Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5904_Библиотеки_им_академика_М_И_Перельмана

This chapter will discuss how recently developed analytical and biotechnological tools have significantly contributed to overcome the challenges posed by the unique features of glycans and their intricate biosynthetic pathways, thus supporting the structural and functional characterization of protein glycosylation in the context of human neoplastic transformation and the successful establishment of glycan‐based biomarkers and therapeutic targets for the clinical management of cancer patients (Figure6.1).

Over the past decades, numerous studies have sought to comprehensively charac­terize both the nature and impact of glycosylation alterations occurring within malignant cells and tissues (reviewed in[9]). In particular, the advent of hybridoma technology just under 50 years ago has supported the generation of a virtually unlimited catalog of highly specific mAbs for the reliable detection of any type of biological antigen, including glycans, and has, therefore, revolutionized the field of cancer biomarker discovery[15]. Indeed, the development of highly specific mAbs targeting TACAs, such as the prematurely truncated O‐glycan determinant STn, allowed the unprecedented disclosure of their tissue-based cancer‐specific expres­sion pattern [16–18]. In the hybridoma system, activated immunoglobulin‐secreting B cells, previously challenged with an isolated and structurally defined antigen of interest (ideally), are fused with myeloma cells, generating a hybrid, isogenic, and immortalized cell line with antibody‐producing capacity [19]. Generally, mAb‐ based tissue mapping of entirely peptidic epitopes with a known sequence can be performed with reasonable certainty, as the specificity of the used mAb can be accu­rately determined. However, the signals produced by carbohydrate‐binding mAbs often reflect the expression of a variety of distinct protein carriers, which hinders the precise identification of the target glycan epitope[20]. The same limitation applies to other glycan‐recognizing molecules whose carbohydrate specificity is not fully determined, including polyclonal antibody mixtures and other GBPs. The poor affinity of lectins, usually in the low micromolar range, frequently limits their appli­cation for histochemical staining purposes[1, 21]. Indeed, these glycan‐ binding molecules do not provide insights on complete monosaccharide compositions, gly­can tridimensional conformation, glycosylation site occupancy, microheterogene­ity, or the precise location of glycan chains on the backbone of specific glycoproteins. Such affinity reagents facilitate the identification of broader glycosylation traits (e.g. N‐glycan branching, core fucosylation, and linkage‐specific sialylation) rather than elucidating the exact tridimensional structure of individual glycan species. In fact, such information may only be accurately retrieved through the implementation of increasingly complex mass spectrometry (MS)‐based methods. However, the major­ity of glycan‐directed analytical techniques, including MS‐based workflows, require analyte extraction from the target tissue, which, in turn, leads to the loss of informa­tion on glycan spatial distribution and tissue histopathological architecture.

format reduces intersample technical variability and further allows the IMS‐based multiplexed analysis of larger cohorts of clinical samples, which is of extreme rele­vance to the cancer biomarker discovery field, particularly when conducting retro­spective studies[36]. Moreover, the proven applicability of IMS in the analysis of FFPE tissues is of extreme significance since these samples can be easily archived at room temperature for several years in tissue banks and biorepositories and are more widely available than cryopreserved clinical specimens. Additionally, by directly linking detailed and spatially resolved structural data to well‐defined histopatho­logical regions at the individual sample scale, IMS‐based analysis of whole tissue sections provides invaluable molecular insights on intratumor heterogeneity. This is of particular relevance when defining tumor margins and interfaces. IMS analysis of neoplastic tissues has clearly demonstrated the expression of tumor‐associated molecular signatures in apparently healthy histological regions. The comprehensive comparative characterization and relative quantification of glycan species from nontransformed adjacent mucosa, premalignant lesions, and fully transformed neo­plastic regions may lead to the identification of robust glycan‐based biomarkers of malignant transformation capable of accurately discriminating patient clinical out­comes and tumor subtypes. The MALDI‐IMS technology has been successfully used to illustrate the astonishing differences in the glycosylation patterns between cor­responding healthy and malignant tissues across multiple epithelial cancers, includ­ing prostate, pancreatic, ovarian, gastric, and hepatocellular carcinoma, as well as myxoid liposarcoma[24–28, 33, 35, 37]. IMS technology has thus emerged as a novel source of robust cancer‐specific biomarkers, either as individual glycan masses or as more complex panels of combined mass spectra from multiple glycan species, for unequivocal tissue region identification.

due to heterogeneous or reduced expression of the target protein and the require-

of the target glycoprotein’s glycan component. The successful implementation of the PLA system requires several methodological considerations, such as the optimi­zation of histochemical staining protocols for each individual target antigen and possible incompatibilities with antigen retrieval steps. Furthermore, for the same target epitope, multiple affinity reagents may have to be tested since the oligonu­cleotide conjugation reaction may sterically hinder their antigen‐binding capacity. Although the exact minimal distance required for the generation of a positive PLA signal has yet to be fully investigated, such values may be estimated and optimized based on the dimensions of the selected affinity reagents and lengths of the oligonu­cleotide strands. Despite its high selectivity and nanometric resolution, in situ PLA provides only indirect evidence on molecular interactions or protein modifications[41].

Importantly, the in situ PLA technology holds tremendous translational potential as it may be readily incorporated into the routine workflows of cancer‐dedicated pathology laboratories as a robust source of novel glycoprotein‐based biomarkers bearing both diagnostic and prognostic utility, thus improving patient stratification and clinical management.

Although the exact dimension of the cellular glycome remains a matter of debate, it is estimated to be in the range of 100 Such structural diversity has created the need for molecular tools capable of linking the structure of a given glycan to its biological function. The development of glycan microarrays, just under 20