Pancreatic ductal adenocarcinoma (PDAC) is a significant global health burden, being the third leading cause of cancer-related deaths in the United States with continued increase in incidence and mortality. On a global scale, data from 2022 also reflect the pervasive impact of PDAC – being the 6^th leading cause of cancer death, emphasizing the need for enhanced diagnostic and therapeutic strategies. According to recent statistics on PDAC, the five-year overall survival rate for PDAC in the USA is currently only 13%, reflecting the aggressive nature and late-stage diagnosis common with this cancer. For 2024, it is projected that there will be approximately 66,440 new cases of PDAC in the USA, with an estimated 51,750 deaths attributed to the disease. These figures underscore the critical need for advancements in early detection and treatment strategies (1). The prognosis for PDAC is particularly poor, especially in cases where the disease has metastasized. At the time of initial diagnosis, approximately 50% of PDAC patients present with synchronous distant metastases. A recent study analyzed data from the SEER database and confirmed that, at the initial diagnosis, many PDAC patients present with metastases, primarily in the liver which significantly impacts their prognosis. These patients have a drastically reduced overall survival rate, often less than 6 months, compared to those without metastases, whose median survival can extend beyond a year.

Galectin-9 (Gal-9) is a 36 kDa protein with its two carbohydrate recognition domains (CRDs), connected by an unstructured linker peptide (2, 3). These CRDs form glycan-binding pockets, which confer different affinities and biological activities depending on their glycan partners (3, 4). The structural specificity of these CRDs is crucial, as variations significantly influence receptor recognition, signaling pathways, and cell death mechanisms in immune responses (4, 5). In normal physiology, Gal-9 is involved in various cellular functions, including cell aggregation, adhesion, and apoptosis (6). Gal-9 is expressed in various cell types, including T cells, fibroblasts, and activated endothelial cells, and is notably present in tumor tissues, where it can contribute to tumor progression and immune evasion (3).

This review aims to provide a comprehensive analysis of the TIME in PDAC and the roles of Gal-9 within this context. PDAC is known for its poor prognosis and resistance to conventional therapies, largely due to its complex TIME, which includes various immune cells, cytokines, cancer-associated fibroblasts (CAFs), and the extracellular matrix. Understanding the interactions and signaling pathways within this microenvironment is crucial for identifying new therapeutic targets. This review will detail the structure and function of Gal-9, highlighting its dual role in immune modulation and tumor pathogenesis, and its specific contributions to PDAC and other cancers. Additionally, the review will explore the potential of Gal-9 as a therapeutic target in immunotherapy, addressing current challenges and the promise of combination strategies to enhance treatment efficacy and overcome resistance. By integrating insights into PDAC biology, Gal-9 functions, and immunotherapeutic approaches, this review seeks to offer a detailed understanding of potential advancements in PDAC treatment.

Gal-9 is a negative regulator of anti-tumor immunity, suppressing the immune response by targeting T cells and inducing Tregs, which are known for their immunosuppressive capabilities in the TME (15). The suppression of effector T cell activity by Gal-9 is crucial in the context of PDAC, where the TME is notoriously immunosuppressive.

One of the critical mechanisms by which Gal-9 facilitates immune evasion in tumors is through its interaction with the immune checkpoint receptor Tim-3 on T cells. The Gal-9/Tim-3 pathway inhibits T cell function and induces apoptosis of effector T cells, thereby reducing the anti-tumor immune response and contributing to the creation of an immunosuppressive tumor microenvironment (9). In addition to Tim-3, Gal-9 binds to PD-1 which promotes the survival of exhausted T cell populations. (16). Gal-9 can also specifically interact with V-domain Ig-containing suppressor of T cell activation (VISTA), adding another layer to Gal-9’s role in immune suppression (9, 17). In the context of acute myeloid leukemia (AML), VISTA binds to Gal-9 secreted by AML cells as a ligand. Importantly, soluble VISTA released by AML cells enhances the effect of Gal-9, likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells, leading to the activation of granzyme B inside cytotoxic T cells and resulting in apoptosis (17). The immune-evasive effects of Gal-9 are also associated with other immunoregulatory molecules such as the amino acid L-kynurenine (LKU), which also suppresses anti-cancer T cell functions (18). This cooperation is also highly relevant to PDAC since IDO1, the enzyme which regulates LKU generation, is expressed by pancreatic cancer cells (19).

Further research has demonstrated that Gal-9 and VISTA co-expression on T cells is associated with a terminally exhausted phenotype, particularly noted in HIV-1 patients and virus associated tumors (20, 21). This phenotype is characterized by impaired effector functions and the upregulation of exhaustion markers such as EOMES, Blimp-1, and Glut-1. Gal-9 and VISTA co-expression leads to a profound reduction in T cell activity, significantly attenuating the immune response against tumors. This exhausted T cell phenotype includes effector T cells (CD45RA+, CD45RO-/lo, CD62L-, CD27lo) with high T-bet expression, indicating a dysfunctional state that is likely mirrored in tumor immunology. (9, 21, 22).

Interaction of Gal-9 with CD44 enhances the stability and function of adaptive regulatory T cells, reinforcing immunosuppression within the tumor microenvironment through TGF- β and Smad3 activation (23).

These findings suggest that the interaction between Gal-9 and VISTA might also contribute to immune evasion in solid tumors, reinforcing the immunosuppressive environment and promoting tumor progression (9, 21).

Gal-9 expression in PDAC is elevated not only in tumor cells but also in myeloid cells, including TAMs and MDSCs, particularly in areas adjacent to tumors (8, 11, 24). This upregulation correlates with poor prognosis, as Gal-9 contributes to tumor growth, immune evasion, and the modulation of myeloid cell behavior within the tumor microenvironment (25).

PDAC presents numerous challenges in its treatment, largely due to inherent barriers and resistance mechanisms that reduce the efficacy of both chemotherapy and immunotherapy (30, 31). Particularly, the dense stromal environment of PDAC acts as a physical barrier. This fibrotic environment can inhibit angiogenesis, resulting in a sparse and dysfunctional vascular network (32). Consequently, the delivery of chemotherapeutic agents to the tumor cells is limited. Additionally, the tumor immune microenvironment (TIME) supports various immunosuppressive mechanisms that protect the cancer cells from immune attack, leading to resistance against immunotherapies (33, 34).

One of the primary approaches in immunotherapy is the use of immune checkpoint inhibitors (ICI), such as PD-(L)1 and CTLA-4 inhibitors, that target effector T cells. These inhibitors have revolutionized the treatment of various cancers by blocking pathways that inhibit T cell activation, thus enhancing the immune system’s ability to attack tumor cells. In cancers such as melanoma and lung cancer, these treatments have shown substantial success. However, their effectiveness in PDAC has been limited as the immunosuppressive TIME hampers the efficacy of these checkpoint inhibitors (16, 35, 36). A detailed comparison of Gal-9 with other immune checkpoints in PDAC, including mechanisms, roles, and clinical implications, is summarized in Table 1 . Consequently, these traditional ICIs have shown limited success in clinical trials for PDAC patients, underscoring the need for alternative therapeutic targets (48).

Gal-9 has already shown significant therapeutic potential in various cancers. Enhanced expression of Gal-9 has been associated with improved overall survival in various solid cancers, including colon and hepatocellular cancer, as well as improved disease-free survival in gastric and non-small-cell lung cancer (49). Gal-9 mediates its tumor-suppressive effects through multiple mechanisms, including the induction of apoptosis in tumor cells, modulation of immune responses, and inhibition of metastasis. Particularly, increased levels of caspase-cleaved keratin 18 levels, as a measure of apoptosis, have been observed in Gal-9 treated colon cancer cells. Gal-9 dependent increase of caspase activity has also been observed in HCC cells in vitro and in vivo (50). Anti-metastatic potential of Gal-9 has been observed in triple negative breast cancer (TNBC), HCC (51) and melanoma (52). Additionally, in triple-negative breast (TNBC) cancer Gal-9 expression correlates with increased immune cell infiltration and positive PD-L1 expression on tumor cells. This dual role of promoting tumor cell death while modulating the immune environment makes Gal-9 a compelling target for therapeutic intervention. Furthermore, combining anti-Gal-9 therapy with anthracycline-based chemotherapy, such as doxorubicin, significantly enhances antitumor activity in breast cancer. This combination therapy leverages the complementary mechanisms of action of both treatments (53).

In the context of PDAC, preclinical studies have demonstrated that targeting Gal-9 can disrupt its interactions with Tim-3 and other immune modulators, thereby enhancing anti-tumor immune responses. This approach aims to mitigate the immunosuppressive effects of Gal-9, allowing the immune system to effectively target and eliminate cancer cells (8, 26). Moreover, the blockade of Gal-9 can potentially enhance the efficacy of other immunotherapeutic agents, such as PD-(L)1 and CTLA-4 inhibitors, by relieving the suppression of immune cell activity within the tumor microenvironment. Blockade of Gal-9 can be achieved with neutralizing antibodies or lactose (54–56).

Given the limitations of current immunotherapy in PDAC, targeting components of the TIME with nanomaterials can significantly enhance immunotherapeutic efficacy and optimize drug delivery outcomes. Therefore, combining anti-Gal-9 therapies with nanoparticle-based delivery systems may further enhance this approach in PDAC. Nanoparticles can precisely target tumor cells and modulate the immune response, providing a dual attack on the cancer. This method improves drug delivery, reduces off-target effects, and increases the overall efficacy of the treatment (34, 57). Early studies suggest that these combination therapies may improve patient outcomes by reducing tumor growth and metastasis, representing a novel and promising approach to overcoming the inherent barriers in PDAC treatment and enhancing the overall effectiveness of existing therapies (8, 16). Additionally, in a mouse model, T7 peptide-decorated exosome-based nanoparticles delivering Gal-9 siRNA significantly inhibit glioblastoma and enhance antitumor immunity.

Overall, the therapeutic potential of Gal-9 in cancer treatment is substantial, particularly in PDAC where traditional therapies have limited effectiveness. The development of anti-Gal-9 antibodies and other Gal-9-targeted therapies holds promise for improving outcomes in patients with PDAC and other malignancies by harnessing the immune system’s power to combat cancer. Continued research is necessary to fully understand the mechanisms by which Gal-9 modulates tumor and immune cell interactions and to translate these findings into effective clinical treatments. Despite the significant therapeutic potential of Gal-9, a search in ClinicalTrials.gov revealed only two current clinical trials that are interventional (see Table 2 ), while the other registered trials involving Gal-9 are observational (see Table 3 ). This underscores the need for expanded research and a greater number of clinical trials to fully explore and harness Gal-9’s therapeutic capabilities. Increasing the focus on Gal-9 in clinical settings is crucial for unlocking its potential in cancer treatment and other diseases.

Combining various immunotherapeutic agents can help overcome the limitations of single-agent therapies and address mechanisms of resistance that tumors often develop. Interestingly, a phase II study in PDAC using combination of radiation therapy and anti-PD-1 or anti-CTLA-4 antibodies, indicated increased protein levels of circulating FasL, Gal-1 and Gal-9. Conversely, Gal-9 and PD-L1 combination treatment inhibits PDAC tumor progression in a murine PDAC model (37).

A promising strategy might thus involve the simultaneous blockade of Gal-9, Tim-3 or other ICIs including other galectins, to prevent compensatory mechanisms and resistance. Tumors often exploit multiple pathways to evade immune detection and destruction, so targeting several of these pathways at once can be more effective. For example, blocking Gal-9 and Tim-3 can disrupt the immunosuppressive signals that inhibit T-cell function, while ICIs like anti-PD-1 can further enhance T-cell activity against the tumor. However, ICI combination therapy can increase the risk of immune-related adverse events. In a meta-analysis of 18 studies spanning 10 cancer types, combination ICI therapy was linked to a modestly increased risk of all-grade adverse events and a significantly higher risk of grade 3 or more severe adverse events compared to ICI monotherapy (58).

Another innovative approach is the use of anti-Gal-9 in combination with reprogrammed macrophages or chimeric antigen receptor macrophages (CAR-M). These therapies aim to transform TAMs, which typically promote tumor growth and suppress immune responses, into a phenotype that supports anti-tumor immunity. Reprogrammed macrophages or CAR-M can enhance the phagocytosis of cancer cells and stimulate other immune cells to attack the tumor (59–62). When combined with anti-Gal-9, these therapies can further disrupt the immunosuppressive environment created by the tumor and enhance the overall immune response.

These combination strategies highlight the potential of leveraging multiple facets of the immune system to combat cancer more effectively. By targeting different pathways and using innovative delivery methods, these approaches aim to improve patient outcomes in PDAC and other challenging cancers. Continued research and clinical trials are necessary to optimize these combinations and determine the most effective protocols for different patient populations.