Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5383_Библиотеки_им_академика_М_И_Перельмана

research field. The aberrant glycan signatures expressed at the cell surface of neo­plastic cells actively support the establishment of an immune‐suppressive microen­vironment that facilitates tumor onset and progression [12]. Cancer‐specific abnormal glycan antigens act as natural ligands for specific membrane‐bound or secreted immune receptors. These receptors, which include Siglecs, galectins, and C‐type lectins, constitute GBPs bearing unique glycan specificities that tightly con­trol the activation threshold of several immune cell populations. Importantly, gly­can microarrays have been used to determine the degree of N‐glycan branching as well as the Neu5Ac linkage specificity and sulfation status regulating the binding preferences of several receptors of the Siglec family[58, 81–83]. The same strategy was applied to characterize the binding specificity of distinct galectins, including their branching preferences and linkage‐dependent tolerance to Neu5Ac motifs [58, 69, 84]. The comprehensive profiling of these multivalent interactions, in par­ticular the identification of which glycan epitopes are bound by specific immune receptors, is fundamental for the establishment of functional associations between specific glycosylation signatures of cancer cells and well‐defined outputs of immune response. Such knowledge can not only be the stepping stone for the design of novel anticancer immunotherapies but also an invaluable source of predictive and strati­fying biomarkers of immunotherapeutic response. The glycan microarray technol­ogy has also been extensively applied for cancer biomarker validation by allowing the miniaturized, multiplexed, and parallel screening of large numbers of analytes, samples, and replicates in a single experiment. In particular, glycan microarrays constitute useful tools to screen and compare the serum antibody repertoire of healthy individuals and cancer patients, which is indispensable for the development of highly specific anticancer vaccines targeting tumor‐associated glycan antigens and the identification of glycan‐based cancer biomarkers[75, 85–90]. A microarray of aberrantly glycosylated MUC1 O‐glycopeptides has been developed to assess the antigenicity of these tumor‐associated structures through the screening of MUC1‐ targeting mAbs and cancer patient serum samples[91, 92]. The glycan microarray technology has been applied to the rapid screening of hybridomas for the selection of the most specific mAb candidates against a target glycan antigen[75]. Of rele­vance to the cancer research field, glycan microarrays have been successfully used to infer the substrate specificity of individual glycosyltransferases (GTs) [93, 94]. Furthermore, this technology may be used to assess the efficacy of therapeutic inhibitors targeting glycan‐dependent molecular interactions[95].

The nontemplate‐driven and highly dynamic nature of protein glycosylation poses several challenges to the study of the functional role of complex carbohydrates in biological systems. Additionally, the partially overlapping substrate specificities and compensatory activity of related GTs hinder the systematic dissection of specific

glycosylation pathways and the establishment of robust associations between one enzyme’s activity and the unequivocal expression of specific cancer‐associated gly­can epitopes. Such limitations have motivated the development of glycoengineered invitro models of disease, in which the glycome’s complexity and heterogeneity are simplified so that the exact phenotypical contributions of specific glycosylation pat­terns can be pinpointed[96]. The precise editing of genomic loci codifying for vari­ous elements of the cellular glycosylation machinery (e.g. GTs, glycosidases, molecular chaperones, and monosaccharide transporters) has generated an unprec­edented catalog of isogenic cell lines depicting the loss or gain of selected glycosyla­tion capacities[97–99]. These invitro platforms can be exploited for the systematic functional dissection of the human glycome at the single‐cell level[100]. Moreover, glycoengineering strategies have found wide applicability in the design and recom­binant expression of carbohydrate‐based therapeutics, such as enzymes and immu­noglobulins, which require highly controlled, well‐defined, and homogeneous glycosylation profiles [101–104]. Furthermore, glycoengineered mammalian cell lines in which the expression of a target gene of interest has been either silenced or activated represent appealing platforms for the screening of selective inhibitors tar­geting specific glycosylation pathways and enzymes.

Initial studies based on random mutagenesis, plasmid‐based overexpression, and targeted homologous recombination knock‐out (KO) of glycosylation‐related genes in both mammalian cell lines and animal models have led to the identification of GTs playing essential roles in early embryonic development, homeostasis, and dis­ease[96, 105, 106]. Furthermore, these studies have provided invaluable knowledge on which glycosylation steps are catalyzed by individual GTs and which ones can be performed by partially redundant isoenzymes. Additionally, site‐directed mutagen­esis has been extensively used for the selective and site‐specific abrogation of glyco­sylation in target peptidic sequences to interrogate the functional contribution of glycans to the oncogenic or tumor‐suppressive features of several relevant cancer‐ associated glycoproteins, including the E‐cadherin adhesion molecule and the epi­dermal growth factor receptor (EGFR) RTK[47, 107]. However, such approaches are extremely laborious and generate highly unpredictable and inconsistent genomic and phenotypical outcomes. Additionally, noncontrolled plasmid‐based GT overex­pression often produces unforeseen compensatory perturbations in multiple glyco­sylation pathways, which prevents the unequivocal assignment of observed glycophenotypes to the function of specific GT‐coding genes. On the other hand, RNA interference‐based strategies frequently lead to incomplete downregulation of the target protein, which translates into highly variable glycophenotypes.

of a target genomic locus. In the CRISPR/Cas9 system, a complementary short guide

RNA (gRNA) molecule is used to direct the activity of the Cas9nuclease to a pre­specified site of the target nucleotide sequence[111]. The produced double‐strand breaks are then repaired by the error‐prone nonhomologous end joining (NHRJ) cellular mechanism, which results in mutations or small insertions/deletions (indels) at the cleavage site that disrupt the expression of the target gene by shifting its open reading frame (ORF). Currently, multiple engineering events, including both KOs and knock‐ins (KIs), can be easily and rapidly performed in the same cell line, which allows the generation of catalog invitro models depicting virtually any desired glycophenotype [99]. Importantly, the successful application of genetic engineering strategies requires comprehensive knowledge of the target glycosyla­tion pathways in the selected cell host, namely the cell line‐dependent expression profiles of the target GTs, but also of possible isoenzymes catalyzing redundant bio­synthetic steps. This data not only aids in the prediction of structural glycomic out­comes but also facilitates the rational selection of the most directed and appropriate genome editing pipeline. Recently, comprehensive lentiviral and plasmid‐based libraries of validated high‐efficiency gRNAs for the specific targeting of every known GT‐coding gene have been constructed and made globally available to the scientific community [98, 112]. Moreover, the CRISPR/Cas9 system has been adapted to allow the transcription activation of target dormant genes through the fusion of an inactive Cas9nuclease with specific transcription factors. This strategy has been successfully used to activate the expression of ST6GAL1 and MGAT3 GT‐ coding genes in the CHO cell line[113].

(VVA) and have led to the unprecedented identification of thousands of O‐glycopro­teins and O‐glycosites across multiple invitro cancer models. The same strategy has been applied to the identification of O‐mannosylated substrates[119].

One of the major drawbacks of glycan microarrays is related to the inability of such platforms to present the printed and immobilized glycan ligands in their natu­ral cell surface context. The wide application of precise genome editing technologies targeting virtually all known mammalian glycosylation‐related genes has allowed the establishment of comprehensive cell‐based libraries composed of isogenic cell lines systematically modified to depict any intended glycosylation features by the use of combinatorial KO/KI strategies [21, 82, 120]. The replacement of isolated glycan‐based ligands by glycoengineered cell lines allows for the screening of glycan‐dependent molecular interactions while preserving their natural cell‐ dependent context and is conveniently compatible with high‐throughput cell‐based assays. Cell‐based arrays composed of glycoengineered cell lines have been success­fully applied to gain insights on the functional role and binding specificities of endothelial selectins, galectins, and Siglec receptors[82, 121]. Precise genome edit­ing technology has been successfully used to completely deplete the HEK293 cell line of its sialylation capacities[82]. The generated Neu5Ac‐null HEK293 cells then served as a “white canvas” cellular platform in which the combinatorial loss/gain of individual sialyl‐ and sulfotransferases allowed the comprehensive characterization and structural elucidation of the selective glycan‐binding preferences and protein‐ specific context underlying the Siglec–glycan human interactome. Information on the fine glycan‐binding specificities of human Siglecs is indispensable for the func­tional dissection of molecular resistance mechanisms hampering the clinical devel­opment and implementation of antitumor immunotherapeutic strategies.

live detection of isoform‐specific functions within a complex living experimental

system. Indeed, the compatibility of oligosaccharide metabolic engineering strate­gies with animal models allows for invivo labeling, noninvasive imaging, and track­ing of glycan‐mediated interactions in their native environment[129, 130]. A click chemistry‐based strategy using alkyne‐tagged Neu5Ac monosaccharides has been successfully employed in the live tracking of sialylation to probe the specificity of Siglec‐based interactions in HEK293 cells[82]. A chemoenzymatic glycan editing strategy was successfully employed for the generation of natural killer (NK) cells capable of the multivalent presentation of high‐affinity ligands for the CD22 cell surface receptor overexpressed in B‐cell lymphomas[131].

gastrointestinal tract exhibit a significant increase in the serum levels of the CA 19.9

consequently, the concentration of suppressing molecules through the use of nanoscale ESI setups[160]. Moreover, when compared with MALDI, ESI produces better resolved peaks for glycoconjugates due to the absence of matrix‐derived adduct peaks.

of cancer‐specific serum biomarkers in multiple solid tumors, including pros­tate [182], breast carcinoma [183, 184], and cholangiocarcinoma [185]. The Tn‐binding VVA lectin was used for the affinity capture of simplified mucin‐type O‐ glycosylated peptides generated by the SimpleCell glycoengineering strategy and allowed the identification of over 600 distinct glycoproteins and close to 3000 O‐glycosites[117]. However, regardless of their glycan specificity, no single lectin or combination of lectins can encompass the entire glycoproteome. Other affinity‐based chemical strategies, including titanium dioxide (TiO

ough glycan structural characterization. Detailed fragmentation information from