Thomas W. Powers

MALDI Imaging Mass Spectrometry Profiling of N-Glycans in Formalin-Fixed Paraffin Embedded Clinical Tissue Blocks and Tissue Microarrays

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.

Matrix assisted laser desorption ionization imaging mass spectrometry workflow for spatial profiling analysis of N-linked glycan expression in tissues.

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.

MALDI imaging mass spectrometry profiling of proteins and lipids in clear cell renal cell carcinoma

Reducing the incidence and mortality rates for clear cell renal cell carcinoma (ccRCC) remains a significant clinical challenge with poor 5‐year survival rates. A unique tissue cohort was assembled of matched ccRCC and distal nontumor tissues (n = 20) associated with moderate risk of disease progression, half of these from individuals who progressed to metastatic disease and the other half who remained disease free. These tissues were used for MALDI imaging MS profiling of proteins in the 2–20 kDa range, resulting in a panel of 108 proteins that had potential disease‐specific expression patterns. Protein lysates from the same tissues were analyzed by MS/MS, resulting in identification of 56 proteins of less than 20 kDa molecular weight. The same tissues were also used for global lipid profiling analysis by MALDI‐FT‐ICR MS. From the cumulative protein and lipid expression profile data, a refined panel of 26 proteins and 39 lipid species was identified that could either distinguish tumor from nontumor tissues, or tissues from recurrent disease progressors from nonrecurrent disease individuals. This approach has the potential to not only improve prognostic assessment and enhance postoperative surveillance, but also to inform on the underlying biology of ccRCC progression.

Increased bisecting N-acetylglucosamine and decreased branched chain glycans of N-linked glycoproteins in expressed prostatic secretions associated with prostate cancer progression.

Using prostatic fluids rich in glycoproteins like prostate‐specific antigen and prostatic acid phosphatase (PAP), the goal of this study was to identify the structural types and relative abundance of glycans associated with prostate cancer status for subsequent use in emerging MS‐based glycopeptide analysis platforms.

Renal Glycosphingolipid Metabolism Is Dysfunctional in Lupus Nephritis

Nearly one half of patients with lupus develop glomerulonephritis (GN), which often leads to renal failure. Although nephritis is diagnosed by the presence of proteinuria, the pathology of nephritis can fall into one of five classes defined by different forms of tissue injury, and the mechanisms involved in pathogenesis are not completely understood. Glycosphingolipids are abundant in the kidney, have roles in many cellular functions, and were shown to be involved in other renal diseases. Here, we show dysfunctional glycosphingolipid metabolism in patients with lupus nephritis and MRL/lpr lupus mice. Specifically, we found that glucosylceramide (GlcCer) and lactosylceramide (LacCer) levels are significantly higher in the kidneys of nephritic MRL/lpr lupus mice than the kidneys of non-nephritic lupus mice or healthy controls. This elevation may be, in part, caused by altered transcriptional regulation and/or activity of LacCer synthase (GalT5) and neuraminidase 1, enzymes that mediate glycosphingolipid metabolism. We show increased neuraminidase 1 activity early during the progression of nephritis (before significant elevation of GlcCer and LacCer in the kidney). Elevated levels of urinary LacCer were detected before proteinuria in lupus mice. Notably, LacCer levels were higher in the urine and kidneys of patients with lupus and nephritis than patients with lupus without nephritis or healthy controls. Together, these results show early and significant dysfunction of the glycosphingolipid metabolic pathway in the kidneys of lupus mice and patients with lupus nephritis and suggest that molecules in this pathway may serve as early markers in lupus nephritis.

MALDI Mass Spectrometry Imaging of N-Linked Glycans in Cancer Tissues

Glycosylated proteins account for a majority of the posttranslation modifications of cell surface, secreted, and circulating proteins. Within the tumor microenvironment, the presence of immune cells, extracellular matrix proteins, cell surface receptors, and interactions between stroma and tumor cells are all processes mediated by glycan binding and recognition reactions. Changes in glycosylation during tumorigenesis are well documented to occur and affect all of these associated adhesion and regulatory functions. A MALDI imaging mass spectrometry (MALDI-IMS) workflow for profiling N-linked glycan distributions in fresh/frozen tissues and formalin-fixed paraffin-embedded tissues has recently been developed. The key to the approach is the application of a molecular coating of peptide-N-glycosidase to tissues, an enzyme that cleaves asparagine-linked glycans from their protein carrier. The released N-linked glycans can then be analyzed by MALDI-IMS directly on tissue. Generally 40 or more individual glycan structures are routinely detected, and when combined with histopathology localizations, tumor-specific glycans are readily grouped relative to nontumor regions and other structural features. This technique is a recent development and new approach in glycobiology and mass spectrometry imaging research methodology; thus, potential uses such as tumor-specific glycan biomarker panels and other applications are discussed.

Defining the molecular tumor margin regions of clear cell renal carcinoma tissues by MALDI-MS imaging of lipid and glycan species.

421 Background: The frequent tumor recurrence associated with clear cell renal cell carcinoma (ccRCC) suggests that there are underlying molecular processes present in the remaining tissue following nephrectomy that are not identified through conventional histopathological techniques. At the molecular level, transcript, metabolomic, and protein expression patterns have indicated a striking Warburg Effect profile in ccRCC tissues, with major affects on sugar and lipid metabolism. Our group has been applying MALDI mass spectrometry imaging approaches to uniquely profile lipids and glycans associated with disease progression directly in frozen tissue slides. METHODS Frozen ccRCC tissues with tumor, non-tumor adjacent, and tumor margin regions were selected by a pathologist. Lipid profiles from fresh-frozen tissue slides coated in DHB matrix were obtained on a dual source Bruker Solarix 70 FTICR mass spectrometer. Glycans were imaged in a similar fashion in ethanol-washed tissues using on-tissue protein N glycanase F digestion to release surface N-glycans. Detected lipid and glycan ion intensities were converted to a color pixel scale for creating an image of individual peaks, linked directly to the histopathology of the tissue. RESULTS Five groups of lipid and N-glycan species were identified following MALDI tissue imaging, those present in the immediate margin area of non-tumor tissue adjacent to tumor; only in non-tumor regions; only in tumor regions; primarily in tumor regions but extended beyond the margin; and present throughout the tissue. Specific lipid and glycan species associated with margin and tumor regions are being correlated with disease progression and pathology data. CONCLUSIONS Analysis of the periphery of the tumor tissue and the normal parenchyma or capsule regions at this biomolecule level may better define the metastatic potential of the tumor as compared to analysis of the central tumor region. This approach has the potential to not only improve prognostic assessment and treatment choices, but also to inform on the underlying biology of ccRCC metastasis and new rational targets for therapeutic intervention.

FLI1 Levels Impact CXCR3 Expression and Renal Infiltration of T Cells and Renal Glycosphingolipid Metabolism in the MRL/lpr Lupus Mouse Strain

The ETS factor Friend leukemia virus integration 1 (FLI1) is a key modulator of lupus disease expression. Overexpressing FLI1 in healthy mice results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of FLI1 in two lupus strains (Fli1+/−) significantly improved kidney disease and prolonged survival. T cells from MRL/lpr Fli1+/− lupus mice have reduced activation and IL-4 production, neuraminidase 1 expression, and the levels of the glycosphingolipid lactosylceramide. In this study, we demonstrate that MRL/lpr Fli1+/− mice have significantly decreased renal neuraminidase 1 and lactosylceramide levels. This corresponds with a significant decrease in the number of total CD3+ cells, as well as CD4+ and CD44+CD62L− T cell subsets in the kidney of MRL/lpr Fli1+/− mice compared with the Fli1+/+ nephritic mice. We further demonstrate that the percentage of CXCR3+ T cells and Cxcr3 message levels in T cells are significantly decreased and correspond with a decrease in renal CXCR3+ cells and in Cxcl9 and Cxcl10 expression in the MRL/lpr Fli1+/− compared with the Fli1+/+ nephritic mice. Our results suggest that reducing the levels of FLI1 in MRL/lpr mice may be protective against development of nephritis in part through downregulation of CXCR3, reducing renal T cell infiltration and glycosphingolipid levels.

In Situ Imaging of N-Glycans by MALDI Imaging Mass Spectrometry of Fresh or Formalin-Fixed Paraffin-Embedded Tissue

Glycosylation of cell surface, secreted, and circulating proteins is one of the most common types of post‐translational modification. These modifications occur most commonly as one of three major classes: N‐linked glycosylation on asparagine residues, O‐linked glycosylation on serine or threonine residues, or as glycosaminoglycan oligosaccharide polymers on serine. Specifically, for N‐linked glycans, an endoglycosidase enzyme, peptide N‐glycosidase F (PNGase F), cleaves the attached oligosaccharides between the asparagine and first sugar. A method to analyze released N‐glycans and map them to specific locations within a tissue is presented here. The PNGase F is applied by solvent sprayer as a molecular layer on frozen or formalin‐fixed tissues and all released N‐glycans in a given region of tissue are detected using matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (MALDI‐IMS). Using the described MALDI‐IMS protocol, at least 40 or more individual N‐glycans can be mapped to tissue histopathology and extracted for further structural analysis approaches.

Abstract 540: A MALDI imaging mass spectrometry approach using tissue microarrays to identify an N-glycan biomarker panel for pancreatic cancers

We have recently developed a MALDI imaging mass spectrometry (MALDI-IMS) method to spatially profile N-linked glycans in frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMAs). Tissues are incubated with peptide N-glycosidase, and released N-glycans are detected directly using MALDI-FTICR, linked directly with tissue histopathology. Other methods to detect the localization of glycans in tissues rely on detection of broader glycan structural motifs (i.e., lectins or carbohydrate antigen antibodies), whereas our method is able to simultaneously identify and distinguish 40 or more components of the N-glycome on a single slide. To demonstrate the ability of MALDI-IMS to generate biomarker panels, pancreatic cancer tissue blocks and six TMAs containing matched tumor and non-tumor regions from over 70 patients were profiled. Aberrant glycosylation, such as elevated CA-19-9, is well documented in pancreatic cancer, making this an ideal sample set. To best analyze this complex data set, we developed an in-house analysis script that can extract spectra from tissue cores, and return non-biased statistics for observed glycan ions. Panels were generated using a training set of data and tested on external validation data set. The most accurate panel of 12 glycans achieved an overall sensitivity of 92.9% and specificity of 86.7%. Structural identification of N-glycans has been confirmed by techniques such as on-tissue CID, ethylation analysis, sequential glycosidase digestions, and comparison to glycan structural databases. Furthermore, imaging of entire FFPE sections matched with histological analysis revealed a vast diversity of N-glycan localizations, which could distinguish not only tumor from normal tissue, but also regions of pancreatitis and pancreatic intraepithelial neoplasia. The generated glycan tissue maps will also be used to determine specific glycoprotein carriers of biomarker candidate glycans in tissue and biofluid samples. Citation Format: Richard R. Drake, Thomas W. Powers, Benjamin A. Neely. A MALDI imaging mass spectrometry approach using tissue microarrays to identify an N-glycan biomarker panel for pancreatic cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 540. doi:10.1158/1538-7445.AM2015-540