In order for tumor cells to migrate to local and distant sites, tumor and the surrounding stroma cells acquire the ability to proteolytically degrade the basement membrane and underlying collagen matrix. This degradation of, and invasion through, ECM largely depends on the function of filament-like protrusions formed on invading tumor cells, termed invadopodia, and many recent studies suggest a crucial involvement of invadopodia-mediated ECM remodeling during EMT. These structures contain various proteins such as actin regulators cortactin, dynamin and neural Wiskott–Aldrich syndrome protein (N-WASP) [
292]; adhesion proteins including many integrins [
293]; adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5) [
294]; and many MMPs such as MT1-MMP and MMP-2 [
295]. It has been observed many types of cancer cells, including HNSCC, form invadopodia, which has been correlated to their invasive phenotype in vitro and in vivo [
296‐
300]. Invadopodia facilitate the ECM degradation in a variety of cancers through the regulation of various MMPs, primarily MMP-14 (also known as MT1-MMP), MMP-2 and MMP-9 [
301,
302]. MMPs commonly overexpressed in HNSCC include MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-13, and MT1-MMP. The expression of secreted MMP-1, MMP-2, MMP-9 and transmembrane protease membrane type 1 MMP are commonly associated with HNSCC progression. MMP-2 and MMP-9 levels have been reported in correlation with local invasion, cervical nodal metastasis, tumor progression and prognosis of HNSCC patients. In addition, high levels of MMP-9 have been detected at the invasive tumor front (ITF), thus many studies describe MMP-9 as a potential marker of invasive OSCC [
303‐
305]. MT1-MMP, which is involved in the regulation of MMP-2 activity, has been considered a crucial protease in HNSCC, since its expression is dysregulated in 75% to 100% of HNSCC tumors. The activity of MMPs is regulated by tissue inhibitors of metalloproteases (TIMPs) [
306], secreted mainly by fibroblasts in the stroma. These molecules serve as inhibitors of the catalytic activity of MMPs, as well as activators of pro-MMPs, the latter represented by TIMP-2 required for activation of pro-MMP-2. Among the most commonly identified TIMPs in HNSCC have been TIMP-1 and TIMP-2. Upregulated levels of TIMP-1 expression have been associated with poor survival, while levels of TIMP-2 have been often reported to be unchanged between HNSCC tumors and adjacent tissue. Regarding the invasion and migration of cancer cells, invadopodia formation and secretion of MMPs, the overexpression of neural precursor cell expressed developmentally downregulated 9 (NEDD9) has been suggested as a biomarker of tumor agressiveness in many types of cancer, including oral cancer. Lucas et al. demonstrated that VEGF-stimulated HNSCC cell migration and invasion was NEDD9-dependent, while immunohistochemical analysis revealed that NEDD9 co-localized to invadopodia with MT1-MMP [
307]. Their following studies investigated the role of NEDD9 in secretion of MMPs, MMP-9 and MMP-2 in particular, the formation of invadopodia, as well as the interactions of NEDD9 with vimentin and non-muscle myosin IIA [
308,
309]. Consistent with their findings, the high-throughput gene expression profiling of HNSCC tumor samples has shown that overexpression of NEDD9 is associated with invasive HNSCC [
310]. Recent studies examined the potential involvement of stromal cells on the invadopodia formation and EMT induction in HNSCC. A study conducted by Gao et al. demonstrated that HNSCC cells were able to recruit and educate monocytes into M2 macrophages in a co-culture system via the CCL2/CCR2 axis, and these M2 macrophages then enhanced the invadopodia formation, thus invasion and migration of HNSCC cells. This study also implicated macrophages to be crucial for the induction of EMT in HNSCC cells, since the majority of macrophages have been detected at the leading front of the scratch during the wound healing assay [
311]. In a follow-up study Gao et al. implicated that upregulated levels of EGF and TGF-β secreted by TAMs in direct and indirect co-culture systems with HNSCC cells induce EMT of HNSCC cells via activation of the EGFR/ERK1/2 signaling pathway [
312]. Another study investigated the role of M1 and M2 macrophages in EMT induction in a co-culture system with tongue carcinoma cells, in which they showed that the interaction between cancer cells and M2 macrophages induces migration and invasion in 3D model. Macrophages as well as cancer cells exhibited altered secretome, such as upregulated expression of TGF-β, EGF and M-CSF [
313]. In contrast, a study by Smirnova et al. showed that although macrophages invade together with tumor cells in vivo, the invasion of HNSCC cells was not macrophage-dependent [
314]. TAMs produce macrophage migration inhibitory factor (MIF), which has been associated with EMT in many types of cancer including HNSCC. Zheng et al. demonstrated that knock-down of MIF inhibited proliferation and migration of OSCC cells [
315]. Another study showed that neutrophils can be recruited by HNSCC-derived MIF via a CXCR2 mechanism in vitro. In addition, MIF promoted invasive phenotype of HNSCC cells via neutrophil-secreted CCL4 and MMP9 [
316]. Trellakis et al. observed that neutrophils from HNSCC patients displayed reduced apoptosis compared with healthy donors, which has been associated with upregulated secretion of HNSCC-derived MIF [
317]. Furthermore, neutrophils have been linked to the invadopodia formation in HNSCC cancer cells. Glogauer et al. demonstrated that a co-culture system of neutrophils and OSCC cancer cells increased the invasiveness of OSCC, invadopodia formation and matrix degradation through increased secretion of TNF-α and IL-8 in a contact-independent manner [
318]. Also, a study conducted by Dumitru et al. has shown that neutrophils promote migration of HNSCC by increasing cortactin phosphorylation in cancer cells in vitro [
319]. The role of MDSCs in EMT induction of HNSCC has not yet been extensively studied. However, being a major source of MMP-9, EGF, bFGF and TGF-β, MDSCs have been heavily implicated with the EMT promotion and neoangiogenesis in several other types of cancer [
320‐
323]. Furthermore, there is increasing evidence MDSCs may play a crucial role in establishing the pre-metastatic niche. The exact mechanism of the pre-metastatic niche formation has not yet been fully described, however, it has been suggested the microenvironment of the distant organ site can be altered by the primary tumor itself prior to tumor cell dissemination. Primary tumor cells promote the formation of supportive metastatic microenvironment via secretion of various cytokines and growth factors, such as VEGF, placental growth factor (PlGF), TGF-β and TNF-α, granulocyte-colony forming factor (G-CSF), versican and lysyl oxidase (LOX) into the circulation to mobilize and recruit other supporting cells that interact with stromal cells and ECM of the secondary site, thus establishing the microenvironment suitable for the formation of metastases [
324]. Sceneay at al. suggested that tumor-derived monocyte chemoattractant protein-1 (MCP-1) regulates the accumulation of MDSC in the pre-metastatic niche. In addition, although also the number the NK cells in the pre-metastatic niche was increased, their cytotoxic effector function was compromised, which resulted in metastasis formation [
325]. Another study conducted by Wang et al. demonstrated that VEGFA secreted by cancer cells stimulates TAMs to produce CXCL1, which results in the recruitment of MDSCs to form the pre-metastatic niche [
326]. Shi et al. reported that mo-MDSCs accumulate in the lungs of tumor-bearing mice before the arrival of tumor cells and that these cells secrete IL-1β to stimulate expression of E-selectin, which results in metastasis formation [
327]. The mechanism of pre-metastatic niche formation in HNSCC, however, has not yet been extensively investigated. It has been demonstrated that MDSCs, as well as neutrophils and macrophages, can be recruited to the tumor site via inflammatory protein calprotectin (S100A8/A9; MRP8/14) [
328‐
330]. During inflammation, calprotectin is actively secreted by many types of cells in the microenvironment, such as neutrophils, macrophages, monocytes and MDSCs to modulate the inflammatory response by pro-inflammatory cytokine secretion, reactive oxygen species (ROS) and nitric oxide (NO) [
331‐
333]. The role of calprotectin in EMT has not yet been fully elucidated; however, it has been implicated in the promotion of metastatic spread by MDSCs [
334]. It has been reported, calprotectin activates the MAPK and NF-κB signaling in cancer cells, thus promoting metastasis [
335‐
337] and is strongly upregulated in several types of cancer [
338]. However, the levels of expression of calprotectin in primary HNSCC are downregulated compared with other types of cancer [
339‐
342]. Silva et al. reported, that in HNSCC calprotectin contributes to the regulation of MMP-2 expression and secretion in the 3D cell culture, thus inhibiting invasion and migration of cancer cells [
343].
Representing the most abundant cell type within the tumor microenvironment, the role of CAFs in the process of EMT in many types of cancer, including HNSCC, has been intensely researched. Many studies show that the presence of CAFs promotes cancer cell invasion [
22,
344‐
349]. It has been reported CAFs enhance the invasion of cancer cells via various mechanisms, such as MMP-mediated ECM degradation and subsequent release of latent growth factors [
22]; matrix stiffening through integrin-mediated mechanotransduction and through actomyosin contractility [
150,
350]; secretion of soluble factors, including HGF and TGF-β [
345,
351,
352]; secretion of exosomes [
55]; and direct cell-cell contact [
353]. The stimulating effect of CAFs on HNSCC invasion has been described by various in vitro assays [
354‐
356]. The possible contribution of CAFs to the EMT induction in HNSCC carcinoma cells has been implicated by immunohistochemical analyses, in which markers associated with EMT in CAFs in paired primary and metastatic OSCC showed that Ki-67+ metastatic carcinoma cells downregulate E-cadherin when in direct contact with CAFs [
357]. In addition, various in vitro studies demonstrated that EMT in HNSCC cells can be induced by CAF-derived molecules, such as SDF-1 via activation of the PI3K-Akt/PKB signaling pathway [
358], TGF-β1 via the TGF-β/Smad signaling pathway [
359], endothelin-1 [
360] and CCL-7 [
17]. Richter et al. demonstrated that TGFβ1/ EGF long-term co-stimulation enhances the invasive phenotype of OSCC, such as significantly upregulated expression of MMP-2 and MMP-9, compared with single growth factor stimulation [
361]. A study conducted by Wu et al. examined the effect of Gal-1 on OSCC cell invasion and migration. It has been observed that blocking Gal-1 expression inhibits cancer cell migration and invasion induced by CAF-conditioned medium via MCP-1/CCR2 signaling pathway. Furthermore, in vivo study revealed that Gal-1 knockdown in CAFs efficiently inhibits metastasis in vivo [
362]. Knowles et al. reported that HNSCC-derived CAFs contribute to the HNSCC invasion and metastasis via activation of the HGF/c-Met signaling axis in vitro [
363]. Their following study showed the effects of CAFs on HNSCC metastasis in a mouse model. The co-injection of CAFs with HNSCC cells resulted in increased tumor growth, disease spread to the lymph nodes and lung metastases when compared to the injection of HNSCC cells alone [
364]. Several studies also report that IL-1 secretion of OSCC cells stimulates TGF-β and HGF production by CAFs, which promotes invasion of cancer cells in vitro [
365,
366]. In addition, Lewis et al. show that cancer cell-derived TGF-β1 directly induced the activated phenotype in CAF, which in turn stimulate the OSCC invasion via the HGF production [
367].
Beside the stromal components of tumor environment, it is reasonable to assume that also hypoxia, a crucial hallmark of cancer, may play a major role in the formation of invadopodia, in the induction of EMT and in promotion of migration and invasion of cancer cells. It has been reported that expressions of EMT promoters, Snail, Slug, TWIST and SMAD nuclear interacting protein-1(SNIP1), which are regulated by HIF-1α, correlate with induction of EMT phenotype in OSCC cells in vitro [
368‐
370]. A study by Huang et al. reported that SLUG regulated the expression of MT4-MMP under hypoxia, which promoted the invasiveness of HNSCC cell lines [
371]. Yang et al. demonstrated that hypoxia-induced TWIST activated BMI1 expression and a knock down of TWIST reversed the EMT and invasive phenotype in HNSCC under hypoxia in vitro [
372]. It has been suggested that hypoxia induces EMT in OSCC via activation of the Notch signaling pathway and the inhibition of the Notch signaling pathway suppresses EMT [
373]. These results are consistent with a study by Diaz et al. showing that hypoxia potentiates the invadopodia formation and ECM degradation in HNSCC in a HIF-1α-dependent manner. Furthermore, their results also implicate that the invasive phenotype of cancer cells is regulated by cell contact-dependent hypoxia-mediated Notch signaling coupled with the paracrine activation of the EGFR, which is mediated by the ADAM12-dependent secretion of HB-EGF [
374]. A recent study suggests that hypoxic conditions promote EMT, metastasis and glycolysis in HNSCC via positive feedback loop between metadherin (MTDH) and HIF-1α. The study showed that hypoxia increased the expression levels of genes associated with glycolysis, such as MCT1, MCT4, GLUT1 and LDHA in HNSCC cells and stimulated uptake of glucose, production of lactate and cell invasion in vitro [
375]. Several studies suggest that targeting the pathways associated with altered tumor metabolism impairs EMT, migration and invasion of HNSCC. A recent study by Li et al. demonstrated that blockage of glycolysis via targeting PFKFB3 suppressed the migration and invasion of HNSCC cells by inhibiting the invadopodia formation of HNSCC cancer cells in vitro and in vivo [
376]. A study by Xu et al. showed that blockage of glycolysis by 2-DG reversed EGF-induced EMT in OSCC in vitro and moreover, the treatment of 2-DG reduced the metastatic spread to regional lymph nodes in vivo [
377]. A report by Wang et al. indicates that HNSCC cell invasion and glucose metabolism is regulated via the transcription factor tripartite motif containing 24 (TRIM24)-mediated GLUT3 induction [
378]. Similar results were shown in a study by Chang et al. which provided evidence that the HNSCC cell migration and invasion are regulated by the activation of the GLUT4-TRIM24 axis [
379].