MON-460 Pasireotide Treatment Inhibits Cytokine Release from Pituitary Adenoma-Associated Fibroblasts: Is This Mechanism Playing a Key Role in Its Effect?

Abstract

Introduction: Tumour-associated fibroblasts (TAFs) are important elements of the tumour microenvironment (TME) well-studied in several cancers, but their role in pituitary adenomas (PAs) has never been shown. We aimed to study the role of TAFs in PAs and its response to somatostatin analogues (SSA). Methods: TAFs were isolated from 11 freshly-cultured NFPAs and 5 GHomas as confirmed by vimentin and αSMA immunostaining. TAF presence in PAs was confirmed by immunohistochemistry, and further assessed with the gene-signature based method xCELL on a different set of samples. Macrophage infiltration, angiogenesis and epithelial-to-mesenchymal pathways were evaluated by immunohistochemistry for CD68, CD163, HLA-DR, E-cadherin, ZEB1 and CD31. TAFs secretome was assessed in vitro on culture supernatants, at baseline and after treatment with pasireotide (10-7M) using Millipore MILLIPLEX human cytokine 42-plex. GH3 cells were treated with TAF-conditioned media (CM) and normal skin fibroblasts-CM (F-CM). Cell morphology (analysed by ImageJ), invasion and epithelial-to-mesenchymal pathways were assessed. Results: CCL2 and eotaxin-1 were identified as the top secreted cytokines in TAF supernatants, followed by VEGF-A, CCL22, IL-6, FGF-2 and IL-8. TAF secretomes from NFPAs and GHomas did not differ significantly. PAs with cavernous sinus invasion had higher TAF-derived IL-6 levels compared to non-invasive PAs (72.7±10.7 vs 43.9±6.3pg/mL; p=0.027), while there was a trend for TAFs from PAs with higher Ki67 to secrete more CCL2 (p=0.058). Correlation between macrophages and TAF-derived FGF-2 was found (r=0.499, p=0.049), and increased FGF-2 and CXCL1 levels were seen in PAs with a macrophage M2:M1 ratio≥2. CCL2 levels were correlated with microvessel area (r=0.672, p=0.004), whereas PDGF-AA was negatively correlated with E-cadherin immunoreactivity (r=-0.564, p=0.023). RT-qPCR analyses indicated that the sst1 receptor is the predominant somatostatin receptor expressed in TAFs, while sst2 and sst5 receptors are poorly expressed. Pasireotide treatment decreased TAF-derived IL-6 by 80% (p<0.001) and CCL2 by 35% (p=0.038). GH3 cells treated with TAF-CM showed epithelial-to-mesenchymal-like morphology and increased invasiveness in comparison to F-CM or untreated cells, as well as E-cadherin downregulation and ZEB1 upregulation. Conclusions: TAFs are present in the TME of PAs and display a biological role. TAF-derived cytokines may influence tumor and non-tumoral cells (such as macrophages) leading to increased invasiveness, as well as influence angiogenesis and epithelial-to-mesenchymal pathways in PAs, with IL-6 and CCL2 emerging as relevant mediators. The inhibitory effect of pasireotide on TAF secretome highlights a promising anti-tumoral effect of SSAs by directly targeting TAFs and thus modulating the TME in PAs.