Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5425_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 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‐ 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.

three‐dimensional (3D) molecular maps of hundreds of distinct glycan species across a wide mass range while also providing information on analyte relative abun­dance and on‐tissue spatial distribution, which can be directly linked to the histo­pathological data from the same clinical specimen [22–29]. The IMS technology provides the mass accuracy and chemical specificity of MS‐based detection and sup­ports further on‐tissue tandem MS fragmentation (e.g. collision‐induced fragmenta­tion) to achieve exact structure identification while still preserving the spatial distribution of individual analytes and the histopathological landscape of the target tissue. The implementation of IMS workflows in the cancer research field is of par­ticular relevance since most clinically approved cancer biomarkers are either glyco­proteins or carbohydrate antigens.