Name: a method to define the effects of environmental enrichment on colon microbiome biodiversity in a mouse colon tumor model
Uploaded: 2018-02-28T13:00:00
Description: Watch this Scientific Journal Video about A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model at JoVE.com

We thank B. Dalley in the University of Utah Genomics core for library sequencing, and K. Boucher in the University of Utah Biostatistics core for statistical advice, and access to these technical cores supported by National Cancer Institute award P30 CA042014. The project described was supported by the National Cancer Institute Grants P01 CA073992 and K01 CA128891 and the Huntsman Cancer Foundation.

All methods described here were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Utah.
1. Experimental Design and EE and Control Cage Setup
Note: For reference, an outline of the experimental design is illustrated (Figure 1).
<ol>
	<li>Set up control (NE) and EE cages (Figure 2).</stron...

<ol>
	<li>Bechard, A., Meagher, R., Mason, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+reduces+the+likelihood+of+alopecia+in+adult+C57BL/6J+mice.">Environmental enrichment reduces the likelihood of alopecia in adult C57BL/6J mice.</a> Journal of the American Association for Laboratory Animal Science : JAALAS. 50 (2), 171-174 (2011).</li><li>Jankowsky, J. 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Illumina Available from: <a target="_blank" href="https://suport.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf">https://suport.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf</a> (2017)</li><li>Illumina Experiment Manager. Illumina Available from: <a target="_blank" href="https://www.illumina.com/informatics/research/experimental-design/illumina-experiment-manager.html">https://www.illumina.com/informatics/research/experimental-design/illumina-experiment-manager.html</a> (2017)</li><li>Caporaso, J. 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This procedure allows for the analysis of microbiota isolated from stool following environmental enrichment of normal or tumor bearing animals. Because these are large experiments which involve breeding to obtain many animals of different sexes and genotypes, normalizing the microbiome between animals prior to commencement of the experiment is essential to avoid non-EE related effects on microbiome biodiversity.
For consistency between NE and EE conditions, the breeding process is conducted to...

Environmental enrichment (EE) studies utilize complex housing parameters to affect social stimulation (large housing cages, larger groups of animals), cognitive stimulation (huts, tunnels, nesting materials, platforms) and physical activity (running wheels). EE has been utilized by many labs to understand the effects of increased activity and improved social and cognitive interactions on disease initiation and progression using a wide array of mouse models, including barbering induced alopecia, Alzheimer&#39;s disease, Rett syndrome, and several tumor and digestive disease models1,2,3,4,5,6.
Several mouse models have been developed to study colon tumorigenesis in mice. Perhaps the most well-defined model is the ApcMin mouse. The ApcMin&#160;mouse was developed in the laboratory of William Dove in 19907, and has been used as a mouse model of mutations in the APC gene that are commonly associated with human colorectal cancer. In contrast to humans harboring APC mutations, ApcMin mice primarily develop small intestinal tumors, with very rare occurrence of colon tumors. However, a Tcf4Het allele with a single knockin-knockout heterozygous mutation in Tcf4, vastly increases colon tumorigenesis when combined with the ApcMin&#160;allele8. Recently, this mouse model of colon tumorigenesis has been used to determine the effects of EE on colon tumorigenesis6. In the Bice et al. study, the physiological and phenotypic effects of EE on males and females of four different mouse lines (wild-type (WT), Tcf4Het/+ Apc+/+, Tcf4+/+ ApcMin/+, and Tcf4Het/+ApcMin/+)) were defined. Perhaps the most interesting finding was that EE significantly increases the lifespan of both male and female colon tumor-bearing animals. This demonstrated that EE may reduce at least some of the symptoms associated with colon tumorigenesis, and improve animal health. Remarkably, this improved lifespan in males is not a direct result of reduced tumorigenesis, and instead was linked to the initiation of a tumor wound healing response, including improved microbiome biodiversity6.
Several EE specific studies have been published with interesting results. However, from a technical standpoint, important results are often not translatable to other laboratories. Maintaining identical EE methodologies between different laboratories is an incredibly complex issue, not only due to enrichment devices and housing used, but also bedding, food, ventilation, breeding, genetics, activity in the room, and animal protocol requirements, among others9,10,11. One example is animal integration, where animals must be stably integrated into the mouse colony, therefore normalizing genetic background and diet composition, to avoid non-treatment related effects. Further, many EE studies have been completed prior to the realization of the importance of the microbiome in disease, and the way that common mouse husbandry practices can affect the composition of the gut microbiome10,12.
Breeding strategy and animal placement in EE can increase stress if not performed properly. Since EE studies utilize large numbers of both male and female animals and multiple genotypes, experimental setup can be difficult given the requirement for animals from several litters to be combined. Therefore, a breeding and weaning strategy was developed to allow for combining of weaned animals of the correct genotype from different litters. The primary rationale for this was to normalize the microbiota among litters and to reduce stress when animals were moved to the experimental environment. The microbiome was transmitted from the dam10. To provide microbial diversity to the colony, females were purchased from Jackson Labs and integrated into the colony for one month before the experiment began9,10,12. To further normalize microbiome biodiversity between animals, females were co-housed prior to breeding. Following breeding, communal housing during rearing and the ability to escape nursing pups improved the stress levels of maternal care13,14, possibly furthering microbiome normalization. To prevent non-EE related effects on the microbiome, this communal housing of all experimental animals prevented fighting and additional stress that occurred when combining several males from different litters into one experimental cage. Finally, equal numbers of animals of all genotypes were included in the cages. This provided the opportunity for improved microbiota biodiversity across genotypes, and removed the contribution of coprophagia (the animal&#39;s tendency to consume stool) or possible genotype-specific behavioral differences to the overall study.
This protocol provides a strategy that expands previous EE studies to include known aspects of microbiome research, including microbiota transmission and animal colony integration for microbiota normalization, to enable more uniform microbiome populations between experimental animals. Heeding these precautions is essential due to the ability of non-treatment related microbiota differences to confound study findings. Eliminating non-EE related microbiota changes will enable researchers to specifically define the role of EE on microbiota composition during disease development and progression.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Teklad Diets/Harlan Labs Chow</td>
			<td >Harlan Labs</td>
			<td >3980X</td>
			<td >Standard irradiated chow formulated by Dr. Mario Capecchi in collaboration with Harlan Labs.</td>
		</tr>
		<tr >
			<td >Cell-Sorb Plus bedding</td>
			<td >Fangman Specialties</td>
			<td >82010</td>
			<td >Autoclave prior to use.</td>
		</tr>
		<tr >
			<td >AIMS Tattooing System For Neonates</td>
			<td >AIMS</td>
			<td >NEO-9</td>
			<td >https://animalid.com/neonate-rodent-tattoo-identification/32. Other animal grade tattoo systems and inks can be used with similar results including the Aramis Micro Tattoo Kit.</td>
		</tr>
		<tr >
			<td >Zyfone One Cage 2100 AllerZone Mouse Micro-Isolator System Complete with cage, AllerZone filter top and modular diet delivery system</td>
			<td >Lab Products</td>
			<td >82120ZF</td>
			<td >Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side.</td>
		</tr>
		<tr >
			<td >Zyfone One Cage 2100 Life Span Enrichment Device</td>
			<td >Lab Products</td>
			<td >82109ZF</td>
			<td >Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side.</td>
		</tr>
		<tr >
			<td >Zyfone One Cage 2100 Cage 13-7/8&#34; Length X 19-1/16&#34; Width X 7-3/4&#34; Depth</td>
			<td >Lab Products</td>
			<td >82100ZF</td>
			<td >Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side.</td>
		</tr>
		<tr >
			<td >Zyfone One Cage 2100 AllerZone Micro-Isolator filter top</td>
			<td >Lab Products</td>
			<td >82101ZF</td>
			<td >Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side.</td>
		</tr>
		<tr >
			<td >Tunnel</td>
			<td >Bio-Serv</td>
			<td >K3323 or K3332</td>
			<td >Connect cages together and use for enrichment</td>
		</tr>
		<tr >
			<td >Grommet to connect Tunnel to cages</td>
			<td >Fabricated by the University of Utah Machine Shop</td>
			<td >n/a</td>
			<td >Be certain the material is resistant to chewing and autoclavable</td>
		</tr>
		<tr >
			<td >Fast-track wheel</td>
			<td >Bio-Serv</td>
			<td >K3250 or K3251</td>
			<td >Use with mouse igloo and floor</td>
		</tr>
		<tr >
			<td >Mouse Igloo</td>
			<td >Bio-Serv</td>
			<td >K3328, K3570 or K3327</td>
			<td >Use with Fast-track wheel and floor</td>
		</tr>
		<tr >
			<td >Mouse Igloo floor</td>
			<td >Bio-Serv</td>
			<td >K3244</td>
			<td >Use with mouse Igloo and Fast-Track</td>
		</tr>
		<tr >
			<td >Mouse Hut</td>
			<td >Bio-Serv</td>
			<td >K3272, K3102 or K3271</td>
			<td ></td>
		</tr>
		<tr >
			<td >Crawl Ball</td>
			<td >Bio-Serv</td>
			<td >K3330 or K3329</td>
			<td ></td>
		</tr>
		<tr >
			<td >Bio-hut</td>
			<td >Bio-Serv</td>
			<td >K3352</td>
			<td >Wood pulp hut used for sheltering and nesting</td>
		</tr>
		<tr >
			<td >Adhesive film&#160;</td>
			<td >VWR</td>
			<td >60941-072</td>
			<td >Use to temporarily cover drilled hole in large cage to prevent mice from escaping</td>
		</tr>
		<tr >
			<td >Laminar Flow Ventilated Rack</td>
			<td >Techniplast</td>
			<td >Bio-C36</td>
			<td >The cabinet we used in this study is not currently supplied. The Bio-C36 is very similar.</td>
		</tr>
		<tr >
			<td >1.5 mL Microfuge Tube- RNAse and DNAse free</td>
			<td >Any supplier</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >QIAamp DNA Stool MiniKit</td>
			<td >Qiagen</td>
			<td >51504</td>
			<td >This kit supplies reagents for 50 DNA preparations. Stool Lysis Buffer=ASL; Guanidinium Chloride Lysis Buffer= AL; Wash Buffer 1 with Guanidinium Chloride= AW1; Wash Buffer 2= AW2; Elution Buffer with EDTA=AE</td>
		</tr>
		<tr >
			<td >Waterbath (capable of heating to 95)</td>
			<td >Any supplier</td>
			<td ></td>
			<td >For 94 degree incubation of stool samples to lyse cells.</td>
		</tr>
		<tr >
			<td >Waterbath (capable of heating to 70 degrees)</td>
			<td >Any supplier</td>
			<td ></td>
			<td >For 70 degree incubation of stool samples&#160;</td>
		</tr>
		<tr >
			<td >Ethanol (200 proof)</td>
			<td >Sigma Aldrich</td>
			<td >E7023</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fluorometer: Qubit</td>
			<td >ThermoFisher Scientific</td>
			<td >Q33216</td>
			<td ></td>
		</tr>
		<tr >
			<td >Qubit dsDNA broad Range Assay Kit</td>
			<td >ThermoFisher Scientific</td>
			<td >Q32850</td>
			<td ></td>
		</tr>
		<tr >
			<td >EB Buffer or 10 mM Tris pH 8.5</td>
			<td >Qiagen</td>
			<td >19086</td>
			<td ></td>
		</tr>
		<tr >
			<td >Experiment specific primers</td>
			<td >Any Supplier</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >PCR grade water</td>
			<td >Any supplier</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >2X KAPA HiFi HotStart Ready Mix&#160;</td>
			<td >Kapa Biosystems</td>
			<td >KK2601</td>
			<td >For Amplicon Amplification (1.25 mL allows 100 rxns).</td>
		</tr>
		<tr >
			<td >Agarose for running diagnostic gels</td>
			<td >Any supplier</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >TapeStation High Sensitivity D1000 Screen Tape Trace</td>
			<td >Agilent</td>
			<td >5067-5583</td>
			<td >TapeStation or Bioanalyzer instruments are common in Institutional Genomics Cores to analyze library quality . Alternatively a Bioanalyzer DNA1000 Chip (Agilent, 5067-1504) can be used.</td>
		</tr>
		<tr >
			<td >Agencourt AMPure XP Magnetic Beads</td>
			<td >Beckman Coulter</td>
			<td >A63880</td>
			<td >Magentic beads For PCR cleanup- 5 mL will clean 250 PCR reactions</td>
		</tr>
		<tr >
			<td >Magnetic stand</td>
			<td >Life Technologies</td>
			<td >AM10027</td>
			<td ></td>
		</tr>
		<tr >
			<td >Library Preparation Guide</td>
			<td >Illumina</td>
			<td ></td>
			<td >Illumina. 16S Metagenomic Sequencing Library Preparation: Preparing 16S ribosomal RNA Gene Amplicons for the Illumina MiSeq System. https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf.</td>
		</tr>
		<tr >
			<td >Unique Dual Indexing</td>
			<td >Illumina</td>
			<td >Illumina Experiment Manager Software</td>
			<td >Freely available at: https://support.illumina.com/sequencing/sequencing_software/experiment_manager/downloads.html</td>
		</tr>
		<tr >
			<td >Nextera XT 96 Index Kit</td>
			<td >Illumina</td>
			<td >FC-131-1002</td>
			<td >Used to add barcodes to amplicons</td>
		</tr>
		<tr >
			<td >MicroAmp Optical 96-well reaction plate</td>
			<td >Applied Biosystems/ThermoFisher</td>
			<td >N8010560</td>
			<td ></td>
		</tr>
		<tr >
			<td >TruSeq Index Plate Fixture</td>
			<td >Illumina</td>
			<td >FC-130-1005</td>
			<td ></td>
		</tr>
		<tr >
			<td >Adhesive clear plate seal</td>
			<td >Applied Biosystems /ThermoFisher</td>
			<td >4360954</td>
			<td >Applied Biosystems/ThermoFisher Microamp adhesive film</td>
		</tr>
		<tr >
			<td >Sequencing by MiSeq with v3 reagents and dual 300 bp reads</td>
			<td >Illumina</td>
			<td >MS-102-3003</td>
			<td ></td>
		</tr>
		<tr >
			<td >PhiX Control Kit</td>
			<td >Illumina</td>
			<td >FC-110-3001</td>
			<td ></td>
		</tr>
		<tr >
			<td >Proteinase K (600 mAU/ml)</td>
			<td >Qiagen</td>
			<td >19131</td>
			<td >Equivalent to 20 mg/ml of proteinase K. Supplied with QiaAmp kit</td>
		</tr>
		<tr >
			<td >Data Analysis Tools</td>
			<td >Qiime</td>
			<td >QIIME software Tools</td>
			<td >Installation may differ based on your system and the QIIME website describes several options (http://qiime.org/install/install.html). For this study, MacQIIME software package 1.9.1 was utilized (compiled by Werner Lab, SUNY, http://www.wernerlab.org/software/macqiime</td>
		</tr>
		<tr >
			<td >Step 13.2.</td>
			<td >Qiime</td>
			<td >FastQ Join method</td>
			<td >&#160;(http://code.google.com/p/ea-utils &#160;).&#160; For this study Multiple join paired ends was used http://qiime.org/scripts/multiple_join_paired_ends.html. Aronesty, E. ea-utils: Command-line tools for processing biological sequencing data. Expression Analysis, Durham, NC. (2011).</td>
		</tr>
		<tr >
			<td >Step 13.3.</td>
			<td >Qiime</td>
			<td >De-Novo OTU picking protocol</td>
			<td >http://qiime.org/scripts/pick_de_novo_otus.html.</td>
		</tr>
		<tr >
			<td >Step 13.3.1.</td>
			<td ></td>
			<td >Open Taxonomic Units (OTUs) using Uclust</td>
			<td >Edgar, R.C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 26 (19), 2460-2461, doi:10.1093/bioinformatics/btq461 (2010).</td>
		</tr>
		<tr >
			<td >Step 13.3.1.</td>
			<td >Pynast</td>
			<td >Pynast</td>
			<td >Caporaso, J.G. et al. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics. 26 (2), 266-267, doi:10.1093/bioinformatics/btp636 (2010).&#160;</td>
		</tr>
		<tr >
			<td >Step 13.3.1.</td>
			<td >Pynast</td>
			<td >Pynast_Greengenes</td>
			<td >DeSantis, T.Z. et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 72 (7), 5069-5072, doi:10.1128/AEM.03006-05 (2006). Greengenes version 13_8 was used in this study</td>
		</tr>
		<tr >
			<td >13.3.1. Note:&#160;</td>
			<td >Qiime</td>
			<td >Multiple Split Libraries</td>
			<td>http://qiime.org/scripts/multiple_split_libraries_fastq.html.</td>
		</tr>
		<tr >
			<td >13.3.1. Note:&#160;</td>
			<td >Qiime</td>
			<td >Pick de novo OTUs script</td>
			<td>http://qiime.org/scripts/pick_de_novo_otus.html&#160;</td>
		</tr>
		<tr >
			<td >Step 13.2.2.</td>
			<td >Qiime</td>
			<td >Create a mapping file</td>
			<td>http://qiime.org/documentation/file_formats.html.</td>
		</tr>
		<tr >
			<td >Step 13.2.2.</td>
			<td >Qiime</td>
			<td >Validate a mapping file</td>
			<td>http://qiime.org/scripts/validate_mapping_file.html.</td>
		</tr>
		<tr >
			<td >Step 13.3.3.</td>
			<td >Qiime</td>
			<td >Link the OTU to sample description to mapping file</td>
			<td>http://qiime.org/scripts/make_otu_network.html.</td>
		</tr>
		<tr >
			<td >Step 13.3.4.</td>
			<td >Qiime</td>
			<td >Summarize Taxa through plots</td>
			<td>http://qiime.org/scripts/summarize_taxa_through_plots.html.</td>
		</tr>
		<tr >
			<td >Step 13.3.5.</td>
			<td >Qiime</td>
			<td >Biome Summarize table</td>
			<td >http://biom-format.org/documentation/summarizing_biom_tables.html&#160; In this study, all samples were rarified to 20,000 OTUs followed by analysis using alpha rarefaction script in QIIME.</td>
		</tr>
	</tbody>
</table>

a method to define the effects of environmental enrichment on colon microbiome biodiversity in a mouse colon tumor model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Several studies have demonstrated that the practice of mind-body medicine improves health outcomes. Similarly, in mice, environmental enrichment improves outcomes including improved lifespan and tumor wound repair6. Therefore, an EE procedure was developed with the aim of defining the role of microbiota in this phenotype while first normalizing the microbiome prior to the initiation of the experiment (Figure 1). Importantly, all breedi...

Watch this Scientific Journal Video about A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model at JoVE.com

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

The main goal of this publication is to present a method for normalizing the microbiome of rodents. In this demonstration, the effect of environmental enrichment on the microbiome is assayed during colon tumor genesis. This protocol shows how normalizing the microbiome prior to an environmental enrichment intervention improves the experimental reproducibility.We also show how to reduce animal stress and improve interactions using an enriched environment. Although we have focused on the effect of environmental enrichment in a colon tumor model, the method can easily be applied to better understand the microbiome's role in the etiology of disease. Set up the cages under sterile conditions working under a ventilated hood.The controls for this experiment consist of conventional cages with bedding and lab tissue for nesting, but otherwise lack enrichment devices. To set up the pup rearing or environmentally enriched cages, connect two large autoclaved cages with a sterilized grommet-secured tunnel and include a sterilized platform in each cage to increase floor space. For EE, also provide sterilized running wheels, tunnels, igloos, huts, crawl balls, and nesting material.Place both the enriched and the control cages in the same rack to provide equal ventilation. For breeding, combine one sire and two potential dams in normal cages. Every morning, check for vaginal plugs which indicate that the animal has mated during the night.If a plug is not seen, resistance to vaginal probing can detect an internalized plug. If a female has a vaginal plug, transfer her to a large pup-rearing cage. Up to 12 pregnant dams can be housed in the environmentally enriched cages with platforms provided they are all expected to deliver during the same week.When the pups are 21 to 28 days old, distribute the males and females by genotype into control environments or enriched environments. Make sure to keep an proportion of each genotype in each cage. Non-enriched cages can support up to five mice whereas enriched cages require at least 20 mice for social stimulation and can support up to 41 mice.Start collecting stool one or two days before euthanizing the mice. Always collect at the same time of day. To collect stool from live animals, carefully scruff the animal over a clean cage.Then collect the defecated stool using sterile forceps and transfer to a sterile microfuge tube. If an animal does not immediately defecate, place it into a clean cage and wait for the animal to defecate which can take up to an hour. To collect stool from a euthanized animal, dissect it to access the colon.Then cut the colon just below the cecum and lay the colon out on a piece of filter paper. Then use forceps to lift the top of the colon tube and open the lumen using scissors. Cut longitudinally distal to proximal to splay open the colon lengthwise.Then collect stool from the distal colon into a sterile microfuge tube using sterile forceps. Store the stool samples at minus 80 degrees Celsius. In addition to the stool, collect other tissue samples from the colon and small intestine, microsomes, adipose tissue, and so forth.Utilize a commercial kit to isolate microbial DNA from the stool following a stool pathogen detection protocol. When doing so, transfer the samples from the minus 80 degree Celsius freezer on dry ice and weigh them. Next, set up a PCR that amplifies the microbial DNA as described in the text protocol.Then clean up the reaction products using magnetic beads. For the clean up, first briefly centrifuge the amplicon PCR plate to pool the reaction products. Next, vortex the magnetic beads to evenly disperse them and transfer 20 microliter aliquots to each reaction well.Mix the beads with the solution thoroughly by pipetting the entire volume slowly 10 times. Then let the beads settle for five minutes. Next, place the PCR plate on a magnetic stand and wait two minutes for the supernatants to clear following magnetic collection of beads.Then remove and discard the supernatants. Next, with the plate still on the magnet, fill each well of beads with 200 microliters of fresh 80%ethanol and wait 30 seconds. Then carefully remove the supernatant.Repeat this wash step once and let the beads air dry for 10 minutes. Now elute the reaction products from the beads by removing the plate from the magnetic stand and adding 52.5 microliters of Tris to each well. Completely mix the beads into solution by pipetting the entire volume slowly 10 times.Then collect the beads on the magnetic stand as before and transfer 50 microliters of the supernatant to a clean PCR plate. Cover the plate with a clear adhesive and store it at minus 20 degrees Celsius for up to one week. Use the collected supernatant to perform an index PCR to attach bar codes to the adapter sequence.Then purify the products and prepare the indexed library for sequencing. In mice, environmental enrichment is known to improve lifespan and tumor wound repair. To define the role of microbiota in this phenotype, breeding animals were integrated into the mouse colony for at least one month to normalize the microbiota.Then they were bred and their progeny were raised in an enriched environment or a normal environment. The progeny were also tumor bearing or wild type. After 16 weeks, the animals were euthanized and stool samples were collected to isolate microbial DNA.Next, PCR was used to amplify the 16S ribosomal subunit in the samples to survey the microbiome components. The reaction products were then identified using bar code indexing. Interestingly, enhanced environments do not improve biodiversity in wild type animals, but they vastly increase biodiversity in tumor-bearing animals.This demonstrates the sensitivity of the method. This increase in biodiversity was attributed to an increased presence of the phylum Proteobacteria with significant increases in the classes alphaproteobacteria and betaproteobacteria and a decrease in pathogenic gammaproteobacteria. The largest increase in the betaproteobacteria class was in the genus Sutterella, a likely commensal involved in secreted immunoglobulin A degradation.After watching this video, you should have a good understanding of how to alter husbandry practices to normalize the microbiome between experimental animals prior to commencing with your interventions of interest. While performing this procedure, remember that animals have established their territory within the environment. Because of this, changes to the environment can cause stress that will affect the animal's microbiome.A sudden change in the animal's behavior is a physical sign of stress. Following this procedure, specific microbes can be isolated and further characterized to test the sufficiency of the identified microbe on disease progression.

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Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...