*5.4. Current Heparan Sulfate-Based Biomarkers Landscape*

New biomarkers to improve cancer diagnosis are needed, thus improving patient outcomes. In contrast to RNA, DNA and protein synthesis, HS biosynthesis is not a template-driven process. Instead, as mentioned previously, HS are assembled by the activity of a series of enzymes–turning this biosynthetic pathway into a source of very distinct and specific modifications with diverse applications. The type of HSPG glycosylation was shown to affect the ability of immune cells to infiltrate tumour tissues and engage in the immune response [259]. In addition to the critical roles in multiple aspects of tumour biology, described in the previous sections, HSPGs also have value for clinical diagnosis and prognosis in various cancer types. The alteration of GAG abundances is reflected in body fluids (like blood and/or urine), and the HS levels in plasma can, therefore, predict patient's prognosis [260]. For all these attributes, HS and HSPGs are promising new biomarkers in cancer since their recognition by other molecules is based in high affinity and exquisite specificity. It is imperative to discriminate among related isomers the specific glycan-binding partner. The development of more sophisticated equipment and techniques have been critical to surpass these limitations. Recently, using Raman micro-spectroscopy it was possible to determine unique and discrete HS profiles of individual live cells, which can be of value for clinical screening purposes. This method can be utilised for identifying specific molecular signatures of HS and HSPGs as markers of cancer [261].

GPCs have been receiving great attention from the research community as promising biomarkers [262]. GPC1 is overexpressed in several cancer types [263,264]. GPC1 can be detected in the urine of prostate cancer patients [265]. Further, GPC1 was shown to associate with the dissemination levels of glioblastoma [148]. The abundance of GPC1 also positively correlates with disease severity of pancreatic cancer patients, independently on surgical treatment, suggesting that GPC1 is a surgery-independent diagnostic biomarker [263,266]. Recently, GPC3 was pointed as a promising candidate for hepatocellular carcinoma diagnosis and immunotherapy, as previously described [267]. Similarly, GPC6 was identified as a putative biomarker for the metastatic progression of cutaneous melanoma, since it is possible to track higher levels of GPC6 in melanoma samples when compared with normal melanocytes [268].

The biological features of EVs, such as long half-life and physical resistance properties, make EVs a unique source of biomarkers [167]. Particularly, the identification of specific PGs in EVs secreted by cancer cells has demonstrated their potential as biomarkers for minimally invasive diagnosis. Two important examples are GPC1 in pancreatic cancer [266] and SDC1 in glioma [269]. Both HSPGs were detected in EVs isolated from patients' plasma, supporting the concept of a minimally invasive biomarker for patients' stratification. GPC1 was proven to distinguish healthy individuals and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Moreover, GPC1-positive EV levels correlated with tumour burden and the patients' survival and were shown as a prognostic marker superior to CA 19-9, the serum biomarker currently employed in pancreatic cancer screening [266]. SDC1 was shown to discriminate between highgrade glioblastoma multiforme and low-grade glioma, and therefore, with the potential to improve the management of brain tumour patients that present high risk of surgeryassociated complications [269].

The recent development of EV analysis platforms based on microfluidics technologies holds promise for the development of high sensitivity and high-throughput assays of EVs analysis with clinical diagnosis purpose [270].
