Protocol to establish an accelerated murine model for Helicobacter-induced gastric cancer

Summary

Helicobacter-induced gastric cancer progresses very slowly, even in animal models, making it difficult to study. Here, we present a protocol to establish an accelerated murine model for Helicobacter-induced gastric cancer. We describe steps for infecting mice with Helicobacter felis, harvesting gastric tissue, assessing disease severity by histopathologic scoring, and performing gene expression studies with RT-qPCR and RNA sequencing. The accelerated model shows rapid progression of the disease, with gastric precancerous lesions developing within 6 months post-infection with Helicobacter.

For complete details on the use and execution of this protocol, please refer to Bali et al.¹

Graphical abstract

Highlights

• •Protocol for developing an accelerated gastric cancer model
• •Steps for performing mouse infections, tissue harvesting, and histology
• •Details for performing cDNA synthesis and RT-qPCR
• •Instructions for performing RNA sequencing and pathway enrichment

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Helicobacter-induced gastric cancer progresses very slowly, even in animal models, making it difficult to study. Here, we present a protocol to establish an accelerated murine model for Helicobacter-induced gastric cancer. We describe steps for infecting mice with Helicobacter felis, harvesting gastric tissue, assessing disease severity by histopathologic scoring, and performing gene expression studies with real-time RT-PCR and RNA sequencing. The accelerated model shows rapid progression of the disease, with gastric precancerous lesions developing within 6 months post-infection with Helicobacter.

Before you begin

Institutional permissions

CRITICAL: All animal procedures for this study have been approved by the Institutional Animal Care and Use Committee at the University of California, San Diego (Protocol Number: S02243) and conducted in compliance with federal regulations regarding the care and use of laboratory animals: Public Law 99–158, the Health Research Extension Act, and Public Law 99–198, the Animal Welfare Act which is regulated by USDA, APHIS, CFR, Title 9, parts 1, 2, and 3.

Six- to ten- week-old wild type C57BL/6 (WT) were purchased from The Jackson Laboratory (Bar Harbor, ME). MyD88 deficient (Myd88^−/−) were from our breeding colony originally provided by Dr. Akira (Osaka University, Japan) and backcrossed 10 times onto a C57BL/6 background, bred, and maintained at our facility.

Note:Myd88^−/− mice are available for purchase from different vendors including The Jackson Laboratory.

Purchase Helicobacter felis, strain CS1 (ATCC 49179) from the American Type Culture collection (Manassas, VA).

Animals

• 1.All mice should be housed in the same facility throughout the study in a biosafety level II (BSL-2) facility.
• 2.The mice should be maintained in a room controlled temperature (23 ± 2°C) and relative humidity (45%–60%).
• 3.All mice should have full access to food and water.

Preparation 1: Growing bacteria to use for infecting mice

Timing: 11 days

• 4.Streak Helicobacter felis, strain CS1 (ATCC 49179) on solid Columbia agar supplemented with 5% laked blood and 1% amphotericin B and incubate the cultures under microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂) at 37°C for 5 days (Figure 1).
• 5.Passaged bacterial cultures every 2–3 days by streaking onto fresh solid media.^2,3,4,5
• 6.Two days prior to infecting mice, culture H. felis in 30 mL liquid brain heart infusion (BHI) broth supplemented with 10% fetal calf serum in a T-75 cell culture flask.
• 7.Maintain the culture for 48 h on an orbital shaker at 50 rpm and incubated at 37°C under microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂) (Methods video S1).
• 8.Count spiral bacteria using a Petroff-Hausser chamber before infections.

Figure 1.

H. felis on Columbia agar

The image shows H. felis growing as a lawn, not as individual distinct colonies.

Preparation 2: Mouse infections

Timing: 6 months, 1 week

• 9.Inoculate mice (Myd88^−/− and WT) with 10⁹ organisms in 300 μL of BHI by oral gavage using a feeding needle.^2,3,4,5
• 10.Repeat inoculations of mice with H. felis at 1-day intervals for a total of three inoculations.

Note: Successful H. felis infections can be detected by performing PCR using mice fecal pellets using Helicobacter specific primers.⁶

• 11.After 3 months post-infection with H. felis euthanize the mice and remove their stomachs.

Note: The level of disease severity is compared with the standard model of gastric cancer, which is WT mice. However, to demonstrate disease progression, we also infect both Myd88^−/− and WT mice with H. felis for 1 or 6 months (Figure 1).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
Helicobacter felis, strain CS1	American Type Culture Collection	ATCC 49179
Biological samples
Human gastric biopsy tissue samples	Biorepository, University of California, San Diego	N/A
Critical commercial assays
Direct-zol RNA MiniPrep kit	Zymo Research	R2050
High-capacity cDNA reverse transcription kit	Thermo Fisher Scientific	4368814
Illumina TruSeq stranded total RNA sample prep kit with Ribo-Zero	Illumina	20020597
Deposited data
GEO accession number: GSE250438	Bali et al., 20241	GSE250438
Experimental models: Organisms/strains
Mouse: WT: C57BL/6	Jackson Laboratory	C57BL/6J
Mouse: Myd88 KO: Myd88 deficient	Dr. Akira’s laboratory	N/A
Oligonucleotides
Primers for qPCR	see Table S1	N/A
Software and algorithms
GraphPad Prism 8.2.0	GraphPad Software	https://www.graphpad.com/scientificsoftware/prism/
HOMER’s analyzeRepeats.pl	Dr. Benner’s lab	N/A
Other
NanoDrop 2000	Thermo Fisher Scientific	ND-2000
Veriti thermal cycler	Applied Biosystems	4375305
StepOne	Applied Biosystems	272001471
Illumina/Solexa HiSeq 2500 sequencer	Illumina	SY-401-2501
SURE-TEK biopsy cassette	Fisher Scientific	22-048-119
10% formalin	Fisher Scientific	SF98-4
UltraPure DNase/RNase-free distilled water	Thermo Fisher Scientific	10977015
MicroAmp 96-well reaction plate	Applied Biosystems	43446906
Biopsy foam pads	Fisher Scientific	22-038-221
Flex 80 dehydrating reagent	Fisher Scientific	22-046344
Ethanol	Millipore Sigma	493511
TRIzol	Invitrogen	15596026
KIMBLE Dounce tissue grinder set	Millipore Sigma	DWK885300-0001
Microfuge tube 1.5 mL	Genesee Scientific	24-285
0.2 mL PCR tubes	Genesee Scientific	22-154
Microcentrifuge, 5425	Eppendorf	5405000719
Agilent 2100 bioanalyzer	Agilent	AG-2100B
Rotary microtome RM 2125 RTS	Leica Biosystems	14045746960
Epredia HistoStar embedding workstation	Fisher Scientific	A81000002
Fisherbrand Isotemp water bath	Fisher Scientific	FSGPD05S24
Axio Imager.A2 microscope	Zeiss	490022-0002-000
AT2 Aperio digital slide scanner	Leica Biosystems	23AT2100
TC-treated dishes 100 × 20 mm	Genesee Scientific	25-202
Fetal calf serum	Thermo Fisher Scientific	16000044
Columbia agar	Becton Dickinson	294420
Bacto agar	Becton Dickinson	214020
Laked Horse blood	Hardy Diagnostic	10052-812
Amphotericin B	Corning	30-003-CF-1
Brain heart infusion broth	Becton Dickinson	237500
Petroff-Hausser counting chamber	Hausser Scientific	3900
Drummond Pipet-Aid	Fisher Scientific	13-681-19
Serological pipettes, 50 mL	Genesee Scientific	12-107
Serological pipettes, 25 mL	Genesee Scientific	12-106
Serological pipettes, 10 mL	Genesee Scientific	12-104
Serological pipettes, 5 mL	Genesee Scientific	12-102
50 mL conical flask	Genesee Scientific	28-106
1,000 μl, barrier tip, racked sterile	Genesee Scientific	23-430
200 μL, barrier tip, racked sterile	Genesee Scientific	23-412
10 μl, barrier tip, racked sterile	Genesee Scientific	23-401
Gilson PIPETMAN P100 pipette	Fisher Scientific	F144057MG
Gilson PIPETMAN P1000	Fisher Scientific	F144059MG
Gilson PIPETMAN P200 pipette	Fisher Scientific	F144058MG
Gilson PIPETMAN P10 pipette	Fisher Scientific	F144055MG
Gilson PIPETMAN P2 pipette	Fisher Scientific	F144054MG
Corning 1 L Pyrex glass bottle	Millipore Sigma	CLS13951L
Corning 500 mL Pyrex glass bottle	Millipore Sigma	CLS1395500
Thermo Scientific orbital shaker	Fisher Scientific	11-676-233
Thermo Forma CO2 incubator	Thermo Fisher Scientific	TH-3120
Oral gavage feeding needle	Millipore Sigma	CAD7909
TC-treated flasks, 25 mL, vent	Genesee Scientific	25-207
Thermo Scientific 1300 Series Class II, Type A2 biosafety cabinet	Fisher Scientific	13-261-304
Xylene histology	Fisher Scientific	60-047-036
Epredia Clear-Rite 3	Fisher Scientific	69-05
Epredia bluing reagent	Fisher Scientific	6769001
Epredia Clarifier 1	Fisher Scientific	22-050-118
Epredia Shandon Eosin-Y stain	Fisher Scientific	6766007
Epredia Histoplast paraffin	Fisher Scientific	83-30
Thermo Scientific oven	Fisher Scientific	11-475-152
Thermo Scientific ultra-low chest freezer	Thermo Fisher Scientific	TDEC39686FA

Materials and equipment

Preparation of Brain Heart Infusion (BHI) Liquid Broth (1 L)

Reagent	Final concentration	Amount
BHI	-	37 g
ddH2O	-	1000 mL
Total		1000 mL

Mix thoroughly and warm to completely dissolve. Sterilize by autoclaving. Store at 15°C–25°C for 6 months.

Preparation of Columbia Agar (1 L)

Reagent	Final concentration	Amount
Columbia Broth	-	35 g
Bacto Agar	-	15 g
ddH2O	-	1000 mL
Total		1000 mL

Mix thoroughly and warm to completely dissolve. Sterilize by autoclaving. Store at 15°C–25°C for 6 months.

Preparation of 70% Alcohol (100 mL)

Reagent	Final concentration	Amount
99% Ethanol	70	70 mL
Water	-	30 mL
Total		100 mL

Store at 15°C–25°C for a year.

Preparation of 95% Alcohol (100 mL)

Reagent	Final concentration	Amount
99% Ethanol	95	95 mL
Water	-	5 mL
Total		100 mL

Store at 15°C–25°C for a year.

• •Nanodrop 2000 (Thermo Fisher Scientific).

Alternatives: Any other spectrophotometer equipped for RNA quantification can be used.

• •Veriti Thermal Cycler (Applied Biosystems).

Alternatives: Any other thermal cycler can be used.

• •StepOne qPCR(Applied Biosystems).

Alternatives: Any other qPCR machine can be used.

• •Agilent 2100 Bioanalyzer (Agilent).

Alternatives: 5300 Fragment Analyzer (Agilent), Experion automated system (Bio-Rad), machine can be used.

• •Illumina/Solexa HiSeq 2500 sequencer (Illumina).

Alternatives: Any other high throughput Next-Gen sequencer can be used.

• •Rotary microtome RM 2125 RTS (Leica).

Alternatives: Any other microtome can be used.

• •Epredia HistoStar Embedding Workstation (Fisher Scientific).

Alternatives: Any other embedding workstation can be used.

• •Axio Imager A2 microscope (Zeiss).

Alternatives: Any other upright microscope can be used.

• •AT2 Aperio Digital slide scanner (Leica).

Alternatives: Any other digital slide scanner can be used.

• •Thermo Forma CO₂ Incubator (Thermo Fisher Scientific).

Alternatives: Any other CO₂ Incubator can be used.

• •Thermo Scientific 1300 Series Class II, Type A2 Biosafety cabinet (Fisher Scientific).

Alternatives: Any other Class II, Type A2 Biosafety cabinet can be used.

• •HistoCore Pegasus Automated Tissue Processor (Leica).

Alternatives: Any other Automated Tissue Processor can be used.

• •Epredia Gemini Autostainer (Fisher Scientific).

Alternatives: Any other Slide stainer can be used.

• •Epredia ClearVue coverslipper (Fisher Scientific).

Alternatives: Any other Coverslipper can be used.

Step-by-step method details

Preparing mouse stomach tissue sections for various analyses

Timing: 6 months

In this section, mice stomachs are harvested at different time points and stomach tissue is sectioned.

• 1.At indicated time points, post-infection with H. felis (Figure 2), euthanize mice with carbon dioxide followed by cervical dislocation.
• 2.Remove the stomach from the abdominal cavity and open the stomach by cutting along the greater curvature.
• 3.Cut the stomach tissue longitudinally into three sections (Figure 3), one section to be processed for histology and two sections snap-frozen separately to use for RNA sequencing and real-time PCR and stored in −80°C deep freezer for later use.

CRITICAL: Immediately freeze the tissue samples to be processed for RNA and store in −80°C deep freezer for later use to maintain integrity of the RNA and for better yield.

Figure 2.

Mouse infection timeline

This illustration shows the timeline of inoculating WT and Myd88^−/− mice with H. felis by oral gavage at an interval of one day for a total of three inoculations. Uninfected mice serve as control for each genetic background. n = 8 mice /group.

Figure 3.

Illustration of mouse stomach sections used for analyses

At indicated time points mice are euthanized and stomachs are removed from the abdominal cavity. Each mouse stomach is opened by cutting along the greater curvature and laid flat on a petri plate. Then it is cut into longitudinal sections and processed for various analyses.

Fixing tissue section before paraffin embedding

Timing: 20 h

In this section, longitudinal stomach tissue sections are fixed.

• 4.Flatten longitudinal sections of stomach tissue from each mouse by placing each tissue between biopsy foam pads in a biopsy cassette.
• 5.Fix the tissue sections in neutral buffered 10% formalin.
• 6.After 18 h of fixation remove the formalin and immerse the cassettes containing tissue section in Flex 80 dehydrating reagent.

Note: In this step you can use 70% ethanol instead of Flex 80.

Preparing gastric tissue sections for paraffin embedding

Timing: 4 h

In this section, stomach tissue sections are paraffin embedded.

• 7.Take the fixed gastric tissue sections and run them on short cycle on automated tissue processor.

Steps of the short cycle on the automated processor.

Short cycle

Station	Solution	Time (min)	Temperature °C	Vacuum	Mix speed
1	70% Alcohol	15	Ambient	ON	FAST
2	70% Alcohol	15	Ambient	ON	FAST
3	95% Alcohol	15	Ambient	ON	FAST
4	100% Alcohol	15	Ambient	ON	FAST
5	100% Alcohol	15	Ambient	ON	FAST
6	100% Alcohol	20	Ambient	ON	FAST
7	100% Alcohol	20	Ambient	ON	FAST
8	Xylene	15	Ambient	ON	FAST
9	Xylene	20	Ambient	ON	FAST
10	Xylene	20	Ambient	ON	FAST
11	Paraffin	20	60	ON	FAST
12	Paraffin	30	60	ON	FAST
13	Paraffin	30	60	ON	FAST

Embed gastric tissue sections

Timing: 1 h

In this section, each stomach tissue section is embedded in a tissue cassette.

• 8.Transfer the tissue from the automated process to an embedding instrument. Embed the gastric tissue on the edge.
• 9.Open one tissue cassette at a time to maintain accuracy of each sample and label. Make one paraffin block for each tissue cassette (Figure 4).

Figure 4.

A paraffin embedded tissue section

A stomach tissue section was paraffin embedded and cut on the edge for histopathology analysis.

Section gastric tissue samples

Timing: 24 h

In this section, paraffin embedded tissues are sectioned, and the flattened tissue sections are placed on slides.

• 10.Place paraffin embedded tissue block in a rotary microtome blade holder.
• 11.Adjust thickness to 5 μm.
• 12.Cut and collect 2 to 6 gastric tissue sections in a ribbon.
• 13.Place the ribbon of tissue sections in a water bath at 38°C–40°C.
• 14.Separate the sections using forceps and place on a clear pre-labeled slide.

CRITICAL: Allow the slides with sections to remain in the water bath until they are flattened. Ensure there are no air bubbles trapped under the gastric tissue. Make sure the gastric tissue sections are free of debris and wrinkles.

• 15.Air-dry the gastric tissue sections for 12 h.

Stain the gastric tissue sections

Timing: 1.5 h

In this section, gastric tissues sections are stained with hematoxylin and eosin.

• 16.Stain the gastric tissue sections with hematoxylin and eosin (H&E) using an automatic staining system.
• 17.Cover the stained gastric tissue with a coverslip using an automated coverslipper instrument.
• 18.Image the H&E-stained gastric tissue sections on the slides using an upright microscope to observe the morphological features.
• 19.Scan the slides at 40X magnification using a digital slide scanner.
• 20.Send the slides to a blinded pathologist to review and score histopathologic parameters.

Steps of Hematoxylin-Eosin staining protocol

S. No.	Protocol	Time	Repetition
1	Oven	60 min	One
2	Clear-Rite 3	3 min	Three
3	100% Ethanol	1 min	Two
4	95% Ethanol	1 min	One
5	Running Water	1 min	One
6	Clarifier 1	5 s	One
7	Running Water	1 min	One
8	Bluing Reagent	30 s	One
9	Running Water	1 min	One
10	70% Ethanol	30 s	One
11	Eosin	3 min	One
12	70% Ethanol	1 min	One
13	95% Ethanol	1 min	One
14	100% Ethanol	1 min	Four
15	Clear-Rite 3	3 min	Three
16	Move the rack to the coverslipper instrument or mount/coverslip slides manually

Pathology scoring

Timing: 14 days

In this section, the H&E stained gastric tissue sections are scored for disease severity.

• 21.Score H&E-stained gastric tissue sections for gastric histopathologies, including inflammation, epithelial defects, gland atrophy, hyperplasia, intestinal metaplasia, and dysplasia following the criteria outlined by Rogers et al.⁷
• 22.Give a score for histologic disease severity on a scale from 0 (no lesions) to 4 (severe lesions).

Note: Scoring the histological characteristics is performed as follows: inflammation—infiltration of inflammatory cells into the mucosa and submucosa; epithelial defects—thinned epithelial surface, epithelial and mucosal erosion and multifocal collapse; oxyntic gland atrophy—loss of oxyntic glands (which contain parietal cells and chief cells) in the gastric corpus; mucous metaplasia- presence of large foamy mucus secreting cells; intestinal metaplasia—replacement of gastric epithelium by intestinal type gastric epithelium; and dysplasia—abnormal cellular and glandular maturation of the epithelium, with score of 3 indicating carcinoma in situ.

Extract RNA from mouse gastric tissue sections

Timing: 1 day

In this section, RNA is extracted from mouse gastric tissue.

• 23.Pulverize the frozen gastric tissue and transfer to a microfuge tube containing 1 mL TRIzol reagent.
• 24.Homogenize the gastric tissue sample using the dounce tissue homogenizer.
• 25.Centrifuge the gastric homogenates at 4°C at 13226 × g for 30 min.
• 26.Transfer the supernatant to complete RNA extraction using the Direct-zol RNA mini kit as per the manufacturer’s instructions (Protocol link).
• 27.Determine the quality of the extracted RNA using a Nanodrop system by measuring absorbance levels at 230, 260, and 280 nm.

CRITICAL: The quality of the RNA is very important. If the 260/230 ratio is less than 1, it indicates the presence of contaminants such as TRIzol. The acceptable value is between 2.0 and 2.2. The 260/280 ratio should be between 1.65 and 2.0 with a ratio of 1.8 considered pure.

• 28.Use the extracted RNA for quantitative real time PCR and RNA sequencing.

Perform cDNA synthesis and quantitative real-time RT-PCR

Timing: 8 h

In this section, the extracted RNA is subjected to cDNA synthesis followed by real time RT-PCR.

• 29.Use 2 μg of RNA extracted from each gastric tissue to reverse transcribe RNA into cDNA using the High Capacity cDNA Reverse Transcription (RT) kit according to manufacturer’s instructions (Protocol link).
• 30.Details of the reagents prepared are shown below.

RT Master Mix prepared using High Capacity cDNA Reverse Transcriptase Kit

Component	Volume (μL)
10X RT Buffer	2.0
25X dNTP mix	0.8
10X RT Random Primers	2.0
MultiScribe Reverse Transcriptase	1.0
Nuclease free water	4.2
Total	10

• 31.The reverse transcription reaction is prepared by mixing 10 μL of RT Master Mix with 10 μL of RNA (2 μg concentration, diluted with nuclease free water to make the final volume of 10 μL), to make a total reaction volume of 20 μL per individual tube.
• 32.The tubes are loaded on to the thermal cycler following the amplification conditions shown below.

Note: After the RT-PCR cycle is complete store the cDNA tubes in −20°C till further use or proceed to the next step.

RT Cycle conditions

Steps	Temperature °C	Time (min)
1	25	10
2	37	120
3	85	5
4	4	Hold

• 33.Use 1 μL of each cDNA sample for real-time PCR per well in a total of 10 μL reaction mix.
• 34.Details of amplification conditions and reagents are shown below.

q-PCR Master Mix prepared using SYBR Green Mix

Component	Volume (μL)
SYBR Green Mix	5.0
Forward Primer (10 μM)	0.5
Reverse Primer (10 μM)	0.5
Nuclease free water	3.0
cDNA	1.0
Total	10

qPCR Cycle conditions

Steps	Temperature °C	Time	Cycle
1	95	5 min	1
2	95	15 s	40
3	60	20 s
4	72	40 s
5	4	Hold	Forever

• 35.Primers are listed in supplementary material (Table S1).

Perform RNA sequencing

Timing: 14 days

In this section, the RNA extracted from gastric tissue samples are subjected to RNA Sequencing.

• 36.Use RNA extracted from mouse gastric tissue sections.
• 37.Measure RNA concentration and integrity using an Agilent 2100 Bioanalyzer, which assigns each sample an RNA integrity number (RIN).

CRITICAL: The quality of RNA is very important. A RIN number of 8 or higher is preferred because it indicates that the RNA is of high quality, which also means it is intact. A lower RIN number indicates that the RNA is degraded. A high RIN value is crucial for a proper library construction.

• 38.Prepare the RNA-seq library for sequencing using the Illumina TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero (Human/Mouse/Rat) according to the manufacturer’s instructions (Protocol link), which encompass ribosomal RNA depletion, fragmentation, cDNA synthesis, and cDNA library preparation.

CRITICAL: Avoid the use of sample prep kits that only measure mRNA (they specifically capture polyadenylated RNAs) as these approaches are much more sensitive to changes in RIN integrity and could result in a 3′ capture bias and increased sample variability.

• 39.Perform RNA-sequencing on an Illumina/Solexa HiSeq 2500 sequencer to generate 75-bp single-end reads for all samples at a depth of approximately 40 million reads per sample.

Perform RNA-seq data analysis

Timing: 2 days

In this section, RNA-seq data analysis is performed.

• 40.Trim RNA-seq reads to remove non-biological library adapter sequences by applying the fastp⁸ program to RNA-seq FASTQ files from each experiment.
• 41.Align the trimmed RNA-seq reads from each experiment to the mouse genome using the program STAR.⁹
• 42.Create HOMER-style tag directories for each experiment using HOMER’s makeTagDirectory program.¹⁰
• 43.Determine gene-level read counts for all experiments by counting the number of reads overlapping exons for all genes using HOMER’s analyzeRepeats.pl tool and the transcriptome annotation from GENCODE.¹⁰
• 44.Use DESeq2 to identify genes that are differentially expressed from the unnormalized read counts across all experiments.¹¹
• 45.Following analysis of RNA-seq data with DESeq2, use the EnhancedVolcano package from R/Bioconductor to construct a volcano plot to visualize the differential expression data.
• 46.Use the DESeq2’s rlog function to normalize the raw gene counts and use the normalized gene expression values to cluster and generate a heatmap to visualize the RNA-seq data.
• 47.Use HOMER’s makeUCSCfile tool to generate normalized genome browser read density files (bedGraph), which can then be visualized using the UCSC Genome Browser (http://genome.ucsc.edu/).

Perform pathway and motif enrichment analysis

Timing: 1 day

In this section, the pathway and motif enrichment analysis is performed.

• 48.Use Metascape,¹² a web-based portal that integrates multiple knowledge bases, including the Gene Ontology, to identify overrepresented pathways in sets of regulated genes.
• 49.Use differentially expressed genes with a fold change of at least 2 and an adjusted p-value of 0.05 as input for Metascape analysis.

CRITICAL: It is important to correct for multiple hypothesis testing for the pathway enrichment data acquired using Metascape.

• 50.Use HOMER, a software suite for DNA motif and next-generation sequence (NGS) analysis⁹ to analyze transcription factor motifs in mouse stomach tissue sections (RNA-seq data).
• 51.Scan known transcription factor motifs from the HOMER database in mouse promoter sequences defined within a range from −300 to +50 bp relative to the GENCODE-defined transcription start site (TSS) locations of differentially regulated genes.
• 52.Calculate their enrichment relative to non-regulated genes in the mouse genome using HOMER’s findMotifs.pl tool.

CRITICAL: The known motif results from this analysis will report a Benjamini-Hochberg procedure corrected q-value that address concerns arising from multiple hypothesis testing.

Expected outcomes

Helicobacter pylori (H. pylori) infection is an established cause of many digestive diseases, including gastritis, peptic ulcers, and gastric cancer.¹² The development of gastric cancer is a very slow process, which complicates the study of this disease. Consequently, there is an urgent need for a Helicobacter-induced model that allows for more rapid study of disease progression. We compare our accelerated murine model of gastric cancer to the standard murine model of gastric cancer. Both models involve infection of C57BL/6 mice with H. felis, a close relative of the human pathogen, H. pylori,^13,14 and have been established as cancer mouse models that closely recapitulate the disease progression observed in humans during H. pylori infection.^{4,15,16,17,18,19} We show that gastric precancerous lesions develop and progress more rapidly and with greater severity in H. felis-infected Myd88^−/− mice (accelerated model) when compared to H. felis-infected WT mice (standard model) (Figures 5 and 6). In addition, the model exhibits activation of common oncogenic pathways and novel pathways/genes. The advantages of using this model include the following; i) it shortens the window for Helicobacter infection disease transformation into premalignant gastric lesions by 6 months, allowing for more rapid study of disease progression, ii) it exhibits activation of common oncogenic pathways and, in addition, the activation of novel pathways/genes, providing valuable insights into gastric cancer development, and iii) importantly, the gastric neoplastic phenotype in this model only manifests following Helicobacter infection,¹ making it highly relevant to human gastric cancer development.

Figure 5.

Representative mice stomachs following infection with H. felis for 1, 3, or 6 months

Mice stomachs exhibit thicker mucosa with larger folds that increase in thickness with time in Myd88^−/−-infected (accelerated model) compared to WT-infected (standard model) mice. n = 8 mice /group.

Figure 6.

Histopathologic stages in mouse gastric tissue in response to H. felis infection

WT and Myd88^−/− mice are infected with H. felis for 1, 3, or 6 months (8 mice /group). Representative mouse stomach tissue sections from WT and Myd88^−/− mice are shown (bar scale: 500 μm) (A). Mice stomachs are processed and scored double-blinded for histologic disease severity on an ascending scale from 0 (no lesions) to 4 (severe lesions) as described in our previous studies^4,20 using criteria outlined by Rogers et al.⁷ (B). Histopathologic scores, which are a measure of disease severity^4,5,7,21 show significant differences with increased disease in H. felis-infected Myd88^−/− mice (accelerated model) compared to H. felis-infected WT mice (standard model). ∗p < .05, ∗∗p < .01, ∗∗∗p < .001.

Limitations

The study used only male mice, which limits generalization of the results. However, this was done to allow for comparison with previous studies that have investigated gastric carcinogenesis using the H. felis infection mouse model that have also used only male mice.^15,22,23 This is the limitation of the study. There is immune infiltration in both the accelerated (H. felis-infected Myd88^−/−) and standard (H. felis-infected WT) mouse models as indicated by the inflammation score in Figure 6. However, the difference is that the disease progresses faster in the accelerated model. Indeed, using the accelerated model we observed high expression levels of genes associated with disease severity including interferon stimulating genes (ISGs), which were similarly elevated in human gastric tumor samples.¹ However, even with the presence of inflammatory response in the accelerated model, due to the absence of MyD88, caution should be exercised when interpreting findings from immunological and immunotherapeutic studies. This is the limitation of the model. Nevertheless, the accelerated model is a valuable asset for carrying out gastric cancer related studies given that it exhibits high activation of genes that are also highly expressed in patient gastric cancer biopsy samples and it shortens the window for Helicobacter infection disease transformation.

Troubleshooting

Problem 1

When growing H. felis in the liquid culture there is a possibility that the bacteria might not be enough to inoculate mice to achieve infection (Preparation 1- Growing bacteria to infect mice, step 6).

Potential solution

Increase the amount of medium by expanding the number of T-75 cell flasks used while maintaining a volume of 30 mL per flask. The percent of fetal calf serum in the liquid medium can also be increased to anywhere from 15%–20% and then repeat step 6 of the section on growing bacteria for infection.

Problem 2

Gastric homogenates are not uniform and homogeneous after centrifugation (RNA Extraction, step 24).

Potential solution

Transfer the supernatant containing the non-uniform homogenate, add TRIzol and repeat step 5 of the RNA extraction procedure.

Problem 3

During gavage of mice there might be some loss of bacteria through spilling or/and mice spitting (Preparation 2 – Mouse infections, Step 9).

Potential solution

Problems with liquid spillage and mouse spitting are usually a result of improper restraining of the mouse. To achieve this, hold the mouse in a vertical position and gently grasp the mouse by the loose skin over the neck and back and immobilize the head.

Problem 4

Gastric tissue lifts or peels off the slide (Fixing tissue sections before paraffin embedding, step 4).

Potential solution

Make sure the slides are very clean. Use charged slides.

Problem 5

Gastric tissue is not completely pulverized (RNA Extraction, Step 23).

Potential solution

Make sure that the gastric tissue is frozen and that the tissue pulverizer is completely frozen before adding the tissue to pulverize.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Marygorret Obonyo (mobonyo@health.ucsd.edu).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to the technical contact, Prerna Bali (pbali@health.ucsd.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All data reported in this paper will be shared by the lead contact upon request.
• •Single-cell RNA-seq data have been deposited at GEO and is publicly available as of the date of publication. The accession number is listed in the key resources table.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was supported by funding from the Department of Defense (DOD) award W81XWH-20-1-0675 to M.O. The graphical abstract and figures were created using Biorender.com.

Author contributions

M.O. supervised and designed the study concept and research; C.B. supervised data analysis; P.B., I.L.-P., and J.H. collected and analyzed data; M.V.E. performed mouse histopathologic scoring; P.B. and I.L.-P. wrote the draft of the manuscript; C.B. and M.O. revised the manuscript. All authors read and approved the manuscript.

Declaration of interests

The authors declare no competing interests.