Sex-based differences in response to immunotherapy have been increasingly recognized in HCC treatment, particularly in the efficacy of immune checkpoint inhibitors (24, 25). Checkpoint blockade therapies, such as PD-1/PD-L1 inhibitors (e.g., nivolumab, pembrolizumab, atezolizumab), have revolutionized HCC treatment, yet their effectiveness varies between male and female patients (26, 27). Female HCC patients generally exhibit better response rates to PD-1/PD-L1 inhibitors than their male counterparts. For instance, a meta-analysis of clinical trials across various cancers, including HCC, found that women had a higher overall survival benefit from anti-PD-1/PD-L1 therapy compared to men. The underlying mechanisms for these disparities may involve differential immune system activity, where females generally have stronger adaptive and innate immune responses. Additionally, estrogen has been shown to enhance T cell function and promote anti-tumor immunity, whereas testosterone may contribute to immune suppression. Beyond ICIs, sex-based variations have also been noted in other emerging immunotherapies, such as CAR-T cell therapy. Although CAR-T therapy is still in its early stages for HCC treatment, preliminary studies indicate that female patients may experience greater T cell persistence and expansion post-infusion, potentially leading to more durable responses. This finding aligns with broader observations in hematologic malignancies, where female patients have demonstrated superior CAR-T cell therapy outcomes. However, further research is needed to validate these findings in solid tumors like HCC.

Sex-specific outcomes in HCC treatment have been observed in several clinical trials evaluating immunotherapy efficacy. The IMbrave150 trial, which established the combination of atezolizumab and bevacizumab as a first-line treatment for HCC, provided some insights into sex-based differences. While the trial did not specifically stratify patients by sex, subgroup analyses suggested a trend toward better outcomes in female patients receiving this combination therapy (28). Similarly, retrospective analyses of nivolumab and pembrolizumab trials indicate that female patients tend to experience longer progression-free survival and overall survival compared to males. Despite these observations, significant limitations exist in the current body of research. Most clinical trials are not explicitly designed to assess sex-based disparities, leading to potential underrepresentation and inadequate statistical power to detect meaningful differences. Moreover, hormonal status, menopausal state, and other sex-specific factors are rarely accounted for in clinical trial designs, limiting our ability to fully understand the impact of sex on treatment outcomes.

Estrogen, a key female sex hormone, plays a significant role in modulating immune responses and influencing cancer progression (29, 30). The immunomodulatory effects of estrogen contribute to the observed sex differences in HCC incidence and response to immunotherapy. Estrogen exerts its protective effect by enhancing anti-tumor immunity. Estrogen promotes the activity of CD8⁺ T cells, which are crucial for immune-mediated tumor clearance. Estrogen enhances interferon-gamma (IFN-γ) production, a cytokine critical for T cell activation and cytotoxicity (31, 32). In contrast, lower estrogen levels in males have been associated with decreased CD8⁺ T cell function and increased immune exhaustion, which may contribute to poorer responses to ICIs. Furthermore, estrogen modulates the function of tumor-associated macrophages (TAMs) within the HCC tumor microenvironment. TAMs exist in two major phenotypes: M1, which is pro-inflammatory and anti-tumorigenic, and M2, which is immunosuppressive and promotes tumor progression (33, 34). Estrogen has been shown to shift macrophage polarization toward the M1 phenotype, thereby enhancing anti-tumor immunity (35, 36). This effect contrasts with the influence of testosterone, which promotes M2 macrophage polarization and immunosuppression.

Estrogen exerts protective effects in HCC through its receptors, mainly estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), which are associated with better overall survival and disease-free survival in patients ( Figure 1 ). Mechanistically, ERα suppresses yes-associated protein (YAP) signaling by enhancing YAP phosphorylation, preventing its nuclear translocation, and inhibiting downstream oncogenic pathways (37). This suggests that ERα inactivation of YAP signaling contributes to its tumor-suppressive role in HCC, making it a potential prognostic marker and therapeutic target ( Figure 1A ). Additionally, estrogen influences HCC progression by modulating liver function and the immune response through ERα signaling. During HEV infection, estrogen-ERαp66 signaling regulates the STAT3 pathway, stabilizing SOCS3 and modulating the innate immune response in hepatocytes (38) ( Figure 1B ). While estrogen does not directly affect HEV replication, blocking STAT3 reduces HEV capsid protein levels, highlighting a potential link between estrogen signaling, liver inflammation, and HCC susceptibility (38). ERα activation also induces lipid accumulation and fibrosis by inducing PNPLA3 expression, which contributes to the higher prevalence and severity of fatty liver disease (FLD) in women (39). This interaction suggests that ER-α and PNPLA3 may play a crucial role in sex-based differences in FLD, potentially influencing the progression of HCC in at-risk individuals. In addition, estrogen signaling modulates ATXN7L3’s function, potentially switching HCC from a tumor-promoting to a tumor-suppressing state by facilitating deubiquitinase swapping, where ATXN7L3 and ENY2 replace USP22 (a tumor-promoting deubiquitinase) with USP27x or USP51 (potential tumor-suppressing deubiquitinases) (40). This mechanism may allow ERα to redirect ATXN7L3’s activity toward pathways that inhibit tumor progression rather than promote it (40).

Moreover, ERβ signaling can suppress pro-inflammatory cytokine production, thereby reducing chronic liver inflammation, a major risk factor for HCC. ERβ activation in macrophages has been shown to inhibit tumor growth in certain cancers by suppressing the JAK1/STAT6 pathway, which may contribute to a less immunosuppressive tumor microenvironment (41) ( Figure 1C ). ERβ plays a crucial role in CD8+ T cell-mediated anti-tumor immunity. TCR activation triggers ERβ phosphorylation, enhancing downstream TCR signaling via a non-genomic mechanism. Mutation of the ERβ phosphotyrosine switch impairs CD8+ T cell function and promotes tumor growth, while the ERβ agonist S-equol boosts TCR activation and enhances anti-PD-1 immunotherapy, suggesting ERβ as a potential target for improving cancer immunotherapy (42). Recent studies have revealed that ERβ plays a crucial role in the development of uterine corpus endometrial cancer (UCEC) by modulating the miR-765/PLP2/Notch signaling axis. This mechanism is influenced by the exosomes released by CD8+ T cells, which regulate the miR-765/PLP2 pathway, potentially limiting estrogen-driven tumor progression. Similar interactions between estrogen signaling and immune cell-derived exosomes might be present in liver cancer, suggesting a complex interplay between hormonal signaling and immune responses that could influence HCC progression (43).

G protein-coupled estrogen receptor 1 (GPER1) is a non-classical estrogen receptor that mediates rapid, non-genomic estrogen signaling (44). Unlike the classical estrogen receptors ERα (ESR1) and ERβ (ESR2), which function as nuclear transcription factors, GPER1 is a membrane-bound receptor that activates intracellular signaling cascades in response to estrogen binding. GPER1 was found to play a protective role against HCC tumorigenesis. GPER knockout in a mouse tumor model accelerated liver tumor formation, accompanied by increased immune cell infiltration, fibrosis, and elevated inflammatory factors such as IL-6 (44), activating GPER could be a potential strategy for HCC prevention and treatment. GPER1 also plays a key role in restricting macrophage proliferation in HCC. Lower GPER1 expression in macrophages correlates with increased macrophage proliferation and tumor progression, while activation of GPER1 signaling suppresses macrophage proliferation via the MEK/ERK pathway and reduces PD-L1 expression, delaying tumor growth (45). In addition to its role in macrophages, GPER1 signaling has been implicated in modulating neutrophil recruitment and NK cell cytotoxicity, which may further contribute to its protective effects against HCC (46). These findings highlight a potential therapeutic strategy targeting GPER1 to modulate the tumor microenvironment in a sex-specific manner.

Besides functions via its receptors, estrogen also exerts protective effects against HCC by upregulating lecithin cholesterol acyltransferase (LCAT), which enhances high-density lipoprotein cholesterol (HDLC) production and uptake, thereby suppressing cholesterol biosynthesis and inhibiting tumor growth through an ESR1-dependent pathway ( Figure 1D ). Additionally, HDL-C synergizes with lenvatinib to enhance anti-tumor efficacy, suggesting that targeting the LCAT/HDL-C axis could improve immunotherapy and treatment outcomes in HCC (16).

In contrast to estrogen, testosterone—the primary male sex hormone—has been associated with immunosuppressive effects that may contribute to the higher incidence and poorer prognosis of HCC in males (47, 48). Testosterone has been shown to influence immune responses by modulating androgen receptor (AR) signaling, which affects various immune cells in the tumor microenvironment ( Figure 2 ). Testosterone induces long-term changes in liver gene expression, including the upregulation of HCC-related genes such as Lama3 and Nox4, while suppressing immune response genes like IFNγ. These changes may contribute to the masculinized liver metabolism and immune evasion, creating an environment that favors HCC progression in males, suggesting that testosterone may play a role in promoting HCC by impairing immune surveillance and enhancing tumor-promoting pathways (49). AR mediates sex differences in cancer progression by promoting CD8⁺ T cell exhaustion in males. Specifically, AR influences the development of antigen-experienced progenitor exhausted CD8⁺ T cells in a sex-specific manner. Inhibiting the androgen-AR axis reprograms the tumor microenvironment, enhancing effector T cell differentiation and improving the response to anti-PD-1 immunotherapy, highlighting the potential for targeting androgen signaling in cancer treatment (13). Testosterone also suppresses immune responses is by downregulating the activity of CD8⁺ T cells (50). Blocking AR enhances CD8 T cell function and sensitizes tumor-bearing hosts to effective immune checkpoint blockade. This inhibition prevents T cell exhaustion, increases IFNγ expression, and improves the responsiveness to PD-1 targeted therapy, highlighting a novel mechanism of immunotherapy resistance (51). Male CD8⁺ T cells exhibit impaired effector and stem cell-like properties compared to female CD8⁺ T cells, with AR inhibiting their activity and stemness through epigenetic and transcriptional regulation (52). Androgen-axis blockade (via androgen deprivation therapy and enzalutamide) combined with anti-PD-L1 treatment synergistically restricted tumor growth in male mice (52, 53). In humans, higher AR expression in tumor-infiltrating CD8⁺ T cells correlates with T cell exhaustion, suggesting that sex-biased differences in T cell stemness contribute to cancer progression and responses to immunotherapy. Androgen receptor activation upregulates USP18, inhibiting NF-κB in antitumor T cells (54) ( Figure 2A ). Castration or abiraterone treatment enhances T-cell activity and improves the effectiveness of anti-PD-1 immunotherapy in male mice (54), suggesting that inhibiting androgen signaling could be a promising strategy to enhance immunotherapy in males.

Testosterone also exerts its effects on Tregs, which are crucial for maintaining immune tolerance but can suppress anti-tumor immunity in the context of cancer. Increased testosterone levels have been linked to the expansion of Tregs within the tumor microenvironment, contributing to immune evasion by HCC cells. This immunosuppressive effect may contribute to the lower response rates observed in male patients undergoing checkpoint inhibitor therapy. In HCC, testosterone signaling has been shown to stabilize Foxp3⁺ Tregs, enhancing their suppressive function and limiting the number of effector T cells (55) ( Figure 2B ). Additionally, androgen signaling decreases the expression of Gata2, a transcription factor for ST2⁺ Tregs, and reduces IL-33 production from airway epithelial cells in asthma models (55). These findings suggest that testosterone-induced Treg accumulation and function dampen the immune response against tumors, potentially contributing to the reduced efficacy of immunotherapies in males.

Moreover, testosterone influences macrophage polarization by promoting the M2 phenotype, which is associated with tumor progression and immune suppression. Androgens, specifically dihydrotestosterone, promote M2 macrophage polarization in allergic asthma by enhancing IL-4-induced M2 differentiation in vitro and in vivo. In mice lacking AR in macrophages, reduced M2 polarization led to decreased eosinophil recruitment and lung inflammation (56), highlighting the critical role of androgen/AR signaling in regulating macrophage polarization in diseases like asthma and prostate cancer. M2 macrophages secrete cytokines such as IL-10 and TGF-β, which inhibit the activation of cytotoxic T cells and natural killer cells, thereby reducing the overall effectiveness of immunotherapy. Testosterone-driven M2 polarization has been observed in various malignancies, including HCC, where M2 macrophages support tumor progression by creating an immunosuppressive microenvironment conducive to cancer cell survival and metastasis ( Figure 2C ).

Beside suppressing the immune response, testosterone -AR signaling also modulates HCC progression via other mechanisms. In HCC, AR upregulates EZH2 expression by binding to its promoter, thereby stimulating its transcriptional activity. This leads to increased levels of H3K27me3, which silences wingless//Int-1 (Wnt) signaling inhibitors and activates the Wnt/β-catenin pathway (57). The activation of this pathway promotes cell proliferation and tumorigenesis, correlating with tumor progression and poor prognosis in HCC patients. Furthermore, AR interacts with the mechanistic target of rapamycin complex 1 (mTORC1), which phosphorylates AR at S96 in response to nutrient and mitogenic stimuli (58). This phosphorylation enhances the stability, nuclear localization, and transcriptional activity of AR, further promoting lipogenesis, hepatocyte proliferation, and hepatocarcinogenesis. High AR S96 phosphorylation is associated with poor overall survival and disease-free survival in HCC patients, highlighting the cooperative role of AR and mTORC1 in driving liver tumor progression and providing a potential target for HCC treatment (58) (Figure 2D).

In conclusion, sex hormones play a critical role in shaping immune responses and influencing the outcomes of HCC immunotherapy ( Table 1 ). While estrogen enhances anti-tumor immunity and improves responses to checkpoint inhibitors, testosterone promotes immune suppression and may contribute to resistance to immunotherapy. Understanding these hormonal effects is essential for developing personalized treatment strategies that account for sex-based differences in HCC immunotherapy. Targeting androgen signaling in combination with immune checkpoint inhibition holds promise for improving outcomes in male HCC patients.

The X chromosome is unique because females have two copies of it, while males have only one. This genetic difference results in a differential expression of genes linked to the immune system. Many immune-related genes reside on the X chromosome, and their expression can have significant effects on the immune system. Key genes such as FOXP3, TLR7/8, and others are of particular interest in understanding the gender differences in immune responses.

The Y chromosome is another key player in sex-based immune differences. Unlike the X chromosome, which contains many immune-related genes, the Y chromosome has fewer immune-regulatory genes (89). However, some of these Y-linked genes, particularly those related to male sex determination and spermatogenesis, may also influence immune function.