Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
Department of Pathology, STHF, Skien, Norway.
Acta Neuropathol Commun. 2019 Nov 8;7(1):172. doi: 10.1186/s40478-019-0830-3.
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" (CD68CD4FOXP3CD20) 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.
肿瘤微环境（TME）中的非肿瘤细胞影响肿瘤的增殖、侵袭和血管生成。关于垂体神经内分泌肿瘤（PitNETs）的 TME 知之甚少。我们旨在研究 TME 在 PitNETs 侵袭性行为中的作用，重点关注免疫细胞和细胞因子。使用包含 42 种细胞因子的免疫测定试剂盒，评估了 16 例临床无功能 PitNETs（NF-PitNETs）和 8 例生长激素细胞瘤的细胞因子分泌谱。与正常垂体（NPs，n=5）相比，评估了巨噬细胞（CD68、HLA-DR、CD163）、T 淋巴细胞（CD8、CD4、FOXP3）、B 淋巴细胞（CD20）、中性粒细胞（中性粒细胞弹性蛋白酶）和内皮细胞（CD31）的含量。在体外，通过 GH3（垂体肿瘤）和 RAW264.7（巨噬细胞）细胞系的条件培养基（CM）评估肿瘤-巨噬细胞相互作用对形态、迁移/侵袭、上皮-间充质转化和细胞因子分泌的影响。IL-8、CCL2、CCL3、CCL4、CXCL10、CCL22 和 CXCL1 是 PitNET 衍生的主要细胞因子。巨噬细胞和中性粒细胞含量增加的 PitNETs 具有更高水平的 IL-8、CCL2、CCL3、CCL4 和 CXCL1。CD8+T 淋巴细胞与更高水平的 CCL2、CCL4 和 VEGF-A 相关。PitNETs 中的巨噬细胞比 NPs 多（p<0.001），CD163:HLA-DR 巨噬细胞比例增加了 3 倍。PitNETs 包含更多的 CD4+T 淋巴细胞（p=0.005），但中性粒细胞较少（p=0.047），CD8:CD4 比值降低了 2 倍。NF-PitNETs 分泌的细胞因子更多，中性粒细胞比生长激素细胞瘤多 9 倍（p=0.002）。Ki-67 较高的 PitNETs 中 FOXP3+T 细胞更多，CD68:FOXP3、CD8:CD4 和 CD8:FOXP3 比值更低。具有“有害免疫表型”（CD68CD4FOXP3CD20）的 PitNETs 的 Ki-67≥3%。CD163:HLA-DR 巨噬细胞比值与微血管密度呈正相关（p=0.015）和面积呈正相关（p<0.001）。GH3 细胞-CM 增加了巨噬细胞的趋化性，而巨噬细胞-CM 改变了 GH3 细胞的形态、侵袭、上皮-间充质转化和分泌的细胞因子。PitNETs 的特点是 CD163:HLA-DR 巨噬细胞增加，CD8:CD4 和 CD8:FOXP3 T 细胞比值降低。PitNET 衍生的趋化因子促进巨噬细胞、中性粒细胞和 T 细胞进入肿瘤，这可以决定侵袭性行为。
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