Spatial Distribution of Endogenous Tissue Protease Activity in Gastric Carcinoma Mapped by MALDI Mass Spectrometry Imaging

Abstract

Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue's endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity - with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies. Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue's endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity - with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies. Proteases control cell and tissue protein homeostasis (1Klein T. Eckhard U. Dufour A. Solis N. Overall C.M. Proteolytic cleavage-mechanisms, function, and “Omic” approaches for a near-ubiquitous posttranslational modification.Chem. Rev. 2018; 118: 1137-1168Crossref PubMed Scopus (112) Google Scholar). They influence cell proliferation, tissue morphogenesis and remodeling, and they are therefore associated with many pathological conditions including cancer. Cellular and secreted proteases play important roles during tumor initiation, growth, as well as metastasis, and their activity is not restricted to tumor cells, but affects tumor-surrounding tissue in favor of tumor expansion (2Olson O.C. Joyce J.A. Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response.Nat. Rev. Cancer. 2015; 15: 712-729Crossref PubMed Scopus (379) Google Scholar, 3Merchant N. Nagaraju G.P. Rajitha B. Lammata S. Jella K.K. Buchwald Z.S. Lakka S.S. Ali A.N. Matrix metalloproteinases: their functional role in lung cancer.Carcinogenesis. 2017; 38: 766-780Crossref PubMed Scopus (116) Google Scholar). Hence, they are key drug targets (4Drag M. Salvesen G.S. Emerging principles in protease-based drug discovery.Nat. Rev. Drug Discov. 2010; 9: 690-701Crossref PubMed Scopus (428) Google Scholar). In the past, various optical methods including (near infrared; NIR) fluorogenic and bioluminescence-based substrate- or activity reporter probes have been used to visualize protease activity in vitro, in cultured cells and in vivo (5Baruch A. Jeffery D.A. Bogyo M. Enzyme activity–it's all about image.Trends Cell Biol. 2004; 14: 29-35Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 6Edgington L.E. Verdoes M. Bogyo M. Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes.Curr. Opin. Chem. Biol. 2011; 15: 798-805Crossref PubMed Scopus (139) Google Scholar). Analytical probes typically enable fluorescence quenching, fluorescence resonance energy transfer (FRET) 1The abbreviations used are:FRETfluorescence resonance energy transferABCammonium bicarbonateCANacetonitrileAEBSF4-(2-aminoethyl)benzenesulfonyl fluoride hydrochlorideBCAbicinchoninic acid assayBSAbovine serum albuminCAAchloroacetamideDHB2,5-dihydroxxybenzoic acidEDTAethylenediaminetetraacetic acidFAformic acidFTICRfourier-transform ion cyclotron resonanceH&Ehematoxylin & eosinITOindium tin oxideLFQlabel-free quantificationMALDI MSImatrix-assisted laser desorption/ionization mass spectrometry imagingNIRnear-infraredPBSphosphate buffered salinePCAprincipal component analysisPIMprotease-inhibitor mixtureRTroom temperatureSDSsodium dodecylsulfateTCAtrichloroacetic acidTCEPtris(2-carboxyethyl)phosphineTFAtrifluoroacetic acidTPAAtissue protease activity assay., and other optical read-outs. Whereas several probes have been described for cell studies and in vivo molecular imaging (7Weissleder R. Molecular imaging in cancer.Science. 2006; 312: 1168-1171Crossref PubMed Scopus (985) Google Scholar), direct visualization and biochemical investigation of protease activity in tissue sections remains an underexplored field of research. In a notable exception, Withana et al. topically applied fluorescence-quenched activity-based probes to fresh-frozen tissue sections and visualized protease activity at cellular resolution (8Withana N.P. Garland M. Verdoes M. Ofori L.O. Segal E. Bogyo M. Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes.Nat. Protoc. 2016; 11: 184-191Crossref PubMed Scopus (43) Google Scholar). fluorescence resonance energy transfer ammonium bicarbonate acetonitrile 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride bicinchoninic acid assay bovine serum albumin chloroacetamide 2,5-dihydroxxybenzoic acid ethylenediaminetetraacetic acid formic acid fourier-transform ion cyclotron resonance hematoxylin & eosin indium tin oxide label-free quantification matrix-assisted laser desorption/ionization mass spectrometry imaging near-infrared phosphate buffered saline principal component analysis protease-inhibitor mixture room temperature sodium dodecylsulfate trichloroacetic acid tris(2-carboxyethyl)phosphine trifluoroacetic acid tissue protease activity assay. In this study, we utilize a different analytical approach, matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI), for spatially-resolved analysis of endogenous protease activity in fresh-frozen tissue. MALDI-MS imaging is label-free, and it rapidly localizes biomolecules without prior knowledge of their presence. Sample preparation for MALDI-MS imaging is comparatively fast and, hence, it is of increasing interest for clinical pathology (9Casadonte R. Caprioli R.M. Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry.Nat. Protocols. 2011; 6: 1695-1709Crossref PubMed Scopus (214) Google Scholar, 10Aichler M. Walch A. MALDI Imaging mass spectrometry: current frontiers and perspectives in pathology research and practice.Lab. Investigation. 2015; 95: 422-431Crossref PubMed Scopus (270) Google Scholar). MSI is frequently used for analysis of the in situ distribution of proteins (11Balluff B. Elsner M. Kowarsch A. Rauser S. Meding S. Schuhmacher C. Feith M. Herrmann K. Rocken C. Schmid R.M. Hofler H. Walch A. Ebert M.P. Classification of HER2/neu status in gastric cancer using a breast-cancer derived proteome classifier.J. 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Inhibition of rho-associated kinase 1/2 attenuates tumor growth in murine gastric cancer.Neoplasia. 2016; 18: 500-511Crossref PubMed Scopus (27) Google Scholar), drugs (14Hinsenkamp I. Schulz S. Roscher M. Suhr A.M. Meyer B. Munteanu B. Fuchser J. Schoenberg S.O. Ebert M.P. Wangler B. Hopf C. Burgermeister E. Inhibition of rho-associated kinase 1/2 attenuates tumor growth in murine gastric cancer.Neoplasia. 2016; 18: 500-511Crossref PubMed Scopus (27) Google Scholar), as well as peptides (15Cillero-Pastor B. Heeren R.M. Matrix-assisted laser desorption ionization mass spectrometry imaging for peptide and protein analyses: a critical review of on-tissue digestion.J. Proteome Res. 2014; 13: 325-335Crossref PubMed Scopus (83) Google Scholar), and N-glycans (16Drake R.R. Powers T.W. Jones E.E. Bruner E. Mehta A.S. Angel P.M. MALDI mass spectrometry imaging of N-linked glycans in cancer tissues.Adv. Cancer Res. 2017; 134: 85-116Crossref PubMed Scopus (90) Google Scholar, 17Heijs B. Holst S. Briaire-de Bruijn I.H. van Pelt G.W. de Ru A.H. van Veelen P.A. Drake R.R. Mehta A.S. Mesker W.E. Tollenaar R.A. Bovée J.V.M.G. Wuhrer M. McDonnell L.A. Multimodal mass spectrometry imaging of N-glycans and proteins from the same tissue section.Anal. Chem. 2016; 88: 7745-7753Crossref PubMed Scopus (69) Google Scholar). A recent study with a neuropeptide, dynorphin, spotted onto rat brain slices suggested that it may be possible to detect the levels of substrate and peptide bioconversion products simultaneously by MSI (18Bivehed E. Stromvall R. Bergquist J. Bakalkin G. Andersson M. Region-specific bioconversion of dynorphin neuropeptide detected by in situ histochemistry and MALDI imaging mass spectrometry.Peptides. 2017; 87: 20-27Crossref PubMed Scopus (18) Google Scholar). Here, we take this approach one step further and use spray-coating techniques for application of a protease substrate tracer, substance P, onto tissue for effective monitoring of endogenous enzyme activity and of the transient emergence of cleavage products over time at high spatial resolution. We utilize fast MALDI-TOF and high resolving power MALDI-Fourier Transform Ion Cyclotron Resonance (FTICR) MSI to visualize for the first time tissue protease activity in mouse gastric carcinoma using MALDI MSI. Substance P from trichloroacetic acid & bovine bovine serum albumin and chloroacetamide from indium tin oxide and peptide from MALDI acid from and trifluoroacetic acid and from from acetonitrile and from and from sodium acid and all from and sodium from dodecylsulfate from and phosphate buffered saline from from ammonium bicarbonate from Analytical acid and formic acid from from from the and in from about and with gastric carcinoma described J. T. G. A. van H. A mouse that gastric carcinoma a for tumor J. Cancer. 86: PubMed Google Scholar). studies in with of and by of and tissues and at further MALDI MSI tissue 10 slices a at a temperature of and onto In slices with ammonium and tissue for at To in situ one and one control tissue of endogenous protease activity tissue and for a of MSI protease of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride ethylenediaminetetraacetic acid and applied to tissue. tissue as studies of concentration-dependent protease activity tissue, a of in and one of spotted onto tissue at at all In using mouse tumor an tissue with using the and the in in of tissue for at room temperature or mouse tissues the with the protease substrate substance P in using the as described for for a with substance P. the the a the in for at and simultaneously at and in the for or in time-course and for in tissue for at as protease activity in mouse tissue in tissue, but as tissue. of the substance P and with MALDI-TOF MSI with as (14Hinsenkamp I. Schulz S. Roscher M. Suhr A.M. Meyer B. Munteanu B. Fuchser J. Schoenberg S.O. Ebert M.P. Wangler B. Hopf C. Burgermeister E. Inhibition of rho-associated kinase 1/2 attenuates tumor growth in murine gastric cancer.Neoplasia. 2016; 18: 500-511Crossref PubMed Scopus (27) Google Scholar). tissues analyzed using an in and a mass of at a resolution of using peptide for all and using and mouse a MALDI-TOF used at a resolution of for the MALDI-TOF the same analyzed using a MALDI the used in ion in an of with a of and laser Ion transfer Cell of time of mouse tumor using to an resolving power of at in a of detected mass resolution at using a mass of using ion from of substance P and peptide analysis using and is with To we tissue with or without and control of M. P. M. a for analysis of mass Chem. 2010; PubMed Scopus Google for and of the of interest analysis in analysis of the mouse tumor tissue, T. A. I. Heeren R.M. A. N. M. B. A. for the and of mass spectrometry imaging Proteomics. 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Region-specific bioconversion of dynorphin neuropeptide detected by in situ histochemistry and MALDI imaging mass spectrometry.Peptides. 2017; 87: 20-27Crossref PubMed Scopus (18) Google Scholar). observed and transient of such products that substance P is not the tissue, but MSI of of and to protease in been a fast and in ion in without of the protease attenuated for to the and to In in a tissue for protease substance P by observed in cleavage and in to observed for cleavage products and that the be generated by distinct or proteases that are by for in different tissues and to of of tissue the from ion of substance P and peptide cleavage products in mass of tissue with and without protease onto onto a for and analyzed by MALDI-TOF MSI with are MALDI-TOF of with and without the molecular To the of substance P and transient of cleavage products for and tissue we for substance P and ion of and for and and In and of substance P In in tissues with fast of the and the high of the at time analysis of the cleavage products the that transiently in the of and not and Whereas this may that of proteases play roles in the and studies be to the can be to protease established the transient of cleavage products as an of a enzyme being or we of cleavage we used different of the and to different of a and spotted various onto tissues and for the an concentration-dependent of substance P and to tissue substance P in all with protease activity. other tissues a of substance P ion with increasing We P the of substance P ion at the and in the the suggesting that at the of substance P not be as the tissue with the protease substance P in and be with To this of protease we a protease activity assay for tissue the by an of active from bovine and as in and as a protease substrate substance P not we tissue protease activity for tissue. observed with MALDI the activity of the of protease in with the one derived from MSI we to this for visualization of endogenous protease activity to studies in mice. We used that gastric in situ in MSI studies of drugs and their and the of as a tumor in To spatial distribution of endogenous protease activity and by we a gastric from a mouse and a control from a fresh-frozen mouse tissue protease activity tissue from a Hence, we the time to of of endogenous peptides with to substance P or cleavage products in tumor or mouse as such observed in the of substance P to first a MALDI-TOF and a To the of the peptides analyzed this study and we of the the mouse tissue and their from substance P of the the and the of the tissue by in the distribution of the tumor the of the in studies with imaging but as well as with the to resolving power and mass with the Substance P ion and cleavage products and in tissue with and the peptide in the tumor but in with results in tissues, attenuated substance P of cleavage products but at of analysis of and tissue the that protease as by in tumor tissue and to by protease activity of and at spatial resolution and the distribution of peptides activity is to the tissue and To possible of the protease we the tissue prior to application of substance P with ammonium at or activity at but at at of to the we differential activity by MSI in tumor and tissue in mouse with differential of proteases in To this mouse tumor and tissues to from a mouse as a To differential protease we a with label-free quantification and with the in the mouse and the a of proteases proteases with their associated in a principal component analysis distinct to their tissue same analysis with analysis all proteins, with or by analysis results that levels of proteases tumor and tissues, and that their is a for tissue the of the the of probes for monitoring of endogenous tissue protease is a for label-free methods that are not to fluorescence and other and that enable visualization of transient Here, we have a at high spatial and high mass resolution to that of tissues spray-coated with substrate peptides such as substance P monitoring of protease activity in various is the of this study to the of the proteases in cleavage of the to the that be proteases that have substance P as substrate and the peptides or are to to by proteases to the of R. of of including and their by J. 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In tumor tissue levels of and as well as and are of proteases as molecular imaging for gastric cancer in and vivo 2017; Google Scholar). levels protein levels activity of as proteases are secreted as and proteomics differential protease in tumor and tissue is but to of proteases be for of substance P cleavage it is from various proteomics studies that tissues are to not at but in and B. K. K. C. S. M. M. R. P. R. P. of temperature in proteomics and of the and Cell. Proteomics. 2011; 10 Full Text Full Text PDF PubMed Scopus Google Scholar), we results be by this be it that such proteolysis proteases in substance P cleavage in of the tissue but not in to in the distribution of substance P cleavage In using has been suggested for MALDI MSI to A. used for and of animal and tissue sections for MALDI imaging and Proteomics. PubMed Scopus Google Scholar). In that study, a peptides that in the of the of peptides it is that peptides to substance P but originating from tissue have the same as substance P cleavage products and MALDI In we for by in tissue that not with substance P to current clinical to are described visualize protease activity with high spatial a and for or protein levels tissue and not or protein tissue. methods have a high spatial possible with the MSI be proteomics and MALDI MSI have been to the the with the and A. R. A. H. B. J.A. A for mass spectrometry imaging Chem. 2015; PubMed Scopus (27) Google Scholar, J.A. A. N. J.A. J. I. G. T. R. H. of the and Res. 2016; PubMed Scopus Google Scholar). by have been A. P. I. R. A. Fuchser J. S. C. M. T. for imaging mass spectrometry.Nat. 2017; 14: PubMed Scopus Google by the without protease with protease without protease with protease with