Applications and continued evolution of glycan imaging mass spectrometry.

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.

Development of an Antibody-Based Platform for the Analysis of Immune Cell-Specific N-linked Glycosylation.

N-linked glycosylation plays an important role in both the innate and adaptive immune response through the modulation of cell surface receptors as well as general cell-to-cell interactions. The study of immune cell N-glycosylation is gaining interest but is hindered by the complexity of cell-type-specific N-glycan analysis. Analytical techniques such as chromatography, LC-MS/MS, and the use of lectins are all currently used to analyze cellular glycosylation. Issues with these analytical techniques include poor throughput, which is often limited to a single sample at a time, lack of structural information, the need for a large amount of starting materials, and the requirement for cell purification, thereby reducing their feasibility for N-glycan study. Here, we report the development of a rapid antibody array-based approach for the capture of specific nonadherent immune cells coupled with MALDI-IMS to analyze cellular N-glycosylation. This workflow is adaptable to multiple N-glycan imaging approaches such as the removal or stabilization and derivatization of terminal sialic acid residues providing unique avenues of analysis that have otherwise not been explored in immune cell populations. The reproducibility, sensitivity, and versatility of this assay provide an invaluable tool for researchers and clinical applications, significantly expanding the field of glycoimmunology.

Bioorthogonal Chemical Labeling Probes Targeting Sialic Acid Isomers for N-Glycan MALDI Imaging Mass Spectrometry of Tissues, Cells, and Biofluids.

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 α2,3- and 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 N-linked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffin-embedded 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.

Imaging Mass Spectrometry and Lectin Analysis of N-Linked Glycans in Carbohydrate Antigen-Defined Pancreatic Cancer Tissues.

The early detection of pancreatic ductal adenocarcinoma (PDAC) is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers carbohydrate antigen 19-9 (CA19-9) and sialylated tumor-related antigen (sTRA) are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and noncancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry (IMS) approach was used to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of patients with PDAC represented by tissue microarrays and whole-tissue sections. Orthogonally, these same tissues were characterized by multiround immunofluorescence that defined expression of CA19-9 and sTRA as well as other lectins toward 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 biantennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9-expressing tissues tended to be biantennary, triantennary, and tetra-antennary structures with both core and terminal fucose residues and bisecting GlcNAc. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored triantennary 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-immunohistochemistry and lectin-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.

Desert hedgehog-primary cilia cross talk shapes mitral valve tissue by organizing smooth muscle actin.

Non-syndromic mitral valve prolapse (MVP) is the most common heart valve disease affecting 2.4% of the population. Recent studies have identified genetic defects in primary cilia as causative to MVP, although the mechanism of their action is currently unknown. Using a series of gene inactivation approaches, we define a paracrine mechanism by which endocardially-expressed Desert Hedgehog (DHH) activates primary cilia signaling on neighboring valve interstitial cells. High-resolution imaging and functional assays show that DHH de-represses smoothened at the primary cilia, resulting in kinase activation of RAC1 through the RAC1-GEF, TIAM1. Activation of this non-canonical hedgehog pathway stimulates α-smooth actin organization and ECM remodeling. Genetic or pharmacological perturbation of this pathway results in enlarged valves that progress to a myxomatous phenotype, similar to valves seen in MVP patients. These data identify a potential molecular origin for MVP as well as establish a paracrine DHH-primary cilium cross-talk mechanism that is likely applicable across developmental tissue types.

An N-glycome tissue atlas of 15 human normal and cancer tissue types determined by MALDI-imaging mass spectrometry.

N-glycosylation is an abundant post-translational modification of most cell-surface proteins. N-glycans play a crucial role in cellular functions like protein folding, protein localization, cell-cell signaling, and immune detection. As different tissue types display different N-glycan profiles, changes in N-glycan compositions occur in tissue-specific ways with development of disease, like cancer. However, no comparative atlas resource exists for documenting N-glycome alterations across various human tissue types, particularly comparing normal and cancerous tissues. In order to study a broad range of human tissue N-glycomes, N-glycan targeted MALDI imaging mass spectrometry was applied to custom formalin-fixed paraffin-embedded tissue microarrays. These encompassed fifteen human tissue types including bladder, breast, cervix, colon, esophagus, gastric, kidney, liver, lung, pancreas, prostate, sarcoma, skin, thyroid, and uterus. Each array contained both normal and tumor cores from the same pathology block, selected by a pathologist, allowing more in-depth comparisons of the N-glycome differences between tumor and normal and across tissue types. Using established MALDI-IMS workflows and existing N-glycan databases, the N-glycans present in each tissue core were spatially profiled and peak intensity data compiled for comparative analyses. Further structural information was determined for core fucosylation using endoglycosidase F3, and differentiation of sialic acid linkages through stabilization chemistry. Glycan structural differences across the tissue types were compared for oligomannose levels, branching complexity, presence of bisecting N-acetylglucosamine, fucosylation, and sialylation. Collectively, our research identified the N-glycans that were significantly increased and/or decreased in relative abundance in cancer for each tissue type. This study offers valuable information on a wide scale for both normal and cancerous tissues, serving as a reference for future studies and potential diagnostic applications of MALDI-IMS.

Molecular and translational biology of the blood-based VeriStrat® proteomic test used in cancer immunotherapy treatment guidance.

Introduction: The VeriStrat® test (VS) is a blood-based assay that predicts a patient's response to therapy by analyzing eight features in a spectrum obtained from matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of human serum and plasma. In a recent analysis of the INSIGHT clinical trial (NCT03289780), it was found that the VS labels, VS Good and VS Poor, can effectively predict the responsiveness of non-small cell lung cancer (NSCLC) patients to immune checkpoint inhibitor (ICI) therapy. However, while VS measures the intensities of spectral features using MALDI-TOF analysis, the specific proteoforms underlying these features have not been comprehensively identified. Objectives: The objective of this study was to identify the proteoforms that are measured by VS. Methods: To resolve the features obtained from the low-resolution MALDI-TOF procedure used to acquire mass spectra for VS DeepMALDI® analysis of serum was employed. This technique allowed for the identification of finer peaks within these features. Additionally, a combination of reversed-phase fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was then used to identify the proteoforms associated with these peaks. Results: The analysis revealed that the primary constituents of the spectrum measured by VS are serum amyloid A1, serum amyloid A2, serum amyloid A4, C-reactive protein, and beta-2 microglobulin. Conclusion: Proteoforms involved in host immunity were identified as significant components of these features. This newly acquired information improves our understanding of how VS can accurately predict patient response to therapy. It opens up additional studies that can expand our understanding even further.

Rewiring of the N-Glycome with prostate cancer progression and therapy resistance.

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.

Acquired Drug Resistance Enhances Imidazoquinoline Efflux by P-Glycoprotein.

Multidrug-Resistant (MDR) cancers attenuate chemotherapeutic efficacy through drug efflux, a process that transports drugs from within a cell to the extracellular space via ABC (ATP-Binding Cassette) transporters, including P-glycoprotein 1 (P-gp or ABCB1/MDR1). Conversely, Toll-Like Receptor (TLR) agonist immunotherapies modulate activity of tumor-infiltrating immune cells in local proximity to cancer cells and could, therefore, benefit from the enhanced drug efflux in MDR cancers. However, the effect of acquired drug resistance on TLR agonist efflux is largely unknown. We begin to address this by investigating P-gp mediated efflux of TLR 7/8 agonists. First, we used functionalized liposomes to determine that imidazoquinoline TLR agonists Imiquimod, Resiquimod, and Gardiquimod are substrates for P-gp. Interestingly, the least potent imidazoquinoline (Imiquimod) was the best P-gp substrate. Next, we compared imidazoquinoline efflux in MDR cancer cell lines with enhanced P-gp expression relative to parent cancer cell lines. Using P-gp competitive substrates and inhibitors, we observed that imidazoquinoline efflux occurs through P-gp and, for Imiquimod, is enhanced as a consequence of acquired drug resistance. This suggests that enhancing efflux susceptibility could be an important consideration in the rational design of next generation immunotherapies that modulate activity of tumor-infiltrating immune cells.

Glycan Imaging Mass Spectrometry: Progress in Developing Clinical Diagnostic Assays for Tissues, Biofluids, and Cells.

N-glycan imaging mass spectrometry (IMS) can rapidly and reproducibly identify changes in disease-associated N-linked glycosylation that are linked with histopathology features in standard formalin-fixed paraffin-embedded tissue samples. It can detect multiple N-glycans simultaneously and has been used to identify specific N-glycans and carbohydrate structural motifs as possible cancer biomarkers. Recent advancements in instrumentation and sample preparation are also discussed. The tissue N-glycan IMS workflow has been adapted to new glass slide-based assays for effective and rapid analysis of clinical biofluids, cultured cells, and immunoarray-captured glycoproteins for detection of changes in glycosylation associated with disease.

Defining the human kidney N-glycome in normal and cancer tissues using MALDI imaging mass spectrometry.

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.