The early detection of pancreatic ductal adenocarcinoma is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers CA19-9 and sTRA are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and non-cancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry approach was utilized to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of PDAC patients represented by tissue microarrays and whole tissue sections. Orthogonally, these same tissues were characterized by multi-round immunofluorescence which defined expression of CA19-9 and sTRA as well as other lectins towards carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated bi-antennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9 expressing tissues tended to be bi-, tri- and tetra-antennary structures with both core and terminal fucose residues and bisecting N-acetylglucosamines. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored tri- and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-IHC and IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes.

Clear-cell renal cell carcinoma (ccRCC) presents challenges to clinical management because of late-stage detection, treatment resistance, and frequent disease recurrence. Metabolically, ccRCC has a well-described Warburg effect utilization of glucose, but how this affects complex carbohydrate synthesis and alterations to protein and cell surface glycosylation is poorly defined. Using an imaging mass spectrometry approach, N-glycosylation patterns and compositional differences were assessed between tumor and nontumor regions of formalin-fixed clinical ccRCC specimens and tissue microarrays. Regions of normal kidney tissue samples were also evaluated for N-linked glycan-based distinctions between cortex, medullar, glomeruli, and proximal tubule features. Most notable was the proximal tubule localized detection of abundant multiantennary N-glycans with bisecting N-acetylglucosamine and multziple fucose residues. These glycans are absent in ccRCC tissues, while multiple tumor-specific N-glycans were detected with tri-and tetra-antennary structures and varying levels of fucosylation and sialylation. A polycystic kidney disease tissue was also characterized for N-glycan composition, with specific nonfucosylated glycans detected in the cyst fluid regions. Complementary to the imaging mass spectrometry analyses was an assessment of transcriptomic gene array data focused on the fucosyltransferase gene family and other glycosyltransferase genes. The transcript levels of the FUT3 and FUT6 genes responsible for the enzymes that add fucose to N-glycan antennae were significantly decreased in all ccRCC tissues relative to matching nontumor tissues. These striking differences in glycosylation associated with ccRCC could lead to new mechanistic insight into the glycobiology underpinning kidney malignancies and suggest the potential for new therapeutic interventions and diagnostic markers. K E Y W O R D Sclear-cell renal cell carcinoma, fucosylation, MALDI imaging, N-glycosylation, polycystic kidney disease

Glycosylation is an important posttranslational modifier of proteins and lipid conjugates critical for the stability and function of these macromolecules. Particularly important are N‐linked glycans attached to asparagine residues in proteins. N‐glycans have well‐defined roles in protein folding, cellular trafficking and signal transduction, and alterations to them are implicated in a variety of diseases. However, the non‐template driven biosynthesis of these N‐glycans leads to significant structural diversity, making it challenging to identify the most biologically and clinically relevant species using conventional analyses. Advances in mass spectrometry instrumentation and data acquisition, as well as in enzymatic and chemical sample preparation strategies, have positioned mass spectrometry approaches as powerful analytical tools for the characterization of glycosylation in health and disease. Imaging mass spectrometry expands upon these strategies by capturing the spatial component of a glycan's distribution in‐situ, lending additional insight into the organization and function of these molecules. Herein we review the ongoing evolution of glycan imaging mass spectrometry beginning with widely adopted tissue imaging approaches and expanding to other matrices and sample types with potential research and clinical implications. Adaptations of these techniques, along with their applications to various states of disease, are discussed. Collectively, glycan imaging mass spectrometry analyses broaden our understanding of the biological and clinical relevance of N‐glycosylation to human disease.

Sialic acid isomers attached in either α2,3 or α2,6 linkage to glycan termini confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences in traditional mass spectrometry experiments. Multiple derivatization strategies have been developed to stabilize and facilitate the analysis of sialic acid isomers and their glycoconjugate carriers by high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry workflows. Herein, a set of novel derivatization schemes are described that result in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3and α2,8-linked sialic acids. These chemical modifications were validated and structurally characterized using model isomeric sialic acid conjugates and model protein carriers. Use of an alkyne-amine, propargylamine, as the second amidation reagent effectively introduces an alkyne functional group into α2,3-linked sialic acid glycoproteins. In tissues, serum, and cultured cells, this allows for the detection and visualization of Nlinked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffinembedded prostate cancer tissues and pancreatic cancer cell lines were used to characterize the numbers and distribution of alkyne-modified α2,3-linked sialic acid N-glycans. An azide-amine compound with a poly(ethylene glycol) linker was evaluated for use in histochemical staining. Formalin-fixed pancreatic cancer tissues were amidated with the azide amine, reacted with biotin-alkyne and copper catalyst, and sialic acid isomers detected by streptavidin-peroxidase staining. The direct chemical introduction of bioorthogonal click chemistry reagents into sialic acid-containing glycans and glycoproteins provides a new glycomic tool set to expand approaches for their detection, labeling, visualization, and enrichment.

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An understanding of the molecular features associated with prostate cancer progression (PCa) and resistance to hormonal therapy is crucial for the identification of new targets that can be utilized to treat advanced disease and prolong patient survival. The glycome, which encompasses all sugar polymers (glycans) synthesized by cells, has remained relatively unexplored in the context of advanced PCa despite the fact that glycans have great potential value as biomarkers and therapeutic targets due to their high density on the cell surface. Using imaging mass spectrometry (IMS), we profiled the N-linked glycans in tumor tissue derived from 131 patients representing the major disease states of PCa to identify glycosylation changes associated with loss of tumor cell differentiation, disease remission, therapy resistance and disease recurrence, as well as neuroendocrine (NE) differentiation which is a major mechanism for therapy failure. Our results indicate significant changes to the glycosylation patterns in various stages of PCa, notably a decrease in tri- and tetraantennary glycans correlating with disease remission, a subsequent increase in these structures with the transition to therapy-resistant PCa, and downregulation of complex N-glycans correlating with NE differentiation. Furthermore, both nonglucosylated and monoglucosylated mannose 9 demonstrate aberrant upregulation in therapy-resistant PCa which may be useful therapeutic targets as these structures are not normally presented in healthy tissue. Our findings characterize changes to the tumor glycome that occur with hormonal therapy and the development of castration-resistant PCa (CRPC), identifying several glycan markers and signatures which may be useful for diagnostic or therapeutic purposes.

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