Chemokines modulate the tumour microenvironment in pituitary neuroendocrine tumours

Abstract

Non-tumoural cells within the tumour microenvironment (TME) influence tumour proliferation, invasiveness and angiogenesis. Little is known about TME in pituitary neuroendocrine tumours (PitNETs). We aimed to characterise the role of TME in the aggressive behaviour of PitNETs, focusing on immune cells and cytokines. The cytokine secretome of 16 clinically non-functioning PitNETs (NF-PitNETs) and 8 somatotropinomas was assessed in primary culture using an immunoassay panel with 42 cytokines. This was correlated with macrophage (CD68, HLA-DR, CD163), T-lymphocyte (CD8, CD4, FOXP3), B-lymphocyte (CD20), neutrophil (neutrophil elastase) and endothelial cells (CD31) content, compared to normal pituitaries (NPs, n = 5). In vitro tumour–macrophage interactions were assessed by conditioned medium (CM) of GH3 (pituitary tumour) and RAW264.7 (macrophage) cell lines on morphology, migration/invasion, epithelial-to-mesenchymal transition and cytokine secretion. IL-8, CCL2, CCL3, CCL4, CXCL10, CCL22 and CXCL1 are the main PitNET-derived cytokines. PitNETs with increased macrophage and neutrophil content had higher IL-8, CCL2, CCL3, CCL4 and CXCL1 levels. CD8+ T-lymphocytes were associated to higher CCL2, CCL4 and VEGF-A levels. PitNETs had more macrophages than NPs (p < 0.001), with a 3-fold increased CD163:HLA-DR macrophage ratio. PitNETs contained more CD4+ T-lymphocytes (p = 0.005), but fewer neutrophils (p = 0.047) with a 2-fold decreased CD8:CD4 ratio. NF-PitNETs secreted more cytokines and had 9 times more neutrophils than somatotropinomas (p = 0.002). PitNETs with higher Ki-67 had more FOXP3+ T cells, as well as lower CD68:FOXP3, CD8:CD4 and CD8:FOXP3 ratios. PitNETs with “deleterious immune phenotype” (CD68hiCD4hiFOXP3hiCD20hi) had a Ki-67 ≥ 3%. CD163:HLA-DR macrophage ratio was positively correlated with microvessel density (p = 0.015) and area (p < 0.001). GH3 cell-CM increased macrophage chemotaxis, while macrophage-CM changed morphology, invasion, epithelial-to-mesenchymal transition and secreted cytokines of GH3 cells. PitNETs are characterised by increased CD163:HLA-DR macrophage and reduced CD8:CD4 and CD8:FOXP3 T cell ratios. PitNET-derived chemokines facilitate macrophage, neutrophil and T cell recruitment into the tumours which can determine aggressive behaviour.

Cell Motility and Cancer

“…Tumor cells at the border of infiltration display a collective behavior via E-cadherin between tumor cells themselves and with the microenvironment [61]. In this sense, macrophages, lymphocytes and neutrophils are modulated by chemokines in pituitary neuroendocrine tumors [62]. Also, tumor-associated fibroblasts increase local tumor aggressiveness favoring the development of metastases, as it has been reported in different tumors [63][64][65].…”

The Immune Profile of Pituitary Adenomas and a Novel Immune Classification for Predicting Immunotherapy Responsiveness

“…However, authors of studies on the TIIC distributions in PAs have not reached an agreement. Marques et al ( 56 ) reported that macrophages identified in an immunohistochemical analysis using the marker CD68 were more abundant in PAs than in normal pituitaries. Yeung et al ( 20 ) analyzed the gene expression data of PAs and demonstrated that CD4 + memory T cells and M2 macrophages were the most common types of TIICs in PAs and that CD8 + T cells dominated the corticotroph PA microenvironment.…”

Diagnosis and Treatment of Parasellar Lesions

“…Different treatment strategies need to be actively discussed, since surgery (usually the first-line treatment) can be limited due to the proximity of vital structures (i.e., ICA or cranial nerves within the CS). Recent studies have identified gene mutations or signaling pathway modulations that can confer parasellar lesions a more aggressive behavior (i.e., higher invasiveness of the surrounding structures) [63,[74][75][76] and, therefore, could represent potential candidates for the development of targeted therapies.…”