests
The authors have declared that they have no competing interest. The sources that funded this study played no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript.       de ned as MDSCs in this study. (E-F) Representative density plots showed the MDSC population in the peripheral blood of healthy donors (HD) or CC patients, as well as in the immune cells from tumour tissues (TIL) or tumour-adjacent tissues (NIL). A statistical graph is included for the comparison between the indicated groups. (G-H) Representative immunoblotting shows the expression of METTL3 in the peripheral blood, TILs and NILs. A statistical graph is included for the comparison between the indicated groups. The experiments in (E, F) were performed at least three times, and the data were plotted as the mean ± SEM. Statistics were conducted with an unpaired Student's t test, *P < 0.05, and ***P < 0.001 vs. the corresponding control.

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