Proteases and Cancer: Methods and Protocols (Methods in Molecular Biology, 2747)

Preface
Contents
Contributors
Chapter 1: Web-Based Resources to Investigate Protease Function
	1 Introduction
	References
Chapter 2: N-Terminomics/TAILS of Human Tumor Biopsies and Cancer Cell Lines
	1 Introduction
	2 Materials
		2.1 Reagents
	3 Methods
		3.1 Sample Preparation (Fig. 1)
			3.1.1 Tissue Homogenization
			3.1.2 Primary Cancer Cells or Immortalized Cancer Cell Line Lysis
			3.1.3 Blood, Serum, or Plasma
			3.1.4 Sonication of Cell- and Tissue-Related Lysates
		3.2 Pre-enrichment TAILS and TAILS (Fig. 2)
			3.2.1 Day 1: Sample Preparation and Amine Labeling
			3.2.2 Day 2: Protein Precipitation and Trypsinization
			3.2.3 Day 3: PreTAILS and Polymer Selection
			3.2.4 Day 4: TAILS Enrichment and Sample Filtration
			3.2.5 Alternatives for the TAILS Polymer
			3.2.6 Data Processing, Data Transformation, and Statistical Analysis
	4 Notes
	References
Chapter 3: A New Affinity-Based Probe to Profile MMP Active Forms
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Activation of mMMP12 of HEK Supernatant
		3.2 Labeling of Active mMMP12
		3.3 Capture of Biotin-Tagged mMMP12
		3.4 Analysis of Labeled Supernatant Before and After Streptavidin-Based Enrichment
			3.4.1 Electrophoresis
			3.4.2 Transfer to PVDF Membrane
			3.4.3 Western Blot
				Streptavidin-HRP Detection
				AF 3467 Detection
		3.5 On-Beads Digestion by Trypsin
		3.6 Mass Spectrometry Analysis
			3.6.1 Selection of mMMP12 Tryptic Peptides
			3.6.2 Identification of mMMP12 from HEK Cells´ Supernatant
	4 Notes
	References
Chapter 4: N-Glycan Insertion for Probing Protein-Protein Interactions and Epitope Mapping
	1 Introduction
	2 Materials
		2.1 Molecular Modeling
		2.2 Site-Directed Mutagenesis to Introduce an N-Linked Glycosylation Motif
		2.3 Agarose Gel Electrophoresis and Gel Extraction of the Mutated Plasmid
		2.4 Bacterial Transformation and Plasmid Purification (Maxi- and Minipreps)
		2.5 Recombinant Protein Expression
	3 Methods
		3.1 Inspecting 3D Protein Models for Appropriate Glycan Insertion Sites
		3.2 Site-Directed Mutagenesis
		3.3 Agarose Gel Electrophoresis and DNA Purification from the  Gel
		3.4 Bacterial Transformation and Plasmid Purification
		3.5 Sanger Sequencing and Maxiprep
		3.6 Recombinant Protein Expression
	4 Notes
	References
Chapter 5: Expression and Purification of Recombinant ADAMTS8
	1 Introduction
	2 Materials
		2.1 ADAMTS8 Large-Scale Transient Transfection
		2.2 ADAMTS8 Purification
		2.3 ADAMTS8 SDS-PAGE and Immunoblot
		2.4 ADAMTS8 Concentration and Quantification
		2.5 Osteopontin Cleavage Assay
	3 Methods
		3.1 ADAMTS8 Large-Scale Transient Transfection
		3.2 ADAMTS8 Purification
		3.3 ADAMTS8 SDS-PAGE and Immunoblot
		3.4 ADAMTS8 Concentration and Quantification
		3.5 Osteopontin Cleavage Assay
	4 Notes
	References
Chapter 6: Expression and Purification of Active Monomeric MMP7
	1 Introduction
	2 Materials
		2.1 Expression of Recombinant MMP7 in E. Coli
		2.2 Purification of Recombinant MMP7
		2.3 Activity Titration of Purified MMP7
	3 Methods
		3.1 Expression of Recombinant MMP7 in E. Coli
		3.2 Purification and Refolding of Recombinant MMP7
		3.3 Activity Titration of Purified MMP7
	4 Notes
	References
Chapter 7: Mapping the Binding Sites of MMPs on Types II and III Collagens Using Triple-Helical Peptide Toolkits
	1 Introduction
	2 Materials
		2.1 MMPs
		2.2 Biotinylation of  MMPs
		2.3 Collagen Toolkits
		2.4 Solid-Phase Binding Assay
	3 Methods
		3.1 Biotinylation of  MMPs
		3.2 Toolkit Screening
	4 Notes
	References
Chapter 8: Determination of Versikine Levels by Enzyme-Linked Immunosorbent Assay (ELISA)
	1 Introduction
	2 Materials
		2.1 Generation of Versikine Standard Curve
		2.2 ELISA
	3 Methods
		3.1 Generation of Versikine Standard Curve
		3.2 Elisa
		3.3 Data Analysis
	4 Notes
	References
Chapter 9: Methods to Investigate Thrombin Cleavage of Osteopontin (OPN)
	1 Introduction
	2 Materials
		2.1 Plasticware, Chemicals, and Reagent Preparation
		2.2 Mouse and Human OPN-FL
		2.3 Mouse and Human OPN-R and OPN-L
		2.4 Thrombin
		2.5 Antibodies
		2.6 RAW and B16 Cells
		2.7 Basic Carboxypeptidases (CPB)
		2.8 OPN Peptides
	3 Methods
		3.1 Cleavage of OPN to Generate N-Terminal Fragments: OPN-R and OPN-L
		3.2 Western Blot Analysis of Human OPN-FL, OPN-R, and OPN-L
		3.3 Quantification of Human OPN, OPN-R, and OPN-L by ELISA
		3.4 Quantification of Mouse OPN, OPN-R, and OPN-L by ELISA
		3.5 Direct ELISAs
		3.6 Activity Assays for Thrombin-Cleaved OPN Fragments on RAW or B16 Cells
		3.7 Cell Adhesion Assay
		3.8 Cell Growth Assay
		3.9 Cell Migration Assay
		3.10 Apoptosis Assay
		3.11 Production of PGE2 by RAW Cells in Response to OPN Fragments
	4 Notes
	References
Chapter 10: Using siRNA Silencing to Analyze ADAM17 in Macrophages
	1 Introduction
	2 Materials
		2.1 Isolation of Primary Human Monocytes
		2.2 siRNA Knockdown by Electroporation
		2.3 RNA Isolation and cDNA Synthesis
		2.4 qPCR
		2.5 TNF ELISA
	3 Methods
		3.1 Isolation of Primary Human Monocytes from Leukocyte Cones and Differentiation into Macrophages
		3.2 siRNA Knockdown of ADAM17 by Electroporation
		3.3 RNA Isolation and cDNA Synthesis
		3.4 Validation of Knockdown by RT-qPCR Analysis of ADAM17 Expression
		3.5 Assessing ADAM17 Activity by TNF ELISA
	4 Notes
	References
Chapter 11: Preparation and Characterization of Collagen-Hyaluronic Acid (Col-HA) Matrices: In Vitro Mimics of the Tumor Micro...
	1 Introduction
	2 Materials
		2.1 Preparation of Collagen (Col) and Collagen-Hyaluronic Acid (Col-HA)  Gels
		2.2 Cryo-scanning Electron Microscopy (SEM) Images of Collagen and Col-HA  Gels
		2.3 Confocal Imaging of Collagen and Col-HA  Gels
		2.4 Rheological Characteristics of Collagen and Col-HA  Gels
		2.5 Probing Cancer Cell Invasion Inside Col/Col-HA  Gels
	3 Methods
		3.1 Preparation of Stock Solutions
		3.2 Preparation of Col and Col-HA  Gels
		3.3 Cryo-SEM Imaging of Col/Col-HA  Gels
		3.4 Confocal Imaging of Col/Col-HA  gels
		3.5 Rheological Characterization of Col/Col-HA  Gels
		3.6 Probing Cancer Cell Invasion Inside Collagen and Col-HA Matrices
	4 Notes
	References
Chapter 12: Assessment of Invadopodium Formation and Gelatin Degradation in Vitro
	1 Introduction
	2 Materials
	3 Methods
		3.1 Coating Coverslips with Fluorescent Gelatin
		3.2 Gelatin Degradation Assay
		3.3 Invadopodia Formation Assay
		3.4 Invadopodia Formation Assay with Protein Probing
	4 Notes
	References
Chapter 13: Investigation of MT1-MMP Activity in Cancer Cells
	1 Introduction
		1.1 ProMMP2 Activation
		1.2 Gelatin Film Degradation
		1.3 Collagen Film Degradation
		1.4 Collagen Invasion
	2 Materials
		2.1 ProMMP2 Activation: Zymography
		2.2 Gelatin Film Degradation
		2.3 Collagen Film Degradation
		2.4 Transwell Collagen Invasion Assay
		2.5 Microcarrier Bead Collagen Invasion Assay
	3 Methods
		3.1 proMMP2 Activation: Zymography
		3.2 Gelatin Film Degradation
			3.2.1 Preparation of Alexa488-Labeled Gelatin
			3.2.2 Coating Slide Chamber with Alexa488-Gelatin
			3.2.3 Culture and Visualization
		3.3 Collagen Film Degradation
			3.3.1 Preparation of Collagen Film in 12-Well Plates
			3.3.2 Culture and Visualization
		3.4 Transwell Collagen Invasion Assay
			3.4.1 Preparation of Collagen-Coated Transwell Chambers
			3.4.2 Invasion Assay and Data Acquisition
		3.5 Microcarrier Beads Collagen Invasion Assay
			3.5.1 Preparation of Cytodex 3 Beads
			3.5.2 Attaching Cells to Beads
			3.5.3 Collagen Invasion and Analyses
	4 Notes
	References
Chapter 14: Gelatin In Situ Zymography to Study Gelatinase Activity in Colon Cancer Cells Treated with Platelet Microparticles...
	1 Introduction
	2 Materials
		2.1 General Buffers and Solutions
		2.2 Platelet-Derived Microparticle Isolation Buffers, Reagents, and Materials
		2.3 Cell Culturing
	3 Methods
		3.1 Preparation of PMPs from Platelet Concentrates
		3.2 Preparation of PMPs-Activated Cells.
		3.3 Preparation of Surface Coated with DQ Gelatin, Fluorescein Conjugate, and Measurement of Gelatinase Activity
	4 Notes
	References
Chapter 15: Interrogating the Impact of Protease Activity on Tumor Progression Using 3D Spheroid Models
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Formation of Spheroids
		2.3 Embedding of Spheroids into Collagen  Gels
		2.4 Fixation and Immunofluorescent Staining of Spheroids
	3 Methods
		3.1 Formation of Spheroids
		3.2 Embedding of Spheroids into Collagen  Gels
		3.3 Sphere Analysis
		3.4 Fixation and Immunofluorescent Staining of Spheroids
		3.5 Example  Data
	4 Notes
	References
Chapter 16: Analysis of Matrix Metalloproteinase Activity and Inhibition in Cancer Spheroids
	1 Introduction
	2 Materials
		2.1 Automated Peptide Synthesis
		2.2 Solid Phase for Peptide Synthesis
		2.3 Amino Acids and Other Peptide Synthesis Reagents
		2.4 Organic Solvents and Solutions
		2.5 HPLC Characterization and Purification
		2.6 Mass Spectrometry
		2.7 Circular Dichroism (CD) Spectroscopy
		2.8 Peptide Lyophilization
		2.9 Cell Imaging
		2.10 Enzymatic Assay Buffers, Stock Solutions, and Reagents
		2.11 Cell Culture
		2.12 Western Blot Reagents
	3 Methods
		3.1 Peptide Synthesis
			3.1.1 General Amino Acid Coupling Cycles
			3.1.2 General Fmoc Deprotection Cycle
		3.2 HPLC Purification and Analysis
		3.3 Absorption Spectrophotometry
		3.4 Characterization of fTHPs
		3.5 Activation of proMT1-MMP
		3.6 Enzymatic Assays Using fTHPs
		3.7 Production of Tumor Cell Spheroids
		3.8 Spheroid Inhibitory Kinetic Assay
		3.9 CellTiter-Glo Assay to Determine Cell Viability
		3.10 Invasion Matrix Preparation (Optional)
		3.11 Inhibitor and FAM-fTHP-9 Substrate Addition
		3.12 Kinetic FAM-fTHP-9 Substrate Reading and Tumor Invasion Imaging Assay
		3.13 FAM-fTHP-9 Substrate Reading and Tumor Invasion Imaging Hit Pick Assay Performance
		3.14 Image Processing
		3.15 Tumoroid Invasion Analysis
		3.16 Western Blot
			3.16.1 Preparing the Samples
			3.16.2 Preparing the 10% SDS-Polyacrylamide  Gel
			3.16.3 Transfer
			3.16.4 Blocking
			3.16.5 Secondary Antibody
	4 Notes
	References
Chapter 17: Intravital Microscopy to Study the Effect of Matrix Metalloproteinase Inhibition on Acute Myeloid Leukemia Cell Mi...
	1 Introduction
	2 Materials
		2.1 AML Cells and MMP Inhibition Reagents
		2.2 Surgery and Intravital Microscopy Reagents and Equipment
		2.3 Image Processing and Analysis
	3 Methods
		3.1 AML Experimental Model
		3.2 Prinomastat Treatment
		3.3 Surgery
		3.4 Placing the Mouse on the Microscope Stage
		3.5 Imaging
		3.6 Image Processing
		3.7 Image Analysis: Cell Migration Tracking (2D)
	4 Notes
	References
Chapter 18: Fluorescence-Based Peptidolytic Assay for High-Throughput Screening of MMP14 Inhibitors
	1 Introduction
	2 Materials and Equipment
	3 Methods
		3.1 Determination of the Michaelis-Menten Constant (KM)
		3.2 Determination of Enzyme Concentration
		3.3 Reproducibility and Z′-Factor Determination Prior to HTS
		3.4 Primary High-Throughput Screening (HTS)
		3.5 Inhibitory Activity (IC50) Determination
	4 Notes
	References
Chapter 19: Generation of Protease Inhibitory Antibodies by Functional In Vivo Selection
	1 Introduction
	2 Materials
		2.1 Plasmid Construction
		2.2 Determination of Selection Windows
		2.3 Selection of Protease Inhibitory Antibodies
			2.3.1 Preparation of Electrocompetent E. coli Cells
			2.3.2 Initial Selection
			2.3.3 Secondary Screening
		2.4 Characterizations of Isolated Clones
			2.4.1 Fab Cloning, Expression, and Purification
			2.4.2 Binding Affinity Measurements
			2.4.3 Inhibition Potency Measurements
	3 Methods
		3.1 Plasmid Construction
			3.1.1 Construction of pm9TEM-cd9 and pm9TEM-cd9E402A
			3.1.2 Construction of pHPK-Fab Library Plasmids
		3.2 Determination of Selection Windows
		3.3 Selection of Protease Inhibitory Antibodies
			3.3.1 Preparation of Electrocompetent E. coli Cells
			3.3.2 Initial Selection
			3.3.3 Secondary Screening
		3.4 Characterizations of Isolated Clones
			3.4.1 Fab Cloning, Expression, and Purification
			3.4.2 Binding Affinity Measurements
			3.4.3 Inhibition Potency Measurements
	4 Notes
	References
Chapter 20: Engineering Selective TIMPs Using a Counter-Selective Screening Strategy
	1 Introduction
	2 Materials
		2.1 Laboratory Equipment
		2.2 Laboratory Glassware and Plasticware
		2.3 Expression of MMP-cd
		2.4 Extraction of Inclusion Body and Solubilization of MMP-cd
		2.5 Purification of MMP-cd
		2.6 Refolding of MMP-cd
		2.7 Reconcentration, Activation, and Desalting of MMP-cd
		2.8 Biotinylation of MMP-cd
		2.9 Preparation of TIMP1 Library
		2.10 Yeast Surface Display and Cell Growth
		2.11 Cell Preparation for Flow Cytometry
		2.12 Flow Cytometry
		2.13 DNA Preparation and Evaluation
		2.14 Expression of Soluble TIMP1
		2.15 TIMP1 Variant Binding and Inhibition
	3 Methods
		3.1 MMP Expression, Purification, Refolding, Solubilization, and Biotinylation
			3.1.1 pro-MMP3-cd and pro-MMP10-cd Protein Expression and Extraction
			3.1.2 pro-MMP3-cd and pro-MMP10-cd Protein Purification and Refolding
			3.1.3 pro-MMP3-cd and pro-MMP10-cd Protein Reconcentration, Activation, and Desalting
			3.1.4 MMP3-cd and MMP10-cd Biotinylation
		3.2 Preparation of TIMP1 Targeted Library
			3.2.1 Inserting the TIMP1 Gene with Random Mutations in Interacting Loops into the pCHA Yeast Display Vector
			3.2.2 Electrotransformation of the TIMP1 Targeted Library into EBY100 Cells
		3.3 Counter-Selective Screening of TIMP1 Targeted Library for Improving Binding Selectivity
			3.3.1 TIMP1 Variant Library Passaging and Induction
			3.3.2 Yeast Displayed TIMP1 Variant Library Immunolabeling
			3.3.3 Binding of Biotinylated MMP3-cd and Unlabeled MMP10-cd to the TIMP1 Variant Displayed Protein and Strep-AF647 Labeling
			3.3.4 FACS for Counter-Selective Screening of the Library of TIMP1 Variants
			3.3.5 Recovering of Sorted TIMP1 Variant Library
			3.3.6 Testing the Sorted TIMP1 Variant Library with High Binding Selectivity for MMP3
		3.4 Expression of Soluble WT TIMP1 and TIMP1 Variant Proteins Using HEK FreeStyle 293-F suspension Cells
		3.5 Measuring TIMP1 Variant Binding and Inhibition
			3.5.1 Determine the Concentration of Active TIMP1 by Titration
			3.5.2 Measuring TIMP1 Inhibition
	4 Notes
	References
Chapter 21: Engineering New Protease Inhibitors Using α2-Macroglobulin
	1 Introduction
	2 Materials
		2.1 A2M Expression
		2.2 A2M Purification
		2.3 Native Pore-Limited PAGE
	3 Methods
		3.1 Designing A2M with Engineered Bait Regions
		3.2 Cloning of Vectors for Mammalian A2M Expression
		3.3 Expression of A2M in HEK293 FreeStyle Cells
		3.4 Purification of A2M
		3.5 Zn2+ Immobilized Metal Affinity Chromatography (IMAC)
		3.6 Anion Exchange Chromatography
		3.7 Size Exclusion Chromatography (SEC)
		3.8 Characterization of New A2M Variants
		3.9 PAGE-Based A2M Characterization
	4 Notes
	References
Chapter 22: Design of Bioengineered Peptides/Proteases as Anti-cancer Reagents with Integrated Omics and Machine Learning Appr...
	1 Introduction
	2 Materials
		2.1 Acquisition of Venom
		2.2 Total RNA Isolation
		2.3 Total RNA Quantification and Purity Assessment
		2.4 TruSeq Stranded mRNA Library Preparation
		2.5 mRNA Sequencing
		2.6 Protein Extraction, Purification, and Quantification
		2.7 Protein Alkylation and Digestion
		2.8 Liquid Chromatography (LC)-Mass Spectrometry (MS)/MS and Protein De Novo Sequencing
		2.9 Comparison Between Transcriptome and Proteome
		2.10 Peptide Identification and Selection via Machine Learning
		2.11 Additional Materials and Equipment
	3 Methods
		3.1 Acquisition of Venom
		3.2 Total RNA Isolation
		3.3 Total RNA Qualification and Quantification
		3.4 TruSeq Stranded mRNA Library Preparation
			3.4.1 mRNA Purification
			3.4.2 mRNA Fragmentation
			3.4.3 Synthesis of First Strand  cDNA
			3.4.4 Synthesis of the Second Strand  cDNA
			3.4.5 Adenylation of 3′Ends
			3.4.6 Ligation of Adapters
			3.4.7 Enrichment of DNA Fragments
			3.4.8 Check Libraries
			3.4.9 Normalization and Pooling of Libraries
		3.5 mRNA Sequencing
		3.6 Protein Extraction, Purification, and Quantification
		3.7 Protein Alkylation and Digestion
		3.8 LC-MS/MS and Protein De Novo Sequencing
		3.9 Comparison Between Transcriptome and Proteome
		3.10 Peptide Identification and Selection via Machine Learning
	4 Notes
	References
Index