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 nucleus of tumour cells and tumour-infiltrating immune cells (Figure 1 A and B), while CD33+ cells were scattered mainly in the tumour stroma (Figure 1 C and D), and isotype IgG was used as a control (Figure 1 E). The levels of METTL3 and CD33+ MDSCs in tumour tissues were 63.45% (125/197) and 52.28% (103/197), respectively (Figure 1). Among the 197 patients with CC, the median survival time was 96 months (range: 0 to 120 months); the 10-year DFS rate and 10-year OS rate were 88.83% and 86.80%, respectively (Figure S1 A and B). 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-infiltrating immune cells was linked to advanced tumour stage (P = 0.04 and 0.02, respectively).
In addition, we analysed the relationship between METTL3 expression in tumour cells and in tumour-infiltrating immune cells and the number of CD33+ MDSCs via Spearman’s correlation coefficient and linear regression. The expression of METTL3 in tumour cells was positively correlated with that in tumour-infiltrating immune cells (R = 0.264, P< 0.001) (Figure 2 A). The number of CD33+ cells was positively correlated with the expression of METTL3 in tumour cells (R = 0.145, P = 0.041) and tumour-infiltrating immune cells (R = 0.182, P = 0.011) (Figure 2 B and C).
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 significantly 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-infiltrating 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
Univariate analysis showed that in addition to lymph node and clinical stage, high levels of METTL3 in tumour cells (HR: 4.244, P = 0.002) and in tumour-infiltrating 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 also found that high levels of METTL3 in tumour cells (HR: 5.502, P = 0.001) and in tumour-infiltrating immune cells (HR: 6.021, P = 0.001) and a high density of CD33+ MDSCs cells (HR: 5.755, P = 0.001) were associated with decreased OS. As shown in Table 3, 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 the multivariate Cox proportional hazards regression analysis, we included all the significant 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-infiltrating 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).
METTL3 and CD33+ MDSCs have predictive value for patients with early and advanced disease stages
We further divided the 197 patients into two subgroups based on the clinicopathological stage: 127 of the total patients were in early disease stage (stage I), while 70 of the total patients were in advanced disease stage (stage II-IV). Through Kaplan–Meier method, we found that the high expression of METTL3 in tumour-infiltrating immune cells was significantly correlated with poor DFS (P = 0.033) and OS (P = 0.019) (Figure S2 C and D) in patients with early disease stage, while there was no significant association between the high expression of METTL3 in tumour cells (P = 0.400 vs P = 0.183) and the number of CD33+ MDSCs (P = 0.393 vs P = 0.227) with the DFS and OS of patients with early-stage disease (Figure S2 A, B, E and F). For patients with advanced-stage disease (n = 70), a high level of METTL3 in tumour cells was dramatically correlated with decreased DFS (P < 0.001, Figure 4 A) and OS (P < 0.001, Figure 4B), and a high level of METTL3 in tumour-infiltrating immune cells was negatively correlated with DFS (P = 0.004, Figure 4 C) and OS (P < 0.001, Figure 4D); the increased number of CD33+ MDSCs was dramatically correlated with poor DFS (P < 0.001, Figure 4 E) and OS (P < 0.001, Figure 4F). Using multivariate Cox regression analysis in the 70 patients with advanced-stage disease, METTL3 expression in tumour cells (HR: 6.725, P = 0.010) was an independent prognostic factor for DFS, while METTL3 expression in tumour cells (HR: 5.140, P = 0.021) and CD33+ MDSCs (HR: 8.802, P = 0.037) were independent prognostic factors for OS (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 infiltration, we calculated significance 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 tumour-infiltrating immune cells and CD33+ MDSCs were included in the combined low expression group, those with only one of the two biomarkers with high levels 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 high combination of METTL3 in and CD33+ MDSCs was clearly related to unfavourable DFS (P < 0.001, Figure 5E) and OS (P < 0.001, Figure 5F).
Indeed, compared to METTL3 or CD33+ MDSCs, the combination of METTL3 and CD33+ MDSCs can improve the prognostic stratification of survival for CC patients, especially those in advanced disease stages. Based on a total of 197 patients, the high combination of METTL3 levels and CD33+ MDSCs was a predictor of worse patient prognosis, including DFS [HR (95% CI): 4.672 (2.149-10.156), P < 0.001] and OS [HR (95% CI): 4.890 (2.369-10.093), P < 0.001]. In the patients with early-stage disease, we found that the high combination of METTL3 levels and intratumoural CD33+ MDSCs was negatively correlated with OS [HR (95% CI): 3.071 (1.056-8.931), P = 0.039], but there was no significant association with DFS. Importantly, we found that the high combination of tumour METTL3 and intratumoural CD33+ MDSCs was significantly correlated with poor DFS [HR (95% CI): 7.673 (2.420-24.324), P = 0.001] and OS [HR (95% CI): 7.286 (2.667-19.902), P < 0.001] in patients with advanced disease stages (Table 3), suggesting that the high combination of METTL3 levels and CD33+ MDSCs improved patient prognostic stratification in those with advanced-disease.