Murine Models of Helicobacter (pylori or felis)‐associated Gastric Cancer

Carrie A. Duckworth1, Michael D. Burkitt1, Jonathan M. Williams2, Bryony N. Parsons2, Joseph M.F. Tang2, D. Mark Pritchard3

1 Authors share first authorship, 2 Department of Cellular and Molecular Physiology, Gastroenterology Research Unit, Institute of Translational Medicine, University of Liverpool, Liverpool, 3 Correspondence: Prof D. Mark Pritchard, Department of Cellular and Molecular Physiology, Gastroenterology Research Unit, Institute of Translational Medicine, University of Liverpool, Ashton St, Liverpool, L69 3GE. Tel: 0151 794 5772; e‐mail: dmpritch@liv.ac.uk
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.34
DOI:  10.1002/0471141755.ph1434s69
Online Posting Date:  June, 2015
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Abstract

Gastric adenocarcinoma is the fifth most common cancer and third most common cause of cancer‐related death in the world. The majority of these cancers develop in genetically susceptible individuals who are chronically infected with the Gram‐negative bacterium Helicobacter pylori. Often these individuals have also been exposed to certain environmental factors that increase susceptibility, such as dietary components. Murine models of Helicobacter‐induced gastric cancer are valuable tools for investigating the mechanisms responsible for the stepwise pathological changes of chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric adenocarcinoma. Helicobacter felis colonization greatly accelerates the development of gastric neoplasia in mice, and causes pathologies similar to those observed with Helicobacter pylori‐associated gastric carcinogenesis in humans. These mouse models are therefore useful for investigating genetic and environmental factors that may be involved in the pathogenesis and treatment of gastric cancer. Detailed in these protocols are procedures for inducing Helicobacter‐associated carcinogenesis in mice as well as the histological analysis and interpretation of gastric pathology in these animals. © 2015 by John Wiley & Sons, Inc.

Keywords: helicobacter; felis; pylori; gastric cancer; gastric atrophy; parietal cells; gastric inflammation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Helicobacter felis: Culture, Harvest, Freezing, and Passaging
  • Alternate Protocol 1: Culture of H. pylori
  • Basic Protocol 2: Gastric Instillation of H. felis into Mice
  • Basic Protocol 3: Verification of H. felis Colonization in Mice
  • Basic Protocol 4: Verification of H. felis Colonization by Antral Histology
  • Basic Protocol 5: Verification of Helicobacter felis Colonization by Rapid Urease Test
  • Basic Protocol 6: Quantitative PCR to Validate H. felis Colonization in Mice
  • Alternate Protocol 2: Standard Curve PCR to Identify and Monitor H. felis
  • Support Protocol 1: Isolation of Bacterial DNA from In Vitro Cultured Bacteria
  • Support Protocol 2: Extraction of Bacterial DNA from Stool Samples
  • Support Protocol 3: Gastric Tissue Harvest and DNA Extraction
  • Basic Protocol 7: Gastric Tissue Harvest and Processing from Helicobacter‐Colonized Mice
  • Alternate Protocol 3: Gastric Tissue Harvest: Multiple Sample Types
  • Basic Protocol 8: Quantification and Semi‐Quantitation of Gastric Pathology Resulting from Helicobacter Colonization in Mice
  • Support Protocol 4: APES Coating of Microscope Slides
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Helicobacter felis: Culture, Harvest, Freezing, and Passaging

  Materials
  • Columbia chocolate agar with chocolated horse blood and bacitracin (Oxoid Limited, cat. no. PB0220A)
  • Helicobacter felis (ATCC #49179)
  • Tryptone soy broth powder (Sigma, cat. no. 22092)
  • Distilled water
  • H. felis cell suspension
  • Laminar flow hood (optional; Bunsen burner may be used on bench if experienced in aseptic technique)
  • Micropipets (20 to 200 μl) and tips (Eppendorf or equivalent)
  • Microbiological spreaders and 1‐μl loops (Technical Services)
  • 2.5‐liter anaerobic culture jars (Oxoid Limited, cat. no. AG0025)
  • Microaerophilic gas‐generating sachets, CampyGen 2.5‐liter (Oxoid, cat. no. CN0025)
  • Standard microbiology incubator set at 37°C
  • Autoclave
  • Spectrophotometer capable of reading absorbance at 600 nm
  • Cuvettes for spectrophotometer
  • Sterile cotton buds (Technical Services)
  • 30‐ml universal container (STARLAB)
  • Protect, microorganism cryopreservation system (cryopreservation tubes containing medium and beads, together with freezer storage box (Technical Services, cat. no. TS/80‐YE)

Alternate Protocol 1: Culture of H. pylori

  Materials
  • Female C57BL/6 mice (or strain of interest), aged 5 to 6 weeks
  • 0.5 ml Helicobacter felis suspension (OD>1.0) per mouse (from protocol 1)
  • Curved 22‐G, 3.8‐cm long gavage needle (Kent Scientific)
  • 1‐ml graduated syringes
  • Laminar flow hood

Basic Protocol 2: Gastric Instillation of H. felis into Mice

  Materials
  • Mouse (e.g., from protocol 3)
  • Sterile Dulbecco's phosphate‐buffered saline without CaCl 2 and MgCl 2 (PBS; Sigma‐Aldrich, cat. no. D8537)
  • Columbia chocolate agar with chocolated horse blood and bacitracin (Oxoid Limited, cat. no. PB0220A)
  • Dissection kit including:
    • Scissors
    • Forceps
  • Rotorstator homogenizer or bead‐beating tissue disagregator (Qiagen TissuelyserII or equivalent) or microcentrifuge tube tissue grinder pestle (e.g., Bel‐Art 199230000 Disposable Tissue Grinder Pestle)
  • 1.5‐ml microcentrifuge tubes
  • 96‐well plates
  • Laminar flow hood
  • Micropipets and tips
  • 2.5‐liter anaerobic culture jars (Oxoid Limited, cat. no. AG0025)
  • Microaerophilic gas‐generating sachets, CampyGen 2.5 liters (Oxoid Limited, cat. no. CN0025)
  • Standard microbiology incubator set at 37°C

Basic Protocol 3: Verification of H. felis Colonization in Mice

  Additional Materials protocol 12)
  • Mice
  • Hematoxylin and eosin
  • Light microscope

Basic Protocol 4: Verification of H. felis Colonization by Antral Histology

  Materials
  • Mouse
  • Dulbecco's Phosphate‐buffered saline without CaCl 2 or MgCl 2 (PBS; Sigma‐Aldrich, cat. no. D8537), pH 7.4
  • Dissection kit containing:
    • Toothed forceps
    • Scissors
    • Scalpel
  • Filter paper for blot drying tissue
  • Rapid urease test slides (e.g., ProntoDry)

Basic Protocol 5: Verification of Helicobacter felis Colonization by Rapid Urease Test

  Materials
  • LightCycler 480 Probes Master Mix (Roche Applied Science)
  • UPL probe #120 (Roche Applied Science, cat. no. 4693540001)
  • 100 μM Forward and reverse primers (see Table 14.34.3)
  • Nuclease‐free water (Roche Applied Science)
  • Positive control DNA
  • Laminar flow hood with UV light (recommended)
  • Micropipets and appropriate filter tips
  • LightCycler 480 Multiwell Plate 96 (Roche Applied Science)
  • Centrifuge
  • Real‐Time PCR machine (LightCycler480, Roche Applied Science, UK)
Table 4.4.3   MaterialsFlaA PCR conditions and Reagents

PCR Mix components
Component Stock concentration Final Concentration Volume for 1 well Volume for 96‐well plate
FAM Probe Master Mix 7 μl 740 μl
FAM Probe 10 μM 200 nM 280 nl 30 μl
Forward primer 10 μM 400 nM 560 nl 59 μl
Reverse primer 10 μM 400 nM 560 nl 59 μl
Nuclease‐free water 5.6 μl 590 μl
PCR Cycling conditions
Stage Temperature Duration Cycles
Pre‐Incubation 95 600 1
Amplification 95 10 45
60 30
72 1
Cooling 40 30 1
FlaA targeting primers and probes
Forward Primer Reverse Primer UPL Probe
GTT‐TCT‐GGG‐GCC‐AAT‐GTA‐AG GCC‐TTT‐GGC‐AAA‐ACC‐AAT‐AG 120

Basic Protocol 6: Quantitative PCR to Validate H. felis Colonization in Mice

  Materials
  • H. felis cell suspension in PBS
  • Sterile Dulbecco's phosphate‐buffered saline without CaCl 2 and MgCl 2 (PBS; Sigma‐Aldrich, cat. no. D8537)
  • Spectrophotometer
  • PCR machine‐compatible 96‐well plate
  • Thermal cycler
  • Benchtop centrifuge for 96‐well plates

Alternate Protocol 2: Standard Curve PCR to Identify and Monitor H. felis

To generate a standard curve for real‐time PCR, bacteria must be cultured and their DNA extracted. We perform crude bacterial DNA extractions using the following protocol to achieve this.
  • Bacterial colonies
  • Nuclease‐free water (Roche Applied Science)
  • 1 ‐μl microbiology loops
  • Heat block
  • Microcentrifuge

Support Protocol 1: Isolation of Bacterial DNA from In Vitro Cultured Bacteria

  Materials
  • 10% bleach solution70% (v/v) ethanol
  • Mice subjects
  • DNA extraction kits (either QIAamp DNA Stool Mini Kit or PSP Spin Stool DNA Plus kit; Stratech, cat. no. 1038110300)
  • Animal cages
  • Autoclave, optional
  • 2 × 25 ml universal containers
  • Forceps
  • 1.5‐ml microcentrifuge tubes

Support Protocol 2: Extraction of Bacterial DNA from Stool Samples

  Materials
  • ATL buffer (Qiagen, cat. no. 19076)
  • DX buffer (Qiagen, cat. no. 19088)
  • Frozen tissue sample from protocol 12or protocol 13
  • QIAamp cador Pathogens mini kit (Qiagen, cat. no. 54104)
  • Pathogen lysis tubes (small beads with DX buffer) (Qiagen)
  • Tissue Lyser II (Qiagen)
  • Centrifuge

Support Protocol 3: Gastric Tissue Harvest and DNA Extraction

  Materials
  • 4% formaldehyde (10% neutral buffered; i.e., 1:10 of 37.5% stock made with PBS)
  • Mouse subject
  • 4‐0 sofsilk‐coated suture (Tyco Healthcare)
  • 70% (v/v) ethanol
  • Paraffin wax (Thermo Fisher Scientific)
  • 30‐ml universal tube filled with 4% formaldehyde (also known as 10% NBF; see above)
  • 2‐ml syringe equipped with a 26‐G needle
  • Dissection scissors
  • Curved forceps
  • Fume hood
  • Safety glasses
  • Cutting mat or board
  • Scalpel blade Swann‐Morton size 22
  • Scalpel blade holder
  • Scalpel blade remover
  • Sharps disposal bin
  • 10‐ml syringes
  • Tissue‐embedding cassettes (Thermo Fisher Scientific)
  • Pencil
  • Tissue processor (e.g., Shandon Hypercenter XP, Thermo Fisher Scientific)
  • Tissue‐embedding center (e.g., Shandon Histocenter, Thermo Fisher Scientific)
  • 2 × 2 cm plastic embedding molds
  • Laboratory oven
NOTE: In the absence of a tissue processor, a revolving platform may be used at the lowest setting to provide slight agitation to tissue cassettes.

Basic Protocol 7: Gastric Tissue Harvest and Processing from Helicobacter‐Colonized Mice

  Materials
  • Mouse
  • Dulbecco's phosphate‐buffered saline without CaCl 2 or MgCl 2 (PBS; Sigma‐Aldrich, cat. no. D8537)
  • 4% formaldehyde (10× dilution with PBS from a 40% stock)
  • Liquid nitrogen (optional)
  • Dissection scissors
  • Petri dish
  • Scalpel blade number 22 (Swann‐Morton)
  • Scalpel blade holder
  • Scalpel blade remover
  • Forceps
  • 1.5‐ml microcentrifuge tube with lid removed
  • 30‐ml universal tubes
  • Microscope slides

Alternate Protocol 3: Gastric Tissue Harvest: Multiple Sample Types

  Materials
  • Distilled water
  • Tissue blocks containing stomach rings (see protocol 12 and Fig. )
  • Ice block
  • Xylene (Sigma‐Aldrich)
  • Ethanol (Sigma‐Aldrich)
  • Hydrogen peroxide (H 2O 2; Sigma‐Aldrich)
  • 1% Triton X‐100 in phosphate‐buffered saline (PBS) (1 ml of Triton X‐100 and 99 ml PBS)
  • Dulbecco's phosphate‐buffered saline without CaCl 2 or MgCl 2 (PBS; Sigma‐Aldrich, cat. no. D8537)
  • Normal goat serum (Dako)
  • Tween 20 (Sigma‐Aldrich)
  • H+/K+ ATPase rabbit polyclonal primary antibody (Merck Biosciences)
  • Goat anti‐rabbit biotin conjugated secondary antibody (Dako)
  • Vectastain ABC Elite (Vector Laboratories)
  • 3,3′‐diaminobenzidine (DAB) tablets (Sigma‐Aldrich)
  • Gill's hematoxylin
  • Xylene‐based mounting medium such as DPX (Sigma‐Aldrich)
  • Water bath set at 42°C
  • Microtome
  • Microtome blades
  • Forceps
  • Paintbrush (bristles ∼ 0.5 cm total diameter of all bristles)
  • APES‐coated glass microscope slides (see protocol 15)
  • Absorbent paper
  • Slide rack (Cole‐Parmer)
  • Pencil
  • General purpose laboratory oven set at 37°C
  • Slide tray
  • Glass trough
  • Moist chamber (e.g., StainTray 20 from Newcomer Supply)
  • Catering roll (e.g., WYPall; VWR Jencons, cat. no. 115‐2001)
  • Pastette
  • Peroxidase Anti‐Peroxidase (PAP) pen (Dako)
  • Suitably sized glass coverslips (Thermo Fisher Scientific)
  • Dropper pipet
  • Standard light microscope with 20× and 40× objective lenses
NOTE: Use a standard laboratory microtome to cut tissue sections. While this procedure is described briefly below, in all cases the investigator must follow the manufacturer's instructions in the microtome manual.

Basic Protocol 8: Quantification and Semi‐Quantitation of Gastric Pathology Resulting from Helicobacter Colonization in Mice

  Materials
  • 3‐Aminopropyltriethoxysilane (APES; Sigma‐Aldrich)
  • Acetone
  • Distilled water
  • Glass histology staining jars (Cole‐Parmer)
  • Standard glass microscope slides (Sigma‐Aldrich)
  • Slide rack (Cole‐Parmer)
  • General purpose laboratory oven set at 37°C
NOTE: APES is extremely corrosive and will precipitate on the histology staining jars. Disposal of APES must be through a licensed route. It must not be poured down the sink.
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Figures

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Literature Cited

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