Phenotypic diversity of macrophages in ovarian cancer

Ovarian cancer (OC) is one of the most common gynecological malignancies. OC has the worst prognosis and the highest mortality rate, not only amid gynecological cancers, but also compared to the most spread breast cancer. High mortality rate is associated to asymptomatic course of the disease, tumor evolution and high tumor aggressiveness. OC differs from other tumors in its ability to implantation metastasis through the peritoneal fluid. The frequency of metastasis is increased by the accumulation of ascitic fluid. Both tumor cells and stromal cells facilitate the accumulation of ascitic fluid. They secrete factors that elevate the permeability of the capillaries in the abdominal cavity for diverse proteins and fluid. The main factors involved in these processes are vascular endothelial growth factor (VEGF) and cytokines IL-6 and IL-8. An increased concentration of common protein leads to a violation of the normal oncotic pressure between the peritoneal fluid and plasma, followed by the flow of fluid into the abdominal cavity. In addition, tumor cells in peritoneal cavity disrupt lymph flow through blockage of lymphatic vessels that also contributes to the accumulation of excess fluid due to impaired reabsorption. Ascitic fluid consists of cellular components and products of their metabolism: cytokines, chemokines and growth factors. The cellular component of ascitic fluid includes tumor cells, stromal component and immune cells. Tumor cells detach from the primary tumor and enter the peritoneal cavity, where they move along with the flow. In ascitic fluid, tumor cells can be represented by free-floating single cells, but more often, they interact with each other or the surrounding stromal and immune components to form multicellular conglomerates - spheroids. The tendency to form spheroids is related to inability of single floating cells to survive due to anoikis - a specific type of apoptosis that occurs due to the interruption of cell adhesion. The tumor microenvironment (TME), where immune cells are one of the most important components, determines tumor progression and affects the effectiveness of chemotherapy. The key cells of innate immunity in the TME are tumor-associated macrophages (TAMs). In several OC patient cohorts, the balance of M1/M2 macrophages in tumor tissue has been shown to have a prognostic value for predicting metastasis and recurrence. A number of studies have demonstrated a positive correlation of the total number of CD68-positive TAMs in tumor tissue with a poor prognosis. A meta-analysis of nine studies including 794 patients found that a higher M1(iNOS+ or HLA-DR+)/M2(CD163+) ratio was associated with a favorable outcome in OC. In addition, an increased M1/M2 ratio predicted better progression-free survival (PFS) and 5-year survival for patients with OC. In contrast, lower PFS correlated with a high density of CD163+ TAMs and a higher CD163/CD68 ratio. The density CD206+ macrophage was not predictive, but a higher CD206+/CD68+ cell ratio was strongly associated with worse PFS and overall survival (OS). An association of specific subpopulations of macrophages, expressing various markers, with clinical and pathological parameters in OC has also been found. In the peripheral blood of OC patients, the proportion of PD-L1+ CD68+ cells among all CD68+ cells and the intensity of PD-L1 staining for CD68+ cells were significantly higher compared to the healthy group. Immunohistochemical and immunofluorescence analysis of ovarian tumor samples showed that a reduced M1(HLA-DR+ or iNOS+)/M2(CD163+ or VEGF+) ratio and an increased density of COX-2+ macrophages were predictors of poor survival. Microarray analysis showed that human TAMs express significantly higher levels of insulin-like growth factor 1 (IGF1) than undifferentiated myeloid cells. Under in vitro conditions, TAMs can enhance the proliferation and migration of ovarian tumor cells by increasing IGF1. The infiltration of CD163+TAMs correlates with higher expression of ZEB1, which controls the epithelial-mesenchymal transition (EMT) in OC cells. CD68+ TAM infiltration and HMGB1 expression strongly correlated with lymph node metastasis and poor survival. Macrophages in ascitic fluid reside both in a free unicellular state and as part of tumor spheroids, forming the core of the latter. The M2 subpopulation of macrophages is predominant in the composition of spheroids. Soluble factors produced by macrophages protect tumor cells from anoikis, prepare the premetastatic niche, and support tumor cell proliferation. The results of flow cytometry performed eight weeks after the injection of tumor cells into the peritoneal cavity of mice showed an increased accumulation of F4/80+, CD11b+ and CD68+ macrophages, expressing M2 macrophage markers (CD163, CD206, CX3CR1), in the peritoneal fluid. Analysis of patients' ascitic fluid revealed the presence of large spheroids composed of EGFR+ tumor cells surrounding EGF+ macrophages located in the center, thus explaining a possible model of spheroid formation. In ascitic fluid, EGF secreted by macrophages induces the migration of EGFR+ tumor cells. EGF promotes adhesion of EGFR+ tumor cells to macrophages through the interaction of ICAM1 molecules and аМв2 in-tegrin. Another mechanism of spheroid formation can be related to macrophage-produced CCL18 that activates the EMT in tumor cells. In vivo, tumor spheroids overexpressing ZEB1 (an EMT marker) and containing TAMs in their structure, demonstrated a rapid ability to disseminate. Transcriptomic analysis of tumor cells and TAMs isolated from the ascitic fluid of patients with high-grade serous ovarian cancer (HGSOC) showed several signaling molecules that ensure the interaction between tumor cells and macrophages. They include cytokines, that induce STAT3 signaling (IL-10, IL-6, LIF), and TGFe1, which are mainly expressed by TAMs, and WNT7A, expressed by tumor cells, as well as various genes belonging to the S100 family, chemokines, ephrins, and their receptors. TGFe1, tenascin C (TNC) and fibronectin (FN1) produced by TAMs in ascites activate tumor cell migration. The main factors produced by macrophages are shown in the Table. Thus, in ovarian cancer, TAMs have clinical significance both due to infiltration into the tumor mass and due to close interaction with tumor cells in ascitic fluid. Therefore, the search for new markers associated with TAMs is required to predict an effect of anti-cancer therapy and a prognosis of OC for individual patients. Understanding the mechanisms of macrophage-induced tumor progression will allow finding new potential targets for blocking metastasis to improve OC outcome.

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