Matrix‐based molecular mechanisms, targeting and diagnostics in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a head and neck cancer (HNC) with a high mortality rate. OSCC is developed in the oral cavity and it is triggered by many etiologic factors and can metastasize both regionally and distantly. Recent research advances in OSCC improved our understanding on the molecular mechanisms involved in and the initiation of OSCC metastasis. The key roles of the extracellular matrix (ECM) in OSCC are an emerging area of intensive research as the ECM macromolecular network is actively involved in events that regulate cellular morphological and functional properties, transcription and cell signaling mechanisms in invasion and metastasis. The provisional matrix that is formed by cancer cells is profoundly different in composition and functions as compared with the matrix of normal tissue. Fibroblasts are mainly responsible for matrix production and remodeling, but in cancer, the tumor matrix in the tumor microenvironment (TME) also originates from cancer cells. Even though extensive research has been conducted on the role of ECM in regulating cancer pathogenesis, its role in modulating OSCC is less elucidated since there are several issues yet to be fully understood. This critical review is focused on recent research as to present and discuss on the involvement of ECM macromolecular effectors (i.e., proteoglycans, integrins, matrix metalloproteinases) in OSCC development and progression.


| INTRODUCTION
Oral squamous cell carcinoma (OSCC) is a malignancy originating from the squamous cells lining the oral cavity, including the lips, buccal mucosa, gingiva and alveolar ridge, tongue, the floor of the mouth, hard and soft palate, retromolar trigone and the anterior tonsillar pillar.OSCC is the most common malignant tumor type in the oral cavity (90%) and along with the counterpart of the oropharynx, is the common cancer in the whole head and neck region. 1 Specifically, smoking and alcohol remain the most common risk factors for OSCC, especially in the Western world.In South Asian countries, smokeless tobacco products and areca nut products are the most important etiologic factors associated with OSCC.In India, OSCC is the most diagnosed cancer in male patients, whereas it accounts for 1%-4% of all cancers in the Western world. 2,3According to the latest worldwide cancer data, OSCC is responsible for more than 370,000 new cancer cases and 170,000 cancerrelated deaths. 4Despite great efforts in treating OSCC, the 5-year survival rate is still <50%.The main reasons for the poor prognosis may be related to recurrence and the metastasis, which could be due to incomplete resection of the tumor and neglected metastases. 5nhealthy oral conditions, and persistent viral infections, such as human papillomavirus (HPV), are also included in OSCC risk factors (Figure 1). 1 Lack of knowledge, environmental factors and behavioral risk factors indicate large disparities in incidence worldwide and increased mortality rates. 1 Diagnosis of cancer at an early stage is a key factor in preventing further physical, psychological and financial losses to the patient.Early diagnosis can initiate timely and appropriate treatment, which can increase survival rates by up to 90%. 6 With advances in science and technology, many new techniques are being developed that offer advantages over currently practiced conventional diagnostic methods.
The extracellular matrix (ECM) is a threedimensional (3D) network of macromolecules that provides structural and biochemical support to surrounding cells and plays critical roles in cancer development and progression.ECM plays a crucial role in OSCC development and propagation. 7,8ECM consists of proteins/ glycoproteins, proteoglycans (PGs), matrix-degrading enzymes and several other molecules that provide structural integrity to the TME and contribute to cellular signaling. 9Dynamic changes in the ECM conformation and organization affect tumor behavior and evolution in OSCC.ECM effectors are molecules, including matrix metalloproteinases (MMPs), integrins and various growth factors that interact with the ECM and modulate its structure, organization and functional properties. 10These effectors guide ECM turnover via enzymatic degradation, crosslinking and de novo synthesis of ECM components.They play a critical role in ECM remodeling, tissue homeostasis and cellular responses to the ECM environment.These effectors also regulate tumor behavior by influencing angiogenesis, invasion, migration and cancer cell survival and thus contributing in OSCC pathogenesis and evolution. 10Considering the significant factors related to abnormal ECM remodeling, integrin activation and growth factor-mediated signaling, insights might be provided on their pivotal contribution to the aggressiveness, invasion, metastasis and poor prognosis of OSCC. 11oreover, understanding the significance of the ECM in OSCC is crucial for decoding the complex interplay between tumor cells and their microenvironment.Unravelling the role of ECM in OSCC can provide insights into novel diagnostic and therapeutic strategies targeting ECM effectors and signaling pathways. 11,12This review article discusses on the role and involvement of ECM and its effectors in OSCC development, as well as on potential matrix-based diagnostic, prognostic and therapeutic strategies for OSCC management.

| MATRIX COMPOSITION, STRUCTURE AND FUNCTION IN OSCC
ECM remodeling is a dynamic procedure that can be precisely controlled as a result of the ECM's ability to continuously change its composition in response to biochemical or mechanical signals. 10,13Persistent matrix dysregulation can result in life-threatening pathological conditions because tightly regulated ECM homeostasis is necessary for development, wound healing and normal organ homeostasis. 14,15Cancer development and progression necessitate extensive restructuring of the ECM, a crucial structural component of the TME.Solid tumors are complex structures composed from tumor cells, stroma, blood vessels, ECM and various immune cells.The TME makes up most of the total tumor mass and is critical to tumorigenesis and cancer progression.The ECM of solid tumors differs considerably from that in normal tissues. 15Intratumoral signaling, transport mechanisms, metabolism, oxygenation and immunogenicity are strongly affected when not controlled by the ECM.To understand the specific features of the ECM in solid tumors is necessary to develop approaches to influence its negative effects.Both the ECM itself and the mechanical stimuli is triggered by ECM stiffening and can activate cell membrane receptors and several mechanosensors, thereby modulating the malignant phenotype of tumor and stromal cells. 13,16By directly stimulating cell survival, migration, proliferation and differentiation, the abnormal ECM influences the development of cancer. 15,17By deregulating stromal cell behavior and modulating signaling cascades mediated by interactions with cell surface receptors, the remodeled ECM and its degradation fragments may promote the emergence of an oncogenic microenvironment.The ECM controls connections between cells or between cells and the matrix, acting as a mechanical support for tumor cells.Hypoxia, acidosis, oxygen free radicals produced by invading inflammatory cells, proteases secreted by tumor or stromal cells and others can all cause changes in the ECM (Figure 2). 18Cell invasion and metastatic spread, for instance, are influenced by changes in PG expression and localization in TME.Furthermore, the degradation of ECM components by proteases can lead to the secretion of angiogenic growth factors but also to the release of PGs or other protein fragments called matricryptins or matrikines. 19,20enerally, oral cancers metastasize to regional lymph nodes before metastasizing to distant sites, and this ability of regional lymph node metastasis is considered to be a factor that determines and influences the disease survival rate in OSCC patients. 21The disruption of the basement membrane (BM)-which is a thin layer of specialized ECM underlying cells, composed largely of collagen IV, along with laminin, elastin, fibronectin and various types of PGs-is defined as a critical event of tumor invasion that marks the beginning of the metastatic cascade, so it is possible to better understand how cancer cells acquire the invasive phenotype by observing how they interact with the BM. 15,22Several substances within the ECM, such as laminins and fibronectins, also contribute to tumor development. 23During tumorigenesis, degradation of the ECM may lead to the release of growth factors and cytokines that are sequestered by the ECM, thereby triggering tumor cell growth, angiogenesis and inflammation.Degradation of ECM and BM via the MMPs action contributes to the OSCC tumorigenesis and its establishment. 12It is worth noticing that MMPs are heavily involved in ECM degradation and remodeling, enabling tumor invasion and metastasis, by breaking down barriers and releasing bioactive molecules. 24Integrins as cell surface receptors, mediate the cell-matrix interactions influencing adhesion, migration and signal transduction in HNC and OSCC cells. 25,26ertain growth factors and cytokines can modify OSCC cell behavior. 27Notably, transforming growth factor beta (TGF-β), can induce epithelial-to-mesenchymal transition (EMT) and enhance tumor invasiveness and metastasis, 28 whereas epidermal growth factor (EGF) could enhance cell proliferation and survival. 29These ECM effectors contribute to OSCC progression by activating intracellular signaling pathways and affecting cellular responses.The ECM degradation enhances the motility of cancer cells within the TME and further supports the spreading of tumor cells into adjacent spaces.Cell motility, a prerequisite for tumor cell invasion, is therefore subject to a coordinated balance between cell adhesion receptors and the ECM. 7Notably, oral fibroblasts highly express hyaluronan synthase 3 (HAS3) and in addition increased levels of MMP-2 and -3, which have been shown to contribute to ECM and tissue remodeling as well as in oral inflammatory diseases and OSCC. 7Several ECM effectors have also been shown to be associated with the development of fibroproliferative disorders.

| FUNCTIONS OF MATRIX METALLOPROTEINASES AND THEIR ENDOGENOUS INHIBITORS IN OSCC
MMPs exert proteinase activity by degrading a plethora of ECM components resulting in ECM turnover.Numerous biological processes rely on MMPs, such as embryogenesis, angiogenesis, tissue remodeling and wound healing. 24The human MMP family of endopeptidases contains 23 different enzymes.MMPs are zinc-containing and calcium-dependent and their major function is to degrade and remodel the proteins/glycoproteins/PGs in the 3D ECM network. 30MMPs are also classified, according to their structure, into eight groups of which five are secreted and three are bound to membranes. 11,30Based on their subcellular distribution and specificity for the ECM components, MMPs are divided into membranetype matrix MMPs (MT-MMPs), collagenases, gelatinases, stromelysins and matrilysins. 10,31 loss of MMP activity control can lead to development of several diseases, among them cancer, atherosclerosis and fibrosis.The activities of most MMPs are very low or negligible in normal tissues, but the expression is The pathophysiological processes that exploit cancer cell plasticity during tumor progression, invasion and metastasis.Cancer cell from the primary tumor site invade the basement membrane and establish the formation of the provisional matrix that radically reprogram primary tumor cells and evoke EMT process to establish the aggressive behavior of cancer cells.The reprogrammed cancer cells intravasate into the blood stream and circulating cancer cells extravasate, accompanied by the platelets.The interplay of the premetastatic niche, through the aberrant ECM deposition (i.e., proteoglycans, growth factors, proteolytic and non-proteolytic enzymes), and the release of matrix-derived microvesicles (i.e., exosomes) creates a favorable microenvironment for the cancer cells to metastasize in distant tissues.Excessive matrix remodeling increased inflammatory response, autophagy and hypoxia are the major traits of the premetastatic niche that enables cancer cell settlement in secondary tumor sites and the initiation of metastasis.
transcriptionally controlled by inflammatory cytokines, growth factors, hormones, cell-cell and cell-matrix interactions. 14,24,30,32The activities of MMPs are tightly regulated at the level of transcription, activation of precursor zymogens, interaction with specific ECM components and inhibition by the endogenous inhibitors of MMPs (TIMPs). 7,14,15TIMPs, a family of four members with a molecular weight of 21 kDa, are specific inhibitors involved in the control of local activities of MMPs in tissues. 33They inhibit MMPs by reversible blocking and in this way indirectly regulate ECM remodeling and cell signaling via ECM molecules. 33Their roles are critical in ECM remodeling since an imbalance between active MMPs and TIMPs in favor of increased MMP activity may lead to an inappropriate loss of ECM, or conversely, an imbalance in favor of TIMPs may abrogate MMP activity and lead to excessive deposition of ECM.The presence or absence of TIMPs may influence this MMPdependent growth factor regulation. 34 Increased production of MMPs in the ΤΜΕ, appears to be the key event in tumor invasion. 24,34An emerging concept in the field of tumor invasion is that proteolytic activity on ECM substrates reveals or releases cryptic sites of ECM macromolecules that modulate cellular responses.Among these ECM macromolecules, laminin 5 is a factor that stimulates tumor invasion.MMPs such as MMP-3, MMP-7, MMP-9 and MMP-12 can also degrade plasminogen and generate the N-terminal fragment angiostatin that inhibits endothelial cell proliferation. 35The detection of large amounts of MMPs in many cancers and their association with poor prognosis has underscored their role as potential therapeutic targets. 11Therefore, new therapeutic approaches using anti-MMP agents in combination with classical chemotherapy have been developed in the last 10 years.Unfortunately, these new treatments have not yielded significant results to date because they are associated with significant side effects.Indeed, MMPs have numerous physiological functions that can be altered by the action of these new drugs.Therefore, special efforts are being made to target anti-MMP drugs to the TME selectively.A more targeted effect against MMPs more specifically involved in tumor invasions, such as MMP-2 and MMP-14, could also improve therapeutic approaches. 24,34,36Nonetheless, a few reports highlight the tumor-suppressive roles of specific MMPs, including MMP-8 in non-metastatic breast cancer cells, MMP-9 in node-negative breast cancer, MMP-12 in hepatocellular and colon carcinoma and MMP-26 in diverse hormone-dependent malignancies. 37Notably, the collagenase MMP-8 is the only tumor-suppressive MMP in OSCC and tongue SCC, since it favors better prognosis and lower mortality. 38Salivary MMP-8 levels are reduced in OSCC patients as compared with disease-free samples. 39It is established that the major MMP-8 activator is the cysteine switch mechanism that is mediated by MT1-MMP, 40 suggesting the potential role of MT1-MMPevoked MMP-8 tumor-suppressive properties in OSCC.
Various studies have been conducted to access the possible usefulness of MMPs and their inhibitors in OSCC.Kurahara et al., 41 first examined whether MMPs immunohistochemically expressed in 96 cases of primary OSCC had any association with metastatic and invasive potential.They found that increased expression of MMP-1, -2 and -9 was significantly correlated with a decrease in ECM staining.Moreover, MMP-1, -2, -3, -9 and MT1-MMP were prominently expressed in invasive and metastatic cases.TIMP-1 levels were significantly increased in metastatic cases.According to the authors, tumor development depends on cancer cells' ability to degrade ECMs and metastatic tumor potential is controlled by MMPs overproduction rather than TIMP production.In another study, both TIMP-1 and -2 were expressed in tumoral and stromal OSCC tissues and were also significantly correlated with cyclin D1 and p53 expressions. 42Notably, TIMP-2 was significantly correlated with advanced disease, local recurrence and poor survival.Chen et al. 43 studied MMPs, TIMP-2 and TGF-β1 in normal oral mucosa, lichen planus and OSCC specimens.They found a progressively increased expression of all effectors from normal tissue till OSCC.They suggested that the imbalance between MMPs and their inhibitors may be responsible for the cancerization process of oral lichen planus to OSCC and that MMP-9 could be a useful biomarker indicating the potency of this malignant transformation.
Subsequent studies confirmed similar results, especially for MMP-2 and MMP-9 and their inhibitors. 44,45The activated MMP-2 could be the main gelatinolytic factor in OSCC, as the MMP-2 activated form/proform ratio is higher in advanced OSCC clinical stage groups.Moreover, the proportion in lymph node metastasis cases has also been found significantly higher than that in non-lymph node metastasis cases. 46MMP-2 expression was related to various signal-transduction pathways like extracellular regulatory mitogen-activated kinase (ERK1/2) and Wntβ-catenin pathways. 47Specifically, the ERK1/2-driven stimulation of MMP-2 was related with the invasion and metastatic capacity of cancer.Moreover, treatment of epigallocatechin-3-gallate/mitogen extracellular kinase (EGCG/MEK) inhibitor (PD98059) reduced MMP-2 activity and invasion/migration potential in OSCC patients.Yin et al., 48 using a BALB/c nude mouse tonguexenografted metastasis model, demonstrated that MMP-9 knockdown could significantly suppress cancer cell migration and proliferation, tumor growth, angiogenesis and interactions between endothelial cells.Moreover, it could considerably inhibited OSCC cell metastasis to regional and distal lymph nodes.The authors concluded that MMP-9 plays a key role in OSCC development and spread through a potential mechanism mediated by Ras homolog family member C (RhoC) and Sarcoma (Src) gene expression.][51] Several other MMPs have been reported to demonstrate higher expression in OSCC patients.Cai et al. 52 reported high expression of several angiogenic factors and MMP-1, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13 and TIMP-2, in tumor tissues but also in saliva of OSCC patients.Using bioinformatic analysis demonstrated the correlation of these effectors with patient survival and cancer functional states, suggesting them as potent noninvasive diagnostic/prognostic tools and therapeutic targets of OSCC.According to a recent review, the most promising salivary markers for OSCC diagnosis were MMP-1, MMP-3 and MMP-9, but larger studies are needed to confirm their clinical utility. 53MMP-12 and MMP-21 have been demonstrated to play an essential role in tumor evolution and may be useful non-invasive early diagnostic tools for detecting OSCC, [54][55][56] as well as therapeutic interventions in the future.Zong-Yun and Feng 57 reported that downregulation of growth inhibitory factor 4 (ING4) could stimulate upregulation of hypoxiainducible factor-1α (HIF-1α) and MMP-14, promoting development, EMT, invasion and metastasis of OSCC, thus suggesting that these effectors could be used as significant prognostic markers.High MMP-14 expression levels have also been detected in metastatic neck lymph nodes specimens and precisely predicted extranodal extension and poor prognosis through immuno-histochemical assessment of small biopsies.Specifically, this study confirmed high MMP-14 enzymatic activity in extranodal extensions of OSCC cells that facilitate lymph node metastasis.Thus, MMP-14 could be also used in routine pathological diagnosis and proper treatment planning. 58

| ECM IN CELL SIGNALING, REMODELING AND METASTASIS IN OSCC
Numerous growth factors directly interact with ECM biomolecules, which then control cellular responses.Growth factors can be stored in the ECM and then released, free to diffuse through the tissue until they meet the respective growth factor receptor.Growth factors can be also bound to the ECM.In this way ECM can prevent growth factors from degrading or assist in the formation of concentration gradients, which are crucial for guiding cell migration to a specific location within the tissue.ECM binding controls cellular reactions to growth factors in this manner.Immune cells secrete cytokines (i.e., IL-4, IL-13 and IL-33), as well as growth factors (i.e., TGF-β), which promote the synthesis of ECM components. 59During ECM remodeling growth factors and active fragments can be released.Therefore, several significant cytokines, such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF) and vascular endothelial growth factors (VEGFs), are released during the processing of the ECM. 60Due to their central roles in lymphangiogenesis and angiogenesis, these molecules are essential for the development of tumors. 61In head and neck squamous cell carcinoma (HNSCC), lymph node metastasis and a poor prognosis are linked to high expression of VEGF-C and VEGF-D, as well as of other cytokines like angiopoietins, IGF-I and FGF. 62ntegrins are typical transmembrane gatekeeper proteins that regulate the extracellular-intracellular signaling process mediated by the ECM.Upon the engagement with particular ECM molecules, the activation of integrin results in the recruitment, assembly and phosphorylation of intracellular signaling complexes, such as focal adhesion kinase (FAK) and the non-receptor tyrosine kinase, Src.FAK and Src both play important roles in mediating cell motility in response to the ECM and adhesion dynamics.FAK/Src activation may modify the activity of the MAPK and PI3K/Akt pathways, both of which control transcription to mediate cell proliferation, differentiation and migration.The interaction between integrins, particularly integrin α5, and the ECM, which activates the FAK/PI3K/Akt signaling cascade and protects cells from drug-induced apoptosis, is a crucial mechanism through which the ECM impacts the SCC chemoresistance of upper aerodigestive tract. 11,63,64In fact, SCC tumors frequently exhibit altered integrin expression, which affects the activation of the MAPK/ERK signaling pathways.For instance, the upregulation of integrin α5 is associated with altered PI3K/Akt activation, whereas the T188I mutation of integrin β1 results in sustained ERK activation. 65he "Disease-free Survival Rate" in patients with OSCC is thought to be governed and influenced by the ability of cancer cells to form regional lymph node metastasis before metastasizing to distant sites. 20Throughout the metastatic cascade, the BM confronts connective tissue barriers at various critical locations.These BMs lack pores that would permit passive tumor cell migration and are made up of a dense meshwork of collagen type IV, laminin and heparan sulfate PGs.Therefore, metastatic spread is a multistep process that is characterized by the invasive phenotype that is dominated by tumor cells' capacity to adhere to the ECM, break down matrix elements, and then pass through these matrix defects. 66ccording to reports, the frequency of laminin and collagen IV discontinuity from healthy epithelium to hyperplastic, dysplastic and OSCCs has gradually increased. 67aminin and collagen IV staining patterns in primary OSCCs were found to be like those in metastatic nodules by Harada et al., who also noted that the cellular population of the deep areas displayed the invasive and metastatic potential of oral carcinoma. 68In OSCC, the laminin isoform Ln-5 has been studied well.Due to its dual functionality, Ln-5 promote both adhesion and migration.Whether Ln-5 is an adhesive or migratory factor is determined by proteolytic processing, which controls Ln-5's dual function. 69,70It has been shown that OSCC exhibits loss of Ln-5 expression, which favors the binding of other ligands like collagen or fibronectin that may be more favorable to tumor growth.In addition to stimulating tumor growth, the unoccupied Ln-5 receptors can also bind laminin isoforms that promote keratinocyte proliferation, such as Ln-10/11, which binds to the same integrin receptors as Ln-5. 71he ECM plays an integral role in tumor invasion and metastatic development, particularly in OSCC. 12Signals from host cells that typically reduce MMP proteolytic activity and matrix turnover and thereby provide protection against tumor growth, allow OSCC cells to utilize the ECM to their advantage and lead to OSCC progression. 72While TIMPs regulate proenzyme activation that results in an invasive phenotype, 73 OSCC cells synthesize and secrete MMPs, which play a significant role in the release of bioactive fragments of ECM proteins.The proteolytic activity of MMPs on ECM components in the oral epithelium results in the release of peptides termed as "matrikines," 18 which have profound implications on cell proliferation, migration, apoptosis and angiogenesis, furthering the metastatic potential of OSCC. 12,74Matrikines, produced during the Ln-5 precursor proteolytic processing, such as the α3 and β3 chains, have been identified to promote migration. 74The γ2 chain of the Ln-5 precursor and its N-terminal proteolytic fragments have been located in patients with pancreatic cancer 75 and at the invasive fronts of OSCC, 76 highlighting the ECM's active role in OSCC progression.There is growing evidence that neutrophil-mediated ECM remodeling accelerates the invasiveness of OSCC tumors. 77eutrophils facilitate the creation of invadopodia and the degradation of the collagenous matrix by OSCC cells, a process potentiated by neutrophil-derived TNF-α after IL-8 recruitment. 78etastasis, the primary cause of cancer-related mortality, is a result of unregulated growth of tumor cells within healthy tissue.The ability of OSCC cells to metastasize depends on their ability to adapt to varied tissue environments.The functional adaptations that cancer cells make to survive in an altered environment are referred to as "cancer cell phenotypic plasticity" and play a role in OSCC progression within the ECM milieu. 79mong these, EMT and its reverse, MET, are thoroughly studied. 80,81In EMT, epithelial cells lose their polarity and adhesion, giving them invasive capabilities to become mesenchymal stem cells. 82In the context of OSCC, increased motility, expression of ECM remodeling enzymes as well as enhanced cell survival are all associated with this transformation. 83The dedifferentiation of cancer cells, which endows them with stem cell characteristics, is a crucial step of this ECM-based phenotypic plasticity and allow them to remain dormant, eluding the immune system and treatments. 82,84Figure 2 summarizes the pathophysiological processes of OSCC metastasis that require cancer cell phenotypic plasticity, which is achieved through ECM continuous adaptation.
Normal interactions between parenchyma and stroma are characterized by (i) two-way communication through molecular messengers secreted into the TME, (ii) biochemical and biophysical signals emanating from the ECM and (iii) direct cell-cell contact that allows two-way signaling between the two cell types. 85These interactions control tissue homeostasis and the formation of niches with distinct microenvironmental features that facilitate the maintenance of specialized cell types, including stem cells.Under abnormal conditions, when parenchymal cells acquire tumor-causing genetic lesions, the TME (both cellular and acellular ECM components) is remodeled under the influence of the growing tumor and organism, resulting in abnormal tissue homeostasis and disruption of specialized niches. 79hese changes in the TME have a major impact on disease progression.All of the aforementioned are important adaptations required for BM crossing by the tumor cell and promotion of abnormal tumor cell-ECM interactions, intravasation and survival in the circulation. 86

| ECM EFFECTORS AND IMMUNE MODULATION IN OSCC
Matrix effectors modulate multiple signaling cascades associated with inflammatory responses in patients with OSCC.Cox regression analysis identified three immune cell types that may be important for the prognosis of OSCC patients. 87The nomogram showed that high infiltration of myeloid dendritic cells, CD4 + T cells with central memory and common myeloid progenitor cells were associated with favorable prognosis.OSCC patients were divided into high-risk and low-risk groups based on the mean risk scores for the three immune cell types.Immune cell infiltration differed in the high-risk and low-risk groups and the risk score used to predict the prognosis of OSCC patients.In addition, the low-risk group had a significantly higher survival probability than the high-risk group. 88n the past, the prevailing belief was that the evasion of cancer cells from the immune system depended solely on cancer cells, however, there is increasing evidence that the tumor stroma cancer-associated fibroblasts (CAFs) can also promote evasion of cancer cells from the immune system in the TME. 899][90] In this way, CAFs can attract, retain and influence multiple immune cell subtypes into the TME, including macrophages, dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), natural killer (NK) cells and various subsets of T cells.New evidence supports the hypothesis that this is not a unidirectional effect. 90Tumor-infiltrating immune cells can also alter the stromal compartment, suggesting a high degree of interactions between stromal and immune cells. 91Moreover, CAFs can produce hyaluronan synthases (HASes), including HAS2, which modulate TME function.It has been suggested that HAS2 could be a critical regulator responsible for CAF-mediated OSCC development, acting by modulating the balance between MMP-1 and TIMP-1. 92

| MATRIX BIOMARKERS AND NOVEL TOOLS IN OSCC ASSESSMENT
Biomarkers are biological characteristics that can be identified and objectively assessed as indicators of normal or pathological biological processes. 93,94A prognostic biomarker can predict whether disease progression will be favorable or not independent of therapy, while a predictive biomarker can indicate how well therapy will work.Recent research has focused on the use of potential molecular biomarkers as reliable predictors of tumor aggressiveness based on phenotypic changes in OSCC cells.Increasing understanding of OSCC has revealed the crucial role played by ECM alterations during tumor growth, invasion and metastatic spread. 95,96CM is a significant factor in cancer and therefore ECM-derived biomarkers associated with OSCC progression and patient prognosis should be included in diagnostic and prognostic marker panels in clinical practice.Additionally, the mechanical properties of the ECM, driven by enzymatic crosslinking and remodeling, can also impact cancer cell behavior and can be potential areas for novel biomarkers discovery.96,97 Notably, the interplay between ECM and the TME cannot be understated.In a recent study, Wahbi et al. 98 investigated how HNSCC cells interact with different matrices, which simulate the TME.Four cell lines were tested on five different matrices and their growth, migration, invasion, morphology and gene profiles were studied.Results displayed that the matrix type significantly influenced the cancer cells behavior.Different matrices affected cell proliferation, migration and invasion variably, often depending on the specific cell line.The findings emphasize on the importance of selecting the appropriate matrix for in vitro studies to closely mimic in vivo conditions.In another study, a novel ECM named Lymphogel was developed to simulate the TME of metastatic carcinoma cells.Lymphogel's performance was compared with other matrices using oral tongue squamous cell carcinoma cell lines in various functional tests.Results indicated that cell lines showed varying adhesion and invasion patterns based on the matrix used.Overall, Lymphogel derived from human pre-metastatic neck lymph nodes seems to offer a promising model for studying metastatic OSCC cells in vitro.99 Some of the selected TME-associated biomarkers which are detailed in Table 1, illustrate the rapidly advancing frontiers in this area.Translational oncology is particularly augmented by the advent of ECM biomarkers, which can guide therapeutic interventions.Particularly in immunotherapy, assessing ECM dynamics can serve as an indicator of therapeutic response.This is evidenced by the recent inventions like "liquid biopsies" which permit for non-invasive monitoring of ECM turnover in response to treatments like immune checkpoint inhibitor (ICI) therapies in OSCC. 100 With these advancements, it is anticipated that ECM-derived biomarkers will pave the way for more personalized and effective therapies for OSCC in the near future.Among these, spheroids, organoids and organotypic cultures are particularly useful when studying OSCC.102,103 Spheroid cultures, as an example, are widely used for assaying tumor-sphere formation, enabling researchers to examine the clonal evolution of tumor cells and their self-renewal potential.104 Furthermore, organoids derived from different types of human tumors provide a platform for testing drug sensitivity and resistance, providing a close representation of in vivo tumor heterogeneity and complexity.[105][106][107] As the 3D model technology and understanding have matured, their potential for advancing oral cancer research has increased.In addition to providing a more physiologically accurate representation of in vivo tumor tissues, such models are also helpful when optimizing anticancer drug dosage and assessing drug efficacy.108 Although 3D culture techniques were introduced to OSCC research somewhat later than expected, their rapid assimilation in recent years underlines their potential for transforming the field. Its noteworthy that the first organoids derived specifically from OSCC cells were only produced in 2018, illustrating the relatively new, but powerful intersection of this technology with oral cancer research.109,110 In addition to cellular structures, 3D models like organoids have been crucial in exploring nuances of oral tissue, TME dynamics, cancer features and even ECM biomarkers.As research progresses, these models promise to reveal complex microenvironmental interactions, especially concerning the role of ECM in cancer progression and metastasis.111 The growing innovations in 3D modeling platforms offer profound capabilities for emulating the in vivo pathology of OSCC.In the evolving landscape of cancer research, such advancements are poised to pave the way for unprecedented therapeutic exploration and intervention.112 T A B L E 1 Selected TME-associated ECM biomarkers.

Biomarker Function
Type The main strategies for treating OSCC are surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy or a combination of these modalities, depending on the severity of the disease. 5Despite the compromise to the function and aesthetics of the orofacial region, surgery remains the most effective treatment for OSCC.Surgery with radiotherapy, have difficulty in dealing with metastatic tumors. 113Chemotherapy could inhibit rapidly growing cells, even in metastatic areas, by inhibiting cell growth and division, while monoclonal antibodies and immune checkpoint inhibitors are very promising new therapeutic methods. 114Another promising treatment approach may involve inhibiting ECM elements that support tumor progression and metastasis. 115Through its regulatory control, the ECM influences not only the tumor's development, but also its response to therapy.A better comprehension on the way that ECM stiffness regulates tumor progression will contribute to new therapeutics.The regulators and effectors of ECM stiffness represent potential therapeutic targets for cancer.Stiffening of the ECM may activate the mechanosensor Piezo1, which mediates cancer metastasis triggered by mechanical forces. 116Understanding these interactions may help implement better therapeutic regimens for cancer treatment, where a combination of strategies appears to be more effective than single modalities, as tumor heterogeneity results from a multitude of signaling pathways/ crosstalks that exists in the network of communicating cancer cells.Suppressing the synthesis of ECM elements with protumor properties is one possible strategy.In a recent study on breast cancer cells, it was discovered that 4-methylumbelliferone can significantly lower estrogen receptor cell migration, adhesion and invasion as well as the expression and activity of several pro-tumorigenic, matrix degrading enzymes and pro-inflammatory molecules.According to the findings, 4-methylumbelliferone may be a new treatment depending on the presence of estrogen receptors by preventing the production of HA and controlling HAS2, CD44, matrix-degrading enzymes and inflammatory mediators. 117lthough various studies have been conducted exploring the possibility of ECM targeting in several cancers, little is known about the potential of ECM-based pharmacological targeting to improve response to anticancer therapy in OSCC.Considering the role of MMPs in OSCC progression, their inhibition as a therapeutic strategy still remains an option.However, challenges related to specificity and side effects have to be elucidated.Future antineoplastic approaches could either target directly ECM constituents/provisional matrix/premetastatic niche or to be used in combination with other established tumor-directed therapies.Understanding the interactions between cancer cells, ECM and TME may help implement better therapeutic regimens for cancer treatment, where a combination of strategies appears to be more effective than single modalities, as tumor heterogeneity results from a multitude of signaling pathways/ crosstalks that exists in the network of communicating cancer cells. 118Therefore, to improve the diagnostic efficiency and obtain a better prognosis, a comprehensive study on the molecular mechanism of OSCC should be conducted, and novel diagnostic tools and precise treatment methods should be explored.

| CONCLUSIONS
OSCC is a common HNC with a high rate of regional and distant spread.Research advances in OSCC improved our understanding on the molecular mechanisms involved in its invasion and metastasis.It is known that multiplex cell signaling mechanisms are regulated by ECM and its effectors.Several MMPs, integrins and various growth factors and cytokines interact with the ECM and modify its structure, organization and functional properties through enzymatic degradation, and de novo synthesis of matrix components.They play a vital role in ECM remodeling and stiffness, tissue homeostasis and cellular responses to matrix environment.These effectors also control tumor behavior by influencing angiogenesis, lymphangiogenesis, invasion, migration and cancer cell survival and therefore contributing to OSCC pathogenesis and evolution.As a result of a multitude of signaling pathways/crosstalk within the complex ECM network of communicating cancer cells, understanding these interactions might help develop novel cancer treatment regimens, where a combination of strategies appears more effective than a single therapy.ECMderived biomarkers have great potential as diagnostic and prognostic tools in translational oncology.Nonetheless, the development of novel 3D platforms may provide revolutionary capabilities for studying TME in OSCC through targeting specific biomarkers within the ECM.The improved tools for studying cancer evolution mirror the in vivo pathology of the disease, granting significant opportunities for exploring novel therapeutic approaches.literature review and writing; Alexander Delides, Evangelos Giotakis, Miranda Alexopoulou: conducted literature review and manuscript editing; Nikos K. Karamanos contributed to conceptualization, editing and supervision of the manuscript preparation.All authors read and approved the final manuscript.

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I G U R E 1 Key features related to the development of OSCC.Tobacco use, excessive alcohol use, unhealthy oral conditions and persistent viral infections predispose to developing OSCC.