In this study, we investigated the correlation between the infiltration level of M2-polarized TAMs in BMC and clinical pathological factors (including PD-L1 expression, EGFR expression and CD8 + T cell levels). The results showed that M2-polarized TAM infiltration increased in BMC tissues, and the degree of M2-polarized TAM infiltration in female patients was higher than that in male patients. The degree of infiltration of M2-polarized TAMs is positively correlated with the T stage, N stage, and clinical stage of BMC patients. The infiltration of M2-polarized TAMs was positively correlated with the expression of PD-L1 and EGFR.
BMC has often been analysed together with other oral cancers, making it impossible to obtain specific conclusions about BMC [23]. However, the buccal mucosa is the most common site of oral cancer in South Asians [24], so it is necessary to conduct further independent research on BMC.
As a significant component of the tumour microenvironment (TME), the metabolic changes of TAMs are associated with the promotion of malignant cancer progression [25], and TAMs can support the growth, invasion, metastasis and angiogenesis of cancer [26], which is closely related to the poor outcomes of cancer patients [27, 28]. For example, the increased infiltration of M2-polarized TAMs is correlated with the progression of prostatic adenocarcinoma [29], liver cancer [30], colorectal cancer [31] and breast cancer [32]. However, the difference in M2-polarized TAMs infiltration between BMC samples and non-BMC samples has not been comprehensively reported before.
In present study, we revealed the upregulation of M2-polarized TAMs markers (CD68, CD163, CD206) in BMC tissues with IHC, which was consistent with the results of previous studies. In terms of the clinical characteristics of M2-polarized TAMs, Fan Guo et al. indicated that the level of M2-polarized (CD68+) TAM infiltration was related to the N stage of cervical carcinoma, and the M2-polarized (CD163+) TAM density was related to lymph node metastasis and FIGO stage [33]. Our results indicated that there was no association between the TAM infiltration and clinicopathological characteristics such as age. Compared with those in male patients, the infiltration levels of M2-polarized (CD68 + and CD163+) TAMs were significantly increased in female patients. In addition, high infiltration of M2-polarized (CD206 + and CD163+) TAMs was correlated with larger tumour size, lymph node metastasis, and more advanced clinical stage of BMC. These findings indicated that the infiltration of M2-polarized TAMs may be associated with the progression of BMC.
Furthermore, in order to explore the probability of targeting M2-polarized TAMs in combination with immunotherapy, we also investigated the correlation between M2-polarized TAMs and PD-L1, EGFR, and CD8 + T cells. As an oncogenic gene, EGFR is considered one of the targets for precise treatment of multiple malignant cancers with EGFR mutations [34, 35], and has been found to be associated with a poor prognosis in various malignant cancers [36–38]. Based on many reliable clinical trials, multiple PD-L1 antibodies have been widely used for treating malignant tumours [39]. Thus, it is worthwhile to explore the correlation between M2-polarized TAMs and EGFR/PD-L1 in BMC patients. Our study found that the expression/infiltration of PD-L1, EGFR, and CD8 + T cells was increased in BMC tumour tissues vs. normal tissues. We also found a significant increase in the expression of an M2-polarized TAM marker (CD68+) in the high PD-L1 TPS group and a significant increase in the expression of another M2-polarized TAM marker (CD206+) in the high EGFR expression group.
It was reported that M2-polarized (CD206+) TAMs might play a momentous role in the progression of OSCC and ovarian cancer by secreting EGF [40, 41], and PD-L1 expression was increased in M2-polarized (CD68+) TAMs [42, 43], indicating that high infiltration of M2-polarized (CD206+) TAMs in BMC tissues may become a predictive marker for the response to EGFR-targeted treatment, while the high infiltration of M2-polarized (CD68+) TAMs may be an indicator of immunotherapy response. One study identified that the deficiency of N6-methyladenosine methyltransferase Mettl14 in TAMs can inhibit CD8 + T-cell infiltration and promote tumour growth [44].
Unfortunately, our study did not find a significant relationship between the expression of TAMs in BMC tissues and the degree of CD8 + T-cell infiltration. This result may be related to a small sample size and single experimental method.
Overall, our current work applied IHC to detect the infiltration level of M2-polarized
TAMs in BMC and further explored the clinicopathological significance of M2-polarized TAMs. In addition, this study found that the expression of PD-L1 and EGFR was related to the infiltration of M2-polarized TAMs. Nevertheless, there are also some limitations to our study. First, experients such as flow cytometry and single-cell RNA sequencing need to be carried out to verify the expression levels of M2-polarized TAM markers in BMC. Moreover, the prognostic significance of M2-polarized TAM infiltration needs to be studied in a larger sample size. The potential mechanism of M2-polarized TAMs in BMC also needs to be assessed in further studies.