The complex role of IL-10 in malignant ascites: a review

Malignant ascites (MA) is defined as the peritoneal fluid collection containing malignant cells in a cancer patient. Substantial amount of ascites can lead to abdominal swelling, nausea, early satiety, edema, pain, and even dyspnea, decreasing quality of life and usually indicating a unfavorable prognosis [1‐3]. MA is commonly associated with primary abdomen or pelvic malignancies such as liver, colon, or ovarian cancers; it can also manifest in metastatic diseases. The precise molecular mechanisms underlying MA remain elusive, but increased vascular permeability and peritoneal lymphatic obstruction are recognized as key pathophysiological processes involving multiple cytokines. Notably, vascular endothelial growth factors (VEGF), a prominent vascular permeability factor, exhibit heightened expression in MA [4‐7]. Additionally, other cytokines like tumor necrosis factor-\(\alpha\), interleukin-6, interleukin-8, and interleukin-10 (IL-10) are elevated in the context of ovarian cancer (OC) patients with MA [8‐10]. The intricate interplay of these cytokines may contribute to the impediment of fluid absorption and drainage, as tumor cells disseminate and proliferate within the peritoneal cavity, potentially obstructing lymphatic vessels [5, 11]. In addition to these primary mechanisms, neovascularization induced by matrix metalloproteinase (MMPs) and the retention of hypertonic fluids secreted by tumor may also play pivotal roles in the formation of MA [12‐16] (Figure S1).

There is no clear guidance on managing MA, but the treatments mainly fall into either relieving symptoms or controlling the primary diseases. Intermittent paracentesis is the most widely used intervention for controlling MA clinically, and its modified technique, cell-free and concentrated ascites reinfusion therapy is applied in Japan, which is thought to avoid protein loss compared with conventional paracentesis [17]. However, paracentesis relieves symptoms temporarily, with the ascites rapidly reaccumulating, increasing the risks of rehospitalization and deteriorating the quality of life (QOL) [16]. Long-term drainage approaches include indwelling catheters, indwelling peritoneal ports, and peritoneovenous shunts (PVSs). Palliation rates of long-term drainage approaches achieve 97%, while their complication rates all exceed paracentesis, and PVSs had the highest complication rate (26–55%) [18]. Since the presence of MA usually indicates a more advanced disease status, targeting the primary diseases can be quite challenging. Intraperitoneal chemotherapy (i.p. chemotherapy) is considered to be effective for controlling MA, and the efficacy and safety of various agents have been investigated. Catumaxomab is a mouse/rat monoclonal antibody targeting epithelial cell-adhesion molecule (EpCAM) and has been approved for MA treatment by European Union in 2009 [19]. A phase II/III trial found that paracentesis plus catumaxomab could increase progress-free survival and improve QOL in patients with MA compared to paracentesis alone [20, 21]. A phase II clinical trial results demonstrated the safety and efficacy of the combination chemotherapy comprising of intravenous paclitaxel (PTX) and intraperitoneal S-1 in pancreatic cancer patients with MA [22]. Moreover, bevacizumab, a VEGF inhibitor, is considered as an option for recurrent OC patients with ascites, but with increased risk of GI perforation [23, 24]. A multicenter double-blind, placebo-controlled phase II trial explored intraperitoneally applied bevacizumab in patients with advanced gastrointestinal cancer and MA, and the results showed that bevacizumab was well tolerated but without efficacy in controlling ascites-related symptoms [25]. Hyperthermic intraperitoneal chemotherapy (HIPEC) is another i.p. chemotherapy technique which is thought to increase chemotherapy penetration and enhance cancer cells sensitivity to chemotherapy throughout the peritoneal cavity by heated solution delivery. It is recommended by the NCCN guidelines to use in FIGO stage III OC patients who undertook neoadjuvant chemotherapy (NACT) and interval debulking surgery (IDS) [23, 26]. Moreover, cytoreductive surgery with HIPEC is recommended for patients with gastric cancer and limited peritoneal metastases [27]. However, the application of HIPEC in colon cancer patients with peritoneal metastases remains controversial as several trials found the technique increased morbidity and mortality [28]. Both palliation therapies and methods targeting primary diseases have shown improvements in QOL, but did not demonstrate obvious advantages in survival of patients with MA. As a result, therapies with less complications and higher efficacy are warranted.

IL-10, a pleiotropic cytokine, plays a crucial role in both inflammation and immunity within tumor microenvironment. It is primarily produced by innate immune cells, including monocytes, dendritic cells (DCs), macrophages, and natural killer cells (NK cells), and adaptive immune cells such as CD4+ T cells, CD8+ T cells, Th17 cells, and B cells [29, 30]. Studies also indicate that tumor cells can secrete IL-10 [31, 32]. Cancer patients, in comparison with healthy people and those with benign tumors, exhibit elevated levels of IL-10 in both serum and ascites, with this correlating with advanced clinical staging and poor outcomes [33‐36]. Notably, IL-10 in OC contributes to ascites-mediated apoptosis resistance and transcriptomic data identifying IL-10 in ascites as a robust indicator of poor outcomes for OC patients [37, 38]. Nevertheless, the precise role of IL-10 in oncogenesis remains controversial, with reported anti- and pro-tumor effects observed in various malignancies such as thyroid cancer, non-small cell lung cancer, breast cancer, and colorectal cancer [39‐47]. IL-10 predominantly exerts its inhibitory function on macrophages, while its impact on T cells is complex, involving both the inhibition of T cell responses and the stimulation of CD8+ T cell proliferation [48‐50]. Despite extensive research on MA, a comprehensive review focused on IL-10 in MA is notably absent. Consequently, this review aims to summarize the role of IL-10, spanning its levels to therapeutic targeting in MA, with the intent of providing evidence for the potential application of IL-10 in MA from bench to bedside.

The IL-10 signaling pathway is presumed to play a role in the development of immunosuppressive malignant ascites within the tumor microenvironment. Nevertheless, there is a scarcity of studies focusing on the downstream reactions of IL-10. A comprehensive understanding of IL-10 signal transduction in the development of MA can contribute to the integration of immunotherapeutic strategies. The receptor for IL-10 forms a functional complex consisting of two ligand-binding subunits, IL-10 R1 and IL-10 R2. Ligand engagement and binding are primarily facilitated by IL-10 R1, which exhibits high affinity, while IL-10R2 is responsible for transducing subsequent signals. In addition to being expressed on immune cells, Rabinovich et al. observed elevated IL-10 receptor expression on both OC tumor tissue and cells [31]. At the cell membrane surface, IL-10 binding to IL-10R1 and IL-10R2 activates Janus Kinase 1 (JAK1) and Tyrosine Kinase 2 (TYK2), phosphorylating the receptor complexes Y446 and Y496 located at the cytoplasmic tail. Subsequently, Signal Transducer and Activator of Transcription 3 (STAT3), a key transcription factor downstream of the IL-10 signaling pathway, is assembled and phosphorylated. It then translocates into the nucleus, binding to the promoter elements of relevant target genes and ultimately participating in the expression of pro-tumor and anti-apoptotic genes such as BCL2, TWIST, thereby exerting tumor-promoting effects [97‐99]. Notably, IL-10 can also phosphorylate STAT1 and STAT5, perpetuating downstream signaling [100, 101]. Apart from the JAK1-STAT3 cascade, IL-10 is hypothesized to boost cancer stemness through the JAK1-STAT1-NF-\(\kappa\) B pathway, thereby promoting tumor progression and metastasis [102]. However, contradictory findings exist, as some studies suggest that IL-10-mediated STAT1 activation induces pro-apoptotic and anti-proliferative effects in macrophages pre-treated with IFN-γ or IFN-α, contradicting IL-10-mediated STAT3 activation. The mechanism and switches of IL-10-mediated STAT pathway in MA remain unclear, with ongoing investigations into the involved mechanisms [103]. Additionally, Zhou et al. demonstrated that IL-10 could activate the Phosphatidylinositol 3-Kinase (PI3K) -AKT pathway, thereby regulating cell apoptosis [104]. Furthermore, Reitamo et al. discovered that under the influence of IL-10, the expression of MMPs, associated with neovascularization in MA, increased, suggesting a potential mechanisms by which IL-10 contributes to the formation of MA [105] (Figure S2).

To harness IL-10 signaling, both recombinant IL-10 protein and the blockade of IL-10 and/or IL-10R are being explored for the MA treatment [106, 107]. The potential anti-tumor effects of IL-10 involve upregulating CD8+ T cells cytotoxicity, thereby reducing macrophage infiltration, increasing NK cell infiltration, resisting angiogenesis, and stabilizing intrinsic apoptosis [108]. Guo et al. demonstrated that a half-life-extended IL-10/Fc fusion protein could directly invigorate terminally exhausted CD8+ tumor-infiltrating lymphocytes (TILs) via oxidative phosphorylation and the process is in absence of the progenitor cells, making it a promising complementary therapy to immune checkpoint blockade in the treatment of patients with poor or none tumor infiltration of progenitor-exhausted CD8+ TILs [109]. Consequently, pegilodecakin, a pegylated form of recombinant IL-10 sustaining high concentration, has entered clinical trials. In a phase 1/1b trial (NCT02009449), one ovarian cancer patient and 22 pancreatic cancer patients were recruited to evaluate the safety and efficacy of pegilodecakin (pegylated IL-10) as monotherapy, and the results showed that patients did not have obvious clinical responses [110]. The result of SEQUOIA (NCT02923921), a phase 3 study evaluating pegilodecakin combined with folinic acid, fluorouracil, and oxaliplatin (FOLFOX) in patients with metastasis pancreatic ductal adenocarcinoma (PDAC), demonstrated that the addition of pegilodecakin did not increase efficacy of FOLFOX [111]. Recombinant IL-10 protein exerted promising anti-tumor effects on solid tumors such as renal cell carcinoma and melanoma, but ideal therapeutic effects did not be observed on patients with MA-associated cancer types such as ovarian cancer and pancreatic cancer, and the agent’s direct efficacy on MA remains unknown, impeding its further clinical application [107]. The complex tumor microenvironment and small sample size may account for these limited effects. Future research should specifically investigate recombinant IL-10 protein efficacy on MA.

Blocking IL-10 signaling also presents an immunotherapy strategy due to IL-10’s tumor immunosuppressive effect. Direct blockade via anti-IL-10 antibodies or indirectly use of anti-IL-10R antibodies can interfere with IL-10 function. IL-10/IL-10R neutralization has demonstrated more anti-tumor effects when combined with other immunotherapeutic strategies [112, 113]. Lamichhane et al. found that the combination of PD-1 blocking and IL-10 (R) neutralization activated more T and B cells, decreasing MDSCs in the ascites of ID8 tumor-bearing mice, leading to tumor burden alleviation and survival prolongation [114]. Adams et al. engineered a tumor vaccine by isolating ascites-derived monocytes from ID8 tumor-bearing mice and stimulating with Toll-like receptor (TLR) 4 lipopolysaccharide, TLR 9 CpG-oligonucleotides, and IL-10R antibody. The vaccine suppressed tumor growth and ascites generation in mice, and the proposed mechanism was macrophage activation and increased T cell response [115]. Additionally, IL-10 antibody enhanced the efficacy of OK-432’s locoregional immunotherapy for mouse breast cancer ascites [94]. Hu et al. showed that arsenic trioxide exerted a potential therapeutic response by decreasing IL-10 and TGF-β levels in TILs from ascites of gastric cancer patients, with the proportion of CD4+CD25+CD127-Tregs decreasing and the CD8+ T cells frequency and IFN-γ level increasing [116].

Furthermore, Martincuks et al. found that PARP inhibitors induced their therapy resistance against OC by activating STAT3 pathway, which closely related to the downstream pathway of IL-10. Thus, neutralization of IL-10/IL-10R in combination with PARP inhibitors could reduce therapeutic resistance [117]. Chimeric antigen receptor-T cell (CAR-T) therapy is defined as administrating reengineered chimeric T cells that can recognize tumor cells from cancer patients, enhancing T cell proliferation and reducing apoptosis. CAR-T therapy is different with conventional therapies as it can directly bypass the processing and presentation of tumor antigens by DCs, so it can avoid part of the immunosuppressive effect in the tumor microenvironment. In vitro experiments found that mesothelin CAR-T cells reacted with SKOV-3 cells, promoting the secretion of granzyme B and IFN-γ, which have important roles in producing cytotoxicity and killing OC cells. Blocking IL-10 signaling pathway can further increase granzyme B and IFN-γ levels. Therefore, blocking IL-10 signaling pathway has the potential to reverse the immunosuppressive effect in MA and enable stronger cytotoxicity produced by mesothelin CAR-T therapy [118, 119].

In addition to conventional therapies, emerging medical materials show promise in cancer treatments. Methotrexate-loaded gold nanoparticles (MTX-AuNP) inhibited ascites formation in mice with LL2 ascites tumors [120]. Silver nanoparticles (AgNPs) decreased ascites fluid volume in a lymphoma ascites mice model [121]. α-Tocopheryl succinate nanoparticles (TS-NP) prevented peritoneal dissemination, reducing tumor nodules, tumor weights, and ascites volume via VEGF-A and IL-10 levels decreasing and M2-like TAMs polarization decreasing [122]. Except for nanoparticles, peptide hydrogels stand for another promising drug-loading candidate. Dai et al. created a melittin hydrogel loaded with KN93 (CAMKII inhibitor), resulting in decreased ascites volume and reprogramming the ascites microenvironment in mice with hepatoma and MA [123]. Shamskhou et al. established a hyaluronan and heparin-based system delivery recombinant IL-10 for the treatment of lung fibrosis in vivo [124]. The above results indicate that it is realistic and pragmatic for novel materials like hydrogel system to load with IL-10 protein or IL-10/IL-10R antibody, enhancing their efficacy of immunoregulatory function in MA (Fig. 2).

IL-10 is a pleiotropic cytokine exerting both immunosuppression and immunostimulatory effects within the cancer microenvironment. The levels of IL-10 in MA exhibit considerable variability across different cancer types and even among patients with the same cancer type. Notably, IL-10 levels surpass those found in non-malignant ascites and exceed the serum IL-10 levels within the same patient. Several studies have proposed its potential as a marker for predicting patients' response following neoadjuvant chemotherapy. The cellular origins of IL-10 in MA are diverse, encompassing neutrophils, MDSCs, DCs, TAMs, T cells, B cells, and even cancer cells. Functionally, IL-10, secreted by MA cells, elicits multifaceted effects such as phenotypic alterations and the regulation of cytokines secretion. It is hypothesized that IL-10 could phosphorylate and activate STAT1, STAT3, STAT5, and PI3K pathways, exerting apoptosis regulation and tumor progression functions on immune cells. Systemic administration of IL-10 holds promise as an immunotherapy, as it can enhance CD8+ T cell cytotoxicity and invigorate terminally exhausted CD8+ TILs. Despite the therapeutic efficacy demonstrated by PEGylated human IL-10 (pegilodecakin) in certain solid tumors like renal cell carcinoma and melanoma, its effects on patients with MA remain unclear. Strategies including the blockade of IL-10 signaling, driven by its immunosuppressive effects, have shown therapeutic promise in both in vitro and in vivo experiments, particularly when combined with other immunotherapies. Effectively harnessing IL-10 pleiotropy is challenging yet essential in the treatment of MA. Further research is warranted in the following directions: (1) Explore cytokines interacting with IL-10 in MA to elucidate the cytokine network in microenvironment of MA. (2) Investigate the IL-10 signaling pathway, with a focus on the STAT1/STAT3 switches in MA. (3) Evaluate the therapeutic effects of recombinant IL-10 protein on patients with MA. (4) Develop appropriate IL-10 blocking agents and delivery systems, assessing their efficacy and safety in preclinical trials.