Polarization of tumor associated macrophages in ovarian cancer ascites: relationship with cancer-related anemia and associated inflammation and iron metabolism changes.

Background: Ovarian cancer is the solid tumor in which cancer-related anemia (CRA) is more severe. The tumor microenvironment of ovarian cancer could be one of the most suitable “ex vivo” model to know the origin of cancer-related anemia. In fact, tumor associated macrophages (TAMs) produce high levels of IL-6.Some papers suggest a role for them in the synthesis of hepcidin; both factors have been significantly related with CRA. Moreover, iron handling is an important signature of macrophage polarization that thus may play a central role in inducing of the development of CRA. Aim of the study: to assess the TAMs polarization in the context of ovarian cancer microenvironment, their ability to release hepcidin beyond Il-6 and their correlation with CRA and associated iron metabolism alterations. Patients and methods: Fifty patients with primary ovarian cancer have been enrolled from June 2015 to January 2017. TAMs were separated from ascites through a discontinuous density gradient. TAM presence and polarized phenotype was determined by staining cells with fluorescent antibodies against CD14 and one of the major M1 biomarker (CD80) or M2 biomarker (CD163). GLUT-1 expression as a marker of M1 polarization related to increased glycolytic activity was also assessed. Data were acquired through a FACScan flow cytometer using the CellQuest software (BD Biosciences). Moreover, TAM were cultured in complete medium at 37°C in a humidified atmosphere with 5% CO2 in the presence of PHA: the synthesis of IL-6 and hepcidin and the progression into the cell cycle (S phase) was evaluated. Moreover, TAMs polarization as well as the synthesis of IL-6 and Hepcidin was correlated with the levels of Hb and the ascitic levels of inflammatory and iron-related metabolism parameters. Results: TAMs isolated from ascites of primary ovarian cancer patients presented a mixed phenotype with a prevalence of CD14+/CD80+ (70±11%) cells, resembling a M1 phenotype, and a low percentage of CD14+/CD163+cells (16±9.5%), with a M1/M2 ratio of 3.3±1.5. The CD80 expression and the M1/M2 ratio were significantly inversely correlated with the Hb levels. All CD80+ cells were Glut-1+; then the percentage of CD80+/Glut-1+ cells among TAMs was high (68±11%) and it was significantly negatively correlated with Hb levels. The percentage of TAMs in S phase at the time of collection was 37±12% (indicating the they were functionally activated) and increased significantly after PHA stimulation. The production of IL-6 and hepcidin by PHA-stimulated TAMs increased significantly in comparison to non-PHA-stimulated cells (+659%, p=0.0005 for IL-6 and +227%, p=0.014 for hepcidin). Notably, such increases in the synthesis of IL-6 and hepcidin were significantly positively correlated with the CD80 expression and the M1/M2 ratio and were significantly negatively correlated with Hb levels. Moreover, we observed that ascitic levels of IL-6, ROS, hepcidin, and ferritin were positively correlated with CD80 expression and the M1/M2 ratio and they were significantly inversely related with the Hb levels, whilst ascitic free iron levels were positively related with Hb. Notably, CD80 expression and M1/M2 ratio were positively related with ascitic IL-6, ROS, hepcidin and ferritin levels, whilst they were inversely related with ascitic free iron levels. Conclusions: Our preliminary results suggest a role of M1 polarized macrophages in the pathogenesis of cancer-related anemia and related iron metabolism disorders in primary ovarian cancer. Future development are to better define the metabolic profile of polarized macrophages through the assessment of the molecular pathway involved in cell energy metabolism (Arginase, Tryptophane, IDO, mTOR pathways) and the correlation between TAM polarization with patient clinical outcome (OS, PFS and response to anticancer treatment).