Richard Drake (Presenter)
Medical University of South Carolina
Bio: Dr. Drake is a Professor in the Department of Cell and Molecular Pharmacology and Experimental Therapeutics at the Medical University of South Carolina and SmartState Endowed Chair in Proteomics. He is also Director of the MUSC Proteomics Center, a mass spectrometry-centric facility with state-of-the-art resources for imaging mass spectrometry, glycobiology and protein PTM applications. He is an experienced protein biochemist and glycobiologist, with particular expertise in tissue imaging of glycosylation, tumor biology and biomarker discovery from clinical fluids and tissues related to urological cancers.
Authorship: Richard R. Drake, Danielle Scott, Connor West, Fred David, Peggi Angel and Anand Mehta
Medical University of South Carolina, MUSC Proteomics Center and Hollings Cancer Center, Charleston, SC, USA
Alterations in cell surface glycosylation during tumorigenesis are well documented, and most current FDA approved cancer biomarkers are glycoproteins or glycan antigens. A MALDI mass spectrometry imaging method to spatially profile N-linked glycans in formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMAs) has been applied to nearly 1000 patient samples of breast and prostate cancer tissues. Analysis was done using MALDI-FTICR MS and a new rapid MALDI-TOF MS TissueTyper. Classes of N-glycans representing the most lethal forms of each cancer will be described. The goal will be to develop the approach as a prognostic assay for disease stratification at the time of diagnosis.
Glycoproteins account for approximately 80% of the proteins located at the cell surface and in the extracellular environment, and changes in cell surface glycosylation during tumorigenesis are well documented, including the major N-linked glycan class of glycosylated proteins. Most current FDA approved biomarkers of cancer are glycoproteins or glycan antigens. We have continued to optimize a MALDI mass spectrometry imaging method to spatially profile N-linked glycans in formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMAs) using MALDI-FTICR MS and a new rapid MALDI-TOF MS TissueTyper. Using primarily breast and prostate cancer tissues, nearly 1000 patient samples representing the clinical spectrum from benign to metastatic tumor tissues have been analyzed. The goal of the study was to define the tumor N-glycans associated with the most lethal forms of each cancer, which will be used to develop a prognostic assay for disease stratification at the time of diagnosis.
Formalin-fixed paraffin embedded prostate cancer tissues representing indolent, metastatic (lymph node and bone), neuroendocrine and small cell carcinoma pathologies were provided by biorepositories and collaborators. Tissues representing triple negative breast cancers, Her2+ cancers and controls were provided by collaborators. Tissue slices (5 um) on slides were processed for antigen retrieval and PNGaseF digestions to release N-glycans. Enzymes and CHCA matrix were sprayed onto tissue using a HTX TM-Sprayer. Samples were analyzed by MALDI-FTICR on a Bruker 7T solariX in positive ion mode, collecting 200 laser shots per pixel with a 100 µm step size. Alternatively, data was collected on a Bruker rapifleX MALD-TOF TIssueTyper. Released N-glycans were visualized in flexImaging 4.1 software and analyzed by SCiLS Lab software.
A series of FFPE prostate cancer tissues and TMAs representing neuroendocrine-like adenocarcinomas and small cell carcinomas were evaluated by N-glycan MALDI-FTICR imaging. These tissue represent primary tumor and metastatic tissues of the lymph node and bone. In comparison to more common prostate adenocarcinoma tissues with indolent outcomes, there was a dramatic increase in the detection of N-glycans with multiple outer arm fucose modifications (up to 8) on tri- and tetra-antennary glycans. Indolent tumors have primarily high mannose and singly fucosylated multi-antennary N-glycans. The ability to detect the highly fucosylated forms present in the advanced tumors by MALDI imaging, combined with SCiLS software analysis, has facilitated the development of a “Fucose Score” for each tumor tissue based on the numbers of outer arm fucoses detected. For breast cancers, N-glycan panels derived from TMAs representing HER2 receptor positive and triple negative breast cancers identified a series of high-mannose and branched glycans associated with tumor regions. In triple negative tissues, detection of a series of polylactosamine glycans were consistently increased in these tumors relative to HER2 positive tissues. Conversely, several branched fucosylated glycans were detected at higher levels in HER2 positive tumors.
Conclusions & Discussion
Structural panels of tissue N-glycans determined by imaging mass spectrometry can be used to distinguish genetic subtypes of human breast cancers and the most lethal forms of prostate cancer. The generated tissue glycan maps can be used for targeted proteomic analysis of carrier glycoproteins and immunohistochemistry correlates. Analysis of larger sample cohorts is warranted for clinical biomarker assessment and assay development. The new rapid MALDI-TOF instrument makes N-glycan tissue imaging assays feasible for use in the clinical laboratory, and could be used in combination with other molecular targets in development.
References & Acknowledgements:
Drake RR, Powers TW, Jones EE, Bruner E, Angel P. (2017) MALDI mass spectrometry imaging of N-linked glycans in cancer tissues. Advances in Cancer Research,134, 85-116.
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