Macrophages play a pivotal role in regulating tumor cell proliferation and progression by releasing various inflammatory factors and cytokines. Notably, they are the most commonly encountered immune infiltrating cells within the tumor microenvironment. Of particular concern in CCA is the presence of high levels of M2 macrophages, which have been shown to be strongly associated with carcinogenesis and poor outcomes in with CCA, as evidenced by prior studies (42-44). TAMs, a subtype of M2 macrophages, exert potent pro-tumor effects. The shift of macrophages toward this alternative M2 phenotype is primarily orchestrated by the actions of specific signaling pathways, notably involving IL6/STAT3 and PCAT6/miR-326/RohA pathway (43,45). Notably, Kitano et al demonstrated an association between TAM infiltration, increased levels of Tregs and TANs, and a poor recurrence-free survival (RFS) (46). TAMs participate in remodeling the ECM via secretion of MMPs and the release IL-4, IL-8, IL-10, chemokine ligand (CCL)2, CCL22 and CCL17 to recruit immunosuppressive cells, such as TANs, myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs), to form the suppressive immune microenvironment (47).

TAMs play a multifaceted role in promoting tumorigenesis and can exert their tumor-promoting effects by interacting with CCA cells and TANs (Fig. 2). CCA cells can produce IL-6 and TGF-β, which are involved in the activation of TAMs. In a reciprocal interaction, TAMs release significant amounts of IL-10, which, can activate the STAT3 pathway in CCA cells. This activation, in turn, enhances the migration and invasion of tumor cells, largely through the process of EMT (48). Additionally, activated TAMs are capable of producing molecules, including VEGF-A, angiopoietin, IL-8, cyclooxygenase-2 and inducible nitric oxide synthase, to promote tumor angiogenesis (43,49). Furthermore, CCA cells express certain Wnt ligands, including Wnt3, Wnt5a, and Wnt7b, which have the capability to recruit and activate TAMs. Subsequently, TAMs release Wnt, which in turn stimulates the Wnt/β-catenin pathway, leading to increased tumor cell proliferation (50,51). Moreover, in vitro experiments involving the inhibition of Wnt signaling using a Wnt inhibitor have revealed a significant reduction in CCA proliferation and an increase in apoptosis. These effects have been observed in mouse and rat models of CCA, ultimately resulting in tumor regression (52). TAMs secret IL-6 to promote the activation of TANs and CCA cells can express epithelial-derived neutrophil-activating peptide-78 (ENA-78) to recruit TANs mediated by the PI3K-AKT and ERK1/2 signaling pathways (53). The interaction between TANs and TAMs leads to the production of oncostatin M (OSM) and IL-11 by TANs and TAMs, respectively. Both OSM and IL-11 have been found to stimulate the STAT3 pathway in CCA cells, resulting in increased tumor cell proliferation and invasion. Of note, when STAT3 is knocked down, it mitigates the pro-tumor effects of TANs and TAMs in iCCA (54).

The role of TANs in tumorigenesis and development is still under investigation. TANs are likely to assume the N2 subtype, which is distinct from N1 neutrophils that are activated in normal tissue (55,56). The activation of the N2 subtype neutrophils is mainly induced by TGF-β and granulocyte colony-stimulating factor (CSF) (57). Activated N2 neutrophils can release various growth factors, enzymes and cytokines to promote tumor growth, shape the TIME and stimulate angiogenesis (58). The high level of TAN infiltration tends to be associated with a poor prognosis, as it is related to decreased overall survival (OS) and RFS of patients with CCA (46,59). However, a recent study proposed a contrasting view, suggesting that patients with biliary tract cancer (BTC) with a higher neutrophil infiltration exhibit an improved prognosis (60). Owing to the limited amount of evidence and the conflicting findings, it remains challenging to arrive at a definitive conclusion regarding the prognostic significance of TAN infiltration. High levels of TAN infiltration have been shown to promote the growth and invasion of tumors in vivo, although they do not appear to alter the in vitro proliferative and invasive abilities of iCCA cells (53).

MDSCs are a group of heterogeneous immune cells which exert potent immunosuppressive effects that can inhibit various immune cell activities. Chronic inflammation functions as a stimulus for MDSCs, prompting them to synthesize molecules such as arginase, reactive oxygen species, inducible nitric oxide synthase and indoleamine 2,3-dioxygenase. Furthermore, MDSCs release immunosuppressive factors, such as TGF-β and IL, which act to curtail the function of cytotoxic T-lymphocytes, natural killer (NK) cells and their respective subpopulations, thereby achieving a state of immunosuppression (61). The accumulation of MDSCs in the TIME has been linked to heightened immune evasion and increased resistance to immunotherapy in various types of cancers (62,63). Studies on primary hepatocellular carcinoma (HCC) have shown that MDSCs play a role in promoting the development of Tregs, the inactivation of CD8⁺ T-cells, and the suppression of the cytotoxic activity of NK cells. Furthermore, elevated levels of mononuclear MDSCs in the peripheral blood have been shown to be associated with the poor OS of patients with HCC (64). Nevertheless, the precise functions of MDSCs in the context of CCA remain incompletely understood. Recent research has indicated that the gut microbiome can induce the accumulation of CXCR2⁺ polymorphonuclear MDSCs through TLR4-dependent CXCL1 production, thus facilitating the establishment of an immunosuppressive environment that promotes CCA progression (65). The level of CD33⁺ MDSCs in the blood and tumor tissues of patients with iCCA has been found to be increased and to be associated with a poor clinical outcome (66). The relevance between CAFs and MDSCs has been brought to light. Specifically, CAFs have been found to secrete IL-6 and IL-33, which in turn stimulate MDSCs to upregulate the expression of 5-lipoxygenase (5-LO). Notably, one of the metabolites of 5-LO, known as LTB4, has been shown to activate the PI3K/Akt-mTORC1 signaling pathway in iCCA cells through the interaction with BLT2. This activation ultimately contributes to the promotion of cancer stemness in iCCA (66). Additionally, Loeuillard et al (67) highlighted the significance of the interaction between TAMs and MDSCs. They identified an abundance of programmed death ligand 1 (PD-L1)-positive TAMs in both human CCA samples and CCA mouse models. Elevated levels of PD-L1⁺ TAMs were associated with an enhanced cancer progression. However, attempts to block TAMs alone have not effectively reduced CCA tumor burden (67). This lack of a response may be attributed to the compensatory accumulation of granulocyte MDSCs (G-MDSCs), which impair the T-cell response and induce immune evasion. Notably, the simultaneous inhibition of G-MDSCs and TAMs has shown promise in enhancing the efficacy of anti-PD-1 therapy for CCA (67). Collectively, that study underscored the role of MDSCs as mediators that collaborate with other components within the CCA TIME to promote tumor progression (67).

NK cells are known for their potent antitumor activity, as they possess the ability to induce tumor cell apoptosis directly by releasing molecules, such as perforin, cytotoxic factors and TNF (68). Previous findings have suggested that enhancing NK cell function or increasing the numbers of NK cells delays CCA progression, which may be a potential therapeutic target for CCA. Anti-Globo H antibody VK9 can enhance the activation and increase the presence of NK cells in the TIME, resulting in the inhibition of iCCA rat tumor growth (69). In vitro studies have demonstrated that the cytotoxic effect of activated NK cells on CCA cell lines may be augmented by cetuximab and cordycepin, effectively restraining CCA cell growth (70,71). Additionally, in a xenograft mouse model of CCA, the transplantation of ex vivo-expanded human NK cells has been found to inhibit tumor growth (72). Fukuda et al (73) observed that low numbers of tumor-infiltrating NK, cells regulated by endogenous CXCL9, were associated with a large tumor size and the poor survival of iCCA (73). The activating receptor natural-killer group 2D (NKG2D), predominantly expressed on NK cells, holds promise as a therapeutic target. A high expression of the NKG2D receptor in patients with CCA has been linked to an improved prognosis (74). Moreover, it is worth noting that variations in NKG2D have been found to be associated with bile duct tumorigenesis in patients with primary sclerosing cholangitis (75).

The adaptive immune system is the predominant defense system against tumors. The major components of the adaptive immune system in the CCA TIME are TILs, including B-lymphocytes, CD4⁺ helper T-lymphocytes, CD8⁺ cytotoxic T-cells and Tregs. While various studies have examined the spatial distribution of TILs in CCA tissue (49,76,77), it remains a topic of debate and discussion. According to the current literature, it appears that CD3⁺, CD4⁺ and CD8⁺ T-cells predominantly reside in the peritumoral region, regardless of the CCA subtype. However, as for Foxp3⁺ T-cells and B-cells, the exact distribution location has not been definitively determined (6). Recent studies have demonstrated that Foxp3⁺ T-cells have been observed to accumulate in the tumor border area (76,77). Another previous study indicated that Foxp3⁺ T-cells were distributed in the intratumoral area (78), while another study failed to found the distribution difference of Foxp3⁺ T-cells (79). In the case of B-cells, two separate studies have reported their presence in the peritumoral area (78,80). These varying observations regarding the distribution of Foxp3⁺ T-cells and B-cells may be attributed to distinct contexts or factors, warranting further investigation for a comprehensive understanding. The potential association between TILs and various signaling pathways in tumor promotion has been revealed by numerous studies. Recent research has demonstrated that a low abundance of tissue-resident memory T-cells is associated with a significantly increased expression of genes related to the Wnt/β-catenin and TGF-β signaling pathways (81). Carnevale et al (82) made an intriguing discovery where the expression of cellular FADD-like IL-1β-converting enzyme-inhibitory protein in iCCA cells significantly increased following co-culture with human peripheral blood mononuclear cells. This led to the activation of the Fas/FasL pathway, inducing the apoptosis of T-cells and NK cells, ultimately resulting in tumor immune escape (82). Another study highlighted the significance of the B7-H1/PD-1 signaling pathways in the induction of CD8⁺ TIL apoptosis and the inhibition of antitumor immune responses (83). Isocitrate dehydrogenase 1 mutations drive the activation of interferon γ (IFN-γ)-responsive genes in iCCA cells through a TET2-dependent mechanism. This occurs by impeding the recruitment of activated CD8⁺ T-cells and the expression of IFN-γ (84).

It has been proven that the number or density of TILs in the TIME of CCA affects prognosis. High levels of CD8⁺ T-cells have consistently been associated with an improved survival and reduced invasion into surrounding tissues in several studies (76,78,85,86). A substantial infiltration of CD4⁺ T-cells has similarly been linked to a favorable OS and RFS (77,78). Foxp3⁺ T cell infiltration, however, has generated mixed results in terms of prognosis. While the majority of studies suggest that it is associated with a poor prognosis of patients with CCA (49,76,87), Goeppert et al (78) indicated that patients with Foxp3⁺ T cell infiltration experience improved outcomes. Thus, the prognostic value of Foxp3⁺ T-cells remains uncertain, necessitating further studies to clarify their specific impact on long-term results. Current studies have revealed that patients with CCA benefited from high levels of B-cell infiltration (78,88). Nevertheless, due to limited available evidence, the association between B-cells and the prognosis of patients with CCA also warrants further investigation through additional research.

Of note, Tregs play an essential role in establishing the suppressive TIME (Fig. 3). Tregs are the major immune cells in the TIME of advanced-stage cancer, overexpressing FoxP3 and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) (89-91). The transcription factor Forkhead box M1 has been shown to increase FoxP3 expression, leading to the recruitment of Tregs. This recruitment attenuates the killing ability of CD8⁺ T-cells on iCCA cells, contributing to immune escape and the progression of CCA (92). Recent studies have shown that Tregs can adapt to the glucose-depleted tumor microenvironment by shifting their glucose metabolism to fatty acid metabolism, allowing them to survive in the TIME (93). The activation of the EGFR/PI3K/Akt signaling pathway, triggered by mucin protein 1 (MUC1), induces the enrichment of Tregs to bolster the malignant phenotypes, including tumor growth and metastasis (94). Moreover, natural Treg-like CD4⁺CD25⁻ cells activated by CCA cells can produce numerous TGF-β, and elevated levels of TGF-β downregulate the expression of miR-29a, which, in turn, suppresses antitumor immune responses and eventually causes adverse clinical outcomes (95).

DCs are essential components of the immune system, functioning as professional antigen-presenting cells (APCs) that bridge innate and adaptive immune responses. Their presence in tumor tissues can trigger robust antitumor immune responses, potentially improving cancer patient outcomes. The abundance of DCs in the peripheral blood of patients is significantly decreased compared to healthy individuals (96). It has been shown that patients with a higher number of DCs infiltrating the tumor margin experience a lower rate of lymph node metastasis and better prognosis (97). DCs may affect the number of TILs within the TIME. Junking et al (98) found that DCs pulsed by CCA cells induced the differentiation of PBMCs into DCs. This process increased and activated CD3⁺ CD8⁺ T-cells, empowering them to induce tumor cell apoptosis (98). DCs generated by the stimulation of CCA cells can utilize activated lymphocytes as anti-CCA effector cells. IL-12 and TGF-β in TIME can impair the functions of DCs. The application of specific neutralizing antibodies that block the IL-10 and TGF-β receptors on DCs, or the knockdown of TGF-βRII and IL-10RA mRNA, has been shown to enhance the performance of effector T-cells (99,100). Sung et al (101) discovered that ABL501, a bispecific antibody targeting lymphocyte-activation gene 3 (LAG-3) and PD-L1, induced CD8⁺ T-cell activation by promoting DC maturation. This activation ultimately amplifies the cytotoxic effects of CD8⁺ T-cells against tumor cells (101). CTLA-4 is a transmembrane protein encoded by the CTLA-4 gene as well as a homologous protein of CD28, which is expressed in activated CD4⁺ and CD8⁺ T-cells (102), which presents with higher affinity competes with CD28 for binding to B7 (CD80/86) to impair T-cells. Mature DCs have been demonstrated to significantly release CTLA-4 into the extracellular compartment by vesicular transport, which can inhibit the antibody binding of B7, eventually resulting in the dysfunction of T-cells (103). In addition, the activation of the CD40/CD40L pathway can enhance the function of DCs and induce cytotoxic effects to CCA cell (104). CD40/CD40L is significantly involved in the maturation, proliferation and survival of DCs, which were triggered by activating p38 MAPK, PI3K-Akt and NF-κB pathways (105). In addition, CD40 signaling triggered the expression of Bcl2l1 to reduce the caspase activation and apoptosis, eventually contributing to maintaining classical type 1 DCs survival during the initiation of anti-tumor immune responses (106). The signaling pathways associated with DCs exerting antitumor effects are summarized in Table SI. In summary, these findings underscore the potential of DC-based immunotherapies as promising approaches for improving outcomes in the context of CCA treatment.

The clinical trial NCT01876511 exhibited that 86 (including CCA) patients with dMMR or MSI-H were treated with pembrolizumab and achieved satisfactory treatment outcomes (139). In the phase 1b trial KEYNOTE-028, 20 patients with advanced-stage CCA and 4 patients with advanced-stage gallbladder cancer (GBC), all of whom tested positive for PD-L1, received pembrolizumab monotherapy (142). A total of 3 patients with CCA and 1 patient with GBC achieved stable disease (SD). Grade 3 toxicities were observed in 17% of cases, with no grade 4 events reported. The objective response rate (ORR) was 17%, and the median progression-free survival (mPFS) and median OS (mOS) were 1.8 and 6.2 months, respectively. Notably, that study confirmed that pembrolizumab was well-tolerated, displayed excellent antitumor activity, and exhibited manageable safety and effectiveness (142).

In a phase 2 study involving 54 patients with BTC pre-treated with at least one line, but no more than three lines of systemic therapy, the anticancer activity of nivolumab in advanced refractory BTC was evaluated (143). More than half of the patients had well-controlled conditions, resulting in a mOS of 14.22 months and a mPFS of 3.68 months. However, Ueno et al (144) suggested a poor response rate to nivolumab monotherapy. In summary, the effectiveness of nivolumab monotherapy for CCA remains uncertain due to the cohort size, and further research is required to provide a clearer picture. Durvalumab, atezolizumab and avelumab are also approved by the FDA for the treatment for various solid tumors. However, studies have indicated that these monoclonal antibodies have limited efficacy when used as monotherapy for CCA. For instance, Doki et al (145) reported that among 42 patients with BTC treated with durvalumab alone, the median OS was 1.5 months, the mPFS was 8.1 months and the ORR was 4.8%.

Apart from ICIs targeting a single immune checkpoint, there is growing interest in ICIs that can simultaneously act on two immune checkpoints. A promising example is ABL501, which can inhibit both LAG-3 and PD-L1 concurrently, demonstrating higher antitumor activity compared to a combination of anti-LAG-3 and anti-PD-L1 treatments (101). ABL501 has emerged as a promising candidate in cancer immunotherapy and is currently undergoing its initial human trial (NCT05101109). Another innovative approach is represented by M7824, a novel bifunctional fusion protein. M7824 comprises a monoclonal antibody against PD-L1 fused to the extracellular domain of human TGF-β receptor II. This design allows M7824 to serve a dual function by blocking PD-L1 and sequestering TGF-β molecules (146). Research has explored the response of M7824 in Asian patients with CCA, revealing an ORR of 23%. However, it is essential to note that treatment-related adverse events (TRAEs) have been observed in 63% of patients (147). Further investigations are warranted to assess the overall safety and efficacy of this approach in CCA immunotherapy.

Given the limited effectiveness of ICI monotherapy for CCA, there is a growing focus on exploring combination immunotherapies to enhance treatment responses and overcome immune tolerance. Combination therapy involving two ICIs has demonstrated promising outcomes in various solid tumors. In a phase 2 study evaluating the combination of nivolumab and ipilimumab in 39 patients with advanced-stage BTC (148), the trial reported an ORR of 23% and a disease control rate (DCR) of 44%, highlighting the potential superiority of dual ICI combination therapy compared to monotherapy. A phase 1 study investigated the combination of durvalumab and tremelimumab in advanced-stage BTC (145), revealing an ORR of 10.8% in a cohort of 65 BTC patients. The study reported a mPFS of 1.6 months and a mOS of 10.1 months. However, grade 3 or higher TRAEs were observed in 23.1% of patients. Nevertheless, it is worth mentioning that durvalumab plus tremelimumab combination therapy, when tested in Japanese patients with HCC and BTC, yielded less encouraging response rates and survival outcomes (149). Considering the mixed results, the efficacy of durvalumab and tremelimumab combination therapy in patients with CCA remains unclear, and the safety profile of this approach warrants further refinement. It is important to exercise caution when interpreting these conclusions due to the limited sample sizes in these studies. Therefore, while combination immunotherapies hold promise, particularly in the context of dual immune checkpoint inhibition, continued research with larger and more diverse patient cohorts is essential to establish the true effectiveness and safety of these approaches in the treatment of CCA.

The trial NCT03046862 classified 124 patients with advanced-stage BTC into three treatment cohorts and revealed that ICIs combined with gemcitabine and cisplatin (GS) hold promise as an effective treatment for advanced BTC (150). Another study supported the strategy of GS plus immunotherapy for BTC. The TOPAZ-1 trial represents a significant advancement in the field, being the first phase 3 study designed to investigate PD-L1 inhibitors in combination with chemotherapy for progressive CCA (151). In that trial, 685 patients were randomly assigned to either the durvalumab + GS group or the placebo + GS group. The interim analysis revealed encouraging results, with a mOS of 12.8 months and a mPFS of 7.2 months in the durvalumab + GS group, compared to 11.5 and 5.7 months in the placebo + GS group, respectively. Notably, while TRAEs were observed in 62.7% of patients in the treatment group and 64.9% of patients in the control group, the combination of chemotherapy regimens for advanced-stage BTC with ICIs appeared to enhance survival and other efficacy outcomes without significantly increasing the risk of treatment toxicity. This suggests a promising avenue for improving treatment outcomes in advanced-stage BTC.

In addition, several trials have evaluated the therapeutic efficacy and safety of GS in combination with nivolumab (11,144,152), pembrolizumab plus capecitabine and oxaliplatin (CAPOX) (12) and paclitaxel with durvalumab and tremelimumab (153) in BTC (Table I). Apart from the trial NCT03704480, the results from these studies have been consistently positive. This collective body of evidence suggests a promising treatment approach that combines chemotherapy with ICIs in patients with BTC, offering not only enhanced efficacy, but also an acceptable safety profile.

The combination of molecular targeted therapy and immunotherapy has demonstrated synergistic effects. The capacity of immunotherapy to eliminate immunosuppression can extend the remission effect induced by molecular targeted therapy, thereby enhancing the overall effectiveness of targeted therapy.

The trial NCT02443324 assessed the benefits of pembrolizumab in combination with ramucirumab (a VEGFR-2 monoclonal antibody) in 26 patients with locally advanced, unresectable or metastatic BTC (154). However, the results were quite disappointing, with only one patient achieving a partial response, and a mOS of 6.4 months. The trial NCT03201458 compared the efficacy of atezolizumab monotherapy with atezolizumab in combination with cobimetinib (MEK inhibitor) in patients with BTC who had failed first/second line therapy (155). While the combined treatment group exhibited a slightly better mPFS, both groups had extremely low ORRs. Similarly, a single-arm study investigated the efficacy and tolerability of pembrolizumab + lenvatinib in the treatment of patients with BTC who had failed prior systemic treatments (156). That study reported that 25% of patients responded to treatment and the DCR was 78.1%, with a clinical benefit rate of 40.5% (156). Additional studies exploring the combination of ICIs with targeted therapies are listed in Table I.

Local treatment may be an effective option for patients with BTC who are not eligible for surgery or have advanced lesions. Techniques such as local ablative therapy and radiation therapy can effectively kill tumor cells, boost the production of tumor neoantigens and enhance the immune recognition response. Consequently, there is a theoretical synergy between local therapy and immunotherapy. The trial NCT01853618 revealed that in the evaluable patients who received combination therapy with tremeliumab + microwave ablation, 12.5% achieved a PR, 31.2% achieved SD, with a mPFS of 3.4 months, and a mOS of 6 months (157). Furthermore, studies have suggested that the immune response triggered by antigens released from dead tumor cells following radiation therapy can extend to distant metastatic lesions (140). This phenomenon underscores the potential of combining local therapies with immunotherapy for more comprehensive cancer treatment.

The dense fibrotic matrix found in CCA tissue can impede the effectiveness of antitumor drugs and immune cell infiltration. Therefore, reducing stromal fibrosis in CCA may enhance the response to therapy. The trial NCT03267940 is currently investigating the effectiveness of hyaluronidase when combined with ICIs and chemotherapy for progressive BTC. As aforementioned, increasing the number of NK cells can inhibit tumor growth. A phase 1/2a trial explored the safety and efficacy of pembrolizumab combining allogeneic NK cells in chemotherapy-refractory BTC patients (158). That study reported an overall ORR and DCR of 17.4 and 30.4%, all without severe TRAEs. That study demonstrated that pembrolizumab plus allogeneic NK cells represents a promising therapeutic approach, exhibiting an improved efficacy and a favorable safety profile (158).