Melanoma in the Eyes of Mechanobiology

fcell-08-00054 February 11, 2020 Time: 16:43 # 1 REVIEW published: 11 February 2020 doi: 10.3389/fcell.2020.00054 Edited by: Shamik Sen, Indian Institute of Technology Bombay, India Reviewed by: Zhizhan Gu, Dana-Farber Cancer Institute, United States Takashi Kato, Johns Hopkins University, United States *Correspondence: Manfred Radmacher [email protected] †ORCID: Pedro L. Granja orcid.org/0000-0003-2761-4929 Specialty section: This article was submitted to Cell Adhesion and Migration, a section of the journal Frontiers in Cell and Developmental Biology Received: 15 July 2019 Accepted: 21 January 2020 Published: 11 February 2020 Citation: Brás MM, Radmacher M, Sousa SR and Granja PL (2020) Melanoma in the Eyes of Mechanobiology. Front. Cell Dev. Biol. 8:54. doi: 10.3389/fcell.2020.00054 Melanoma in the Eyes of Mechanobiology M. Manuela Brás1,2,3, Manfred Radmacher4*, Susana R. Sousa1,2,5 and Pedro L. Granja1,2,3† 1Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal, 2Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal, 3Faculdade de Engenharia, Universidade do Porto, Porto, Portugal, 4Institute for Biophysics, University of Bremen, Bremen, Germany, 5Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Porto, Portugal Skin is the largest organ of the human body with several important functions that can be impaired by injury, genetic or chronic diseases. Among all skin diseases, melanoma is one of the most severe, which can lead to death, due to metastization. Mechanotransduction has a crucial role for motility, invasion, adhesion and metastization processes, since it deals with the response of cells to physical forces. Signaling pathways are important to understand how physical cues produced or mediated by the Extracellular Matrix (ECM), affect healthy and tumor cells. During these processes, several molecules in the nucleus and cytoplasm are activated. Melanocytes, keratinocytes, fibroblasts and the ECM, play a crucial role in melanoma formation. This manuscript will address the synergy among melanocytes, keratinocytes, fibroblasts cells and the ECM considering their mechanical contribution and relevance in this disease. Mechanical properties of melanoma cells can also be influenced by pigmentation, which can be associated with changes in stiffness. Mechanical changes can be related with the adhesion, migration, or invasiveness potential of melanoma cells promoting a high metastization capacity of this cancer. Mechanosensing, mechanotransduction, and mechanoresponse will be highlighted with respect to the motility, invasion, adhesion and metastization in melanoma cancer. Keywords: melanoma, mechanobiology, melanocytes, keratinocytes, invasion, adhesion, migration, metastization MELANOMA Melanoma is one of the most severe skin cancers in humans with a mortality rate of 80% (Hofschroer et al., 2017), due to the high resistance of this tumor to radiotherapy and chemotherapy (Uong and Zon, 2010). The resistance to those treatments is due to high number of mutations (approx. 75%) affecting specific genes, which lead to an increasing proliferation and survival (Kozar et al., 2019). Immunotherapy is being exploited and investigated, and can be grouped in five types: (i) targeted antibodies (Sanlorenzo et al., 2014;Herzberg and Fisher, 2016); (ii) adoptive cell therapy (Sanlorenzo et al., 2014); (iii) oncolytic virus therapy (Franklin et al., 2017); (iv) cancer vaccines (Tuettenberg et al., 2007;Rodriguez-Cerdeira et al., 2017); and (v) immunomodulators (Johnson et al., 2014;Faries, 2016;Cancer Research Institute, 2019). Chemotherapy and immunotherapy, can be combined (biochemotherapy) regarding the stage disease (Sanlorenzo et al., 2014;Kozar et al., 2019). The disease appears due to the malignant transformation of melanocytes located in Frontiers in Cell and Developmental Biology | www.frontiersin.org 1February 2020 | Volume 8 | Article 54 fcell-08-00054 February 11, 2020 Time: 16:43 # 2 Brás et al. Melanoma in the Eyes of Mechanobiology the stratum basale of the epidermis although it is not clear how a melanocyte becomes malignant (Uong and Zon, 2010). Bandarchi et al. (2010) suggested that a combination of up and down regulation of factors in several molecular pathways, seem to be the mechanism of transformation of normal into malignant melanocytes. If the disease is detected in an early stage, there is a high probability of disease control, however, when metastization occurs, the 5 years life expectancy drops to 14%. Some epidemiological evidence related to melanoma incidences in white and dark skinned population (Bandarchi et al., 2013) was presented by the authors whereas Bradford (2009) presented epidemiological data about the total numbers of melanoma incidences in the world. Dark skin results in a lower incidence of skin cancer due to the protection of the increased epidermal melanin, which filters UV light twice as good as white skin of Caucasian people (Bradford, 2009). Malignant melanoma are diagnosed by the ABCD rule, where A stands for asymmetry, B for borders irregularity, C for color variation and D for diameter higher than 6 mm (Bandarchi et al., 2010). Melanoma shows three perceptible steps in tumor progression: (i) one only affecting the epidermis; (ii) a second one affecting both the epidermis and the superficial papillary of the dermis; and (iii) a third one affecting the epidermis, dermis and hypodermis (vertical growth melanoma) (Bandarchi et al., 2010). Haass et al. (2005) divided the process of melanoma development in the following steps: (i) the damage of adhesion between keratinocytes by down-regulation of E-cadherin, P-cadherin, desmoglein, and connexins, which is influenced by growth factors produced by fibroblasts or keratinocytes; (ii) the interaction of melanoma with fibroblasts and/or melanoma with melanoma cells normally not found in melanocytes, is up-regulated by MCAM and N-cadherin; and (iii) alteration of integrin expression, inducing the loss of basement membrane anchorage (Haass et al., 2005). According to the At Melanoma Foundation (AIM), melanoma develops in a similar way than other cancers: the DNA of a gene that controls cell division and proliferation is mutated. This damaged gene results in a lack of control of cell division and growth. This DNA mutation is passed to the daughter cell during division. Melanoma is originated from a melanocyte that experienced too many mutations growing in an abnormal way. In the case of melanoma, this DNA mutation is caused by an overexposure to UV radiation, affecting the melanocytes (At Melanoma Foundation, 2014). When there is DNA damage, several genes may be affected, resulting in encoding errors of several molecules hampering their function (At Melanoma Foundation, 2014;Kunz, 2014) and there is a risk for a melanocyte to generate a melanoma. If the melanoma is not suppressed or treated, it will spread along the epidermis, after penetrating the dermis and hypodermis, getting in contact with the lymph and the blood vessels (At Melanoma Foundation, 2014). The main function of melanocytes is to produce melanin (responsible for skin pigmentation) that absorb UV light to avoid keratinocytes’ DNA damage (Hirobe, 2014). Keratinocytes will distribute melanin to the upper parts of the skin layer (Wang et al., 2017). Wang et al. (2017) also mentioned that hormonal factors, family history and cosmetics are the three main triggering and aggravating factors for melanoma development. When a primary melanoma is formed, it activates some signals, which will provide conditions that make the primary tumor strong such as: (i) attachment to the wall of the blood and/or lymphatic vessels, enabling the movement through a new organ; (ii) enough nutrients to grow; and (iii) it is resistant to the immune system. With the three conditions mentioned before the primary tumor has the capacity to promote the metastization (At Melanoma Foundation, 2014). Why can melanoma develop in some body parts that are not exposed to sunlight? It was suggested that there are different changes occurring in genes of tissue cells exposed, compared with the changes of genes of tissue cells not exposed to UV light. These mutations are transmitted into the next generations. Some genes appear during melanoma development, being not genetic. The transformation of melanocytes into melanoma cells is a very complex process, which involves several signaling pathways reviewed by Paluncic et al. (2016). Briefly, melanocytes are formed from their precursors: melanoblasts. Melanoblasts differentiate into melanocytes, which get mature and start melanin production on melanosomes, which will be transferred to keratinocytes. Melanomagenesis depends on the microenvironment, genetic and environmental factors. Although genetic factors are crucial, they are not sufficient to induce melanomagenesis (Paluncic et al., 2016). Melanoma tumors are different from patient to patient. The environment, where melanoma tumors grow, is very complex depending on the interactions with ECM, microvasculature, fibroblastic cells and changing concentration of growth factor, cytokines, and nutrients like glucose, oxygen, etc (Paluncic et al., 2016). Some signaling pathways are more involved in tumor development, whereas other signals are more crucial in the metastization process. The description of biochemical pathways signaling is beyond of this review, however the readers can find more information in different sources (Dissanayake et al., 2007;Straussman et al., 2012;Moriceau et al., 2015;Stark et al., 2015;Wang et al., 2016; Ahmed and Haass, 2018;Kodet et al., 2018). Melanoma can be pigmented or not pigmented. The color of melanoma is due to the existence of melanin, secreted and stored in melanosomes in the cytoplasm of melanocytes. There are two types of melanin: eumelanin (has a black color) and pheomelanin (has a red/yellow color). Normal melanocytes may or may not secret either type of melanin (Lassalle et al., 2003). Mechanical properties and cell pigmentation are correlated (Sarna et al., 2013, 2014). As melanoma needs a high amount of iron and copper, the development of substances that target these ions could be another methodology (Gorodetsky et al., 1986). MECHANOBIOLOGY OF SKIN CELLS Currently, mechanobiology brings together biologists and biophysicists in order to better understand the role of mechanical forces in cell motility, adhesion and invasion. For developing therapies that avoid the invasiveness of cancer cells, it is crucial to understand the mechanisms of tumor invasion. The mechanical stiffness of tumors is correlated with its invasiveness, which was demonstrated by a study with cancer ovarian cells: more invasion corresponds to softer cells resulting in deformation and shape Frontiers in Cell and Developmental Biology | www.frontiersin.org 2February 2020 | Volume 8 | Article 54 fcell-08-00054 February 11, 2020 Time: 16:43 # 3 Brás et al. Melanoma in the Eyes of Mechanobiology changes, being suitable for metastization (Swaminathan et al., 2011). Makale (2007) raised three hypotheses correlating cancer and cell mechanobiology: (i) the cell biology of tumor invasion is a key to understand cancer treatment; (ii) cancer cells activate physiological mechanisms and migratory processes that are used by cells usually during embryo morphogenesis; and (iii) forces that are sensed by the tissue and cells trigger the differentiation and spreading of tumors. In fact, mechanical forces are generated by tissues, being sensed by cells, during embryogenesis and tumor expansion (Makale, 2007). In melanoma the Epithelial to Mesenchymal Transition (EMT) switching, and forces behind this process (Wei et al., 2015) are crucial processes regarding the loss of connections between melanocyte and keratinocytes; melanocytes acquired the embryonic phenotype, promoting cells invasion, their intravasation in body fluids and the extravasation to other organs. Arriving at their target destination, tumor cells are reverted to their original phenotype known as Mesenchymal to Epithelial Transition (MET), establishing secondary tumors. The production of metalloproteinases (MMP) will induce ECM degradation, favoring metastization (Paluncic et al., 2016). Migration of collective cells interact with the extracellular environment via integrins (Hegerfeldt et al., 2002). When normal cells are compressed because of a tumor, these forces will be sensed by cell body, through membrane molecules, the integrins, which are connected to the cytoskeleton, and to the nucleus. All cells respond to these external forces organizing cellular components in the cell and generating biochemical signals that will help them to adapt to external physical pressure. The normal ECM produced by fibroblasts also induces pressure on tumor cells via its cytoskeleton, resulting in cell and nucleus deformation. A consequence of nucleus compression could be the modulation of gene expression that may induce changes in cell viability, promoting locomotion, due to the adoption of a more malignant phenotype. In spite of high tumor stroma stiffness, tumor cells are deformable and have some degree of plasticity (Makale, 2007). In melanoma, the relation among melanocytes, keratinocytes, fibroblasts and the ECM mediate and control this disease. In the next paragraphs, mechanobiology of these cell types and ECM contribution to melanoma will be addressed. The Role of Melanocytes Melanocytes are located in the basal layer of the epidermis, eyes, ears, hair and meninges (Bandarchi et al., 2013). Melanocytes have a dendritic shape (Figure 1). They are originated from migratory embryonic cells, called neural crest cells, which have a high capacity of migration (Uong and Zon, 2010). Melanocytes are the cells responsible for skin pigmentation, through the production of melanin, also named melanogenesis (Lassalle et al., 2003). They are present in the epidermis and exhibit long dendritic protrusions, which are extended through the epidermal keratinocytes. Melanocytes synthesize melanosomes and transport them to the keratinocytes (Zhang et al., 2004). Melanocytes adhere to keratinocytes through E-cadherins, Desmoglein (DSg)/Desmocollin (DSc), Connexins (CX) molecules (Li et al., 2003) and to the basement membrane through integrins (Wang et al., 2016). The ratio of melanocytes and keratinocytes is around 1:36/1:40. 8 to 12% of malignant melanoma are associated with mutations on CDKN2A (cyclindependent kinase Inhibitor 2) gene (Bandarchi et al., 2013). Uong and Zon (2010) also mentioned that the pathways involved in melanocyte development and proliferation start to be subverted in melanoma formation. BRAF and NRAS (a gene of the RAS family identified for the first time at human neuroblastoma) (Pugh et al., 2013) overactive mutations are found in 65 and 20% of melanomas, respectively. CDKN2A is lacking by melanoma cells, consequently the encoding suppressor genes (INK4a and ARF) are not active (Bandarchi et al., 2010). Malignant melanocytes are characterized by changes of biophysical properties (like shape, elasticity and stiffness) which will contribute to the cell locomotion, invasion and metastization in other organs. In this case, the plasticity of melanocytes is very important, since they need to be highly deformable. Since the mechanical properties of melanoma cells may be affected by the production of melanin, this has to be considered (Sarna et al., 2013). Melanin and melanogenesis may act in two different directions: melanin act as a protection shield to radiation, however it also attenuates the effectiveness of radiotherapy, chemotherapy and phototherapy in the case of melanotic melanoma. This characteristic can provide tumor growth and progression (Slominski et al., 2015). AFM was used to observe the shape of human epidermis melanocytes in culture and to observe the melanosome transfer in vivo. Filopodia originated from the dendrite tips of the cell body of melanocytes, which contained melanosomes have been observed (Zhang et al., 2004). Van Den Bossche et al. (2006) hypothesized different mechanisms for melanin transfer: (i) keratinocytes phagocyte the melanocyte dendrites; (ii) the melanosomes release melanin into the extracellular space by melanosome exocytosis, being internalized by the keratinocytes; (iii) melanosomes are transported via membrane nanotubes; and (iv) the melanocytes enriched of melanosomes are phagocytosed by the keratinocytes (Van Den Bossche et al., 2006). Wu et al. (2012) studied melanocytes of mice lacking myosin Va (Myo5a). This protein is involved in the accumulation of melanosomes in the melanocyte dendrites. When myosin Va is present, melanosomes stay concentrated in the centre of cancerous melanocytes reducing melanosome transfer to keratinocytes (Wu et al., 2012). Wäster et al. (2016) studied the relation between UV skin radiation and skin pigmentation response. They concluded that when pigmented skin is exposed to UVA, there is a spillage of extracellular vesicles from the melanocyte’s plasma membrane. This does not happen when pigmented skin is exposed to UVB (Wäster et al., 2016). AFM was used to study the morphological changes of melanocytes exposed to the melanocyte stimulated hormone α-MSH, which induces melanization (Shin et al., 2014). The authors found that the roughness inside the pore-like structures on dendrites increased and MSH produced an increase in the size and density of the melanosomes. However, regarding the response to the presence of α-MSH, more studies are needed to correlate morphological shape alterations with the movement of melanosomes (Shin et al., 2014). Human melanocyte’s pigmentation in melanoma cells are also important in regulation Frontiers in Cell and Developmental Biology | www.frontiersin.org 3February 2020 | Volume 8 | Article 54 fcell-08-00054 February 11, 2020 Time: 16:43 # 4 Brás et al. Melanoma in the Eyes of Mechanobiology FIGURE 1 | Schematic representation of skin and cell population. Melanocytes are located in the basal layer of epidermis. Each melanocyte is connected to several keratinocytes. Melanosomes (black spots) are distributed in the dentrites, produce and store melanin. The dendrites allow the transfer of the melanin to keratinocytes, which will be transferred to the stratum corneum layer of the epidermis. of elastic properties according to Choi et al. (2014). The authors studied the effect of substrates with different stiffness on the pigmentation, using normal human melanocytes (NHM) and melanoma cells (MNT1) cultured on polydimethylsiloxane (PDMS) coated with laminin. They observed a reduction of dendrite formation in both cell type as well as a difference on the transference rate of melanosomes for both cell type on softer substrates. Cells cultured on stiffer substrates showed lower migration capacity once traction forces formation are more evident through via focal adhesion integrin complexes (Choi et al., 2014). Based on some studies of cell elasticity, cancer cells typically show a decrease in stiffness relatively to normal cells. Sarna et al. (2013) described a study of elastic properties comparing melanoma cells and healthy melanocytes. Basically, the authors used AFM to determine the stiffness of human melanoma cells SKMEL-188 with different stages of pigmentation and compared with SKMEL-188 cells not pigmented and with NHMs healthy human melanocytes (Sarna et al., 2013). They found that healthy melanocytes were softer than cancer cells. This feature could be related with the amount of melanin present in melanoma cells, resulting in some limitations for cancer diagnosis using the stiffness. Lazova and Pawelek (2009) mentioned that melanoma cells do not excrete the pigment like healthy melanocytes do, which may result in pigment accumulation in melanoma cells in relation to melanocytes. This could explain the higher stiffness of cancer cells, since they are often highly pigmented (Lazova and Pawelek, 2009). In conclusion, by AFM nanoindentation assays it is not possible to distinguish melanoma cells from melanocytes. AFM can be used as a complementary technique, however, this study was useful in understanding better pigmented cell elasticity. Sarna et al. (2014) also mentioned the need to understand if there is a correlation between the pigmentation of melanoma cells and the metastization phenotype process. In this process, malignant cells must pass the epithelial barrier, having the capacity to deform their cell body, including the nucleus to migrate through tissue. Cells with low Young’s moduli exhibit a great potential to invade tissue (Sarna et al., 2014), so this is why biophysical properties, as cell elasticity, must be taken into account. The authors showed that melanin granules inhibit the transmigration potentialities of melanoma cells. For non-pigmented melanoma cells, the stiffness was lower, and the transmigration efficiency was higher. This study contributed with new insights about the relationship between pigmentation and elasticity of melanoma cells in terms of migration and invasion. Some substances are responsible for the retraction of melanocyte dendrites. Ito et al. (2006) have studied the effect of dendrite retraction using a flavonoid substance. In this case, TYRP-1 signaling pathway is interrupted, once this molecule is important for melanosome migration. Rho signaling molecule, when activated induces dendritic retraction, reducing the melanosome transfer to the keratinocytes but does not affect the amount of melanin production. However, this melanin will not be transferred due to the dendrite’s retraction. The mechanism of dendrite retraction via RhoA involves changes in cytoskeleton and microtubules organization due to actin and tubulin-βdisassembly. These changes will induce stress fiber formation promoting actin polymerization and reorganization of microtubules (Ito et al., 2006). Rac is involved in melanocyte dendrite growing, inducing the appearing of lamellipodia (Scott, 2002). The dendrite tips of melanocytes are also stimulated by the α-MSH and endothelin – 1 proteins from keratinocytes that are surrounding the melanocytes and the ECM produced by the keratinocyte (Kippenberger et al., 1998). Kippenberger et al. (1998) demonstrated that co-cultures of melanocytes and keratinocytes in the presence of high concentration of calcium, showed sheets of keratinocytes formation, surrounding by melanocytes with polar dendrites extended around the differentiated keratinocytes. Differentiated keratinocytes will have stable areas of contact with melanocytes due to the calcium gradient. The normal melanocytes that lose their dendrites soon will be in an apoptosis state (Kippenberger et al., 1998). The senescence of primary melanocytes could also be important in the melanoma appearance (Feuerer et al., 2019), however, there are no studies that correlate melanocytes’ senescence with mechanical properties measured by AFM. Some studies showed Frontiers in Cell and Developmental Biology | www.frontiersin.org 4February 2020 | Volume 8 | Article 54 fcell-08-00054 February 11, 2020 Time: 16:43 # 5 Brás et al. Melanoma in the Eyes of Mechanobiology a correlation between the melanocytes aging and senescence biomarkers (Wang and Dreesen, 2018) and exposition of melanocytes to light (Bennet et al., 2017). AFM is a powerful tool to assess the morphological and elastic properties regarding the interface between primary melanocytes and primary melanoma with and without senescence. In opposition to the melanoma cell lines that are immortalized and thus, submitted to several passages, the primary melanocytes and primary melanoma cells should be assessed in order to evaluate what passage the senescence will not interfere with mechanical properties. The quantification of senescence level can be performed using the quantification of the Population Double Level (PDL) method (Kruse and Patterson, 1973). The Role of Keratinocytes Keratinocytes are the predominant cell type in the epidermis, most of them (90%) being located at the outmost epidermis layer (McGrath et al., 2004). Keratinocytes are connected to each other through actin cell junctions (Hsu et al., 2018). The main function of keratinocytes is to form a barrier against pathogenic microorganisms, heat, UV radiation and water loss. They also produce cytokines that attract leukocytes to the location of pathogen invasion (Hall et al., 2014). Keratinocytes also produce different keratins. Keratins (type 1, 5, 10, and 14) are the main structural protein of their cytoskeleton, all of which play an important role in the mechanics of these cells (Ramms et al., 2013). Keratins become altered when responding to stresses, resulting in its modification and reorganization (Karantza, 2011). Keratinocytes become corneocytes, when they are completely differentiated, losing their nucleus and cytoplasmic organelles (Mi, 2009). Differentiation of keratinocytes is driven by a calcium gradient, which is established between the interior of the cell and the ECM (Proksch et al., 2008) and regulated by vitamin D3 (Bikle, 2004), cathepsin E (Kawakubo et al., 2011), TALE homeodomain transcription factors (Jackson et al., 2011), and hydrocortisone (Rheinwald and Green, 1975). Keratinocytes are affected by senescence, losing their biological function with age (Orioli and Dellambra, 2018). Keratin, actin filaments and microtubules form the cytoskeleton network of keratinocytes, providing shape and structure of cells allowing transmission of mechanical loads (Silver and Siperko, 2003). For keratinocytes migration one of the most important parameters is the substrate stiffness (Ramms et al., 2013), since it is expected that the keratinocytes must change their shape during migration through the epidermis until they reach the outmost layer and become corneocytes (Zarkoob et al., 2015). This change in shape will affect also the components existing inside the cytoplasm, namely the cytoskeleton, in which keratin fibers are crucial (Ramms et al., 2013). Ramms et al. (2013) used AFM to probe keratinocytes (in the nuclear region and in the cell body) deficient in keratins finding that they are softer than healthy ones. In addition, with magnetic tweezers, the viscous contribution to the displacement of magnetic beads increased, corroborating the results achieved by AFM. These authors also found that the softening was reversible and could depend on the re-expression of two types of Keratins (K4 and K15) only (Ramms et al., 2013). Bar et al. (2014) reported the skin fragility in mice and hampered desmosomes adhesion when there is a lack of all keratins Bar et al., 2014. AFM showed a reduction of adhesive forces and membrane stability in murine keratinocytes when lacking keratin filaments (Vielmuth et al., 2018). Adherent junctions (AJ) have functions in mechanosensing and transducing mechanical forces between the plasma membrane and the actomyosin cytoskeleton. Desmosomes and intermediate filaments promote the mechanical stability to maintain tissue architecture and integrity when they are submitted to mechanical stress. Both (AJ and desmosomes) connect the actin and keratin filament network of adjacent cells. Desmosomes are important for intercellular cohesion, where keratins determine cell mechanics but are not involved in tension generation (Hatzfeld et al., 2017). Zarkoob et al. (2015) investigated the role of substrate stiffness on keratinocyte colony formation (in vitro) during epithelial formation, due to calcium exchange during keratinocyte cell culture. Keratinocytes cultured on soft polyacrylamide substrates (E= 1.2 KPa) presented small contact areas, an increase in migration velocity and in colony formation compared to those cultured on stiff polyacrylamide gels (E= 24 KPa) (Zarkoob et al., 2015). How melanoma cells escape the keratinocyte’s control invading other tissues and form metastasis, is still unclear. In a healthy tissue, there is a homeostasis maintained by each cell. In this normal tissue, keratinocytes are the “surveyors” of melanocytes, controlling their growth and behavior through complex paracrine growth factors and cell-cell adhesion molecules. When alterations occur to this homeostatic balance, cell-cell adhesion and cell communication molecules change and induce melanoma development resulting in the damage of the epidermal melanin unit, inducing the continuous melanocyte proliferation, and eventually leading to melanoma development (Haass et al., 2005). Chung et al. (2011) suggested keratinocytes-derived ECM factors may act as regulators of melanocytes. The authors showed that laminin-332, a component of basement membrane, plays a crucial role in the adhesion and migration of melanocytes and melanoma. It is known that keratinocytes regulate the behavior of melanocytes such as proliferation, melanin synthesis and dendrite formation and the ECM also regulates different cell behaviors (Chung et al., 2011). The Role of Fibroblasts and the ECM Fibroblasts are produced in the bone marrow. They synthesize the components of the ECM, for instance collagen, glycosaminoglycans and elastic fibers. Fibroblasts also produce a structural network called “stroma” in animal tissues (connective tissue), playing an important role in cancer and in wound healing (Desjardins-Park et al., 2018). Kwa et al. (2019) mentioned that cancer is associated with fibroblasts and how these fibroblasts affect metastization. Fibroblasts produce high amounts of ECM molecules, cytokines and growth factors contributing to the stroma, cancer progression and metastization due to several pathways. These pathways could be a target for new cancer treatment strategies to be developed. However, it is difficult to characterize this subtype of fibroblasts and to identify specific molecules involved in this process (Kwa et al., 2019). Mechanosensing has a large impact in Cancer-Associated Fibroblasts (CAFs). Fibroblasts sense Frontiers in Cell and Developmental Biology | www.frontiersin.org 5February 2020 | Volume 8 | Article 54 fcell-08-00054 February 11, 2020 Time: 16:43 # 6 Brás et al. Melanoma in the Eyes of Mechanobiology mechanical stresses and activate mechanosignaling processes, which use mechanoreceptors located in the cell membrane, the cytoskeleton and transcription factors. The ECM stiffness is determinant in the mechanosignaling, which by itself can activate the mechanosensing pathways in CAFs, leading to ECM production and stiffness increasing, supporting different CAFs differentiation (Kwa et al., 2019). The study of mechanosignaling and fibroblasts response to the ECM stiffness was conducted using hydrogels, to mimic pathologically the ECM of tissues. Viji Babu et al. (2018) have used the AFM to study the stiffness and viscoelasticity of normal, scar and Dupuytren’s disease fibroblasts, growing on top of soft (1 KPa) and stiff (50 KPa) hydrogels. Basically, a MLCTBio pyramidal cantilever (nominal spring constant of 0.01 N/m, Bruker, United States) was used to indent the fibroblasts, using the z-step response methodology, in which the elasticity and viscosity was assessed (Figure 2). The authors wanted to evaluate the effect of TGF-αin the fibroblast’s elasticity. The authors concluded that Dupuytren’s fibroblasts increased their elastic moduli and became stiffer in TGF-β1 presence, whereas they did not find significant changes in the elastic moduli of scar and normal fibroblasts, before and after addition of TGFβ1. Dupuytren’s fibroblasts presented a wide number of wellorganized stress fibers and bundles of stress fibers were even thicker in presence of TGF-β1 (Viji Babu et al., 2018). RhoA signaling is the key for the fibroblast’s reaction to ECM stiffness. RhoA is activated by a stiff matrix, causing cell contraction and actin polymerization, inducing the differentiation of fibroblasts in myofibroblasts. Therefore, CAFs need the activation of this pathway, since it is required for their contractile and invasive properties. It has been mentioned that there are several CAF subtypes, however, there is a lack of information about their plasticity, which could be important for the progression and metastization of cancer (Kwa et al., 2019). AFM could give new insights regarding the characterization of fibroblasts subtype based in their morphometric characteristics (shape factor, volume and area) and mechanical properties. As far as we know, there is not any specific study in literature, which FIGURE 2 | Basic components of (A) AFM and (B) force curve with (C) viscoelastic creep response measurement. (A) The basic four components of AFM (i) a laser diode, (ii) a cantilever of 0.01 N/m spring constant with 30 nm radius AFM pyramidal tip, (iii) a position-sensitive photo detector (PSPD), and (iv) xyz-piezo stage. (B) Sample indentation by the AFM tip obtains the force curve that gives the approach (red arrow) and retract (blue arrow) curve on deflection vs. Z-height graph, and apparent Young modulus was calculated by applying Hertz model to the approach curve. In creep response curve (C), the Z-height profile (i) shows the approach and retract ramp toward the cell for 3 s, and in-between, there is a z step, which is applied at t= 1.5, which is enlarged in panel (ii).(iii) The deflection data show global creep of the cell, which includes the creep after loading and unloading step, which is enlarged in panel (iv), and global creep was determined by the exponential fit (black curve) and was subtracted for qualitative analysis (reproduced with permission from John Wiley and Sons with Copyright Clearance Center’s Right, Journal of Molecular Recognition, 2018) (Viji Babu et al., 2018). Frontiers in Cell and Developmental Biology | www.frontiersin.org 6February 2020 | Volume 8 | Article 54 fcell-08-00054 February 11, 2020 Time: 16:43 # 7 Brás et al. Melanoma in the Eyes of Mechanobiology could be useful for the development of therapies directed to a specific fibroblast subtype related with melanoma. This study could also promote new insights to develop therapeutic strategies to other cancer types. The ECM is composed by (i) the structural proteins collagen and elastin; (ii) adhesion proteins such as fibrillin, fibronectin, vitronectin and laminin; and (iii) the proteoglycans (which have a protein core bound to glycosaminoglycans) (Makale, 2007). The ECM is responsible for cellular support. Variations in its structure and composition alter the mechanical properties of the ECM. Generally, the external forces applied to cell-matrix complexes are transmitted to the interior of the cell by integrins according to three steps (Roca-Cusachs et al., 2009): (i) integrins binding to ECM molecules; (ii) transmission of forces to the interior of the cells, through integrins, where they are transformed into biochemical signals (mechanotransduction); and (iii) integrins binding to cytoskeleton, transmitting the forces all over the cell, and reinforcing their adhesion properties to resist the force. The integrins inside of cell recruit several other molecules, which bind to the cytoskeleton and/or signaling proteins to form what is called “focal adhesion” to resist to the external force (Roca-Cusachs et al., 2009). What are the mechanical cues sensed by cells when forces are applied from the outside? Integrin-associated complexes (IACs) in cellular membranes establish the connection between the ECM and the cytoskeleton of the cell, via actin filaments (Haase et al., 2016). Haase et al. (2016) found that physical forces stemming from the ECM have a huge impact on gene regulation; it has been hypothesized that gene expression is affected by nucleus deformation, which was observed in 3T3 fibroblasts. The actin and microtubule network generates nuclear deformation. The deformation of the nucleus will be anisotropic being observed along the shorter axis and is intrinsic to the nucleus. This behavior is due to the chromatin organization and lamin-A expression (Haase et al., 2016). The ECM uses the concept of “tensegrity,” which employs tensile forces and rigid support structures to distribute and manage the loads mediated by the cellular cytoskeleton complex. In the cytoskeleton microfilaments and microtubules, which are connected to the ECM, there are forces in between (Makale, 2007). This means that the pressure exerted on the ECM will influence the cell’s behavior in terms of differentiation, motility, adhesion, invasion, and metastasis (Makale, 2007). ECM mechanical properties are directly related with elastin fibers, collagens, glycosaminoglycans (GAGs) and proteoglycans. Elastin fibers contribute to cyclic stretching during life; collagen fibers contribute to the tissue stiffness and mechanical strength of tissues and GAGs affect ECM viscoelasticity, forming bridges between the collagen fibers and contributing to the compressive tissue stiffness. Cells sense the forces exerted by the ECM, converting these stimuli into intracellular signal pathway, which further downstream regulate transcriptional changes (Hsu et al., 2018). Several mechanosensors are identified in cells: glycocalyx, lipid raft/caveolin-1, cell adhesion structures (integrin, hemidesmosomes and focal adhesion), ion channels (calcium-sensing receptor), transient receptor potential channels, connexins, intercellular complexes (desmosomes or cadherin) and the cytoskeleton (particularly the actin filaments) (Hsu et al., 2018). Any abnormality that causes changes in the ECM mechanical properties, cell interactions, or cellular response to mechanical stimuli will induce tensional homeostasis, contributing to several diseases like cancer and fibrosis (Hsu et al., 2018). Integrins (transmembrane proteins) are responsible for transmission of forces between ECM and the interior of the cell. Tumor cells expressed the avb3 integrin and the invasiveness of melanoma cells depends on the expression of β-3 and β-1 integrins, which are related with their aggressive properties (Petitclerc et al., 1999;Hegerfeldt et al., 2002). Therefore, one therapeutic strategy could contribute to the reduction of these integrins expression (Makale, 2007). Stiffness of tumors is important since it activates biochemical pathways regarding the cell cycle, EMT, cell motility and leads to compression in neighboring healthy cells. These healthy cells may then activate tumourogenic pathways (Broders-Bondon et al., 2018). Cell spreading depends on substrate stiffness, which, in turn, depends on the complex adhesion type. A7 melanoma cells were seeded in polyacrylamide gels laminated with collagen type I (Coll I), fibronectin (FN) and a mixture of Coll I and FN (Winer et al., 2011). Figure 3 shows the stiffening and spreading of filamin A-expressing A7 human melanoma cells and is compared with different stiffnesses substrates coated with FN or Coll I. Stiffening but not spreading of A7 melanoma cells depends very strongly on whether integrins specific for FN or Coll I are engaged. When A7 cells are plated on gels coated with saturating amounts of either FN or Coll I, they spread to approximately the same extent (Figure 3B), but the cells on Coll I are much stiffer than those adherent to FN (Figure 3A). When both FN and Coll I are present, allowing both α1 and β3 integrins to bind, adherent area increases, but cell stiffness reaches an intermediate value between those found on FN or Coll I alone (Kandow et al., 2007). CELL MIGRATION AND PROGRESSION IN MELANOMA Motility Melanoma starts with the lost binding intercellular connections between melanocytes and keratinocytes (Figure 4). In the process of melanoma development, due to EMT, cells acquired a mesenchymal stem cell phenotype, contributing to cell migration (Bandarchi et al., 2010). The EMT model is very well explained by Nieto et al. (2016) and Brabletz et al. (2018), the forces involved in EMT and its relation with cancer metastasis are described by Wei et al. (2015). After losing their connections, melanocytes move due to a motility process. Motility is the capacity of a living system to move independently, using metabolic energy to perform mechanical work and should not be confused with mobility (Allen, 1981; Figure 5). For cell migration, there is the contribution of the internal forces (which are generated by some structures present inside each cell, such as the cytoskeletal filaments) and the external forces (the shear flow of blood or lymphatic fluids contributions). The motility of cells through tissues is Frontiers in Cell and Developmental Biology | www.frontiersin.org 7February 2020 | Volume 8 | Article 54 fcell-08-00054 February 11, 2020 Time: 16:43 # 8 Brás et al. Melanoma in the Eyes of Mechanobiology FIGURE 3 | Melanoma stiffness (A) and spreading (B) in function of Collagen type I (Coll I) and Fibronectin (FN) presence in hydrogels. When A7 cells are plated on gels coated with saturating amounts of either FN or Coll I, they spread to approximately the same extent (B), but the cells on Coll I are much stiffer than those adherent to FN (A). When both FN and Coll I are present, allowing both α1 and β3 integrins to bind, adherent area increases, but cell stiffness reaches an intermediate value between those found on FN or Coll I alone. Each trace drawing inside the cells corresponds to 1 KPa (based on Kandow et al., 2007). a process mediated by internal forces, which are related with the polarization of the cell body within the ECM. This motion depends on shape changes, cell-substrate interaction, and the generation of forces from the cells mediating through the tissue. The internal forces responsible for cell motility are composed by the following steps: (i) cytoskeletal polarization, forming extensions of actin filaments located in the leading edge; (ii) cell protrusions connect to the ECM scaffold by cell surface adhesion receptors; (iii) contraction of actomyosin motors that are connected with the ECM substrates exerting traction forces; (iv) bipolar tension, which is transmitted through the substrate, allows the retraction of the cell rear completing the movement of the cell; and (v) when cells move, they produce ECM degrading proteases and the transmigrated ECM is remodeled by proteolysis (Tao et al., 2017). Tao et al. (2017) also refers that the actin cytoskeleton exhibits viscous and elastic responses, which can be characterized by the relaxation time observed in a creep response after applying a stress. The relaxation corresponds to the time scale where the cell cytoskeleton can flow and behave in an elastic way. Cytoplasm is more than cytoskeleton: it has a lot of water content (around 70%), which also play a role in cell motion, since water flows in between the cytoplasm organelles. The external forces are related with the water flow and osmotic pressure, as well to the fluid shear stress of the blood and lymph. FIGURE 4 | Melanocytes – keratinocytes adhesion molecules in healthy and in cancer tissues. In a healthy state the melanocyte-keratinocyte interactions are characterized by the existence of E-cadherin adherent junctions. The keratinocytes and melanocytes are connected to the ECM through the integrins. In a cancer state, the E-cadherins are lost and N-cadherins are the adherent junctions stablished between melanocytes. Melanocyte dendrites retracted and melanocytes acquired a round shape phenotype. The water can pass through the cell membrane via aquaporin in both directions, so the cell can change its volume, either forced by external cues like the osmotic pressure difference between inside and outside of the cell or actively controlled by ion pumps (Tao et al., 2017). Cytoskeleton rearrangement occurs through the arrangement of three proteins: Rho, Rac and Cdc42. The Rho GTPase family since they influence the appearance and structure of filopodia, stress fibers and adhesion plaques of melanoma cells (Tao et al., 2017) encourage actin retraction; Rac was treated to encourage lamellipodia extension but block actin depolymerization; Cdc42 was assumed to encourage filopodia elongation and block actin depolymerization (Tao et al., 2017). Motility is resulting from what is calling the mechanotransduction “inside-out” combining with a water flow in and out. In human melanoma, by micropore filtration, it was found that vimentin and keratin intermediate filament expression results in a more migratory and invasive phenotype (Gao, 2014). The authors also found that damage of actin filaments and microtubules increases the deformability contributing to motility and metastization (Gao, 2014). Cell motion requires that the cytoplasm viscosity change in the cell periphery through the transformation of the gel state to a fluidic one. This mechanism also induces formation of protrusions that Frontiers in Cell and Developmental Biology | www.frontiersin.org 8February 2020 | Volume 8 | Article 54 fcell-08-00054 February 11, 2020 Time: 16:43 # 9 Brás et al. Melanoma in the Eyes of Mechanobiology FIGURE 5 | Cell motility: Stage 0 (Steady state) -Cells maintain the connections to the ECM through the integrins; Stage 1 (Protrusion of the leading edge) -internal forces induce the F-actin polymerization and the water flow enters through the cell membrane; the actin polymerization induce the formation of protrusions; Stage 2 (Adhesion of leading edge) - the cortex under tension, accumulates new actin, through the movement of unpolymerized actin to the protrusion edge, and new adhesion molecules appear in the protrusion part. Stage 3 (Movement of the cell body) - there is the contraction of the cell in the same direction of the cell movement, and the water flow exits from the cell in the opposite direction, contributing to cell motility; Stage 4 (Deadhesion of the trailing edge) - Deadhesion on the cell back edge: the integrins in the back of the cell decreases, and new adhesion molecules appeared in the front of the cell in the same direction of cell movement based on Tao et al. (2017). facilitate cell locomotion. Actin filaments are bond to the plasma membrane by a glycoprotein. In the presence of this glycoprotein cells are stable, the protrusions are well organized, occurring cell movement. If this glycoprotein is not present, as in the case of certain human melanoma, locomotion is impaired, and the plasma membrane shows blebbing (because of the cytoskeleton instability). If the presence of the glycoprotein is re-established, blebbing disappears and cell movement is recovered. This glycoprotein also increases the stiffness of normal cells, meaning that in melanoma cancer the lack of this protein increases the deformability and contributes to the efficiency of the migration process of tumor cells (Gao, 2014). The whole cell, including the nucleus, must present a certain plasticity. Inside the nucleus, there is chromatin together with lamins in coordination with the cytoskeleton, maintaining shape and mechanical stability of the nucleus. Chromatin works like an elastic spring and is responsible for the force response to small deformations of the nucleus. Lamin A deforms easily for small extensions and gives stiffness to resist large nuclear deformations (Stephens et al., 2019). Microtubules in the cell cytoskeleton exert forces generated by dynein that can deform or even cause the rupture of the nucleus. Vimentin protects the nucleus giving it stability and perinuclear stiffness, which hinders 3D motility. 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