With the advent of the field of cancer therapeutics, multiple preclinical studies emphasized the pivotal role of Tim3 in cancer immunotherapy (10). To the best of our knowledge, this is the first research that has investigated not only the expression of Tim3 but also its ligand Gal9 in the most prevalent malignancy of Southeast Asia which is oral squamous cell carcinoma.
Tim3 is an emerging immune marker that has been studied extensively in various tumors due to its immunomodulatory role. In the current research, we speculated that the Tim3 protein is mainly expressed on cancer-associated TILs in TME. Higher Tim3 expressions were associated with the degree of tumor differentiation, TNM stages, lymph vascular invasion, and distant metastasis. Moreover, we found patients with positive Tim3 expression had a significantly lower survival rate as compared to those with negative Tim3 expressions. In addition, multivariate analysis revealed Tim3 overexpression in TILs was associated with a poor prognosis of oral cancer. These findings are in line with previous literature. Nora et al, observed high expressions of Tim3 on TILs and speculated the immunosuppressive role of Tim3 in the tumor microenvironment of Oropharyngeal squamous cell carcinoma. (11) Liu et al, postulated that in Head and neck cancer Tim-3 was associated with immune suppression and its blockage leads to the reinvigoration of IFɣ production by CD8 + T cells. It resulted in an increased immune response to tumors. (12) Sahar et al discovered that the amount of Tim-3 positive cytotoxic T lymphocytes increases with tumor grades in breast carcinoma (13) Piao et al investigated Tim-3 expression on lymphocytes via flow cytometry in prostate cancer. He revealed high Tim-3 expression in cancerous patients in contrast to benign prostate hyperplasia cases. Increased Tim3 expression was correlated with Gleason score and PSA levels. He suggested Tim-3 as an indicator of tumor progression. (14) Shayan et al, in Head and neck cancer, observed higher Tim3 expressing TILs in continuously growing tumors. (15) Our findings are consistent with these results. The rationale of Higher tim3 expression on TILs could be because of the constant stimulation from its ligand within the tumor site. Most importantly Cao et al confirmed the findings that downregulation of Tim3 expression in immortal cell lines (Hela cells) significantly inhibits the invasion as well as the migration of tumor cells. (16) In the lite of the above literature it can be speculated that apart from immune suppression against the tumor, Tim3 could directly aggravate the progression of the tumor.
Galectin-9 has been reported to contribute to tumorigenesis by tumor cell transformation, cell-cycle regulation, angiogenesis, and cell adhesion. Recently the immunomodulatory role of Gal9 has also been identified with terminally exhausted T cells (17). Current research identified increased Gal9 expressions in patients with TNM stages, lymph node metastasis, lymph vascular invasion, and distant metastasis. Furthermore, poor survival rates were observed with patients showing increased Gal9 expression. Multiple studies support our findings. In solid tumors, multiple clinical studies have acknowledged a close association of Gal-9 expression with metastasis and tumor recurrence. These include melanoma (18), gastric cancer (19), hepatocellular cancer (20), and lung cancer (21). Liu et al found a positive correlation of Gal-9 levels with the clinical stage of the tumor. (22) Kwong et al, detected a significantly increased number of tumor cells expressing Gal9 as compared to their normal counterparts in NPC cases. Moreover, he reported the association of increased Gal9 expression with shorter overall survival. (23) Liu et al, speculated that patients who had higher levels of Gal-9 were associated with increased susceptibility to develop malignant brain tumors. (24) Furthermore, according to Labrie et al, in epithelial tumors increased Gal-9 expressions were predictable of poor response to treatment (25) Additionally, elevated Gal-9 expression was found in patients with pancreatic ductal adenocarcinoma, which was associated with poor survival after metastasis. (26) In our previous study overexpression of Gal9 was identified in OSCC patients. A significant association of Gal9 expression with clinicopathological parameters was also observed. (9) However, disagreeing exist between the studies regarding alterations in the antitumor environment. Some studies reported that loss of Gal-9 expression occurred as the tumor progresses. (27). Reports have also shown better survival with higher Gal9 expression in gastric and colon carcinoma. (28). Nobumoto et al reported the antimetastatic role of Gal-9. He speculated that tumor expressing Gal9 are less likely to metastasize because of Gal9 cause inhibition of adhesion between endothelium and extracellular matrix. (29) Nevertheless, it has also been postulated that reduction in the attachment of cancer cells to ECM could itself pave the way for tumor cells to easily enter into the circulation from their primary area of location. (30). The variance in studies maybe because of the difference in immune status among individuals, etiological factors of inflammation, and tissue specificness of Gal9 expression.
A growing body of evidence suggested that Tim3/Gal9 pathway is operated by multiple tumors to evade immune cells. (31) In our findings, we speculated that the co-expression of these immune proteins was significantly associated with tumor differentiation and clinical stages. Both increased Gal9 expression HSCORE > 200 and positive Tim3 expression were significantly associated with poor overall survival (p = 0.015, p = 0.006 respectively); the expression of both receptor and ligand was an independent prognostic marker in OSCC. Higher expressions of Tim3 and Gal9 were found in glioma cases as compared to healthy brain cells and their expressions were directly proportional to the disease progress. (24) Therefore, it can be speculated that the expression of the Tim3/Gal9 axis could have been attributed to immune suppression against cancer by killing cytotoxic T cells, thus allowing for disease progression. In gastric carcinoma higher Gal9 expressions and lower Tim3 expressions were associated with better overall survival (19). The inconsistency in the expressions of the marker in question may be due to the heterogenicity of various tumors with different origins, diversity in tumor immunity, and different study design and samples collection processes. These findings indicate that Tim3/Gal9 pathway may play a pivotal role in the progression of the tumor.
Furthermore, we evaluated the prognostic significance of the Tim3/ Gal9 co-expression. We found that patients with high levels of Tim3 positive tumors showed a short survival rate as compared to the Tim3 negative group. These findings are in line with the previously published data. Jia et al in NSCLC found that the prognosis of the patients having high Tim3 expressions on TILs compare showed poor prognosis (32) Rietz et al, in his research found that Tim3 not only cause progression of the tumor but it also causes short survival rates and resistance to immunotherapies (33). Mohsenzadegan et all demonstrated that increased Tim3 expression causes tumor progression in bladder cancer. He also suggested that Tim3 can be a therapeutic target either alone or in combination with other checkpoint inhibitors (34) Similar to this Jia et al also observed poor prognosis with increased Tim3 expressions in non-small cell lung carcinoma (35). In the current study higher expressions of Gal9 were found in patients with advanced tumor stages. This can be due to its role in the adhesion of the cells to the extracellular matrix (ECM). Increased Gal9 expression causes a decrease in adhesion of tumor cells to ECM that helps in the detachment of tumor cells from primary tumor sites (36). Moreover, in our study elevated Gal9 expressions were significantly associated with poor survival. The findings of the current research were in line with previous studies. According to Okoye et al, higher expression of Gal9 on lymphocytes was associated with a poor prognosis of virus-affected tumors (37). In urinary tumor expression of Gal9 implies to poor prognosis. In high grade serous ovarian cancer Labrie et al, reported that increased Gal9 was associated with poor 5-year overall survival (25) Flu et al, also postulated high Gal9 expression predicts poor overall survival in clear cell renal cell carcinoma patients (38) In contrast, multiple studies acknowledge the fact that increased Gal9 expression was associated with better prognosis in hepatocellular carcinoma and colon cancer. A study conducted only on Gal9 expression showed longer overall survival in patients expressing high Gal9 protein in colon tumors (28).
This can lead us to hypothesize that presence of Tim3/Gal9 Axis in OSCC can be a potential immunotherapeutic target as it is involved in immunosuppression leading to inhibition of antitumor immunity. There were certain limitations in our study. Firstly, we did not investigate the HPV status of the OSCC patients. It can be done in future research as it has been revealed in the literature that Gal9 showed an association with HPV status. Secondly, other coinhibitory markers can also be evaluated with tim3 and gal9 proteins to evaluate their interactions with other proteins