Ovarian epithelial malignancy is a prevalent neoplasm in women worldwide, ranking seventh among all malignant tumors (Siegel et al., 2020). It can be categorized into five main histological subtypes with distinct histopathological features and molecular pathogenesis: high-grade serous carcinoma, endometrioid carcinoma, clear cell carcinoma, mucinous carcinoma, and low-grade serous carcinoma. HGSC is the most frequently observed subtype, accounting for 60% of all cases of epithelial ovarian malignancy. OCCC is the second most common subtype, representing 5%–11% of all ovarian epithelial malignancies (Zhu et al., 2021; Torre et al., 2018). On Magnetic Resonance Imaging (MRI), OCCC and HGSC can present as large pelvic cystic masses with solid components, exhibiting regular or irregular shapes. The cystic lesions may appear as single or multiple entities, some of which are separated, and the cyst wall may exhibit masses, nodules, or papillae protruding into the cavity. The solid components mostly demonstrate moderate to marked enhancement during contrast-enhanced scanning. Abdominal fluid accumulation is commonly observed. However, in comparison to HGSC, OCCC typically presents with a more regular shape, larger diameter, unilateral onset, and a higher prevalence of International Federation of Gynecology and Obstetrics (FIGO) stage I disease. Most OCCC tumors exhibit characteristics of cystic-solid composition with predominantly single cysts and internal prominences that are mainly nodular or papillary. High signal intensity within the cystic component on T2-weighted imaging (TWI) is more frequently encountered in OCCC cases, whereas low signal intensity within the solid component on TWI is more common. Furthermore, peritoneal implantation metastases are rare in OCCC cases, while peritoneal effusion may occur.

In contrast to HGSC, OCCC typically lacks P53 mutations and exhibits a lower frequency of Breast Cancer Gene (BRCA)1/2 mutations (6.3%). However, it has a higher incidence of AT-rich interaction domain 1A (ARID1A) and Phosphatidylinositol-4,5-bisphosphate 3 kinase catalytic subunit alpha (PIK3CA) mutation (Zhou et al., 2020). Both OCCC and HGSC are primarily managed through surgical intervention followed by adjuvant chemotherapy, however, OCCC has a poorer prognosis due to its resistance to conventional platinum-based drugs (Itamochi et al., 2008). It has been observed that Hepatocyte Nuclear Factor-1β(HNF-1)βis preferentially activated in OCCC, leading o immunosuppression within the tumor microenvironment through activation of the Signal Transducer and Activator of Transcription 3 (STAT-3) signaling pathway and Nuclear Factor-κB (NF-κB) dependent pathway, resulting in the production of Interleukin-6 (IL-6) and Interleukin-8 (IL-8). Overexpression of NF-κB signaling can be detected both in serum and cancerous ascites from OCCC patients, inducing inhibitory co-stimulators, Programmed Death - Ligand 1 (PD-L1), and B7-H1 as part of the B7 family molecules (Oda et al., 2018). Furthermore, gene expression profiling studies on OCCC tumors have revealed overexpression of Effector memory CD8⁺ T cells, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), PD-1, T cell immunoglobulin and mucin domain 3 (Tim-3), and lymphocyte activation gene 3 (LAG3). Conversely, human leukocyte antigen (HLA) expression was found to be decreased. These findings collectively contribute to an immunosuppressive microenvironment (Oda et al., 2018). Fortunately, the distinct immune microenvironment of OCCC has facilitated the emergence of immunotherapy as a novel therapeutic approach in recent years.

To enhance the prognostic outcomes for patients with OCCC, it is imperative to investigate the cellular and molecular distinctions between OCCC and HGSC to develop more efficacious treatment strategies specifically tailored for OCCC.

At present, because of the scarcity of evidence-supported therapeutic approaches for OCCC, the management protocol adheres to the National Comprehensive Cancer Network (NCCN) guidelines designed for HGSC (Armstrong et al., 2021). In contrast to HGSC, OCCC exhibits greater resistance to conventional platinum-based chemotherapy and a higher likelihood of recurrence (Ye et al., 2015a). The response rate to platinum-based therapy for OCCC ranges from 11% to 56%, compared with approximately 80% for HGSC (Ye et al., 2015b; Jiang et al., 2018; Ye et al., 2019). OCCC can be classified into two transcriptional subtypes: epithelioid and mesenchymal. The mesenchymal subtype is associated with advanced-stage disease, enriched immune-related pathways, and poor prognosis, while the epithelioid subtype is linked to early-stage tumors, frequent mutations in the Yeast mating-type switching/sucrose non-fermenting (SWI/SNF) complex, and a more favorable prognosis (Tan et al., 2019). Epithelial (EpiCC) and mesenchymal (MesCC) subtypes exhibit distinct patterns of immune infiltration. Compared to EpiCC, MesCC shows a higher level of enrichment in both immune-related pathway activity and the presence of tumor-infiltrating lymphocytes. Specifically, EpiCC expresses genes related to the regulatory T cells and the activated dendritic cells, whereas MesCC expresses genes associated with CD4 memory T cells and γδT cells. Moreover, MesCC shows expression of various immune-related genes, including those involved in antigen processing and presentation, such as CD74, HLA-DPA1, HLA-DRA, HLA-DMB, and TAP1. The prognosis of OCCC is positively associated with the expression levels of antigen-presenting genes and negatively correlated with the overexpression of immune checkpoint molecules, particularly PD-1 and CTLA-4. Gene expression profiling studies have demonstrated that effector memory CD8⁺ T cells, as well as immune checkpoint molecules such as PD-1, Tim-3, CTLA-4, and Lymphocyte-activation gene 3 (LAG3), are upregulated in OCCC tumors. Despite these insights, the immune microenvironment of OCCC remains incompletely characterized. Key immune checkpoints in OCCC include LAG-3, PD-1/PD-L1, and CTLA-4, which are expressed on various immune cells. Tumor-infiltrating lymphocytes (TILs) expressing LAG-3 compromise the effector function of PD-1+CD8⁺ T cells. However, combined inhibition of LAG-3 and PD-1 not only restores this effector function but also significantly enhances the antitumor activity (Zaitsu et al., 2023). It has been demonstrated that tumors in patients with early recurrence of OCCC exhibit increased infiltration by mast cells, neutrophils, macrophages, and T cells. This indicates that OCCC is characterized by a higher TIL signal and relatively abundant CD8⁺ T cells compared to Tregs. Furthermore, OCCC with high expression levels of CTLA-4 and PD-1 exhibits a higher recurrence rate (Huang et al., 2023). A study using multiple immunohistochemical approaches to investigate immune cells in OCCC tumors found that increased infiltration of CD8⁺ T cells and macrophages was associated with poor survival outcomes, while high PD-L1 expression correlated with improved survival (Mabuchi et al., 2010). Additionally, low expression of the ARID1A gene is linked to tumor invasion and reduced immune infiltration in OCCC. It shows a negative correlation with CD8⁺ T cells and plasma cells, yet exhibits a positive association with activated CD4⁺ T cells (Jung et al., 2021). Despite OCCC’s high resistance to radiotherapy and chemotherapy, its unique immune microenvironment makes immunotherapy, including immune checkpoint inhibitors, immunomodulatory drugs, and T cell engineering therapies, a promising new treatment modality. Further investigation into the immunological, molecular, and genetic characteristics of OCCC is expected to pave the way for more personalized treatments using specific targeted immunotherapies. For example, pembrolizumab and nivolumab plus ipilimumab induction followed by nivolumab maintenance have shown higher response rates and prolonged progression-free survival in platinum-resistant OCCC patients (Oda et al., 2018). Inhibition of Histone Deacetylase 6 (HDAC6) combined with the immune checkpoint blockade (anti-PD-L1) can treat ARID1A-mutated OCCC by enhancing cytotoxic T cell activity and modulating the tumor immune microenvironment (Fukumoto et al., 2019).

Our GO analysis results indicated that DIRGs are primarily associated with molecular functions such as receptor ligand activity, signaling receptor agonist activity, and cytokine activity. Several studies have investigated NK cells from OCCC patients who exhibit resistance to conventional chemotherapy. These studies demonstrated a cytotoxic interaction between NK cells and autologous tumor cells derived from these patients. In the presence of autologous tumor cells, the degranulation capacity of NK cells isolated from the patient’s peritoneal fluid is compromised due to inhibitory interactions between HLA-I-specific NK cell receptors and HLA-I molecules, as well as impaired interactions between activating NK receptors and their respective ligands (Patrizi et al., 2020). Additionally, KEGG pathway analysis indicated that DIRGs are associated with cytokine-cytokine receptor interaction and the PI3K-AKT signaling pathway. Research has shown that in glioma, Tripartite Motif Containing 6 (TRIM6) modulates the interaction between cytokines and their receptors, contributing to inflammatory responses and immune regulation imbalance, thereby influencing glioma progression (Guo J. et al., 2023). The PI3K/AKT/mTOR signaling cascade has been widely shown to be crucial in the tumorigenesis, chemoresistance, and radioresistance of ovarian carcinoma through various aberrations within the pathway and alterations at multiple regulatory points. Dysregulation of this pathway can contribute to tumor formation, cancer cell migration, invasion, and enhanced chemoresistance. Beyond its involvement in ovarian carcinogenesis, the PI3K/AKT/mTOR signaling pathway also protects primordial follicles from damage during oocyte maturation. The combination of the pan-PI3K inhibitor LY294002 with cisplatin effectively inhibits the proliferation and migration of ovarian cancer cells by downregulating the expression of Matrix Metalloproteinase-2 (MMP-2), TIMP1, and TIMP2 (Ediriweera et al., 2019). Overexpression of progranulin (PGRN) leads to cisplatin resistance in epithelial ovarian cancer cell lines. Studies have shown that the inhibition of the PI3K/AKT/mTOR signaling reduces PGRN expression in OCCC cells (Perez-Juarez et al., 2019). The AKT inhibitor, Perifosine, has exhibited significant antitumor activity in OCCC that has developed resistance to bevacizumab or cisplatin, thereby inhibiting proliferation and inducing apoptosis in OCCC cells (Guo D. et al., 2023). Targeted inhibition of the PI3K/AKT/mTOR signaling pathway holds promise as a therapeutic strategy for addressing OCCC recurrence and drug resistance.

Based on a machine-learning algorithm and diagnostic power analysis, we identified two diagnostic biomarkers, IL4R and NR1H4, which were validated using independent validation sets and immunohistochemical methods. IL4R, a cell surface receptor that targets M2 macrophages in the tumor microenvironment, is classified into two types: Type I and Type II. Type I IL4R is primarily found in certain immune cells and is composed of the IL4Rα and IL2Rγc subunits. IL-4 binds to the alpha subunit of IL4R, subsequently recruiting either the IL2Rγc or IL13Rα subunits (LaPorte et al., 2008). Type II IL4R comprises the IL4Rα and IL13Rα subunits and is significantly upregulated in certain tumor types associated with poor prognosis (Vadevoo et al., 2024). Elevated expression of IL4Rα has been positively correlated with the recurrence of oral squamous cell carcinoma (OSCC) patients (Kwon et al., 2015). High levels of IL4R are also linked to an increased risk of cervical cancer (Muhammad et al., 2021). Studies have demonstrated that high expression of IL4R in breast cancer bone metastasis involves monocyte-derived macrophage subsets promoting metastasis in an IL4R-dependent manner (Ma et al., 2020). Activation of the Extracellular signal-regulated kinase (ERK) pathway via IL4/IL4R signaling plays a critical role in the proliferative phase of preosteoclasts, contributing to colorectal cancer bone metastasis (Sasano et al., 2015). The IL4/IL4Rα signaling axis enhances glucose and glutamine metabolism in breast cancer cells, thereby promoting tumor growth (Jin et al., 2014). Furthermore, IL-4 cytotoxin has shown significant anti-tumor activity against IL4R-expressing ovarian carcinoma in a mouse model of ovarian cancer. The targeted therapy against IL4R has also been effective in eradicating cancer in a mouse model of orthotopic ovarian cancer (Li et al., 2014).

The farnesoid X receptor (FXR), encoded by NR1H4, belongs to the nuclear receptor superfamily and functions as a transcription factor (Ramos Pittol et al., 2020). It is essential for numerous biological processes, like bile acid metabolism, gluconeogenesis, glycogen synthesis, lipogenesis, and ammonia detoxification (Massafra and van Mil, 2018). Furthermore, FXR is proposed to be involved in the development and progression of multiple types of carcinoma, including breast carcinoma, clear cell renal carcinoma, colorectal carcinoma, and hepatocellular carcinoma. There is a notable decrease in NR1H4 expression within pathways involved in the inflammatory response, IL6/STAT3 signaling, IL2 STAT5 signaling, initial estrogen response, and Transforming Growth Factor-β(TGFβ) signaling. This downregulation may be closely linked to biliary atresia (Ma et al., 2022). Elevated NR1H4 levels correlate with poor prognosis in breast cancer patients, promoting tumor cell proliferation and metastasis through CBP-dependent p53 K382 acetylation (Huang et al., 2024). In clear cell renal cell carcinoma (CCRCC), high NR1H4 expression facilitates early diagnosis, while its knockdown significantly inhibits cancer cell proliferation, migration, and invasion. Genetic alterations and DNA methylation of NR1H4 are strongly associated with patient prognosis in clear cell renal cell carcinoma (Huang et al., 2022). In colorectal cancer, NR1H4 activation suppresses the JAK2/STAT3 signaling pathway via trans-activation of the suppressor of cytokine signaling 3 (SOCS3) gene and antagonizes Wnt/β-Catenin signaling, acting as a tumor suppressor (Yu et al., 2020). Additionally, NR1H4 enhances drug-induced cell death in colorectal cancer by modulating myelocytomatosis oncogene (MYC) expression and Myc protein stability (Huang et al., 2022). In hepatocellular carcinoma, the nuclear translocation of transketolase promotes HDAC3 binding to the NR1H4 promoter, thereby inhibiting FXR expression (Li et al., 2020).

We employed the CIBERSORT algorithm to examine the relationship between NR1H4 and IL4R gene expression and immune cell infiltration. The analysis demonstrated that the proportions of plasma cells, M2 macrophages, and neutrophils were significantly elevated in the OCCC group relative to the HGSC group, while the proportion of M0 macrophages was notably higher in the HGSC group compared to the OCCC group. Additionally, tumor tissues from patients with recurrent OCCC exhibited increased expression signals for mast cells, neutrophils, macrophages, and T cells. Notably, IL4R expression levels are significantly higher in M2 macrophages than in M1 macrophages (Movahedi et al., 2010; Shiao et al., 2015; Vadevoo et al., 2017). IL4 and IL4R can induce M2 macrophage polarization, thereby promoting tumor progression and metastasis. Targeting IL4R enhances the efficacy of nab-paclitaxel by facilitating the conversion of M2 macrophages to an M1-like phenotype via the Reactive Oxygen Species (ROS)-HMGB1-TLR4 axis. This approach increases the presence of immunostimulatory cells while reducing immunosuppressive cells within the tumor microenvironment, thus enhancing antitumor immunity and providing a novel strategy for tumor immunotherapy (Vadevoo et al., 2024). The IL-4 signaling pathway, through its receptor IL4R, may be involved in regulating the immunosuppressive microenvironment of ovarian cancer. Studies have shown that the IL-4 signaling may promote tumor immune escape by activating CD4⁺ regulatory T cells (Tregs) and inhibiting the function of NK cells (Mollaoglu et al., 2024). Single-cell sequencing studies have found that the infiltration of natural killer (NK) cells and CD8⁺ T cells in the tumor microenvironment of OCCC is associated with responses to immunotherapy, suggesting that NK cells may exert their effects through direct killing of tumor cells or regulation of other immune cells (Xia et al., 2024). After kidney transplantation, the number of CD56dimCD16+ natural killer (NK) cells co-expressing IL4R (p = 0.038) and CD56bright NK cells with this phenotype increased over time (Zhu et al., 2018). At present, no research has revealed the direct association between IL4R and NK cells or CD4⁺ T cells in OCCC. However, the infiltration levels and functional states of NK cells and CD4⁺ T cells have been confirmed to be closely related to immune escape and treatment response in OCCC. Future studies are required to integrate multi-omics data to deeply explore the potential role of IL4R in the immune microenvironment of OCCC.

NR1H4 expression also correlated with the degree of immune cell infiltration, such as CD4⁺T cells, macrophages/monocytes, and neutrophils. In CCRCC, NR1H4 functions as a mediator of tumor immunity and plays a key role in tumor metastasis and drug resistance. Furthermore, the expression of NR1H4 exhibits a significant positive correlation with the infiltration levels of CD4⁺ T cells, macrophages/monocytes, and neutrophils in CCRCC (Huang et al., 2022). FXR (NR1H4) agonist GW4064 prevents dextran sulfate sodium - parenteral nutrition (DSS-PN) - induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation, and transcription of cytokines in hepatic macrophages stimulated by lipopolysaccharide in vitro (El Kasmi et al., 2022). After treatment with an FXR agonist, bone marrow-derived macrophages exhibit increased expression of M2 polarization-associated signaling molecules (such as those related to the retinoic acid receptor pathway), while inhibition of the retinoic acid receptor pathway blocks FXR-mediated M2 polarization (Jaroonwitchawan et al., 2023). In the intestine, FXR inhibits inflammation-associated tumor growth by regulating macrophage recruitment, polarization, and interactions with Th17 cells. Deletion of FXR in intestinal macrophages exacerbates inflammation, whereas activation of FXR reduces pro-inflammatory cytokine secretion and improves intestinal barrier function (Dong et al., 2024). Additionally, FXR may indirectly influence macrophage phenotypes by regulating bile acid metabolism, such as abnormal bile acids that can trigger pro-inflammatory responses through macrophage-intrinsic FXR (Dong et al., 2024). While no study has directly reported the regulation of resting natural killer (NK) cells by FXR, evidence suggests a potential link between the two. CXCR6+ macrophages enhance interactions between T/NK cells and myeloid cells through the CXCL16-CXCR6 axis (Lu et al., 2024). FXR primarily participates in immune regulation by regulating macrophage polarization, while its effects on NK cells may be mediated indirectly through macrophages. The direct association between resting NK cells and FXR has not been explicitly supported by existing literature and requires further research for validation.

Nevertheless, our study has certain limitations. Owing to the rarity of clear cell carcinoma and the limited availability of clinical cases, the roles of NR1H4 and IL4R in OCCC and their associations with immune cell infiltration require further validation through prospective studies with larger sample sizes. Additionally, because of the current sample size constraints, only IHC was employed for verification. Future research should consider combining single-cell RNA sequencing and multi-color flow cytometry in follow-up studies to deeply dissect the effects of the IL4R and NR1HR on the functions of immune cell subsets and animal models.

In summary, this research utilized machine learning approaches to explore the possible relationship between immune-related genes and OCCC, uncovering a substantial connection. The immune system is crucial for both the development and progression of OCCC. Specifically, NR1H4 and IL4R were identified as key immune genes associated with macrophage M2 polarization. These findings offer novel perspectives for predicting, diagnosing, and elucidating the pathogenesis of OCCC. The importance of specific immune cell subsets within the tumor microenvironment was further highlighted. These results not only afford new insights into the immunological characteristics of OCCC but also have important implications for developing innovative immunotherapy strategies against OCCC. By identifying and validating immune genes closely linked to OCCC development, we have provided valuable information for future research and clinical practice, thereby facilitating the advancement of personalized treatment approaches.