A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in ...frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in ...resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8
T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8
T cells with the resistance or non-responsiveness of cancer to immunotherapies.
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.
Specific alterations in N-linked glycans, such as core fucosylation, are associated with many cancers and other disease states. Because of the many possible anomeric linkages associated with ...fucosylated N-glycans, determination of specific anomeric linkages and the site of fucosylation (i.e., core vs outer arm) can be difficult to elucidate. A new MALDI mass spectrometry imaging workflow in formalin-fixed clinical tissues is described using recombinant endoglycosidase F3 (Endo F3), an enzyme with a specific preference for cleaving core-fucosylated N-glycans attached to glycoproteins. In contrast to the broader substrate enzyme peptide-N-glycosidase F (PNGaseF), Endo F3 cleaves between the two core N-acetylglucosamine residues at the protein attachment site. On tissues, this results in a mass shift of 349.137 a.m.u. for core-fucosylated N-glycans when compared to N-glycans released with standard PNGaseF. Endo F3 can be used singly and in combination with PNGaseF digestion of the same tissue sections. Initial results in liver and prostate tissues indicate core-fucosylated glycans associated to specific tissue regions while still demonstrating a diverse mix of core- and outer arm-fucosylated glycans throughout all regions of tissue. By determining these specific linkages while preserving localization, more targeted diagnostic biomarkers for disease states are possible without the need for microdissection or solubilization of the tissue.
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.
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•N-glycan structures localize to distinct regions in normal and PDAC tumor tissue.•N-glycan composition differs between biomarker-stratified PDAC subtypes.•Modeling with N-glycan IMS and biomarker data improves tumor/normal discrimination.•Multiplexed IMS and lectin staining potentiates new PDAC classification strategies.
N-glycosylation is an attractive target for PDAC biomarker discovery because of its well-understood roles in oncogenesis, cancer maintenance, and metastasis. Using MALDI-IMS, we observed distinct histopathology localized differences in N-glycosylation distributions between healthy and cancerous tissues. We combined the tumor-to-normal ratio of N-glycan changes determined from IMS with biomarker IHC data into modeling which improved PDAC identification over models utilizing either data set individually. This multiplexed approach potentiates the development of cross-disciplinary biomarker panels for pancreatic cancer detection.
A new matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in tissues is ...described. Application of an endoglycosidase, peptide N-glycosidase F (PNGaseF), directly on tissues followed by incubation releases N-linked glycan species amenable to detection by MALDI-IMS. The method has been designed to simultaneously profile the multiple glycan species released from intracellular organelle and cell surface glycoproteins, while maintaining histopathology compatible preparation workflows. A recombinant PNGaseF enzyme was sprayed uniformly across mouse brain tissue slides, incubated for 2 h, then sprayed with 2,5-dihydroxybenzoic acid matrix for MALDI-IMS analysis. Using this basic approach, global snapshots of major cellular N-linked glycoforms were detected, including their tissue localization and distribution, structure, and relative abundance. Off-tissue extraction and modification of glycans from similarly processed tissues and further mass spectrometry or HPLC analysis was done to assign structural designations. MALDI-IMS has primarily been utilized to spatially profile proteins, lipids, drug, and small molecule metabolites in tissues, but it has not been previously applied to N-linked glycan analysis. The translatable MALDI-IMS glycan profiling workflow described herein can readily be applied to any tissue type of interest. From a clinical diagnostics perspective, the ability to differentially profile N-glycans and correlate their molecular expression to histopathological changes can offer new approaches to identifying novel disease related targets for biomarker and therapeutic applications.
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and the cancer with the fastest increase in mortality in the United States, with more than 39,000 cases and 29,000 ...deaths in 2018. As with many cancers, survival is significantly improved by early detection. The median survival of patients with early HCC is >60 months but <15 months when detected at an advanced stage. Surveillance of at-risk patients improves outcome, but fewer than 20% of those at risk for HCC receive surveillance, and current surveillance strategies have limited sensitivity and specificity. Ideally, blood-based biomarkers with adequate sensitivity or specificity would be available for early detection of HCC; however, the most commonly used biomarker for HCC, alpha-fetoprotein, has inadequate performance characteristics. There are several candidate serum proteomic, glycomic, and genetic markers that have gone through early stages of biomarker validation and have shown promise for the early detection of HCC, but these markers require validation in well-curated cohorts. Ongoing prospective cohort studies will permit retrospective longitudinal (phase III biomarker study) validation of biomarkers. In this review, we highlight promising candidate biomarkers and biomarker panels that have completed phase II evaluation but require further validation prior to clinical use.
Hepatocellular carcinoma (HCC) remains as the fifth most common cancer in the world and accounts for more than 700,000 deaths annually. Changes in serum glycosylation have long been associated with ...this cancer but the source of that material is unknown and direct glycan analysis of HCC tissues has been limited. Our laboratory previously developed a method of in situ tissue based N-linked glycan imaging that bypasses the need for microdissection and solubilization of tissue prior to analysis. We used this methodology in the analysis of 138 HCC tissue samples and compared the N-linked glycans in cancer tissue with either adjacent untransformed or tissue from patients with liver cirrhosis but no cancer. Ten glycans were found significantly elevated in HCC tissues as compared to cirrhotic or adjacent tissue. These glycans fell into two major classes, those with increased levels of fucosylation and those with increased levels of branching with or without any fucose modifications. In addition, increased levels of fucosylated glycoforms were associated with a reduction in survival time. This work supports the hypothesis that the increased levels of fucosylated N-linked glycans in HCC serum are produced directly from the cancer tissue.
Changes in the levels and compositions of N-glycans released from serum and plasma glycoproteins have been assessed in many diseases across many large clinical sample cohorts. Assays used for ...N-glycan profiling in these fluids currently require multiple processing steps and have limited throughput, thus diminishing their potential for use as standard clinical diagnostic assays. A novel slide-based N-glycan profiling method was evaluated for sensitivity and reproducibility using a pooled serum standard. Serum was spotted on to an amine-reactive slide, delipidated and desalted with a series of washes, sprayed with peptide N-glycosidase F and matrix, and analyzed by MALDI-FTICR or MALDI-Q-TOF mass spectrometry. Routinely, over 75 N-glycan species can be detected from one microliter of serum in less than 6.5 h. Additionally, endoglycosidase F3 was applied to this workflow to identify core-fucosylated N-glycans and displayed the adaptability of this method for the determination of structural information. This method was applied to a small pooled serum set from either obese or nonobese patients that had breast cancer or a benign lesion. This study confirms the reproducibility, sensitivity, and adaptability of a novel method for N-glycan profiling of serum and plasma for potential application to clinical diagnostics.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a rapidly evolving field in which mass spectrometry techniques are applied directly on tissues to characterize the ...spatial distribution of various molecules such as lipids, protein/peptides, and recently also N-glycans. Glycans are involved in many biological processes and several glycan changes have been associated with different kinds of cancer, making them an interesting target group to study. An important analytical challenge for the study of glycans by MALDI mass spectrometry is the labile character of sialic acid groups which are prone to in-source/postsource decay, thereby biasing the recorded glycan profile. We therefore developed a linkage-specific sialic acid derivatization by dimethylamidation and subsequent amidation and transferred this onto formalin-fixed paraffin-embedded (FFPE) tissues for MALDI imaging of N-glycans. Our results show (i) the successful stabilization of sialic acids in a linkage specific manner, thereby not only increasing the detection range, but also adding biological meaning, (ii) that no noticeable lateral diffusion is induced during to sample preparation, (iii) the potential of mass spectrometry imaging to spatially characterize the N-glycan expression within heterogeneous tissues.
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