Investigating the role of IDO1 in tumors: correlating IDO1 expression with clinical pathological features and prognosis in lung adenocarcinoma patients

Introduction

Lung cancer stood as one of the most prevalent malignant tumors globally, with an incidence rate of 12.4% and a mortality rate of 18.7%, steadily increased year by year (Sung et al., 2021; Bray et al., 2024). Non-small cell lung cancer (NSCLC), comprising 80–85% of all cases, included adenocarcinoma (40%) and squamous cell carcinoma (30%) (Wu et al., 2024; Wei et al., 2023; Sahu et al., 2023). Early-stage symptoms often lacked specificity, resulting in advanced-stage diagnoses where surgical intervention was no longer viable. Consequently, the 5-year survival rate remained low, and systemic chemotherapy and radiotherapy offered limited efficacy (Liu et al., 2022; Cai, Dong & Wang, 2018; Ge et al., 2022). The advent of targeted therapy and immunotherapy introduced novel frontline treatment options for advanced-stage lung cancer patients. Examples included tyrosine kinase inhibitors (TKIs) that targeted the epidermal growth factor receptor (EGFR), and immune checkpoint inhibitors (ICIs) designed to block programmed cell death ligand 1 (PD-L1), both of which had demonstrated promise. However, EGFR-TKIs typically developed resistance in patients after one year, and ICIs exhibited greater efficacy in patients with high PD-L1 expression, with poorer responses seen in those with low or absent expression (Hanna et al., 2017; Nan et al., 2017; Pardoll, 2012; Reck et al., 2016). Therefore, exploring novel immune prognostic markers, identifying immunotherapy targets, and advancing research into new therapeutic agents become paramount research priorities in the field.

Indoleamine 2,3-dioxygenase 1 (IDO1) was a monomeric oxidoreductase and a rate-limiting enzyme in human tryptophan metabolism, responsible for more than 95% of tryptophan (Trp) degradation via the kynurenine (Kyn) pathway (Dolsak, Gobec & Sova, 2021; Zhang et al., 2022). The literature reported heightened IDO1 levels in numerous human tumors, often correlating with increased tumor aggressiveness and poorer patient outcomes (Platten et al., 2019; Yao et al., 2021; Mandarano et al. 2020; Bishnupuri et al., 2019). IDO1 inhibitors were demonstrated to exhibit significant immunotherapeutic efficacy in the treatment of malignant tumors such as metastatic melanoma and renal cell carcinoma (Zhang et al., 2021). Furthermore, studies indicated that IDO1 inhibitors could synergize with conventional cancer therapies and immune checkpoint inhibitors (Li et al., 2018). Moreover, 35 clinical trials were underway investigating various types of IDO1 inhibitors (Du et al., 2019).

In the literature related to lung cancer, we observed that, compared to adjacent normal lung tissues, the expression level of IDO1 was significantly upregulated in stage I, II, and III lung adenocarcinomas (Zhao et al., 2022). In patients with stage III NSCLC who were inoperable and had undergone chemotherapy and radiotherapy, high IDO1 expression was associated with poorer overall survival (OS) and progression-free survival (PFS) (Wu et al., 2022). In studies on IDO1 inhibitors, it was found that these inhibitors could enhance the functionality of NK cells while also inhibiting tumor growth in NSCLC mouse models (Fang et al., 2022). Additionally, IDO1 inhibitors were shown to potentiate the inhibitory effects of conventional fractionated radiotherapy on NSCLC tumors (Lan et al., 2023). When combined with nicotinamide phosphoribosyltransferase (NAMPT), IDO1 inhibitors were effective in treating chemotherapy- and targeted therapy-resistant NSCLC (Wang et al., 2023). These findings suggested that IDO1 might have potential as an immune prognostic biomarker and a novel therapeutic target for immunotherapy or combination therapy in lung cancer. Based on this, we planned to initially investigate the expression patterns and potential roles of IDO1 through bioinformatics analysis, followed by experimental validation to systematically assess its functional and clinical significance in NSCLC.

In this study, we utilized cancer genomics datasets from The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx) project, and the Gene Expression Omnibus (GEO) databases to examine IDO1 expression across various human cancers and its association with cancer prognosis. Furthermore, we investigated the interactions between IDO1 protein and other human proteins. In preliminary experiments, we observed higher IDO1 expression in adenocarcinoma compared to other types of NSCLC. Consequently, we focused our analysis on IDO1 expression levels specifically in adenocarcinoma and its correlations with immune cell infiltration, gene mutations, immune checkpoint markers, and patient prognosis. To explore these relationships further, we conducted immunohistochemistry to evaluate IDO1 expression in lung adenocarcinoma tissues, assessing its potential implications for clinical-pathological characteristics and patient outcomes. Our objective was to evaluate IDO1’s viability as both an immune-related prognostic marker and a potential target for novel immunotherapeutic strategies in lung adenocarcinoma.

Materials and Methods

Results

Pan-cancer analysis results

IDO1 exhibited high expression across various types of cancer patients (Figs. 1A and 1D). Cancers characterized by high IDO1 expression exhibit decreased progression-free survival (PFS) and overall survival (OS) rates (Fig. 2), highlighting a negative prognostic impact associated with elevated IDO1 expression in cancer patients. Furthermore, there existed a correlation between the functional activity and expression of human IDO1 protein with several other human proteins, including TDO2, KYNU, and others (Figs. 1B and 1C).

Bioinformatics analysis results

IDO1 expression was higher in lung adenocarcinoma compared to normal lung tissue (Fig. 3A). Patients with high IDO1 expression in adenocarcinoma showed reduced first progression survival (FP) (Fig. 3B) and OS (Figs. 3C and 3D). Moreover, IDO1 expression in adenocarcinoma correlated with various immune cell infiltrations (Fig. 3E), multiple gene mutations including EGFR (Fig. 3F), and the expression of multiple immune checkpoint genes such as CD274 (Fig. 3G).

Expression and localization of IDO1 in lung adenocarcinoma

Microscopic examination revealed that tumor cells with high IDO1 protein expression were diffusely distributed within tumor tissues. IDO1 protein was prominently localized in the cytoplasm and membrane, exhibiting a brown-yellow granular staining pattern in the cytoplasm (Fig. 4). Additionally, heterogeneous expression of IDO1 was observed among tumor cells, with those closer to normal lung tissue exhibiting higher IDO1 expression levels (Figs. 5A and 5B).

Discussion

IDO1 was a monomeric oxidoreductase that contained heme and comprised two structural domains. The large C-terminal domain (CTD) was essential for IDO1’s catalytic activity, whereas the small N-terminal domain (NTD) primarily served signaling functions. IDO1 was widely distributed across various human tissues and exhibited high expression in antigen-presenting cells such as dendritic cells, monocytes, and macrophages (Zhai et al., 2018; Merlo, Peng & Mandik-Nayak, 2022). Functioning as a rate-limiting enzyme, IDO1 played pivotal biological roles by modulating the levels of tryptophan and kynurenine metabolites. IDO1 was involved in the suppression of CD8+ T effector cells and natural killer (NK) cells, while enhancing the activity of CD4+ regulatory T cells (Tregs), dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs) (Du et al., 2019). IDO1 was involved in peripheral immune tolerance, contributing to maintaining homeostasis by preventing autoimmunity or immunopathology that would result from uncontrolled and overreacting immune responses (Pallotta et al., 2022; Tang et al., 2022). However, overexpression of IDO1 has also been implicated broadly in human pathology, including exacerbating pathogen infections, contributing to poor prognosis in various cancers, promoting autoimmunity, increasing neurodegenerative conditions, and more (Pallotta et al., 2022; Li et al., 2017; Taleb, 2019).

The literature reported that in various types of cancers, including ovarian cancer, colorectal cancer, endometrial cancer, melanoma, cervical cancer, glioblastoma, lung adenocarcinoma, and diffuse large B-cell lymphoma, overexpression of IDO1 in tumor cells or other cells within the tumor microenvironment (TME) was associated with more aggressive cancer phenotypes, advanced disease stages, and poorer clinical outcomes (Platten et al., 2019; Yao et al., 2021; Li et al., 2017; Liu et al., 2018). In our pan-cancer analysis, we also found that IDO1 was highly expressed in multiple human cancers, and cancers with high IDO1 expression—such as GBMLGG, KIRP, LGG, KICH, UVM, and myeloma, among others—demonstrated significantly lower PFS and OS. These findings suggested that high IDO1 expression correlates with increased tumor invasiveness and poor patient prognosis, indicating the potential of IDO1 as a prognostic biomarker and a novel therapeutic target for immune treatment in various cancers. Our research team primarily focused on targeted and immunotherapy approaches for lung cancer. Moreover, the preliminary experiments revealed that IDO1 expression was higher in lung adenocarcinoma compared to other types of NSCLC. Therefore, we focused on analyzing the role of IDO1 in lung adenocarcinoma to explore its potential as an immune prognostic biomarker and a new target for immunotherapy.

In the bioinformatics analysis, we found that IDO1 expression in lung adenocarcinoma was higher than in normal lung tissue. Additionally, patients with high IDO1 expression in adenocarcinoma exhibited lower PFS and OS, which was consistent with reports in the literature (Zhao et al., 2022; Jin et al., 2024). This finding was further validated in subsequent experiments. According to literature (Zhao et al., 2022; Bessede et al., 2023; Schalper et al., 2017; Wu et al., 2023; Kozuma et al., 2018), in NSCLC, including lung adenocarcinoma, clinical TNM staging showed a negative correlation with IDO1 expression. In later stages of clinical progression, IDO1 expression in tumors was found to be elevated, but there was a marked reduction in the infiltration of T lymphocytes, especially CD8+ T cells. The bioinformatics analysis further revealed that IDO1 expression in adenocarcinoma was negatively correlated with tumor heterogeneity, while positively correlated with various immune cell types, particularly with immune suppressive cells such as DCs. These findings suggested that in lung adenocarcinoma, IDO1 might promote tumor immune evasion by inhibiting the infiltration and survival of cytotoxic T cells within the tumor microenvironment, while enhancing the activity of immunosuppressive cells like Tregs and DCs.

According to literature (Zhao et al., 2022; Wu et al., 2022), IDO1 expression was higher in stage III lung cancer compared to stages I and II, and the activity of IDO1 in stage III lung cancer was negatively correlated with OS and PFS. Mandarano et al. (2021) reported that the Kyn/Trp ratio could also serve as a clinical pathological marker for prognosis in NSCLC patients, monitoring cancer invasiveness and progression. Additionally, Pan et al. (2017) indicated that IDO1 expression in lung cancer patients was associated with microvascular density (MVD) marked by CD34 and CD146 and that silencing of IDO1 could inhibit tumor angiogenesis. In the present study, we found that IDO1 expression was positively correlated with lymph node metastasis, pleural invasion, recurrence and metastasis, poor tumor differentiation, solid components, and TNM staging in adenocarcinoma patients. It was also negatively correlated with PFS and OS. Microscopic examination revealed that tumors expressing IDO1 exhibited heterogeneous expression, with tumor cells near normal lung tissue showing higher levels of expression. These findings suggested that IDO1 played a crucial role in immune evasion, neoangiogenesis, and metastasis in lung adenocarcinoma. Furthermore, tumors with positive IDO1 expression demonstrate increased invasiveness and metastatic potential, leading to a higher clinical stage and poorer prognosis for patients. Therefore, we believe that IDO1 holds potential as an immunological prognostic marker for lung adenocarcinoma.

Bioinformatics analysis revealed that the expression of IDO1 was negatively correlated with mutations in genes such as EGFR, while positively correlated with the immune checkpoint protein CD274 (also known as PD-L1). In a previously published study (Jin et al., 2024), we demonstrated that IDO1 inhibitors could suppress the expression of PD-L1 and p-EGFR in the H1975 cell line. In related literature, a co-expression of IDO1 and PD-L1 had been identified, with co-expressing lung adenocarcinomas exhibiting more aggressive characteristics (Kozuma et al., 2018). Dual inhibition of MEK and PD-L1 had been shown to enhance IDO1 expression in tumors, leading to immune escape, however, this effect could be reversed by the addition of IDO1 inhibitors (Della Corte et al., 2022). Furthermore, combination therapy with IDO1 inhibitors and nicotinamide phosphoribosyltransferase (NAMPT) was proposed for the treatment of chemotherapy- and targeted therapy-resistant NSCLC (Wang et al., 2023). These findings suggested that IDO1 interacted with various gene mutations and immune checkpoints, with particular emphasis on its potential synergistic effect with PD-L1. Based on these findings, IDO1 held promise as a new therapeutic target for combination immunotherapy or targeted therapy in lung adenocarcinoma.

This study, in contrast to previous research, incorporated databases such as TCGA for exploratory analysis. Through bioinformatics analysis and experimental validation, we systematically evaluated the role of IDO1 in lung adenocarcinoma. Compared to earlier studies, our study included a larger and more detailed collection of clinical data and provided a more comprehensive description of IDO1 expression and localization under the microscope. For the first time, we reported that IDO1 expression was heterogeneous within tumors, with higher expression in tumor cells adjacent to normal lung tissue compared to other tumor cells. Additionally, IDO1 expression was associated with lower tumor differentiation and the presence of solid components. In this study, we also found that lung adenocarcinoma patients who underwent neoadjuvant chemotherapy exhibited higher IDO1 expression, a finding not previously reported in the literature. This might suggest that chemotherapy drugs could stimulate IDO1 expression in tumor cells, warranting further investigation.

Conclusions

Our study revealed uniform cytoplasmic and membranous expression of IDO1 protein in tumor cells and higher IDO1 expression observed in tumor cells near normal lung tissue. Moreover, high IDO1 expression in adenocarcinoma patients positively correlated with lymph node metastasis, pleural invasion, tumor recurrence, lower tumor differentiation grade, presence of solid components, and advanced clinical TNM staging. Conversely, there was a negative association with patient surgical history, PFS, and OS. These findings suggest that tumors exhibiting elevated IDO1 levels demonstrate increased invasiveness and metastatic potential, often presenting at more advanced clinical stages and associated with poorer prognosis. Additionally, our observations indicated higher IDO1 expression in lung adenocarcinoma tumors following chemotherapy. This study underscores that IDO1 was a promising immune prognostic marker and a potential target for novel immunotherapeutic approaches in lung adenocarcinoma.

Supplemental Information