MON-462 Cytokine Network in Pituitary Adenomas and Its Role in the Tumor Microenvironment: Focus on Macrophages

Introduction: Cytokines are important elements of the tumor microenvironment (TME) coordinating host responses against the tumor, promoting growth, invasion, angiogenesis and regulating non-tumoral cells in TME. Little is known regarding the cytokine network in pituitary adenomas (PAs) and its role in the TME. We aimed to characterise PA-derived cytokines to study their role in the TME of PAs. Methods: The cytokine secretome of culture supernatants from 27 human PAs (16 NFPAs, 9 GHomas, 1 TSHoma, 1 ACTHoma) was assessed using Millipore MILLIPLEX cytokine/chemokine 42-plex. IL-8 and CCL2 expression was further studied by RNAscope. Angiogenesis, macrophages and epithelial-to-mesenchymal transition were evaluated by immunohistochemistry for CD31, CD68, CD163, HLA-DR, E-cadherin and ZEB1. Macrophage infiltration in PAs and its phenotype was further assessed with the gene-signature based method xCELL. We have developed an in vitro model of pituitary cells (GH3) and macrophages (RAW264.7) and studied their interaction via assessing the effect of conditioned media. We documented cell morphology (ImageJ), migration and invasion (Boyden chambers), epithelial-to-mesenchymal transition pathway (RT-qPCR and immunocytochemistry) and secretome changes (cytokine array). Results: IL-8, CCL2, CCL3 and CCL4 are the main PA-derived cytokines, with CXCL1, CXCL10, CCL22 and CX3CL1 also secreted by most of these tumors. RNAscope data showed that CCL2 and IL-8 are mainly synthesised in the pituitary tumour cells. NFPAs secreted more cytokines than somatotropinomas, especially CCL2 (16x more), IL-8 (25x more) and CCL4 (27x more). PAs contained more macrophages than normal pituitary (4.9±0.7 vs 1.2±0.2%, p=0.007). Macrophage infiltration was associated with higher chemokine levels (CCL2, CCL3, CCL4, CXCL1 and IL-8). Macrophages in PAs belong to the CD163+ M2-subtype, while in normal pituitary M1-macrophages predominate, resulting in 3-fold increased M2:M1 ratio in PAs (p<0.001). These findings were confirmed with xCELL on a different set of PA samples. M2:M1 ratio correlated with microvessel density (r=0.509; p=0.008). Macrophage-conditioned media induced numerous effects on GH3 cells: more elongated, epithelial-to-mesenchymal transition-like cells, significantly increased migration and invasiveness, and altered GH3 cell cytokine secretome. On other hand, GH3 cell-conditioned media induced activation-associated morphological changes in RAW264.7 macrophages, increased chemotaxis and altered macrophage cytokine secretome. Conclusions: Primarily M2-subtype macrophages are present in the TME of PAs and their number is correlating with CCL2, IL-8, CCL3 and CCL4 release. NFPAs secrete higher levels of cytokines than somatotropinomas. Our data suggest that there is crosstalk between pituitary tumor cells and macrophages which may determine the biological behavior of PAs.