Friend and foe: the regulation network of ascites components in ovarian cancer progression

Abstract

The tumor microenvironment (TME) and its complex role in cancer progression have been hotspots of cancer research in recent years. Ascites, which occurs frequently in patients with ovarian cancer especially in advanced stages, represents a unique TME. Malignant ascites contains abundant cellular and acellular components that play important roles in tumorigenesis, growth, metastasis, and chemoresistance of ovarian cancer through complex molecular mechanisms and signaling pathways. As a valuable liquid biopsy sample, ascites fluid is also of great significance for the prognostic analysis of ovarian cancer. The components of ovarian cancer ascites are generally considered to comprise tumor-promoting factors; however, in recent years studies have found that ascites also contains tumor-suppressing factors, raising new perspectives on interactions between ascites and tumors. Malignant ascites directly constitutes the ovarian cancer microenvironment, therefore, the study of its components will aid in the development of new therapeutic strategies. This article reviews the current research on tumor-promoting and tumor-suppressing factors and molecular mechanisms of their actions in ovarian cancer-derived ascites and therapeutic strategies targeting ascites, which may provide references for the development of novel therapeutic targets for ovarian cancer in the future.

Introduction

As the deadliest gynecological malignancy, ovarian cancer was estimated to cause 5% of the total female deaths from cancer in the United States in 2021, ranking fifth on the list of cancers by mortality rate (Siegel et al. 2021). The high mortality of ovarian cancer lies predominantly in the difficulty of early detection and diagnosis as well as the extensive intraperitoneal metastasis and chemoresistance. Tumors typically have progressed to an advanced stage in approximately 75% of ovarian cancer patients upon diagnosis due to absence of symptoms in early stages and the lack of effective screening methods. The five-year relative survival rate of patients with advanced ovarian cancer is only 29%, while that of patients with early-stage ovarian cancer is 92% (Lheureux et al. 2019). Therefore, it is of great significance to improve the early diagnosis efficiency of ovarian cancer and explore new targets for inhibiting ovarian cancer metastasis and chemoresistance to ultimately improve the survival rate of patients with ovarian cancer.

The tumor microenvironment (TME) has continued to attract researchers' attention over the past few decades for its complex role in cancer progression. Modifying surrounding stromal and immune environment is one of the means by which malignant tumors survive and thrive. As a result, TME is filled with cytokines, chemokines, suppressive immune cells and other factors that contribute to cancer progression and metastasis(Hinshaw and Shevde 2019; Soysal et al. 2015; Fabris et al. 2021; Ren et al. 2018). However, certain tumor suppressive cellular and acellular factors present in the TME have been discovered with further research, revealing a complicated regulatory network of TME components.

Ascites is an important TME characterized by the build-up of excess fluid in the abdomen primarily due to increased microvascular permeability and blocked lymphatic vessels. Angiogenesis occurs as ovarian cancer progresses in severity, and higher levels of glycoprotein, vascular endothelial growth factor (VEGF), and matrix metalloproteinase (MMP) increase microvascular permeability (Bekes et al. 2016; Herr et al. 2012), resulting in increased fluid exudation. In addition, lymphatic vessels are an important metastasis pathway for tumors; thus, after infiltrating lymphatic vessels, metastatic tumors block lymphatic drainage, eventually leading to fluid accumulation and ascites formation (Adam and Adam 2004).

Ascites occurs in more than one third of ovarian cancer patients (Kipps et al. 2013) and is an excellent material for TME research due to its large volume, wide distribution and accessibility. Studies have found that the composition of ovarian cancer ascites is highly complex and includes cellular components (e.g., cancer, immune, stromal, and mesothelial cells) and acellular components (e.g., cytokines, chemokines, metabolites, and exosomes), both of which play vital roles in tumorigenesis and cancer progression. It is traditionally considered that malignant ascites provides tumors with an immunosuppressive microenvironment that helps cancer cells evade immune surveillance and promotes tumor growth, intraperitoneal dissemination, and metastasis, and it is usually an indication of advanced-stage ovarian cancer and poor prognosis (Giuntoli et al. 2009; Worzfeld et al. 2017; Yeku et al. 2017; Brencicova et al. 2017; Saini et al. 2017; Kim et al. 2016).

In recent years, some studies have highlighted that ovarian cancer-derived ascites also has a bright side (Cohen and Petignat 2014), as the fluid contains tumor-suppressive components in addition to the tumor-promoting components. Understanding the effects and mechanisms of both the tumor-promoting and suppressive factors in ascites will aid in the understanding of tumorigenesis, particularly with respect to ovarian cancer, and the identification of potential therapeutic targets. Ford et al. (2020) and other researchers have summarized impacts of ovarian cancer ascites on cancer prognosis, metastasis and chemoresistance; in this review, we highlight the roles and underlying molecular mechanisms of cellular and acellular components of ascites in the progression of ovarian cancer as well as research progress on therapeutic strategies targeting ascites for the treatment of ovarian cancer. We also pay attention to tumor-suppressing factors in ovarian cancer ascites and the possibility of amplifying their antitumor capacities as a therapeutic strategy.

Tumor-promoting and tumor-suppressing factors in the TME

Immunosuppression, one of the most prominent characteristics of the TME, is caused by a combination of cellular and acellular factors and plays a pivotal role in cancer progression. However, in certain conditions, these factors also exert antitumor effects. For example, a cancer-associated fibroblasts (CAFs) subtype categorized in a scRNA analysis of melanoma, head and neck squamous cell carcinoma and lung cancer samples was reported to highly express proinflammatory and immunosuppressive factors IL33, CXCL12, and CXCL14 (Galbo et al. 2021); Other CAF subtypes were found to positively corelate with cytotoxic T‐cell dysfunction (Wu et al. 2020) and PD-1 and CTLA4 protein levels in Tregs (Kieffer et al. 2020) in breast cancer samples, indicating the potential immunosuppressive role of CAFs in the TME of these cancers. However, the situation appears to be opposite in pancreatic ductal adenocarcinoma (PDAC). An in vivo assay showed that depletion of myofibroblast, a subtype of CAFs, led to increased Tregs and more invasive tumors (Ozdemir et al. 2014), demonstrating that certain CAFs are able to enhance anti-tumor immunity in PDAC. Furthermore, the presence of CD146⁺ CAFs in the TME sustains sensitivity of cancer cells to tamoxifen in ER⁺ breast cancer (Brechbuhl et al. 2017). The underlying mechanism of CAFs’ opposing functions in different TMEs and whether this can be used as a therapeutic target need further research.

Similar to CAFs, some soluble factors in the TME conduct opposing effects on cancer progression in different cancer types. It has been reported that Sonic hedgehog (Shh), a tumor-derived soluble hedgehog ligand in the TME, drives TAM M2 polarization, thereby enabling TAMs to perform immunosuppressive roles in HCC (Petty et al. 2019). Anti-tumor function of Shh was found in PDAC, where Shh was demonstrated to promote generation of an angiogenesis-restraining stroma (Rhim et al. 2014). The circadian regulator CLOCK and its heterodimeric partner BMAL1 serve as tumor suppressors in prostate, lung, and glioma cancers (Shafi and Knudsen 2019). However, studies have also found that CLOCK and BMAL1 heterodimer transcriptionally upregulate chemokine OLFML3, which recruits immune-suppressive microglia into the TME of glioblastoma (GBM) (Chen et al. 2020). Tumor-promoting functions of CLOCK and BMAL1 have been discovered in CRC (Karantanos et al. 2014; Wang et al. 2015) and acute myeloid leukemia (AML) (Puram et al. 2016) as well. MicroRNA miR-149 was found contributing to 5-FU resistance in gastric cancer (Wang et al. 2020a). On the contrary, miR-149 was proved to inhibit TNBC metastasis and possess anti-tumor capacity by regulating cross-talk between cancer cells and macrophages (Sanchez-Gonzalez et al. 2020).

Although the study of tumor suppressing components in the TME could improve the research of tumor microenvironment and provides important information for targeted therapy, only few studies have reported the tumor suppressive effect of TME components, except for the ascites microenvironment. Therefore, this study mainly reviewed the tumor-promoting and tumor-suppressing factors in ovarian cancer-derived ascites, with the aim to provide references for the development of therapeutic targets for ovarian cancer in the future.

Tumor-promoting and tumor-suppressing factors in ovarian cancer-derived ascites

Ovarian cancer-derived ascites is a unique TME of ovarian cancer. Its accessibility has given rise to many in-depth studies on acellular and cellular components of the TME, some of which had become therapeutic targets of cancer. The acellular components of ovarian cancer-derived ascites include cytokines, chemokines, metabolites, and exosomes, which collectively affect tumor progression and regulate the body's antitumor immunity.

Acellular components of ovarian cancer-derived ascites

Tumor-promoting acellular components

The tumor microenvironment comprises myriad different immune cells that engage in various immune responses; therefore, ascites contains abundant cytokines and chemokines. Most cytokines are present in ascites as tumor-promoting factors, among which interleukins constitute one of the most important types. In ascites of ovarian cancer patients, Interleukin-6 (IL-6) is an important tumor-promoting factor that can promote tumor progression through various mechanisms. Studies have found that IL-6 mainly regulates tumor progression by targeting immune cells. For example, IL-6 can bind to leukemia inhibitory factor (LIF), enabling monocytes to consume autocrine or paracrine macrophage colony-stimulating factors (M-CSFs), thereby inducing monocytes to differentiate into tumor-associated macrophage (TAMs)-like cells that promote angiogenesis, tumor growth, and metastasis by upregulating VEGF and MMP9 and downregulating PTX3 expression (Duluc et al. 2007). IL-6 can coordinatively activate the IL-10/STAT3 axis, subsequently upregulating arginase 1 (ARG1) and inducible nitric oxide synthase (iNOS) expression in myeloid-derived suppressor cells (MDSCs), which enables MDSCs to perform immunosuppressive functions (Wu et al. 2017). IL-6 promotes the expression of tumor necrosis factor receptor 2 (TNFR2) in regulatory T cells (Tregs), thereby activating and promoting the proliferation of Tregs, which express functional immunosuppressive molecules such as PD-L1, CTLA-4, and GARP that enhance their immunosuppressive functions. Increased expression of TNFR2 also suppresses the production of IFNγ by effector T cells and inhibits antitumor immune responses mediated by IFNγ (Kampan et al. 2017). In addition, IL-6 in ascites can bind to IL-6 receptors on tumor cell membranes and activate the JAK2-STAT3 signaling pathway, thereby increasing the invasiveness of ovarian cancer cells (Kim et al. 2016). Clinical studies have shown that higher level of IL-6 in ascites is an independent predictor of shorter overall survival in ovarian cancer patients (Rodrigues et al. 2020) and has the potential to be a highly reliable biomarker for detecting the malignancies (Dalal et al. 2018).

IL-8 is another important tumor-promoting cytokine in the ascites microenvironment of ovarian cancer patients. IL-8 promotes ovarian cancer cell migration (Mikula-Pietrasik et al. 2016), enhances the proliferation of ovarian cancer cells in ascites spheroids, and promotes ovarian cancer metastasis and angiogenesis (Uddin et al. 2020). IL-8 is also involved in enabling drug resistance and the recurrence of ovarian cancer. IL-8 paracrined by cancer-associated fibroblasts (CAFs) not only activates normal ovarian fibroblasts, facilitating their proliferation and angiogenesis, but also induces stemness in cancer cells collectively with autocrine IL-8 by activating Notch3, thereby promoting drug resistance and the recurrence of ovarian cancer (Ji et al. 2021).

The upregulation of IL-10 in the ascites microenvironment is associated with increased production of ascites, poor prognosis, and ovarian cancer recurrence. The combination of IL-10 and IL-4 in ascites has the potential for accurately predicting the stage of ovarian tumors (Gening et al. 2021). IL-10 affects ovarian cancer progression mainly by regulating antitumor immunity. Upon STAT3 activation, IL-10 can induce the expression of PD-1 on the surface of dendritic cells (DCs), inhibiting the initial function of dendritic cells in antitumor immunity. IL-10 also maintains an immunosuppressive phenotype of tumor-infiltrating dendritic cells (TIDCs) by upregulating PD-L1 expression on the surface of TIDCs (Lamichhane et al. 2017). IL-10⁺ B cells in ascites can inhibit the production of IFNγ expressed by CD8⁺ T cells through upregulation of IL-10, thereby inhibiting antitumor immunity (Wei et al. 2016). IL-10 secreted by TAMs promotes both T cell differentiation into Tregs and tumor progression by activating Foxp3 (Zhu et al. 2016). Other studies have shown that IL-10 directly and independently alters the phenotype and function of MDSCs and T cells, and tumor-infiltrating MDSCs require IL-10 to achieve their immunosuppressive functions (Hart et al. 2011).

Other interleukins associated with ovarian cancer progression in ascites include IL-1β and IL-12. Studies have found that IL-12 levels in ovarian cancer ascites were negatively correlated with disease-free survival and overall survival (Zeimet et al. 1998), while the levels of IL-1β in ascites were significantly positively correlated with the proportion of MDSCs in ascites, suggesting that IL-1β might promote ovarian cancer progression through the immunosuppressive functions of MDSCs (Wang et al. 2017).

Vascular endothelial growth factor (VEGF) plays an important role in promoting cancer progression. The levels of VEGF were significantly higher in ascites of advanced ovarian cancer patients compared to benign ascites or ascites of other cancers; higher levels of VEGF are typically indicative of poor prognosis and chemoresistance (Bamias et al. 2008; Zhan et al. 2016). VEGF promotes angiogenesis and enhances vascular permeability, leading to intraperitoneal tumor growth, dissemination, peritoneal carcinogenesis, and ascites formation. VEGF is an important regulator of physiological and pathological angiogenesis. VEGF in ascites promotes tumor angiogenesis by binding to VEGF receptors, thereby activating downstream signaling pathways such as PI3K and MAPK, which promotes tumor growth (Yang and Cao 2022). Different cell-to-cell junction types (i.e. tight junctions and adhesive junctions) in the peritoneal vasculature interact with each other to regulate endothelial permeability under the influence of VEGF. VEGF downregulates the expression of the tight junction protein VE-cadherin, which in turn downregulates the expression of the adhesive junction protein claudin 5. These events lead to higher endothelial permeability, which causes ascites to form by allowing a massive flow of fluid into the peritoneal cavity, thereby promoting peritoneal dissemination of ovarian cancer cells (Bekes et al. 2016). VEGF binds to VEGFR2, activating the downstream ERK pathway, which promotes the differentiation of cancer stem cells (CSCs) into endothelial cells and lymphatic endothelial cells (Krishnapriya et al. 2019). VEGF also plays an important role in tumor immunosuppression and enabling tumors to evade immune surveillance. Binding of VEGF to VEGFR2 also directly inhibits the proliferation and function of T cells. Some studies previously observed that inhibiting the binding of VEGF to its receptor restored the proliferation of T cells (Gavalas et al. 2012) and suppressed the progression of ovarian cancer in a mouse model (Tuppurainen et al. 2017). VEGF also hindered the functional maturation of DCs, thereby inhibiting the tumor antigen-specific activation of T cells (Gabrilovich et al. 1996). Inhibiting VEGF restored the immune response of natural killer T cells (NKT) by downregulating the expression of ganglioside (GD3) (Tiper et al. 2016). In clinical studies, drugs targeting the VEGF signaling pathway, such as Bevacizumab and Apatinib, have achieved promising results in the treatment of ovarian cancer patients (Sjoquist et al. 2021; Lan et al. 2018).

Transforming growth factor-β (TGF-β) is another important tumor-promoting cytokine in malignant ascites of ovarian cancer patients. Mainly secreted by TAMs, TGF-β in ascites promotes cancer cell migration, adhesion, chemoresistance, and survival of CSCs (Yang et al. 2017; Uruski et al. 2021; Cao et al. 2012). Increased levels of TGF-β in ascites suggest early recurrence and worse prognosis (Reinartz et al. 2016; Newsted et al. 2019). TGF-β was also found to be indispensable in both Epithelial-Mesenchymal Transition (EMT) and Mesenchymal-Epithelial Transition (MET) processes, playing a vital regulatory role in both spheroid formation and implantation in ovarian cancer-derived ascites (Kan et al. 2020; Rafehi et al. 2016). Epithelial ovarian cancer derived TGF-β in ascites drives mesothelial cells to acquire a mesenchymal fibroblast phenotype, causing them to secrete more VEGF, which facilitates angiogenesis and promotes ovarian cancer progression (Fujikake et al. 2018). TGF-β1 in ascites induces the proliferation, activation, and type I collagen deposition of omental fibroblasts, which in turn increases the response of ovarian cancer cells to other growth factors in ascites such as heparin-binding epidermal growth factor (Fogg et al. 2020). TGF-β also promotes the production of periostin (POSTN) in ovarian cancer cells, which is found at high levels in the ascites of ovarian cancer patients and promotes ovarian cancer progression by recruiting TAMs and regulating the secretion of cytokines by macrophages (Tang et al. 2018).

Other tumor-promoting cytokines in ovarian cancer ascites include epidermal growth factor (EGF), insulin-like growth factor (IGF), and prostaglandin (PG). Within spheroids in ovarian cancer ascites, cancer cells stimulate fibroblasts to produce EGF, which in turn upregulates the expression of ITGA5 in the cancer cells, thereby strengthening the connections between ascites tumor cells (ATCs) and CAFs that reinforce the spheroid structure and promote the intraperitoneal dissemination of ovarian cancer (Gao et al. 2019). The concentrations of IGF-1 and IGF-2 in the ascites of patients with advanced ovarian cancer were higher than those of patients with early-stage ovarian cancer, while PGE2 was only present in malignant ascites. IGFs stimulate the secretion of IL-10 from DCs and inhibit the maturation of DCs by regulating ERK1/2 phosphorylation and p38 dephosphorylation (Huang et al. 2015). On the other hand, PGE2 inhibits the activation of DCs mediated by TLR (Brencicova et al. 2017); therefore, both IGFs and PGE2 suppress antitumor immunity mediated by DCs, which are responsible for enabling antitumor immunity and promoting the maturation of immature lymphocytes, thereby promoting ovarian cancer progression.

Ovarian cancer-derived ascites contains a diversity of metabolites, some of which exhibit cancer-promoting properties. Lysophosphatidic acid (LPA) is an intermediate product of glycerophospholipid metabolism and an important tumor-promoting factor in ovarian cancer-derived ascites. The levels of LPA in ovarian cancer ascites was found to be significantly higher than in plasma, possibly due to the secretion of LPA into the ascites microenvironment by TAMs (Feng et al. 2020; Reinartz et al. 2019). LPA enhances cell survival, proliferation, and motility in the ascites (Yu et al. 2008) and specifically stimulates VEGF promoter activity through transcriptional activation and the mediation of Sp-1 and c-Myc, which induces VEGF expression in ovarian cancer cells (Hu et al. 2001; Song et al. 2009). LPA also stimulates the expression of TNF-α through NF-κB-mediated transcriptional activation, which upregulates pro-inflammatory factors in the ascites microenvironment (Wang et al. 2020b). LPA is also an important factor in ascites because it stimulates the invasion and metastasis of ovarian cancer. LPA activates ERK and AKT signaling pathways by binding to LPAR1 (Yu et al. 2016), LPAR2, LPAR3 and LPAR6 (Yu et al. 2008) receptors, thereby affecting the invasiveness of ovarian cancer cells (Onallah et al. 2019). LPA also maintains the acetylation of HIF1α by downregulating the expression of SIRT1. On the one hand, acetylation of HIF1α activates HIF1α-ZEB1 pathway which promotes EMT and the invasion and metastasis of ovarian cancer cells (Ray et al. 2017). On the other hand, activated HIF1α induces the metabolism of glucose in normal fibroblasts into aerobic glycolysis and causes the normal fibroblasts to acquire the phenotypes of CAFs (Radhakrishnan et al. 2019), which also promotes ovarian cancer invasion and metastasis through the increase in CAFs.

Malignant ascites in ovarian cancer patients contains elevated cholesterol levels. Cholesterol acts as a tumor-promoting factor that reduces the chemosensitivity of ovarian cancer and increases the expression of MDR1 by upregulating nuclear receptor LXRɑ/β and activating the expression of ABCG2 through unclear mechanisms. MDR1 and ABCG2 are both ATP-binding cassette transporters closely related to drug resistance, whose upregulation impels the transport of chemotherapeutic drugs out of tumor cells (Kathawala et al. 2015), reducing the sensitivity of ovarian cancer cells to cisplatin and paclitaxel and ultimately causing chemoresistance and poor prognosis in ovarian cancer patients.

Other cancer-promoting components in ovarian cancer ascites include exosomes, damage associated molecular patterns (DAMPs), and trace elements.

Exosomes are a class of microvesicles about 30–200 nm in diameter that are generated from the plasma and endosomal membranes and contain proteins, lipids, nucleic acids, and glycoconjugates (Pegtel and Gould 2019). Tumor-associated exosomes in ovarian cancer ascites have immunosuppressive effects. Small extracellular vesicles (EVs) containing ARG1 that are found in both ascites and the plasma of ovarian cancer patients transport ARG1 from tumor cells to antigen-presenting cells in secondary lymphoid organs, where ARG1 inhibits antigen-specific T cell proliferation and activation via multiple mechanisms. ARG1 catalyzes the degradation of non-essential ʟ-arginine to ʟ-ornithine and urea, while depletion of ʟ-arginine in the microenvironment blocks T cell cycle progression and IFN-γ production. In addition, ARG1 downregulates the expression of the ζ and ε chains of TCR-associated CD3, which are key components of the T-cell receptor signaling complex, thereby impairing T-cell function. ARG1 also interacts with DCs and impairs the activation potential of DCs, leading to the downregulation of MHC class II molecules necessary for antigen presentation (Czystowska-Kuzmicz et al. 2019). Exosomes secreted by ovarian cancer cells play an important role in tumor metastasis as well by regulating the interaction of extracellular matrix (ECM) receptors which promotes cancer cell migration (Bortot et al. 2021).

Damage-associated molecular patterns (DAMPs), including mitochondrial DNA (mtDNA), also exist in the ascites fluid and promote tumor metastasis and hinder antitumor immunity by affecting neutrophil and platelet responses (Singel et al. 2019). Researchers found that the contents of cobalt, nickel, copper, zinc, arsenic, selenium, and molybdenum were higher in the ascites of patients with malignant and borderline ovarian tumors compared to benign ovarian tumors, and the copper content was found to induce the production of VEGF, thereby promoting the progression of ovarian cancer (Onuma et al. 2021).

Ovarian cancer-derived ascites also contains soluble HLA-G, which acts as an immune tolerance molecule and can be absorbed by the peritoneal tissue and infiltrate immune cells. The production of HLA-G is related to the presence of immune tolerance cells (e.g. Tregs) and the reduction of immune effector cells (e.g. T cells and NK cells) (Ullah et al. 2019).

Tumor-suppressing acellular components

Although non-cellular components of ovarian cancer ascites mainly consist of tumor-promoting factors, there are also tumor-suppressing factors found in ascites; some tumor-promoting factors have also been demonstrated to possess certain tumor-suppressing effects. As mentioned above, IL-12 has been found to promote ovarian cancer progression (Zeimet et al. 1998). However, as a prominent cytokine affecting immune responses generated by Th1 cells, IL-12 also has important tumor suppressor properties. For example, tumor-associated myeloid dendritic cell-derived IL-12 was observed to inhibit angiogenesis in vivo (Curiel et al. 2004a). In addition, Cytokine-induced memory-like (CIML) NK cells induced by IL-12, IL-15, and IL-18 have strengthened the killing ability of NK cells on ovarian cancer cells (Uppendahl et al. 2019). Genetically modified CAR T cells secreting IL-12 to reshape the cytokine microenvironment and improve the efficiency of CAR T cell immunotherapy through increased levels of peritoneal IFN-γ, TNF-α and eradication of peritoneal tumor cells (Yeku et al. 2017).

Most studies have suggested that interferons are important tumor suppressors in ovarian cancer-derived ascites. IFNβ, one of the members of the interferon family, is known to inhibit cell proliferation, promote cell apoptosis, induce antitumor immunity, and suppress tumor angiogenesis (Imamura et al. 2018). Some studies have found that, despite exhibiting certain tumor-promoting properties by helping tumors evade immune surveillance, IFNγ exerts its antitumor role predominantly in the tumor microenvironment (Castro et al. 2018; Rozman and Svajger 2018). IFNγ converts TAMs to immunostimulatory cells by reversing the immunosuppressive and tumor-promoting properties of TAMs; blocks the production of tumor-promoting factors, such as IL-10, VEGF, MMP9, and CCL18 secreted by TAMs; and prevents the generation of TAMs from precursor cells. In addition, IFNγ facilitates tumor elimination by inhibiting tumor cell growth, TAMs-mediated angiogenesis, and the activation of immune cells (Duluc et al. 2009). Upregulation of IFNγ also restores the inhibitory effect of ovarian cancer ascites on IL-12, resulting in better clinical outcomes (Adhikary et al. 2017).

Other tumor-suppressing factors in ascites include fibrin(ogen) degradation products (FDPs) and oxytocin. FDPs in ovarian cancer-derived ascites comprising soluble macromolecular fragments, D-dimers, and fragments D and E, to name a few, have been found to possess anti-angiogenic activities, classifying them as tumor suppressors in ascites (Jandu et al. 2006). Higher levels of oxytocin in ascites is associated with positive psychosocial factors such as positive affect, purpose in life, and social nurturance (Cuneo et al. 2021) and may have an antitumor effect. Clinical studies found that the levels of oxytocin in the ascites of patients with ovarian cancer were higher than in the plasma of those patients; higher oxytocin levels in ascites is an indicator of lower IL-6 levels and a better prognosis because oxytocin reduces the secretion of IL-6 by activating oxytocin receptors, thereby inhibiting inflammatory responses and tumor progression (Cuneo et al. 2019) (Table 1).

Table 1.

Acellular factors in ovarian cancer ascites

Tumor-promoting acellular factors
Name	Classification	Effect on microenvironment	Mechanism of effect	References
Interleukin 6	Cytokine	Promotes migration and invasion of ovarian cancer cells	IL-6R-JAK2-STAT3	Kim et al. (2016)
		Promotes differentiation of monocytes into TAMs-like cells	M-CSF	Duluc et al. (2007)
		Enables MDSCs to execute immunosuppressive functions	STAT3	Wu et al. (2017)
		Promotes proliferation of Tregs	TNFR2	Kampan et al. (2017)
Interleukin 8		Promotes migration of ovarian cancer cells	Unclear	Mikula-Pietrasik et al. (2016)
		Promotes proliferation of ovarian cancer cells in spheroids	Unclear	Uddin et al. (2020)
		Induces stemness of OC cells	Notch3	Ji et al. (2021)
Interleukin 10		Disables DCs and activate immunosuppressive TIDCs	PD-1/PD-L1	Lamichhane et al. (2017)
		Promotes differentiation of T cells into Tregs	Foxp3	Zhu et al. (2016)
		Maintains immunosuppressive phenotype of MDSCs	Unclear	Hart et al. (2011)
Interleukin 12		Negatively correlates with disease-free and overall survival	Unclear	Zeimet et al. (1998)
Interleukin 1β		Positively correlates with ascitic MDSCs level	Unclear	Wang et al. (2017)
VEGF		Angiogenesis	PI3K/MAPK, etc	Yang and Cao (2022)
		Promotes formation of ascites	V/E Cadherin-Claudin 5	Bekes et al. (2016)
		Induces CSCs differentiation into endothelial cells and lymphatic endothelial cells	VEGFR2-ERK	Krishnapriya et al. (2019)
		Inhibits function of T cells	VEGFR2/GD3	Tiper et al. (2016)
TGF-β		Promotes spheroids forming and implantation	EMT/MET	Kan et al. (2020), Rafehi et al. (2016)
		Induces fibroblastic phenotype of mesothelial cells	Unclear	Fujikake et al. (2018)
		Impacts tumor cell proliferation in response to additional ascites growth factors	collagen deposition	Fogg et al. (2020)
EGF		Enhances structure of spheroids	ITGA5	Gao et al. (2019)
IGF		Inhibits maturing of DCs	ERK1/ERK2/p38	Huang et al. (2015)
PEG2		Inhibits activation of DCs	TLR activator	Brencicova et al. (2017)
LPA	Metabolite	Induces VEGF expression in OC cells	Sp-1/c-Myc	Hu et al. (2001), Song et al. (2009)
		Induces TNF-α expression	NF-κB	Wang et al. (2020b)
		Promotes invasion of OC cells	LPAR1/LPAR2/LPAR3-ERK/AKT	Yu et al. (2008), Yu et al. (2016), Onallah et al. (2019)
		Promotes EMT and CAFs induction	SIRT1-HIF1α	Ray et al. (2017), Radhakrishnan et al. (2019)
Cholesterol		Promotes chemoresistance	LXRɑ/β-MDR1/ABCG2	Kathawala et al. (2015)
Exosome	Others	Immunosuppression	ARG1	Czystowska-Kuzmicz et al. (2019)
		Promotes migration of OC cells	ECM receptor	Bortot et al. (2021)
DAMPs		Facilitates metastasis and obstruct anti-tumor immunity	Neutrophil and platelet responses	Singel et al. (2019)
Copper		Induces angiogenesis	Upregulation of VEGF	Onuma et al. (2021)
HLA-G		Correlates with presence of Tregs and reduction of immune effector cells	Unclear	Ullah et al. (2019)

Tumor-suppressing acellular factors
Name	Classification	Effect on microenvironment	Mechanism of effect	References
Interleukin 12	Cytokine	Antiangiogenesis	Unclear	Curiel et al. (2004a)
		Increases killing ability of NK cells	Unclear	Uppendahl et al. (2019)
Interferon		Induces growth arrest and apoptosis	Unclear	Imamura et al. (2018)
		Inhibits generation and tumor-promoting properties of TAMs	Unclear	Duluc et al. (2009)
FDPs	Others	Antiangiogenesis	Binding to VEGF(R)/integrin or activating plasminogen	Jandu et al. (2006)
Oxytocin		Reduces production of IL-6	Oxytocin receptor	Cuneo et al. (2019)

Cellular components of ovarian cancer-derived ascites

The cellular components in ovarian cancer-derived ascites include ovarian cancer cells, immune cells, stromal and mesothelial cells, and tumor stem cells. They exert their influence on ovarian cancer progression through inhibition or activation of their own functions.

Tumor-promoting cellular components

Since cancer cells produce many cytokines, the presence or absence of cancer cells significantly affects the levels of cytokines in ascites in patients with epithelial ovarian cancer (Silva et al. 2017). Multiple pathways, such as the constitutive activation of pSTAT3 by phosphorylation of the Tyr705 residue, are abnormally activated in ovarian cancer cells in ascites. For example, the activation of STAT3 promotes the invasion and metastasis of ovarian cancer cells through mechanisms involving mesenchymal cells and phosphatase receptor kinase (Saini et al. 2017). Peritoneal adipocytes de-differentiate and acquire characteristics of CAFs resulting from the activation of the Wnt/β-catenin pathway. These adipocyte-derived CAF-like cells, known as omental adipocyte-derived fibroblasts (O-ADFs), have demonstrated the ability to promote ovarian cancer progression (Iyoshi et al. 2021).

Ovarian cancer-derived ascites contains a large number of spheroids that structurally are aggregates of cancer and other cells. Metastatic units (Mus) are highly invasive spheroids composed of ITGA5-high ascitic tumor cells recruited by CAFs and are considered an important driver of the intraperitoneal dissemination of ovarian cancer (Gao et al. 2019). Cancer cells within spheroids have less proliferative viabilities but stronger tumorigenic capacities compared to adhering cancer cells (Ding et al. 2021). Spheroids suspended in ascites activate apoptosis-related signaling pathways, such as p27, FAK, Src, PKB/Akt, Bcl-2, and caspase, through conformational alteration of the αvβ3 transmembrane domains of integrin to prevent anoikis (Dolinschek et al. 2021), thus surviving in ascites and metastasize.

Myeloid-derived suppressor cells (MDSCs) are the most critical cells in the immunosuppressive microenvironment in ovarian cancer-derived ascites (Baert et al. 2019). The precursors of MDSCs are immature myeloid cells, which are abnormally activated in tumor microenvironment (Condamine et al. 2015). Previous studies found that IL-6 and IL-10 in ovarian cancer ascites activated STAT3 in MDSCs and subsequently upregulated the expressions of ARG1 and iNOS in MDSCs, which enabled MDSCs to suppress T cell function and exert their immunosuppressive effects (Wu et al. 2017).

Another important subset of immunosuppressive cells in ovarian cancer ascites are Tregs. Not only an increase of Tregs was observed in ovarian cancer ascites compared to peripheral blood (Wertel et al. 2015), Tregs in ascites are also more activated than those in peripheral blood (Landskron et al. 2015). Tregs, recruited by Tumor cell and macrophage-derived CCL22, execute immunosuppressive functions by blocking tumor-specific T cell immunity (Curiel et al. 2004b). TNFR2 expression on Tregs, which can be upregulated by IL-6 in ascites, is positively correlated with expression of immunosuppressive molecules PD-L1, CTLA-4, and GARP on Tregs (Kampan et al. 2017), while blocking TNFR2 inhibited ascitic Tregs (Torrey et al. 2017). These demonstrate a critical intrinsic relationship between TNFR2 and the immunosuppressive properties of Tregs.

In ascites, TAMs play significant roles in tumor growth, metastasis, and immunosuppression (Song et al. 2020). TAMs secrete a series of soluble factors, including IL-1β, IL-6, IL-10, CCL18, CCL22, TGFα, TGF-G, IGF1, and EGF, that enable the TAMs to exert tumor-promoting effects, some of which have been introduced above. TAMs promote tumor cell proliferation, migration, adhesion, and invasion by secreting ECM remodeling-related proteins, including transforming growth factor beta-induced (TGFBI), tenascin C (TNC), and fibronectin (FN1) (Imamura et al. 2018; Steitz et al. 2020). In particular, when spheroids in ovarian cancer-derived ascites are seeded in the peritoneal cavity, selectively activated macrophages within ascites secrete soluble factors to promote the detachment of ovarian cancer cells from spheroids through the JAK2/STAT3/MMP-9 signaling pathway, thereby facilitating the growth of metastases (Fogg et al. 2019).

CAFs exert important tumor-promoting effects in ascites. In the “metastatic unit” mentioned above, CAFs serve as skeletons to recruit ovarian cancer cells in ascites to aggregate, migrate, adhere, invade, and form a matrix at the metastatic site during the formation of metastases (Gao et al. 2019). CAFs also induce EMT in cancer cells through the activation of TGFβ pathway and promote the seeding of metastases at the metastatic site (Kan et al. 2020). Another study found that secreted ligands, such as IL6 and CXCL12 are highly expressed in cells in ascites, such as CAFs and cancer cells, could activate JAK/STAT pathway, while JAK/STAT pathway inhibitors could abrogate the development of ascites and tumor growth, suggesting that CAFs and cancer cells promote ascites formation through JAK/STAT pathway (Izar et al. 2020).

CSCs are tumor-initiating cells that are extremely rare in minor differentiated tumor mass. Current cancer treatments primarily target rapidly proliferating tumor cells to eliminate tumors and relieve symptoms, whereas CSCs possess a static phenotype that renders current conventional cancer treatments ineffective. Therefore, even after the substantial part of a tumor is eliminated by chemotherapy, CSCs persist and cause cancer recurrence, ultimately leading to the deaths of ovarian cancer patients (Ahmed et al. 2018). The PI3K/AKT pathway plays a key role in stemness and chemoresistance of CSCs (Parashar et al. 2022). The ALDH ⁺ CD44⁺ tumor cell subset in ascites developed stem cell properties through the PDK4-mediated STAT3/AKT/NF-κB/IL-8 signaling pathway (Jiang et al. 2020). Genetically modified NK cells recognizing CD133 on CSCs have demonstrated the ability to effectively target and kill CSCs in in vitro experiments (Klapdor et al. 2017). Ovarian cancer CSCs also have the potential to differentiate into vascular endothelial cells, pericytes, and lymphatic endothelial cells under certain conditions, thereby promoting angiogenesis, lymphangiogenesis, and the recurrence and progression of ovarian cancer (Krishnapriya et al. 2019).

Human peritoneal mesothelial cells (HPMCs), which constitute a single layer of squamous epithelium on the surface of peritoneum called the peritoneal mesothelium, are affected by the ascites microenvironment and play an important role in the peritoneal dissemination of ovarian cancer. HPMCs in ascites undergo mesothelial-mesenchymal transition regulated by TNF-α, TGF-β, IL-1β, and IL-6, and may acquire the phenotype of mesenchymal fibroblasts to secret more VEGF and promote angiogenesis, intraperitoneal dissemination, and metastasis (Fujikake et al. 2018; Rynne-Vidal et al. 2017). One study found that elevated GRO-1 and HGF levels in the ascites induced the senescence of HPMCs, while senescent HPMCs produced more HA, uPA, VEGF, IL-8, and MCP-1 to facilitate ovarian cancer cell adhesion, proliferation, and migration (Mikula-Pietrasik et al. 2016).

Tumor-suppressing cellular components

The immunosuppressive microenvironment of an ovarian cancer-derived ascites endows cancer cells with the ability to evade immune surveillance through various mechanisms. The number of CD20⁺ T cells in the ascites of ovarian cancer patients was significantly higher compared to peripheral blood. These CD20⁺ T cells were found to be effector memory T cells that acquired the B cell marker CD20 from interactions between the T and B cells. The CD20⁺ T cells also acquired not only the ability to produce and secrete IFNγ but also a phenotype with better adhesion and less migration. However, the elevated levels of CD20⁺ T cells in ascites were not found to be associated with ovarian cancer progression (Bruyn et al. 2015; Vlaming et al. 2021), but this may require further research for validation. Other studies observed that a high proportion of CD16⁺ mDCs and a low proportion of CD4⁺ T cells in ascites correlated with higher survival in ovarian cancer patients (Wefers et al. 2018). Higher levels of neutrophils in the ascites of KRAS-mutated ovarian cancer patients promoted the proliferation or survival of CD8⁺ T cells, which demonstrate antitumor properties by regulating the tumor microenvironment (Yoshida et al. 2018).

NK cells play an essential role in antitumor immunity; however, NK cells in ascites of ovarian cancer patients have a functionally different phenotype compared to blood NK cells because the NK cells in ascites express less of the DNAM-1 activating receptor and overexpress the inhibitory receptor CD96 (Maas et al. 2020). NK cells in ascites with presence of malignant cells are less sensitive to IL-2 stimulation, undergo less degranulation, and have a weaker killing effectiveness toward cancer cells compared to NK cells in ascites without presence of malignant cells, which contributes to the immunosuppressive microenvironment of the ascites; the reason for this might be related to Tregs for that the amount of Tregs was significantly higher in ascites with malignant cells than ascites without malignant cells (Silva et al. 2017). Higher levels of CD56⁺ NK cells in ascites are typically an indicator of better prognoses in patients with ovarian cancer (Hoogstad-van Evert et al. 2018). CD56^dim NK cells in ascites are easily impaired by immunosuppressors in the tumor microenvironment, while CD56^bright NK cells are sensitive to stimulatory cytokine responses, which are abundant in epithelial ovarian cancer ascites and can be induced to enable antitumor immunity (Tonetti et al. 2021). In one study, the blocking of the inhibitory receptor TIGIT was found to enhance the immune function of CD56^dim NK cells (Maas et al. 2020), while, in another study, the IL-15 superagonist ALT-803 restored the cytotoxicity against ovarian cancer of NK cells in ascites in vitro and in vivo (Felices et al. 2017), both of which revealed the potential of NK cell-based immunotherapies for the treatment of ovarian cancer (Fig. 1).

Fig. 1.

Interactions of acellular and cellular factors in ovarian cancer ascites

The mechanisms that determine the balance of tumor-promoting and tumor-suppressing factors

Malignant ascites is usually an indication of advanced-stage ovarian cancer and poor prognosis. Nonetheless, there has been clinical data showing that certain OC patients survive ascites for a considerable amount of time. A study concluded that survival of OC patients with ascites is associated with ascites volume (Feigenberg et al. 2014). OC patients with smaller ascites volume (equal to or less than 200 cc) showed higher expression of immune genes, more tumor-infiltrated immune cells and better prognosis compared to those with bigger ascites volume (equal to or more than 1000 cc). Unfortunately, patients without ascites were not included in this study. ScRNA sequencing of cells in ovarian cancer-derived ascites found significant differences in the composition and function of ascites cells among different patients including differences in immunomodulatory fibroblast subpopulations and macrophage subpopulations(Izar et al. 2020), which may lead to distinct tumor-promoting and tumor-suppressing factor spectrum among patients.

The balance between tumor-promoting and tumor-suppressing factors may be modulated by a variety of elements, including (a) Tumor metabolism. Glutaminase purified from human ovarian cancer ascites fluid reduced tumor burden and inhibited tumor-induced or heparin mediated angiogenesis in an in vivo experiment, while administration of glutaminase in combination with copper, a tumor-promoting factor described above, amplified such effect (Bhattacharya et al. 2001). Glutamine metabolism restrains generation and recruitment of MDSCs in breast cancer (Oh et al. 2020), providing new evidence for the anti-tumor role of glutamine. A study also revealed close connection between immunome and metabolome in HCC TME (Zhang et al. 2019); (b) Germline and somatic mutations. Increases of CD20⁺ and TIA-1⁺ immune cell infiltrates were observed in OC tumors with BRCA1 or BRCA2 mutations compared with those without mutations (McAlpine et al. 2012), revealing the immunomodulatory role of germline and somatic mutations; (c) Therapeutic strategy. Cisplatin administration elevates immune activity of ascitic monocytes and cytotoxic T-cells in a murine model of epithelial ovarian cancer (Hopkins et al. 2021). On the other hand, treatment with cisplatin or carboplatin enhances the ability of OC cell lines to assist generating tumor-promoting M2 macrophages (Dijkgraaf et al. 2013); (d) Competition between mutant cells and wild-type cells. It has been reported that normal cells in skin tissue possess the capacity of actively eliminating beta-catenin mutant cells and suppressing generation of aberrant structures (Brown et al. 2017). Whether this competition also exist in OC needs further research; (e) Psychosocial factors. The acellular tumor-suppressing factor oxytocin, as described above, is regulated by positive affect, purpose in life, and social nurturance (Cuneo et al. 2021). Therefore, these psychosocial factors may in part influence the tumor-suppressing effect of oxytocin; (f) Intestinal microbiota. Modulation of the intestinal microbiota investigated in liver cancer (Loo et al. 2017) and breast cancer (Modica et al. 2021) was reported to correlate with efficacy of anti-tumor immunity via regulation of T cells, DCs, IFN, IL-12 and PEG2, all of which are found in ovarian cancer ascites and participate in tumor progression; (g) Chronotherapy. As discussed above, the circadian clock and its regulators display distinct functions in different cancer types. Furthermore, it has been discovered that time of cisplatin administration alters patient outcome in prostate, breast, cervical, and ovarian cancer (Kobayashi et al. 2002). This might be a promising area to be explored.

Therapeutic strategies targeting components of ovarian cancer ascites

In addition to the applications of ascitic IL-6, IL-12, and VEGF in ovarian cancer immunotherapy, targeted therapy and as diagnostic or prognostic biomarkers (Yeku et al. 2017; Rodrigues et al. 2020; Dalal et al. 2018; Sjoquist et al. 2021; Lan et al. 2018), more therapeutics targeting tumor-promoting components in ascites have been discovered or studied in various applications. Studies have found that resveratrol, a natural polyphenolic compound produced by plants (in particular, berries and peanuts), was able to mitigate the tumor-promoting effect of LPA in ascites, suppressing tumor metastasis and drug resistance, and has been applied in the treatment of ovarian cancer (Ferraresi et al. 2021). ABBV-085, an antibody–drug conjugate targeting LRRC15, which is a membrane-bound protein that activates the FAK pathway on spheroids and metastatic cancer cells to promote tumor metastasis, was capable of preventing the metastasis of ovarian cancer in preclinical models (Ray et al. 2022). AZD2014, an mTORC1/2 inhibitor, suppressed the accumulation of MDSCs in ovarian cancer ascites, thereby delaying ovarian cancer progression and reducing recurrence by enhancing antitumor immunity (Pi et al. 2021) (Table 2).

Table 2.

Therapeutic strategies targeting ovarian cancer ascites

Therapies targeting factors in OC ascites
Name	Applicaton	Target	References
IL-6	Biomarker	IL-6	Rodrigues et al. (2020), Dalal et al. (2018)
Armored CAR T cell	Immunotherapy	IL-12	Yeku et al. (2017)
Bevacizumab	Chemotherapy	VEGF	Sjoquist et al. (2021)
Apatinib		VEGF	Lan et al. (2018)
Resveratrol	Potential drugs	LPA	Ferraresi et al. (2021)
ABBV-085		LRRC15 on spheroids	Ray et al. (2022)
AZD2014		MDSCs	Pi et al. (2021)

Intraperitoneal bevacizumab (IP-bev) therapy was found to be effective in delaying the re-accumulation of malignant ascites in women with chemotherapy-resistant epithelial ovarian cancer (CR-EOC), reducing the median time from first to second therapeutic ascitic drainage (Sjoquist et al. 2021). Unfortunately, the researchers did not analyze the relationship between IP-bev treatment and survival because the study included only 24 samples. However, further research is expected to deeply explore the relationship between therapies targeting ascites and survival in the future.

Cell-free and concentrated ascites reinfusion therapy (CART) was first reported by Inoue et al. in 1977 and involves the filtering, concentrating, removal of cellular components, and reinfusion of ascitic fluid (Inoue et al. 1977). CART effectively relieves symptoms caused by ascites and improves the general condition of patients (Yamamoto et al. 2021; Kawata et al. 2019). The main goal of current CART is to alleviate symptoms and avoid protein loss caused by frequent paracentesis. With the progress of the research on the elucidation of ascites components, the combination of CART and therapies targeting various components of ascites may provide new avenues for the treatment of advanced ovarian cancer.

Discussion

Myriad studies have correlated the occurrence of ascites to the degree of ovarian cancer progression. Over 90% of patients with advanced ovarian cancer suffer from ascites (Huang et al. 2013), and patients with high-grade serous ovarian cancer are more likely to develop ascites compared to those with low-grade serous ovarian cancer (Krugmann et al. 2019). In addition, volume of ascites in patients who respond to therapy gradually decreases as treatment progresses (Zhang et al. 2018). However, many patients with ascites still have a better prognosis than those without ascites, suggesting that there may be components of ascites that are sensitive to treatment, which can improve the survival rate of patients. Therapeutic strategies targeting ascites and the components thereof have unlimited potential and broad prospects in the treatment of ovarian cancer, especially advanced ovarian cancer.

However, the current understanding of composition of the components of ovarian cancer ascites is still very limited. In recent years, omics analyses of ascites components have discovered many new therapeutic targets and biomarkers. For example, single-cell RNA sequencing of ascites cells found that SDC4 has the potential to be a prognostic marker of ovarian cancer (Kim et al. 2021). Ascites transcriptomic and metabolomic studies found that the WNT signaling was negatively associated with immune cell infiltration and prognosis in ovarian cancer patients and detection of WNT5A levels in ascites could be a prognostic marker for high grade serous carcinoma patients (Arend et al. 2022; Kotrbova et al. 2020). Epigenomic and transcriptomic profiling of tumor biopsies or malignant ascites indicates that the inhibition of DNA methylation may change gene expression patterns related to DNA repair and immune activation and restore platinum drug sensitivity of ovarian cancer (Fang et al. 2018). In addition, ascites peptidomics studies have identified a number of peptides that are differentially expressed in ovarian cancer-derived ascites, but the significance of these peptides needs to be explored in subsequent studies (Shender et al. 2019; Huang et al. 2018). The identification of these peptides is an indication that there are many other acellular and cellular components in ascites that may affect the progression or treatment of ovarian cancer.

Furthermore, omics analyses of ascites have recognized certain patterns and alternations in cell metabolism and autophagy. It has been reported that cancer cells or spheroids in ascites undergo a metabolic shift from aerobic glycolysis and lipolysis to lipophagy and β-oxidation (Swamy et al. 2022; Chen et al. 2019). The level of autophagy has been found to increase in ascites spheroids (Correa et al. 2015) as well as recurrent ascites tumor cells compared to chemosensitive ascites tumor cells (Liu et al. 2018). These integrated studies contribute to the overall understanding of the ascites microenvironment and are instructive to explore the interplay of ascites components. In the future, continued in-depth research on the components of ovarian cancer-derived ascites will help to develop more therapeutic strategies to inhibit the tumor-promoting factors in ascites, rebuild antitumor immunity, and mitigate cancer metastasis, bringing new hope to ovarian cancer patients.

Declarations

Conflict of interest

The authors report no conflicts of interest in this work.