*2.1. Angiogenesis in Thyroid Cancer*

Neovascularization in the cancer microenvironment is a multistep process that is necessary during the progression of solid and hematologic tumors [8,9]. It is a complex and heterogeneous process that includes three different mechanisms: (i) angiogenesis, the sprouting of newly formed vessels from mature preexisting ones; (ii) vasculogenesis, the formation of neovessels starting from endothelial precursor cells, namely hemangioblasts; and (iii) vasculogenic mimicry, the ability of tumor cells or other non-endothelial cells to complete the neovessel wall or to form a complete capillary network without vascular endothelial cells [40,41]. Moreover, tumor-related neovessel formation occurs mainly through sprouting of new capillary vessels out of preexisting ones (angiogenesis), the longitudinal splitting of existing vasculature into two functional vessels (intussusceptive angiogenesis) and the loop-shape expansion of the vessel (looping angiogenesis) involved mainly in wound healing [42–44]. Increased vascularity in the thyroid can occur in hyperplastic goiter, Graves' disease, and cancer [45]. As in other solid and hematologic tumors, the microvessel density (MVD) has been shown to correlate with disease-free survival in thyroid cancers, particularly in PTC and in follicular thyroid carcinoma (FTC) [45–47]. Differences have been demonstrated among tumor types in the patterns of spread and metastasis, probably due to the influence of tumor metastasis route by phenotype, angiogenic or lymphangiogenic, determining a more aggressive behavior [48,49]. Thyroid adenomas, microcarcinomas, PTC, FTC, undifferentiated thyroid carcinomas, and medullary thyroid carcinomas (MTCs) present very different behaviors, clinical outcomes, and metastatic routes (lymphatic or hematic) [48,50]. These differences correlate with different angiogenic regulators released (stimulators or inhibitors), the different expression of receptors, and different extracellular matrix composition in the tumor microenvironment [50].

In thyroid tumors, angiogenesis is activated and maintained by the modulation of the genes involved in angiogenesis and response to hypoxia (HIF1A, TUFT1, BHLHB2), cell survival (RIPK5), proliferation (PTGS2, DUSP5), apoptosis (ZFP36L1, IER3), metabolism (SLCA2A3), cell organization (RAB7B) and protein degradation (SKP1, KLK-4) in the ST surrounding the tumor cells [39]. These alterations are induced and stabilized in the components of the microenvironment through the reciprocal positive and negative interactions between tumor cells and ST (endothelial cells, fibroblasts, macrophages, mast cells) and are mediated by an array of cytokines, receptors, and adhesion molecules [39,51]. Moreover, the communication between cancer cells, ST, and the various stromal cells is mediated by the release of exosomes by the thyroid tumor cells, which contribute to tumor progression, angiogenesis, and metastasis [52].

Evidence indicates that there is a dysregulation of miRNA in thyroid cancer that influences the hallmarks of cancer, including proliferative signaling, evading growth suppressors, resisting cell death, inducing angiogenesis, activating invasion and metastasis, and acquiring the epithelial-mesenchymal transition phenotype [53]. A role in modulating angiogenesis is played by the thyroid hormone (L-thyroxine, T4; 3,5,3 -triiodo-L-thyronine, T3) that represents a valid contributor to this process in thyroid cancers [54]. Thyroid hormone acts by binding the hormone receptor site on αvβ3 integrin and then modulating angiogenic cytokine (VEGFR and bFGFR) release via integrin activation and signaling in blood vessel cells [47,55]. This modulation is also mediated by hypoxia-inducible factoralpha (HIF-1α), a transcription factor whose stabilization in cells is regulated by thyroid hormone via αvβ3 [56,57]. Thyroid-stimulating hormone (TSH), the glycoprotein hormone stimulating the number, size, and activity of thyrocytes as well as the synthesis of thyroid hormone, also contributes to angiogenesis stimulation [40,58,59]. TSH enhances angiogenesis and macrophage recruitment into the thyroid tumor microenvironment and then tumor cell growth through VEGF mRNA and protein induction via the protein kinase C pathway [60,61]. Iodine deprivation causes reactive oxygen species (ROS) production, stabilization of HIF-1α and VEGF release through the activation of signals in the tumor thyrocytes that induce microvascular expansion to facilitate enhanced delivery of iodide [62,63]. Iodine deficiency induces VEGF-A expression by increasing phosphorylation of ribosomal S6 kinase (p70S6K), mediated by mammalian target of rapamycin (mTOR); the latter acts as a positive regulator and AMP-activated protein kinase, in turn stimulating thyroid microvascular activation [64].
