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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.
Recently optimized methods are capable of the simultaneous multimodal acquisition of MALDI‐IMS spectra derived from both N‐glycans and proteolytic peptides
from the same tissue section[38]. The spatial distribution map of identified peptidic
sequences can then be combined with the corresponding N‐glycan map for the identification of overlapping regions. Subsequent bioinformatic analysis may lead to the
identification of glycoprotein candidates, and their spatial distribution can be further assigned to well‐defined histological regions. Such studies may provide mechanistic insights on the functional roles played by particular glycan signatures in
malignant cell transformation, in particular through the identification of proteins
modified with specific aberrant glycan determinants. Furthermore, the combination of N‐glycan‐ and protein‐derived structural data may provide novel combinatorial sets of cancer‐specific biomarkers with improved sensitivity and specificity.
6.4 In Situ Proximity Ligation Assay
The unbiased and comprehensive identification of glycoproteins for biomarker discovery purposes through on‐tissue IMS analysis can be laborious and time‐
consuming. Moreover, glycoprotein validation through direct immunoprecipitation
experiments from cryopreserved clinical specimens can be technically challenging
due to heterogeneous or reduced expression of the target protein and the requirement of considerable amounts of starting frozen material. Furthermore, although
methodologies based on genetically modified cell models represent powerful tools
to dissect the interactome of specific proteins, they are not compatible with patient
samples. On the other hand, traditional immuno‐ or lectin‐based histochemical
staining methods remain limited to the detection and tissue mapping of individual
proteins and carbohydrate signatures while overlooking biologically significant
molecular interactions. In addition, such techniques do not allow the unequivocal
identification of the protein carriers that are modified with a given type of glycan
structure. To circumvent such technical limitations, numerous studies have instead
pursued the direct on‐tissue validation of specific protein glycoform candidates, initially identified in invitro models of malignant transformation, using large cohorts
of tumor clinical samples. As a result, several oncogenic proteins modified with
specific glycosylation signatures may emerge as more sensitive and specific biomarkers of human malignancy when compared to more classical biomarkers targeting fully peptidic or carbohydrate‐based epitopes.
The in situ proximity ligation assay (PLA) allows the on‐tissue detection, imaging, and relative quantification of a plethora of cellular events at single‐molecule
resolution, including protein–protein interactions, protein translation, and degradation, as well as multiple post-translational modifications such as protein phosphorylation and glycosylation. This assay relies on the dual binding of highly specific
affinity reagents, such as primary antibodies or lectins, for the in situ detection of
the molecular proximity between two prespecified target epitopes on either FFPE or
cryopreserved tissue specimens[39, 40]. In this method, a pair of affinity reagents
labeled with single‐stranded DNA molecules, also termed oligonucleotide proximity/detection probes, are bound to their respective target epitopes in a whole tissue
section. If the epitopes recognized by both antibodies/lectins are found in close
molecular proximity (10–40 nm), a proper detection complex is formed, which
allows for the antibody‐bound oligonucleotide molecules to be enzymatically joined
into a circular DNA strand through rolling‐circle amplification. The resulting DNA
ligation product can then serve as a template for PCR‐based multimeric signal
amplification. The final amplification product can then be hybridized with fluorescent or chromogenic oligonucleotide strands (detection probes) for microscopic
visualization and counting of individual spots at single complex resolution. The
requirement for two proximal recognition reactions ensures highly selective mapping of interacting complexes since individual probe binding is insufficient to produce visible detection signals. In the typical approach to identify proteins carrying
specific glycosylation traits, one antibody binds to a peptidic epitope within the target protein, and a second glycan‐ binding affinity reagent (mAb or lectin) targets the
carbohydrate motif. Conveniently, the in situ PLA method can be performed using
labeled secondary affinity reagents, avoiding the need for the conjugation of numerous pairs of primary antibodies or lectins. The PLA methodology is highly versatile,
as a myriad of affinity reagents can be readily and easily converted into proximity
probes. This is of particular significance when studying protein‐specific glycosylation reactions, as it allows the use of carbohydrate‐binding lectins for the detection
1676.4 In Situ
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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].
Over the past decade, several studies have successfully used the PLA technology
to validate, in whole tissue tumor clinical samples, the modification of several
cancer‐associated proteins with aberrant glycan antigens, aiming at the identification of clinically relevant diagnostic and prognostic biomarker candidates. Such is
the example of the MUC2intestinal mucin and the CD44 co‐receptor, both disclosed
by fluorescence‐based PLA as carriers of the short‐truncated O‐glycan epitope STn
in advanced gastric adenocarcinomas[42–44]. The PLA‐based association between
x
the sialyl Lewis x (sLe
) tetrasaccharide and several membrane‐anchored proteins
in gastric cancer tissues, including the RON receptor tyrosine kinase (RTK) and the
carcinoembryonic antigen (CEA) adhesion molecule, has further unveiled the
active role played by aberrant glycans in tuning the malignant features of oncogenic
x
receptors[45, 46]. Of note, the expression of sLe
‐containing CEA proteoforms was
associated with worse patient clinical outcome, portraying the PLA technology as a
valuable source of prognostic markers. Recently, the E‐cadherin cellular adhesion
molecule has been validated as a molecular carrier of highly branched N‐glycan
chains by brightfield PLA analysis of advanced gastric adenocarcinoma tissue sections, highlighting this particular E‐cadherin glycoform as a robust predictive biomarker of patient dismal prognosis[47]. In addition, the use of PLA technology has
demonstrated the modification of the oncogenic ErbB2 RTK, which currently
remains one of the few actionable therapeutic targets in the gastric cancer setting,
with α2,6‐linked Neu5Ac moieties in whole tissue sections of intestinal‐type gastric
carcinomas[48]. In situ PLA analysis has also provided valuable insights on the
glycosylation status of mucin receptors required for Helicobacter pylori (H. pylori)
adhesion to the gastric epithelium of glycoengineered mice models[49]. The combination of glycan metabolic labeling with the PLA‐based analysis of protein–
carbohydrate interactions has allowed the disclosure of the mechanistic contribution
of specific glycan traits (e.g. sialylation and fucosylation) to various functional
aspects of cancer‐relevant proteins at the subcellular scale, including the turnover
and ligand‐induced dimerization of oncogenic cell surface receptors[50].
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.
6.5 Glycan Microarrays
Although the exact dimension of the cellular glycome remains a matter of debate, it
is estimated to be in the range of 100 000–500 000 unique glycan structures [51].
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 years ago, has propelled the high‐throughput systematic
interrogation of glycan‐based molecular interactions[52–55]. By facilitating the fast
and highly reproducible screening of a great number of glycan epitopes and compositions, this technology has allowed the unprecedented elucidation of the
carbohydrate‐binding specificity of a multitude of pathogens, whole cells, and
GBPs. Indeed, the use of glycan microarrays for the comprehensive validation of the
glycan ligand repertoire of several mAbs and plant-derived lectins has solidified
their applicability as invaluable research tools to address the role of glycans in biological systems[54, 56–58]. The Consortium for Functional Glycomics (CFG) has
used glycan microarray technology to disclose the detailed specificity of over 100
plant lectins (http://www.functionalglycomics.org/)[59]. Furthermore, the binding
specificity and affinity data provided by glycan microarrays will pave the way for the
rational design of carbohydrate‐based therapeutic agents. Printed carbohydrate
microarrays enable the simultaneous analysis of thousands of binding events
between a single target analyte (e.g. lectin, soluble ligand, and mAb) and a miniaturized catalog containing hundreds of spatially defined glycan species immobilized
onto a solid phase in a covalent or noncovalent manner[54, 60]. Moreover, the condensation of hundreds of different ligands into a miniaturized format significantly
reduces the required amount of both the target analyte and each unique immobilized ligand. In addition, glycan microarrays represent ideal platforms for the
screening of glycan‐dependent molecular interactions by allowing the multivalent
display of immobilized ligands on a solid surface, which mimics the weak and
reversible nature of cell–cell interactions. Additionally, glycan microarrays are
highly reproducible, cost‐effective, and allow the fast screening of multiple samples.
Depending on the biological question, different types of macromolecules can be
immobilized on the array solid substrate (e.g. glycans, glycoproteins, glycopeptides,
mAbs, or lectins), giving rise to a diversified set of platforms. A standard‐size microscope glass slide represents the original solid surface for ligand immobilization and
remains the most widely used[61]. In the case of glycan microarrays, the immobilized
library of pure carbohydrate structures can be either chemically synthesized or isolated from natural sources[53, 55, 58, 62, 63]. Chemoenzymatic strategies combining
automated glycan assembly with selected enzymatic steps allow the controlled synthesis of glycosaminoglycans, branched N‐glycans, and sialylated structures in a
linkage‐specific manner [64–68]. The selection of the most appropriate strategy for
the covalent or noncovalent immobilization of glycans onto the solid phase depends
on the synthetic method used in the preparation of a given glycan library or the source
from which naturally occurring ligands are isolated. Glycans produced by fully chemical or chemoenzymatic synthesis are derivatized with orthogonal bi‐functional linkers to allow their covalent attachment to a functionalized solid surface[69, 70]. On the
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other hand, naturally occurring carbohydrates, such as milk oligosaccharides and free
reducing glycans released from glycoproteins by enzymatic digestion or chemical
hydrolysis, also require proper derivatization prior to immobilization[55, 62, 71, 72].
Although, in principle, polysaccharides can be directly attached to the array by simple
adsorption, enzymatically released glycans require the introduction of a functional
group at their reducing end, which can be accomplished through different conjugation strategies.
Immobilized ligand libraries are spotted with micrometric resolution onto the
selected surface by automated arraying robots in a prespecified, spatially resolved
manner. For a single carbohydrate ligand, several replicates of serial concentrations
are printed. Since glycans naturally establish low‐affinity interactions with other
macromolecules, the density at which individual glycan structures are spotted onto
the microarray surface is a determinant in the generation of detectable signals and
subsequent data interpretation[73]. After spotting is concluded, a washing step is
performed for the removal of unbound carbohydrate molecules. A single‐array slide
may contain up to 20
000 spots[74]. Despite the significant advances in the chemical
synthesis of structurally defined glycan chains made over the last two decades, both
the number and structural diversity of the glycans that can be immobilized onto a
single microarray remain far from representative of the human glycome’s estimated
dimension[72, 73].
The glycan microarray technology has been most extensively applied in the dissection of protein–carbohydrate interactions, namely in the characterization of the
binding specificity of mAbs and GBPs[54, 75–77]. There are several possible strategies for the detection and quantification of bound molecular partners at the microarray surface. In the case of glycan microarrays, the analytes of interest, either in a
pure isolated form (e.g. single GBP) or as a complex mixture (e.g. serum), are labeled
and incubated on the solid substrate to allow ligand binding. Following the washing
of unbound macromolecules, the signal emitted by the labeled bound analytes can
be detected and quantified in individual spatially resolved spots, each one corresponding to a unique glycan structure. Fluorescence‐based quantification of ligand
binding remains the most widely used method for signal detection due to its high
sensitivity and wide availability of fluorescence scanning equipment[62, 69, 70, 78].
Of note, the labeling of proteins with fluorescent tags may lead to protein denaturation or alterations in their carbohydrate‐binding domain. Alternatively, a fluorescently tagged antibody for the recognition of bound analytes can be used. An
adaptation of this detection strategy, known as sandwich array, relies on the use of
fluorescently labeled secondary antibodies or lectins for the detection of ligands
bound to the microarray solid substrate via a primary set of immobilized antibodies[79]. Due to the limited availability of specific carbohydrate‐recognizing antibodies and lectins, label‐free detection methods, such as quantitative on‐chip MS
and surface plasmon resonance (SPR), have become increasingly popular for signal
quantification[80]. Interestingly, SPR‐based analysis of glycan microarray signals
provides valuable quantitative parameters on the reaction kinetics, including association and dissociation constants. As the volume and complexity of information
generated by glycan microarrays grow exponentially, so does the need for software
tools and algorithms capable of comprehensive and integrative data analysis.
The glycan microarray technology has been successfully applied in the cancer
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].
171 In Vitro, In Vivo, and Ex Vivo
6.6 Glycoengineered In Vitro, In Vivo, and
Ex Vivo Models
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
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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
invitro 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 invitro 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.
The stable genetic engineering of glycan‐related genes has been significantly simplified with the advent of precise genome editing technologies, characterized by
high precision, consistency, and speed at a reduced cost. These include zinc‐finger
nucleases (ZFNs)[108], transcription activator‐like effector nucleases (TALENs)
[109], and the clustered regularly interspaced short palindromic repeats with the
CRISPR‐associated protein 9 (CRISPR/Cas9)[110, 111]. All of the aforementioned
genome editing tools can be used to achieve efficient silencing of a selected gene
through the introduction of site‐specific double‐strand breaks in the DNA sequence
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 Cas9nuclease 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 invitro 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 Cas9nuclease 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].
Nuclease‐based genome editing strategies also support the stable integration, or
KI, of exogenous glycosylation‐related genes provided to the cell system under the
form of a homologous DNA template[99, 101]. This technology has been successfully used in the unbiased dissection of the substrate specificity and functional role
of partially redundant GT isoenzymes, as shown for multiple polypeptide
N‐ acetylgalactosaminyltransferases (ppGalNAcTs) [114]. More recently, a ZFN‐
based KI system was adapted to allow the inducible expression of individual ppGalNAcTs over a KO background in HEK293 cells and demonstrated that isoform‐specific
substrates are glycosylated in a dose‐dependent manner[115]. A similar KO/rescue
system was used for the stable integration of the ppGalNAcT6‐coding gene in colon
cancer cells, which led to the disclosure of this enzyme’s active role in regulating
cellular growth and differentiation[116].
Genetic glycoengineering has proven a useful tool when combined within glycoproteomic pipelines for glycosite and glycopeptide discovery and validation by reducing glycosylation diversity and heterogeneity and, thus, facilitating glycan‐directed
enrichment strategies. In the SimpleCell system, the KO of either the COSMC or
C1GALT1 transferase‐coding genes through precise genome editing prevents the
extension of mucin‐type O‐glycan chains beyond the single N‐acetylgalactosamine
(GalNAc) Tn antigen[43, 117, 118]. These isogenic cell lines depicting simplified
O‐glycosylation were used towards the comprehensive MS‐based mapping and
characterization of the GalNAc‐type O‐glycoproteome by allowing the efficient capture and enrichment of O‐glycopeptides by the Tn‐binding Vicia villosa agglutinin
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(VVA) and have led to the unprecedented identification of thousands of O‐glycoproteins and O‐glycosites across multiple invitro 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.
The combination of chemical and genetic engineering methodologies toward precise metabolic glycan labeling has provided invaluable insights on the dynamics of
the cell surface glycoproteome and allowed the unequivocal assignment of isoform‐
specific substrate preferences depicted by partially redundant isoenzymes, in particular the ppGalNAcT family [122–127]. Glycosylation metabolic labeling and
tagging are based on the use of highly selective bioorthogonal reactions between
two complementary, chemically modified functional groups that do not occur naturally in living systems. These chemically engineered probes must be biologically
inert, i.e. they must not perturb the homeostatic function of target macromolecules
in which they are incorporated while remaining highly selective toward each other,
a strategy otherwise known as “click chemistry”[128]. Such bioorthogonal metabolic labeling probes are equipped with either visualization or enrichment tags that
support a diverse range of downstream applications, including live visualization
through imaging techniques and MS‐based structural analysis. In the “bump‐and‐
hole” strategy, the catalytic pocket of an individual ppGalNAcT enzyme (hole) is
enlarged by targeted amino acid substitutions through genetic engineering to
accommodate a complementary, chemically modified UDP‐GalNAc analog (bump)
that is incorporated by the selected cell host[123]. The fact that this modified substrate is uniquely recognized by the mutant enzyme makes the interacting pair
orthogonal to all other competing GTs within the same cell host, which allows the
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 invivo 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].
Glycoengineered animal models, carrying genetic defects on central glycosylation‐
related genes through homozygous targeted gene mutation, have unveiled the
essential function of several GTs and their respective glycan products in mammalian embryonic development[105, 132–135]. Moreover, the organ‐ and tissue‐specific conditional deletion of glycogenes has disclosed their function in central
physiological and pathological processes, such as neurogenesis[136], fertility[137,
138], immunity[139, 140], and neoplastic transformation[141–148]. The CFG has
comprehensively characterized the phenotype of 36mutant mouse strains carrying
genetic deficiencies in different GTs and GBPs[106]. Despite glycoengineered cell
lines providing important mechanistic insights on glycan regulation and function,
the comprehensive study of the functional role played by cancer‐associated glycan
epitopes and specific GTs in human malignant transformation requires more complex in vivo models in which organ‐specific carcinogenesis can be recapitulated.
Importantly, the crossbreeding of glycogene‐deficient strains with mice models of
spontaneous carcinogenesis has significantly expanded the invivo toolbox for conditional, organ‐specific glycoengineering. Such animal systems have highlighted
TACAs and the enzymes responsible for their biosynthesis as major regulators of
the intricate network of cellular and molecular interactions occurring within the
tumor microenvironment, including the interaction of cancer cells with immune
cell populations and ECM components. Moreover, they offer an integrative and systemic view of the biological activity of glycosylation‐targeting therapeutic agents
and represent, therefore, appealing platforms for their preclinical testing.
Targeted gene mutation of Mgat5, which codifies for a GT catalyzing the β1,6‐
branching of N‐glycan antennae, has disclosed branched complex N‐glycans as
active drivers of tumor growth and metastatic dissemination in a viral‐induced mice
model of breast carcinogenesis[146]. The development of Mgat5‐null mice models
of spontaneous intestinal carcinogenesis has further established the pivotal role
played by β1,6‐branched N‐glycan structures in the immune‐based recognition and
elimination of colorectal cancer cells [144]. The genetic silencing of ST6Gal1 in
mice bearing spontaneous, viral‐induced mammary tumors has functionally implicated this enzyme, responsible for the terminal capping of N‐glycan chains with
α2,6Neu5Ac, in the maintenance of tumor stemness and differentiation[147]. These
observations were further confirmed through the conditional restoration of ST6Gal1
activity in cell lines derived from ST6Gal1‐null tumors. Mice with the inducible
expression of the human FUT3 and β3GALT5 transgenes restricted to the
175 In Vitro, In Vivo, and Ex Vivo
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