*4.3. Heparan Sulfate Roles in Cancer Intercellular Communication*

Cancer cells communication within the tumour microenvironment is key to defeat stromal challenges, settle and colonise distant sites, leading to metastasis. Despite being the main cause of cancer therapy failure and responsible for the greatest number of cancerrelated deaths, metastasis remains poorly understood [158]. It is widely recognised that the process of cancer cell systemic circulation and the development of metastasis requires the participation of several glycoconjugates [86,159].

For years, EVs were thought to be a reservoir of cells undesired material. However, in the last two decades, they have emerged as main players in cellular communication [160,161]. EVs are delimited by a lipid-bilayer and can be classified into different classes, including exosomes, microvesicles and apoptotic bodies. Briefly, EVs are secreted to the environment by all cells and carry bioactive cargo that deliver signals and induce several pathophysiological events in the ECM and recipient cells [162,163]. Regarding cancer, EVs have been demonstrated as important signalling nanoparticles in pre-metastatic niche definition and metastasis [164,165].

The EV cargo includes nucleic acids, proteins, lipids and metabolites [166]. Although several seminal studies have addressed in detail the lipid, protein and nucleic acid contents of EVs, the glycans, and particularly GAGs, remain poorly characterised. However, their biological importance is emerging [160,167,168].

Importantly, HSPGs have been described as key regulators of EVs biogenesis (Figure 3) [162,169]. The biogenesis of EVs depends on the small intracellular adaptor syntenin [170], its interaction with SDC [169] and the endosomal-sorting complex required for transport accessory component ALIX [171–173]. Moreover, HPSE can stimulate intraluminal budding of SDC-syntenin-ALIX complex promoting EVs secretion [174–176]. Recently, it was described that tetraspanin-6 (TSPN6) may act as a negative regulator of exosomes release through the promotion of SDC4 and syntenin degradation. This interaction highlights the importance of the interplay between these membrane glycoproteins to produce exosomes [10].

Furthermore, it has been demonstrated that HSPGs, namely, SDCs and GPCs, are critical internalising receptors of cancer cell-derived EVs and determine their functional activity (Figure 3) [116,160]. Very recently, it was shown that under hypoxia stress, the uptake of EVs is upregulated, through a mechanism dependent on HSPG receptors and lipid raft mediated endocytosis [177].

EVs exhibit several distinctive features, from a longer half-life provided by increased resistance to degradation, therefore offering their cargo a higher stability, to the ability of travelling long distances. Furthermore, EVs can carry multiple cargo possibilities and also exhibit a unique interactive surface area [178], which may establish contact with both cells and components in the ECM microenvironment [179]. HSPGs are herein important mediators with several functions from EV secretion and trafficking to their uptake [60,116,180].

Taking together glycan and EVs functional relevance in cancer development, it is not surprising that glycans in EVs have been implicated in cancer cells proliferation, angiogenesis, therapeutic resistance [181], control of metabolic activity, and immune system evasion mechanisms [160].

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**Figure 3.** Heparan sulfate proteoglycans regulate EV biogenesis and uptake. (**1**) Cell surface HSPGs can bind multiple ligands through their GAG chains [9]. GAGs can be modified by heparanase activity [174]. (**2**) Syndecan is internalised through endocytosis process, leaving the cytosolic domain clear for syntenin and Alix proteins binding [160,171]. The early endosomes generate the MVBs by inward budding of theirmembrane. (**3**) EVs, particularly those enriched in tetraspanin-6 (TSPN6), can end on lysosome [10] (**4**) with consequently degradation of their content. (**5**) Alternatively, EVs generated inside of the MVBs can be expelled from the secreting cell, through exocytosis. (**6**) After fusion with cellular membrane EVs are released to the extracellular milieu. (**7**) HSPGs, and specifically GAGs, are important receptors of the cell membrane-EV surface cluster and are directly involved in EV uptake by recipient cell [116]. (**8**) After, the complex is internalised by the recipient cell. (**9**) EV-endosome membrane fusion occurs, and EV content is released to cytoplasmic compartment of the recipient cell, and new biological information is transferred [162].
