Spheroid Formation and Peritoneal Metastasis in Ovarian Cancer: Comparison
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Please note this is a comparison between Version 2 by Amina Yu and Version 1 by Militsa Rakina.

Ovarian cancer (OC) is one of the most common gynecological cancers, with the worst prognosis and the highest mortality rate. Peritoneal dissemination (or carcinomatosis) accompanied by ascites formation is the most unfavorable factor in the progression and recurrence of OC. Tumor cells in ascites are present as either separate cells or, more often, as cell aggregates, i.e., spheroids which promote implantation on the surface of nearby organs and, at later stages, metastases to distant organs. In linked review we describe current knowledge about the role of fibroblasts and macrophages in tumor spheroid formation, and discuss the specific functions of fibroblasts, macrophages and T cells in tumor peritoneal dissemination and implantation.

  • ovarian cancer
  • spheroid
  • composition
  • malignant ascites
  • tumor-associated macrophages

1. Cellular Composition of Ascites

Malignant ascites (MA) contains cellular (tumor cells, diverse immune cells, fibroblasts, adipocytes, mesothelial cells, and extracellular microvesicles) and acellular (cytokines, growth factors and lipid mediators) components. The latter ensures the interaction among cellular elements [21][1]. There is a lack of quantitative data on the cell composition of ascitic fluid. In one report, the tumor cell population, defined as CD45-EpCAM+, fluctuated from 1 to 85% in ascites samples, whereas the range of immune and mesenchymal-like cells was between 10–30% [22][2]. Another study revealed that the total cellular population in ascitic fluid is comprised of 37% lymphocytes, 29% mesothelial cells, 32% macrophages and also very few (<0.1% of the total) adenocarcinoma cells [23][3].
The cellular component of ascites can be divided into so-called “resident cells”, such as tumor cells and cancer-associated fibroblasts, or stromal cells, and “non-resident cells”, such as immune cells and mesenchymal stem cells [24][4]. The processes of auto/paracrine communication and reciprocal interactions between the stromal component of the tumor microenvironment (TME), tumor stem cells and growing tumor foci induce a pro-inflammatory response, correlating with the number of various autocrine and paracrine molecules (growth factors, cytokines, chemokines, matrix proteases, immunosuppressive factors) potentiating tumor growth [5].
The cellular components of MA can be either in a free-floating state or form spheroids, leading to intraperitoneal metastases [11,25,26][6][7][8]. Spheroids can vary in size and structure, and can be composed solely of tumor cells or contain stromal and immune cells [15][9].

2. Peritoneal Metastasis

Peritoneal dissemination is the main factor determining tumor resectability, and is an unfavorable parameter for survival rates in patients with advanced or recurrent OC [34][10]. The first key process in transcoelomic metastasis is the rupture of the capsule containing the primary tumor. This allows cancer cells to disseminate after their detachment from the primary tumor site and form spheroids [10,35][11][12]. The initial implantation sites are the fallopian tubes and contralateral ovary [36][13]. After that, the most common metastatic sites are the omentum, the parietal and visceral peritoneum, as well adjacent organs via direct implantation [10,36][11][13]. The current scenario of intraperitoneal dissemination in OC includes tumor cell proliferation and epithelial-mesenchymal transition (EMT), the latter of which results in tumor cell migration, and, conversely, mesenchymal epithelial transition (MET), which forces the colonization of tumor cells with the formation of peritoneal implants [5,15,37,38][5][9][14][15].
Omental milky spots (MSs) are the major implantation site for malignant cells during peritoneal dissemination [39][16]. MSs are small, capsule-free specific structures, consisting of macrophages, lymphocytes, blood and lymphatic vessels, which enable fluid exchange between the peritoneal cavity, the blood stream and the adjacent omental tissue [39,40,41][16][17][18]. The important role of MSs in tumor cell dissemination, attachment, invasion and proliferation within MSs has been extensively studied in vivo [42,43][19][20]. In orthotopic ovarian cancer models, OC cells invaded mice omental MSs within minutes after intraperitoneal injection [44,45][21][22].

3. Cancer-Associated Fibroblasts (CAFs)

Cancer-associated fibroblasts (CAFs) play a pivotal role in tumor progression. CAFs enhance cancer cell proliferation [54][23], angiogenesis and lymphangiogenesis [55][24], ECM remodeling, immune cell recruitment [56][25], invasion and metastasis via cytokine and chemokine secretion [57,58,59,60,61,62][26][27][28][29][30][31]. CAFs are a highly heterogeneous subpopulation of stromal cells in TME, originating from different precursors, including resident tissue fibroblasts, bone marrow mesenchymal stem cells, hematopoietic stem cells, epithelial cells and endothelial cells [63,64,65][32][33][34].
In ascites, CAFs originate from recruited fibroblasts and mesothelial cells [65][34]. Here, CAFs usually exist as free-floating cells and rarely as a part of spheroids [66,67][35][36]. Free-floating CAFs, as well as TAMs, form the ecosystem of ovarian cancer ascites and provide a suitable microenvironment for cancer progression [66][35]. Single-cell RNA-seq of ascites samples from high-grade serous ovarian carcinoma (HGSOC) patients revealed subpopulations of CAFs expressing immune-related genes that were categorized as complement factors (C1QA/B/C, CFB), chemokines (CXCL1/2/10/12) and cytokines (IL6 and IL10), which are responsible for the activation of JAK/STAT signaling in tumor cells. Inhibition of JAK/STAT reduced the formation of spheroids and their invasion through a mesothelial monolayer in vitro, and decreased the development of malignant ascites and tumor growth in a patient-derived xenograft model in vivo [68][37].
CAFs in ascites are involved in intercellular interactions with stromal cells and floating tumor cells through mechanisms similar to the ones in tumor tissue: cytokine secretion, ECM remodeling and immune cell recruiting [68,69,70,71][37][38][39][40]. Cancer cells can interact with stromal cells, such as fibroblasts and macrophages, and form heterotypic spheroids in ascites [69][38]. CAFs form the core of such spheroids and can serve as a scaffolding to aggregate floating tumor cells [54,69,72][23][38][41]. Due to their high malignant potential and contribution to peritoneal dissemination, such heterospheroid structures are referred to as metastatic units [69][38]. Surprisingly, ovarian cancer cells in spheroids can express fibroblastic marker αSMA and fibronectin (FN1), which are associated with EMT. The same cells inside spheroids maintain the expression of epithelial marker, EpCAM, indicating that ovarian cancer cells in spheroids may undergo so-called “partial” EMT or “epithelial-mesenchymal plasticity” [66,74][35][42].

CAFs contribute to compact spheroid formation via the activation of cadherins and intergrins, which play the most significant role in cell–cell interactions [65,74,75][34][42][43]. High E-cadherin expression is associated with large, more spherical cells which grow in aggregates compared to small, polygonal or spindle-shaped distributed cells with few or no E-cadherin expression. Cells with high E-cadherin expression demonstrate tighter cellular connections in suspension, resulting in the formation of compact multicellular spheroids with longer lifespans [76][44]. CAFs secrete epidermal growth factor (EGF) that upregulates integrin α5 (ITGA5) expression on tumor cells, as well as TGF-β1, leading to strengthened tumor–stromal interactions inside malignant units. In turn, CAFs activated by ITGA5-overexpressing tumor cells secrete TGFβ-associated factors EGF, IP-10, IGFBP-3, BDNF, Flt-3 LG, FGF-7, IL-12, MIF and leptin (Figure 1B) [69][38]. Multicellular spheroids containing TGF-β1-activated fibroblasts were shown to be smaller in comparison to non-activated spheroids due to the presence of a denser ECM. Fibroblast activation resulted in increased collagen deposition followed by ECM stiffness [70][39]. The formation of compact spheroids with stiffened ECM is a major obstacle to therapy efficacy that mediates enhanced integrin-mediated pro-survival signaling and the high degree of invasiveness of tumor cells [74][42].
       Figure 1.
Tumor cell survival and spheroid formation in malignant ascites.
In addition to the organization of spheroids, fibroblasts are actively involved in the formation of pre-metastatic niches, the promotion of peritoneal adhesion and the implantation of tumor cells [77,78][45][46]. Malignant ascites contains various metastasis-promoting mediators, produced by both tumor cells and CAFs, such as TGF-β1, HGF, GRO-1 and IGF-1. Tumor cells are able to activate peritoneal fibroblasts through TGF-β1 secretion [70,73,78,79,80][39][46][47][48][49]. In an in vitro study, TGF-β1 was found to lead to the activation of mesothelial–mesenchymal transition in human peritoneal mesothelial cells, their transformation into fibroblasts and fibrosis and the creation of a favorable microenvironment for tumor cell dissemination [79][48]. Omental co-culture models consisting of SCOV3 cells cultured with CAFs, normal omental fibroblasts and TGF-β1-activated fibroblasts were established. These models showed that CAFs and activated fibroblasts induce stronger adhesion of tumor cells to a layer of mesothelial cells [73][47]. There was an upregulation of HGF and MMP-2 in activated fibroblasts and CAFs compared to the control, suggesting their key role in the adhesion of tumor cells [73][47]. An in vitro cell adhesion assay showed that tumor cells demonstrate stronger TGF-β1 and IGF-1-related adhesion to peritoneal cells in the presence of malignant ascites [80][49]. α5β1 integrins interact with ECM in cooperation with integrin-linked kinase (ILK). An immunofluorescent analysis showed that TGF-β1 and IGF-1 upregulate the expression of other adhesion-associated molecules, such as ICAM-1 and vimentin, on the surface of ascites-treated tumor cells and peritoneal cells [80][49].

4. Tumor-Associated Macrophages (TAMs)

As suggested by several studies, macrophages are the most predominant population of immune cells in ascites, constituting up to 95% of MA cellular components [21,22,25,68,81][1][2][7][37][50]. Peritoneal macrophages play an essential role in the suppression of inflammation and the regulation of immune response in physiological and pathological conditions [82][51]. They are present in the peritoneal cavity of healthy women, but their number increases in advanced EOC ascites [83][52].
In malignant ascites, TAMs float separately or are located in the center of spheroids surrounded by tumor cells; they possess M2 polarization by the abundant expression of CD163 and CD206 [83,84,85][52][53][54]. Additionally, fewer CD163+ TAMs can be found outside of spheroids in a free-floating state [66][35].
TAMs also protect ovarian cancer cells from anoikis by inducing the secretion of several soluble factors which promote the peritoneal dissemination of tumor cells and support their proliferation via STAT3 signaling [5,83,86,87][5][52][55][56]. In an orthotopic mouse model of ovarian cancer, macrophages promoted spheroid formation and induced the proliferation of free-floating tumor cells in ascitic fluid [84][53]. In this model, the number of infiltrating F4/80+, CD11b+ and CD68+ macrophages increased drastically 8 weeks after injection of tumor cells into the peritoneal cavity, and macrophages displayed M2-polarization markers (by the expression of CD163, CD206 and CX3CR1). Within the large spheroids, EGFR+ tumor cells surrounded the central EGF+ macrophages. Mechanistically, EGF secreted by TAMs induced EGFR+ tumor cell migration and TAM spheroid formation through VEGF-C/VEGFR3 signaling (Figure 1B). EGF facilitated the adhesion of EGFR+ tumor cells with TAMs through ICAM-1–αMβ2 integrin interaction. EGFR blockade using erlotinib decreased the amount of TAM and inhibited spheroid formation and OC progression in vivo. In ovarian cancer patients, the five-year overall survival rate was significantly lower in OC patients with high percentages of CD68+ TAMs (>14.5%) in spheroids compared with low percentages (<14.5%) of CD68-positive cells [84][53]. EGF-secreted TAMs also increased the invasive and migratory capacity of SKOV3 cells isolated from in vitro 3D spheroids, generated by TAMs and tumor cells [85][54]. Spheroid formation was facilitated by the CCL18-ZEB1-M-CSF axis. TAM-derived CCL18 induced EMT in tumor cells. The morphology of SKOV3 cells isolated from spheroids resembled that of mesenchymal cells with increased expression of the mesenchymal markers (ZEB1, SNAIL and TWIST) and decreased E-cadherin expression, compared to SKOV3 cells in a transwell system (Figure 2). In vivo, spheroids containing ZEB1-overexpressed OC cells and TAMs were shown to be critical for transcoelomic metastasis [85][54].
             Figure 2.
 Mechanisms of implantation metastasis.
TAMs also promote endothelial permeability in ascites. Human CD33+CD68+MHCII−CD206+ M2 macrophages, isolated from OC patient ascites, and MHCII-negative M2 macrophages, isolated from murine malignant ascites, induced vascular dysfunction in a VEGF-independent manner [88][57]. Since TAMs are the major source of diverse pro-angiogenic factors in TME, they can regulate EC functions by involving different angiogenic pathways [16][58]. In vivo macrophage blockade by CSF1R inhibitor resulted in a reduction of macrophage number in the ascites and vascular normalization [88][57]. In an orthotopic ovarian cancer model, apoptosis signal-regulating kinase 1 (ASK1) regulated EC permeability in the peritoneal cavity and macrophage transmigration to ascites by regulating EC junctions [89][59]. In vivo ASK1 deficiency decreased the amount of CD68+ macrophages inside the spheroids but not the polarization of TAMs, attenuating TAM-spheroid formation and tumor peritoneal implantation [89][59].
Numerous soluble tumor cell-derived and TAM-derived factors in ascites facilitate tumor progression [36,84,85,90][13][53][54][60]. A transcriptomic analysis revealed several signaling networks providing tumor cell-TAMs interactions in ascites. They involve STAT3-inducing cytokines (IL-10, IL-6 and LIF), TGFB1 mainly expressed by TAMs, WNT7A mainly expressed by tumor cells, multiple S100 genes, semaphorins and their receptors (plexins and neuropilins), ephrins, chemokines and their receptors [91][61]. The gene expression of IL-10, TGFb1, S100A8, S100A9, and IL10RA was increased in TAMs compared to tumor cells isolated from ascites of OC patients [91][61]. Several soluble mediators produced by ascites-derived TAMs, e.g., TGFß1 protein, tenascin C (TNC) and fibronectin (FN1), activated tumor cell migration [26][8]. Ascitic TAMs from OC patients express high levels of CCL18 [92][62], the immunosuppressive factor involved in cancer immune evasion (Figure 2) [93][63]. The CCL18 levels in the ascites of patients with serous OC were significantly higher compared to those in the peritoneal fluid of patients with benign gynecological conditions [94][64]. 
A phenotypic analysis of TAMs in ascites of ovarian cancer patients revealed distinct macrophage subpopulations that possessed pro-tumor functions [95][65]. A flow cytometry analysis of TAMs isolated from the ascites of primary HGSOC showed the presence of both M1 macrophages (CD14+/CD80+/Glut1+) and M2 macrophages (CD14+/CD163+) [95][65]. Patients with high M1/M2 ratios (more than 1.4) had a significantly longer overall survival (OS), progression-free survival (PFS) and platinum-free interval than patients with low M1/M2. Patients with platinum-sensitive tumors showed a significantly higher M1/M2 ratio than those with platinum-resistant tumors [95][65].

5. T-cells

The activation of the immune system against tumor cells is expected to lead to a prolonged survival of cancer patients [98][66]. T lymphocytes play a critical role in the host immune system’s ability to eliminate tumor cells [99][67]. Three main subtypes represent T cells in ovarian cancer ascites: CD8+ effector cells, CD4+ helper cells and regulatory T cells (Tregs) [100,101][68][69]. These contribute to the activation and the regulation of immune response in OC patients [102][70]. In ascitic fluid, T cells are free-floating; there are no data on their contribution to spheroid formation (Figure 1 and Figure 2) [103,104][71][72].

Several studies have demonstrated correlations between ascitic T cells and clinical outcome in patients with HGSOC [103][71]. A high CD8/CD4 ratio in ascites was associated with significantly improved survival in OC patients [103][71]. Tregs, which are abundantly represented in malignant ascites, promote tumor development and progression by inhibiting antitumor immunity [105,106,107][73][74][75]. A higher percentage of Treg cells in peripheral blood before chemotherapy correlated to worse long-term outcome in OC patients [108][76]. Tregs in ascites often demonstrate elevated expression of transcription factor FOXP3 [100,109][68][77], that stimulates PD-L1, a negative immunoregulatory molecule which inhibits effector T-cells [110][78]. Comparing malignant (ovarian cancer) and nonmalignant (idiopathic cirrhosis) ascites, the infiltration of Tregs was significantly higher in the former [106][74].

5. Conclusion

The development of peritoneal dissemination is a very complex process involving multiple cellular and acellular components. CAFs promote the remodeling of the extracellular matrix in ascitic fluid and activate cadherins and intergrins on tumor cells, which play the most significant role in cell–cell interactions. Both CAFs and TAMs can protect ovarian cancer cells from anoikis and can form the core of tumor spheroids. However, the specific mechanisms of these processes are still unknown. TAMs in ascites are polarized into the M2 phenotype by expressing M2 markers, mainly CD163 and CD206, and promote tumor cell invasion and chemoresistance. TAM interaction with other immune cells results in the development of immunosuppression in OC ascites. CAFs and TAMs are also essential for the peritoneal metastasis due to their assistance in the attachment of tumor cells to metastatic sites. The main function of T cells in ascites is supporting an immunosuppressive microenvironment.

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