Oral cancer is one of the most prevalent malignancies of the head and neck region, posing significant challenges to patient well-being and healthcare systems (1). The recent data from the Globocan 2022 report reveals the extensive impact of oral cancer, with a global incidence reaching 389,846 cases and a mortality toll of 188,438 (2). Specifically in Pakistan, oral cancer holds the position of the second most prevalent tumor, contributing to a substantial burden with 15,915 reported cases and 10,181 associated deaths (3). Among head and neck cancer cases, oral cancer holds the leading position with a continuing rise in its incidence (1). Oral cancer is characterized by its highly malignant nature, marked by elevated rates of local recurrence and cervical lymph node metastasis (4, 5). The reported 5-year survival rate for oral cancer is approximately 50% (6). Currently, the preferred treatment for tongue cancer involves a combination of surgery, postoperative radiotherapy, and chemotherapy (7). However, the prognosis for oral cancer remains grim due to short-term recurrence and inadequate therapeutic efficacy, significantly impacting the quality of life for affected patients (8).

Increasing evidence highlights the pivotal role played by the interaction between tongue cancer cells and the surrounding microenvironment in the development and prognosis of tongue cancer (9). Predominantly, TSCC stands out for its marked infiltration of immune cells, characterizing it as an immunogenic tumor (10). Among the prevalent stromal components in tongue cancer, the infiltrated immune cells emerge as key contributors to the carcinogenic process (10).

The concept of cancer immunoediting is widely accepted, and tumor immune escape is considered an emerging hallmark of cancer (10). Cancer immunotherapy utilizing immune-checkpoint inhibitors has emerged as a highly effective therapeutic modality for various cancers, including tongue cancer (11–13). In several malignancies, programmed death ligand-1 (PD-L1) checkpoint blockades have shown substantial clinical efficacy (14–16). Recent evidence has pointed out that the expression of PD-1 and PD-L1 were considerably associated with local recurrence in patients with TSCC (13). Overexpression of PD-L1 on tumor cells inhibits the activation of T cells, leading to the progression of tumors (17–20). PD-L1 overexpression in TSCC correlates with advanced stage and shorter disease-free survival (21). Several ongoing clinical trials have demonstrated efficacy against recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), leading to clinical approval in multiple countries following successful phase III trials (22). Thus, the focus on immune checkpoints represents a new area of investigation for novel cancer therapies (23–25). Another such prospective target in this realm is indoleamine 2,3-dioxygenase (IDO), a checkpoint protein influencing an immunosuppressive tumor microenvironment (26). IDO, a heme-containing enzyme, metabolizes L-tryptophan into kynurenine (27). The localized depletion of tryptophan hinders T-cell cytotoxicity, thereby inhibiting T-cell immune responses through the induction of regulatory T-cell differentiation (28, 29). IDO expression has been reported in many cancers, including breast, colorectal, ovarian, gastric, and oral cancer (28, 30–34). It is also associated with cancer poor prognosis in oral squamous cell carcinoma patients (33). The significance of IDO expression is underscored by its ability to predict tumor responsiveness to anti-IDO immunotherapy. Similarly, high PD-L1 expression is associated with an unfavorable prognosis in diverse tumors. Thus, evaluating the levels of IDO and PD-L1 expressions may offer insights into identifying patients who could potentially benefit from anti-IDO/anti-PD-L1 therapy or combination therapies. In this study’s context, we performed an immunohistochemical analysis to investigate the expressions of IDO and PD-L1 in individuals with TSCC.

TSCC is one of the most prevalent malignancies of the head and neck region, constituting a significant global health concern with a survival rate of about 50% (45–47). Traditionally managed with surgery and radiotherapy or chemoradiotherapy, recent research has focused on immunotherapy to improve outcomes (47). The response rate to PD-1 and PD-L1 antibody treatment remains limited, with only about 20% of patients showing favorable outcomes in most solid tumors (48). PD-L1 is a transmembrane glycoprotein structurally characterized by extracellular IgV- and IgC-like domains (49). Under physiological conditions, PD-L1 is expressed by various immune cells, including macrophages, activated T and B lymphocytes, dendritic cells, and certain epithelial cells, particularly in response to pro-inflammatory signals (50). Within the tumor microenvironment, PD-L1 is frequently overexpressed on malignant cells as an adaptive mechanism to suppress cytotoxic T-cell activity and facilitate immune escape (51). This upregulation is often observed in immune-infiltrated tumors characterized by a high density of CD8+ T cells, Th1 cytokines, interferons, and distinct immunomodulatory gene signatures (52). Multiple oncogenic and inflammatory pathways regulate PD-L1 expression in digestive system cancers including the EGFR/ERK signaling axis in esophageal squamous cell carcinoma (53), the JAK/STAT pathway in gastric cancer (54), and the ERK/MAPK pathway in hepatocellular carcinoma (55). In addition, the IFN-γ–driven activation of the JAK/STAT pathway has been shown to mediate PD-L1 induction in myeloid leukemia as well as pancreatic and gastric cancers (56, 57).

Similarly, another potential focus is on IDO, a checkpoint protein implicated in creating an immunosuppressive environment within tumors (58). IDO is a cytosolic heme-containing enzyme that catalyzes the first and rate-limiting step in the metabolism of tryptophan (Trp) to kynurenine (Kyn) (59). It initiates the oxidative cleavage of the pyrrole ring of L-tryptophan to produce N-formylkynurenine, which is subsequently converted into L-kynurenine by kynurenine formamidase (60). Kynurenine and its downstream metabolites contribute to immunosuppression by inhibiting T-cell responses, enabling tumor cells to evade immune surveillance (61). L-kynurenine is further metabolized to anthranilic acid via kynureninase, then to 3-hydroxyanthranilic acid through kynurenine-3-monooxygenase, ultimately generating 2-amino-muconic acid, picolinic acid, and quinolinic acid (62). These downstream metabolites reinforce immune evasion by promoting regulatory T-cell differentiation and inhibiting effector T-cell activity, thereby fostering an immunosuppressive tumor microenvironment (63).

IDO plays a critical role in the pathogenesis of various conditions, including chronic inflammatory diseases, infections, and a wide range of malignancies (33, 64, 65). Its aberrant overexpression has been reported in several tumor types, including oral, colorectal, hepatocellular, ovarian cancers, and melanomas (33, 65). High IDO expression within the tumor microenvironment contributes to immune evasion, facilitates tumor progression and metastasis, and is strongly associated with poor clinical outcomes and decreased overall survival in cancer patients (66–68). Consistently, preclinical studies have demonstrated that IDO overexpression is linked to poor prognosis across multiple cancer types (69). Elevated IDO levels have been observed across various cancers such as breast, colorectal, ovarian, gastric cancer, and TSCC (30–34, 65), correlating with poorer prognosis in TSCC patients (70). This highlights the necessity for enhanced combined immunotherapeutic strategies and predictive biomarkers to effectively identify patients who could derive optimal benefit from these treatments.

In this study, we evaluated the expression of IDO and PD-L1 in 106 patients with tongue cancer both before and after chemotherapy. Our analysis indicates that chemotherapy significantly influences the expression of IDO and PD-L1. Prior to chemotherapy, the majority of patients were negative for IDO (83%) and PD-L1 (75.5%). Post-chemotherapy, there was an increase in the expression of both markers, with IDO positivity rising to 24.5% and PD-L1 positivity to 29.2%. This suggests that chemotherapy may upregulate these immunosuppressive markers, potentially impacting the tumor microenvironment and immune evasion mechanisms. The increase in IDO and PD-L1 expression post-chemotherapy could imply a chemoresistance mechanism, where tumors adapt to therapeutic pressures by enhancing immunosuppressive pathways.

Regarding the effect of chemotherapy on PD-L1 expression, several studies have documented changes in PD-L1 expression following traditional cancer treatments like chemotherapy, radiotherapy, or their combination. In a study conducted by Park BJ et al, chemoradiation therapy has shown to significantly alter PD-L1 expression in locoregional recurrent squamous cell carcinomas of the head and neck (71). This change in PD-L1 score post-treatment suggests a potential shift in the tumor’s immune microenvironment, which may influence the effectiveness of subsequent immunotherapeutic approaches (71). The findings highlight the importance of re-assessing PD-L1 status in recurrent tumors, as initial evaluations conducted prior to treatment may not accurately represent the post-therapy immune profile. A systematic review by Van den Ende et al. identified 48 studies examining PD-L1 expression changes post-treatment. Statistical analysis indicated that 30 of these studies reported increased PD-L1 expression in post-treatment specimens or treated versus untreated samples (72). However, Karpathiou et al. evaluated the stability of PD-L1 expression in 106 HNSCC tissue specimens over storage periods of 20–48 months (73). They reported a significant decline in PD-L1 tumor proportional score, immune cell expression, and combined positive score over time, indicating loss of antigenicity in archived FFPE samples (73). This degradation may lead to underestimation of PD-L1 expression, potentially influencing treatment decisions and study results depending on the interval between the first and second biopsy. Timely evaluation on freshly processed tissue is therefore recommended for accurate immunotherapy eligibility assessment.

Despite an extensive literature search, we found no studies addressing IDO status before and after chemotherapy specifically in oral squamous cell carcinoma. However, our findings align with other research indicating elevated IDO expression post-chemotherapy (74). IDO-inducing signals may be inherently present in the tumor’s inflammatory microenvironment and could be further triggered by dying cells and tumor antigens released during chemotherapy. Nevertheless, the precise extent of IDO production following chemotherapy remains largely unexplored (74).

Our study found a significant association between IDO and PD-L1 positivity both before and after chemotherapy, reinforcing the notion of an immunosuppressive microenvironment in IDO-positive patients. Multifocal tumors were also significantly associated with IDO positivity prior to chemotherapy, suggesting a potential link between tumor multiplicity and immunosuppressive marker expression. Post-chemotherapy, IDO-positive patients had more comorbidities, highlighting the complex interplay between patient health status and tumor biology.

The overall five-year survival rate for patients in this study was 49%, with a median survival time of 53 months. Despite investigating IDO and PD-L1 expressions, our analysis did not reveal significant differences in survival outcomes related to these markers. This finding suggests that, while IDO and PD-L1 are involved in the immunosuppressive environment of TSCC, their expression levels may not directly impact overall survival within this cohort. Further research could explore additional factors influencing survival and assess whether combining IDO and PD-L1 with other biomarkers might provide a more comprehensive understanding of patient prognosis.

To the best of our knowledge, no study has yet examined the expression of both biomarkers before and after chemotherapy. This highlights the strength of our research, providing valuable insights into the immunosuppressive dynamics within oral squamous cell carcinoma before and after chemotherapy. Future research should focus on longitudinal studies to monitor these markers over time. Additionally, further investigation into the molecular pathways driving these expression changes could uncover novel therapeutic targets.

The current study has some limitations. The retrospective design and reliance on existing patient records may introduce selection bias and limit data quality control. The cross-sectional analysis only captures data at two points, missing dynamic changes. A larger cohort would enhance statistical power and overall impact of the study.

The current study demonstrates that chemotherapy increases IDO and PD-L1 expression in TSCC, suggesting that incorporating immunotherapy could benefit patients undergoing chemotherapy. Future research should emphasize longitudinal studies and the combinatorial analysis of multiple biomarkers to gain a clearer understanding of immune marker expression dynamics in TSCC.