**6. Conclusions and Future Challenges**

The emerging HSPGs biological functions have largely surpassed their classical role as cellular co-receptors and have highlighted HSPGs as main maestros of cancer cell communication and ECM structuring. The HSPGs cellular interactome is vast and is finetuned by the biochemical and structural features of the HS chains. HS lack a template for its biosynthesis, and their structural features result from the dynamic cellular GAGosylation pathways that include the sequential, and in some cases competitive, action of specific enzymes, which may associate to form supramolecular complexes [25]. The structural efforts to produce chemically defined HS oligosaccharides [271] have been crucial for the identification of the molecular determinants of enzymatic activity. However, we are still far from fully understanding the complete regulation of HS structural and functional diversity in health and particularly in cancer.

The recent developments in glycosaminoglycanomics, namely, on the analytical techniques for GAGs profiling in cells and clinical samples (tissues and biological fluids), together with the establishment of computational tools for mining GAG-protein interactions and creation of databases, are contributing to significantly improve the knowledge on the human glycosaminoglycome [19,272]. Moreover, the integration of HS structural features and expression with proteomic and transcriptomic analysis will be crucial for further elucidating HS-ligand interactions and unravel HS structure features associated with specific biological functions [273]. Particularly relevant would be the integration of data on relative abundance and structural features of HS for the definition of cancer-specific profiles. Therefore, it is important to apply the most recent GAG analytical approaches to well characterised clinical samples to identify HS signatures that are cancer-specific. To successfully achieve this aim, it is key to incorporate also knowledge on the dysregulation of HS biosynthetic and post-synthetic modification pathways, as well as on the functional redundancy of different PG core protein families in cancer.

The multidimensional roles of HS and HSPGs in different steps of cancer progression have propelled the development of HS-targeted strategies for cancer diagnosis and treatment [200]. Indeed, in this new period of precision oncology, HS GAGs are currently (un)expectedly emerging as allies to improve cancer clinical management by their potential to detect cancer in early stages, allowing an accurate diagnosis, disease monitoring, patients stratification and improve prognosis.

The HSPGs role in regulating EV release, cargo and uptake is well defined [160], but the implications of altered PG expression and glycosylation features in EV biodistribution and metastasis tropism remain to be discovered. It has become highly relevant to understand the impact of HSPG remodelling, both at the level of the glycan structures and core protein expression, in cancer EV-mediated signalling. Understanding the glycosylation modifications involved in EV-cell interaction and cellular uptake is of major relevance for developing therapeutic approaches targeting EV-HSPG interactions as novel cancer treatment strategies.

**Author Contributions:** Conceptualization I.F.-R., J.P., C.M., and A.M.; writing—original draft preparation I.F.-R., J.P., C.M., J.S.-A. and A.M.; Figures preparation I.F.-R., J.P. and C.M.; writing—review G.M., C.A.R., A.M.; funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financed by FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274) and by the grant POCI-01-0145- FEDER-028489 (to A.M.). J.P. and C.M. are funded by FCT PhD scholarships SFRH/BD/137319/2018 and 2020.06412.BD, respectively. The APC was funded by FCT grant POCI-01-0145-FEDER-028489. The authors acknowledge the support of the COST Action CA18103 INNOGLY.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design and writing of the manuscript, or in the decision to publish.
