Permalink
Cannot retrieve contributors at this time
Fetching contributors…
Cannot retrieve contributors at this time
| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">04186</article-id><article-id pub-id-type="doi">10.7554/eLife.04186</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Registered report</subject></subj-group><subj-group subj-group-type="heading"><subject>Human biology and medicine</subject></subj-group><subj-group subj-group-type="heading"><subject>Microbiology and infectious disease</subject></subj-group></article-categories><title-group><article-title>Registered report: Intestinal inflammation targets cancer-inducing activity of the microbiota</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-17005"><name><surname>Eaton</surname><given-names>Kate</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17006"><name><surname>Yang</surname><given-names>Wanwan</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf3"/></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="group-author-key">group-author-id1</contrib-id><collab>Reproducibility Project: Cancer Biology</collab><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf2"/></contrib><aff id="aff1"><label>1</label><institution content-type="dept">Germ Free Laboratory</institution>, <institution>University of Michigan Medical School</institution>, <addr-line><named-content content-type="city">Ann Arbor</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><label>2</label><institution>ImpeDx Diagnostics Inc.</institution>, <addr-line><named-content content-type="city">Kansas City</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor" id="author-17615"><name><surname>Sears</surname><given-names>Cynthia L</given-names></name><role>Reviewing editor</role><aff><institution>Johns Hopkins University School of Medicine</institution>, <country>United States</country></aff></contrib></contrib-group><contrib-group><contrib contrib-type="author non-byline"><contrib-id contrib-id-type="group-author-key">group-author-id1</contrib-id><name><surname>Iorns</surname><given-names>Elizabeth</given-names></name><aff><institution>Science Exchange, Palo Alto</institution>, <addr-line><named-content content-type="city">California</named-content></addr-line></aff></contrib><contrib contrib-type="author non-byline"><contrib-id contrib-id-type="group-author-key">group-author-id1</contrib-id><name><surname>Gunn</surname><given-names>William</given-names></name><aff><institution>Mendeley</institution>, <addr-line><named-content content-type="city">London</named-content></addr-line>, <country>United Kingdom</country></aff></contrib><contrib contrib-type="author non-byline"><contrib-id contrib-id-type="group-author-key">group-author-id1</contrib-id><name><surname>Tan</surname><given-names>Fraser</given-names></name><aff><institution>Science Exchange, Palo Alto</institution>, <addr-line><named-content content-type="city">California</named-content></addr-line></aff></contrib><contrib contrib-type="author non-byline"><contrib-id contrib-id-type="group-author-key">group-author-id1</contrib-id><name><surname>Lomax</surname><given-names>Joelle</given-names></name><aff><institution>Science Exchange, Palo Alto</institution>, <addr-line><named-content content-type="city">California</named-content></addr-line></aff></contrib><contrib contrib-type="author non-byline"><contrib-id contrib-id-type="group-author-key">group-author-id1</contrib-id><name><surname>Errington</surname><given-names>Timothy</given-names></name><aff><institution>Center for Open Science</institution>, <addr-line><named-content content-type="city">Charlottesville</named-content></addr-line>, <country>Virginia</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>fraser@scienceexchange.com</email></corresp></author-notes><pub-date publication-format="electronic" date-type="pub"><day>17</day><month>03</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>4</volume><elocation-id>e04186</elocation-id><history><date date-type="received"><day>29</day><month>07</month><year>2014</year></date><date date-type="accepted"><day>12</day><month>02</month><year>2015</year></date><fn><p><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Arthur</surname><given-names>JC</given-names></name><name><surname>Perez-Chanona</surname><given-names>E</given-names></name><name><surname>Muhlbauer</surname><given-names>M</given-names></name><name><surname>Tomkovich</surname><given-names>S</given-names></name><name><surname>Uronis</surname><given-names>JM</given-names></name><name><surname>Fan</surname><given-names>TJ</given-names></name><name><surname>Campbell</surname><given-names>BJ</given-names></name><name><surname>Abujamel</surname><given-names>T</given-names></name><name><surname>Dogan</surname><given-names>B</given-names></name><name><surname>Rogers</surname><given-names>AB</given-names></name><name><surname>Rhodes</surname><given-names>JM</given-names></name><name><surname>Stintzi</surname><given-names>A</given-names></name><name><surname>Simpson</surname><given-names>KW</given-names></name><name><surname>Hansen</surname><given-names>JJ</given-names></name><name><surname>Keku</surname><given-names>TO</given-names></name><name><surname>Fodor</surname><given-names>AA</given-names></name><name><surname>Jobin</surname><given-names>C</given-names></name></person-group>. <day>05</day><month>10</month><year>2012</year>. <article-title>Intestinal inflammation targets cancer-inducing activity of the microbiota</article-title>. <source>Science</source> <volume>338</volume>:<fpage>120</fpage>–<lpage>123</lpage>. doi: <pub-id pub-id-type="doi">10.1126/science.1224820</pub-id>.</mixed-citation></p></fn></history><permissions><copyright-statement>© 2015, Eaton et al</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Eaton et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife04186.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.04186.001</object-id><p>The <ext-link ext-link-type="uri" xlink:href="https://osf.io/e81xl/wiki/home/">Reproducibility Project: Cancer Biology</ext-link> seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered report describes the proposed replication plan of key experiments from ‘Intestinal Inflammation Targets Cancer-Inducing Activity of the Microbiota’ by <xref ref-type="bibr" rid="bib2">Arthur et al. (2012)</xref>, published in Science in 2012. Arthur and colleagues identified a genotoxic island in <italic>Escherichia coli</italic> NC101 that appeared to be responsible for causing neoplastic lesions in inflammation-induced <italic>IL10</italic><sup><italic>−/−</italic></sup> mice treated with azoxymethane. The experiments that will be replicated are those reported in Figure 4 (<xref ref-type="bibr" rid="bib2">Arthur et al., 2012)</xref>. Arthur and colleagues inoculated <italic>IL10</italic><sup><italic>−/−</italic></sup> mice with a mutated strain of <italic>E. coli</italic> NC101 lacking the genotoxic island, and showed that those mice suffered from fewer neoplastic lesions than mice inoculated with the wild type form of <italic>E. coli</italic> NC101 (Figure 4). The Reproducibility Project: Cancer Biology is a collaboration between the <ext-link ext-link-type="uri" xlink:href="http://centerforopenscience.org/">Center for Open Science</ext-link> and <ext-link ext-link-type="uri" xlink:href="https://www.scienceexchange.com/">Science Exchange</ext-link>, and the results of the replications will be published by <italic>eLife</italic>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04186.001">http://dx.doi.org/10.7554/eLife.04186.001</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>Reproducibility Project: Cancer Biology</kwd><kwd>methodology</kwd><kwd>intestinal inflammation</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>Laura and John Arnold Foundation</institution></institution-wrap></funding-source><principal-award-recipient>Reproducibility Project: Cancer Biology</principal-award-recipient></award-group><funding-statement>The Reproducibility Project: Cancer Biology is funded by the Laura and John Arnold Foundation, provided to the Center for Open Science in collaboration with Science Exchange. The funder had no role in study design or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2.0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p>In their 2012 Science paper, Arthur and colleagues examined the interplay between colitis and colon cancer. They identified a shift in the composition of the microbiota in <italic>Il10</italic><sup><italic>−/−</italic></sup> mice, which develop chronic colitis; amongst other changes noted, <italic>Escherichia coli</italic> was over 100-fold more represented in <italic>Il10</italic><sup><italic>−/−</italic></sup> mice than wild type. After treatment with azoxymethane (AOM), a carcinogen that induces colon cancer, germ-free <italic>Il10</italic><sup><italic>−/−</italic></sup> mice mono-associated with the colitis-inducing <italic>E. coli</italic> NC101 strain developed invasive mucinous carcinomas, while mice mono-associated with <italic>Enterococcus faecalis</italic>, another colitis-inducing bacterial strain, did not. <italic>E. coli</italic> NC101 harbors a 54 kb <italic>polyketide synthases</italic> (<italic>pks</italic>) genotoxic island encoding several enzymes involved in the production of toxin called Colibactin. This island has been previously shown to induce DNA damage, double strand breaks and aneuploidy (<xref ref-type="bibr" rid="bib7">Nougayrede, 2006</xref>; <xref ref-type="bibr" rid="bib5">Cuevas-Ramos et al., 2010</xref>) and was not found in <italic>E. faecalis</italic> or another non-colitic <italic>E. coli</italic> strain, K12.</p><p>In Figure 4, Arthur et al. inoculated germ-free <italic>IL10</italic><sup><italic>−/−</italic></sup> mice treated with or without AOM with either wild-type <italic>E. coli</italic> NC101, or with a mutant of <italic>E. coli</italic> NC101 lacking the <italic>pks</italic> island (NC1010Δ<italic>pks</italic>). Arthur and colleagues first confirmed that loss of the <italic>pks</italic> island did not impair bacterial growth (Supplemental figure 7, replicated in Protocol 1). Presence or absence of the genotoxic <italic>pks</italic> island had no effect on colonic inflammation in <italic>IL10</italic><sup><italic>−/−</italic></sup> mice alone or treated with AOM (Figure 4A). However, at 12 and 18 weeks, mice treated with AOM and inoculated with <italic>E. coli</italic> NC101Δ<italic>pks</italic> had many fewer neoplastic lesions than mice inoculated with wild-type <italic>E. coli</italic> NC101 (Figure 4B). At 18 weeks, invasion (Figure 4C), tumor burden (Figure 4D) and tumor size (Figure 4E) were all reduced in mice mono-associated with <italic>E. coli</italic> NC101Δ<italic>pks</italic> as compared to NC101. Taken together, the data indicate that loss of the <italic>pks</italic> genotoxic island from <italic>E. coli</italic> NC101 strongly reduces the incidence of colon cancer in mice with chronic colitis. These experiments are replicated in Protocol 3.</p><p><xref ref-type="bibr" rid="bib3">Buc et al. (2013)</xref> performed an experiment similar to Figure 3B (not included for replication in this study), wherein <xref ref-type="bibr" rid="bib2">Arthur et al. (2012)</xref> examined if the <italic>pks</italic> genotoxic island was more prevalent in patients with colorectal cancer. Both the dataset from <xref ref-type="bibr" rid="bib2">Arthur et al. (2012)</xref> and the dataset presented by <xref ref-type="bibr" rid="bib3">Buc et al. (2013)</xref> support the hypothesis that the genotoxic <italic>pks</italic> island is more prevalent in patients with colorectal cancer. <xref ref-type="bibr" rid="bib4">Cougnoux et al. (2014)</xref>, while not performing a direct replication, extended the findings of Arthur and colleagues to explore the mechanism of <italic>pks-</italic>produced colobactin toxicity effects on colorectal cancer. Finally, Arthur and colleagues have since published further work exploring in greater detail the genetic mechanisms behind the association of colorectal cancer with colitis and colonization by <italic>E. coli</italic> NC101 with or without the <italic>pks</italic> island, in which they demonstrate that colon inflammation itself has an influence on the expression of the genotoxic <italic>pks</italic> island (<xref ref-type="bibr" rid="bib1">Arthur et al., 2014</xref>).</p></sec><sec sec-type="materials|methods" id="s2"><title>Materials and methods</title><p>Unless otherwise noted, all protocol information was derived from the original paper, references from the original paper, or information obtained directly from the authors. An asterisk (*) indicates data or information provided by the Reproducibility Project: Cancer Biology core team. A hashtag (#) indicates information provided by the replicating lab.</p><sec id="s2-1"><title>Protocol 1: comparing the growth curves of <italic>E. coli</italic> NC101 and <italic>E. coli</italic> NC101Δ<italic>pks</italic></title><p>This protocol describes how to grow both <italic>E. coli</italic> NC101 and <italic>E. coli</italic> NC1010Δ<italic>pks</italic> to compare their growth curve, as seen in Supplemental figure 7.</p><sec id="s2-1-1"><title>Sampling</title><p><list list-type="bullet"><list-item><p>This experiment will be repeated a total of three times.</p><list list-type="simple"><list-item><p>a. Power calculations were not performed, as no significant difference was reported in the original study.</p></list-item></list></list-item></list></p></sec><sec id="s2-1-2"><title>Materials and reagents</title><p><list list-type="bullet"><list-item><p>Reagents that differ from those used originally are indicated with an asterisk (*).</p></list-item></list><table-wrap id="tblu1" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Reagent</th><th>Type</th><th>Manufacturer</th><th>Catalog #</th><th>Comments</th></tr></thead><tbody><tr><td><italic>E. coli</italic> NC101 strain</td><td>Cells</td><td>Original authors</td><td>n/a</td><td/></tr><tr><td><italic>E. coli</italic> NC101Δpks strain</td><td>Cells</td><td>Original authors</td><td>n/a</td><td/></tr><tr><td>Luria–Bertani (LB) broth*</td><td>Materials</td><td>Sigma</td><td>L3022</td><td>Original unspecified</td></tr></tbody></table></table-wrap></p></sec><sec id="s2-1-3"><title>Procedure</title><p><list list-type="order"><list-item><p>Streak <italic>E. coli</italic> strains NC101 and NC101∆pks on agar plates and incubate at 37°C overnight.</p></list-item><list-item><p>Inoculate 5 ml Luria–Bertani broth (LB broth) with a single colony of each <italic>E. coli</italic> strain picked from the freshly streaked plates and grow for ∼8 hr at 37°C in a shaking incubator.</p></list-item><list-item><p>Inoculate <italic>E. coli</italic> strains at a 1:500 dilution in 150 ml LB broth and incubate at 37°C overnight (12–16 hr) in a shaking incubator.</p></list-item><list-item><p>Inoculate <italic>E. coli</italic> strains at 1:500 dilution in 10 ml LB broth and measure the OD600 of the diluted cultures (timepoint 0). Collect the remaining <italic>E. coli</italic> for genomic DNA extraction in protocol 2.</p></list-item><list-item><p>Incubate at 37°C in a shaking incubator.</p><list list-type="simple"><list-item><p>a. Measure the OD600 of cultures every 20–30 min until saturation phase has been reached for both cultures (∼8 hr).</p></list-item><list-item><p>b. Lab will note instrument make, model and RPM.</p></list-item></list></list-item><list-item><p>Repeat independently two additional times.</p></list-item></list></p></sec><sec id="s2-1-4"><title>Deliverables</title><p><list list-type="bullet"><list-item><p>Data to be collected:</p><list list-type="simple"><list-item><p>a. Raw values for OD600 measurements at each time point for NC101 and NC101∆pks.</p></list-item><list-item><p>b. Semi-logarithmic graph of average OD600 (log) vs time (linear) for NC101 and NC101∆pks.</p></list-item></list></list-item><list-item><p>Sample delivered for further analysis:</p><list list-type="simple"><list-item><p>a. Cultures from Step 3 for use in Protocol 2.</p></list-item></list></list-item></list></p></sec><sec id="s2-1-5"><title>Confirmatory analysis plan</title><p><list list-type="bullet"><list-item><p>Statistical analysis of the replication data:</p><list list-type="simple"><list-item><p>a. Comparison of growth curve fit by nonlinear regression.</p></list-item></list></list-item></list></p></sec><sec id="s2-1-6"><title>Known differences from the original study</title><p><list list-type="bullet"><list-item><p>The 37°C incubator used by the lab may not be the same make and model as used in the original study. The lab will note the make and model of the incubator they use.</p></list-item></list></p></sec><sec id="s2-1-7"><title>Provisions for quality control</title><p>All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (<ext-link ext-link-type="uri" xlink:href="https://osf.io/y4tvd/">https://osf.io/y4tvd/</ext-link>).<list list-type="bullet"><list-item><p>This protocol will confirm that loss of the <italic>pks</italic> genotoxic island does not affect the growth curve of <italic>E. coli</italic> NC1010Δ<italic>pks</italic> as compared to its parental strain, <italic>E. coli</italic> NC101.</p></list-item></list></p></sec></sec><sec id="s2-2"><title>Protocol 2: PCR amplification and sequencing of polyketide synthase (<italic>pks</italic>) genotoxic island in <italic>E. coli</italic> NC101 and NC101∆pks</title><p>This protocol describes the PCR amplification of the 5′ and 3′ ends of the <italic>pks</italic> genotoxic island to confirm its presence in <italic>E. coli</italic> NC101 and its absence in <italic>E. coli</italic> NC101Δ<italic>pks</italic>. This is a quality control step to confirm the absence of the <italic>pks</italic> island in the <italic>E. coli</italic> NC101Δ<italic>pks</italic> strain.</p><sec id="s2-2-1"><title>Sampling</title><p><list list-type="bullet"><list-item><p>This experiment will be performed once.</p><list list-type="simple"><list-item><p>a. Power calculations are not necessary for this PCR screen.</p></list-item></list></list-item><list-item><p>The experiment consists of two cohorts:</p><list list-type="simple"><list-item><p>a. Genomic DNA from <italic>E. coli</italic> NC101.</p></list-item><list-item><p>b. Genomic DNA from <italic>E. coli</italic> NC101Δ<italic>pks</italic>.</p></list-item><list-item><p>c. Each cohort has four PCR reactions run:</p><list list-type="simple"><list-item><p>■ L1 + L2: detects the 5′ end of the <italic>pks</italic> island.</p></list-item><list-item><p>■ R1 + R2: detects the 3′ end of the <italic>pks</italic> island.</p></list-item><list-item><p>■ 16S F + 16S R: control gene for amplification.</p></list-item><list-item><p>■ ClbB-F + ClbB-R: also detects the colibactin gene.</p><list list-type="simple"><list-item><p>i. Additional reaction recommended by original authors.</p></list-item></list></list-item></list></list-item></list></list-item></list></p></sec><sec id="s2-2-2"><title>Materials and reagents</title><p><list list-type="bullet"><list-item><p>Reagents that differ from those used originally are indicated with an *.</p></list-item></list><table-wrap id="tblu2" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Reagent</th><th>Type</th><th>Manufacturer</th><th>Catalog #</th><th>Comments</th></tr></thead><tbody><tr><td><italic>pks</italic> L1</td><td>Primer</td><td>At replicating lab's discretion</td><td>n/a</td><td>Original synthesis provider unspecified</td></tr><tr><td><italic>pks</italic> L2</td><td>Primer</td><td>At replicating lab's discretion</td><td>n/a</td><td>Original synthesis provider unspecified</td></tr><tr><td><italic>pks</italic> R1</td><td>Primer</td><td>At replicating lab's discretion</td><td>n/a</td><td>Original synthesis provider unspecified</td></tr><tr><td><italic>pks</italic> R2</td><td>Primer</td><td>At replicating lab's discretion</td><td>n/a</td><td>Original synthesis provider unspecified</td></tr><tr><td>16S F</td><td>Primer</td><td>At replicating lab's discretion</td><td>n/a</td><td>Original synthesis provider unspecified</td></tr><tr><td>16S R</td><td>Primer</td><td>At replicating lab's discretion</td><td>n/a</td><td>Original synthesis provider unspecified</td></tr><tr><td>ClbB-F</td><td>Primer</td><td>At replicating lab's discretion</td><td>n/a</td><td>Recommended by the original authors</td></tr><tr><td>ClbB-R</td><td>Primer</td><td>At replicating lab's discretion</td><td>n/a</td><td>Recommended by the original authors</td></tr><tr><td>Agarose*</td><td>Reagent</td><td>Sigma</td><td>A9539</td><td>Original unspecified</td></tr><tr><td>Ethidium bromide*</td><td>Reagent</td><td>Sigma</td><td>E1510</td><td>Original unspecified</td></tr></tbody></table></table-wrap></p></sec><sec id="s2-2-3"><title>Procedure</title><p><list list-type="order"><list-item><p>Extract bacterial genomic DNA from the sample collected in Protocol 1.</p><list list-type="simple"><list-item><p>a. Lab will document their methodology for bacterial genomic DNA extraction.</p></list-item><list-item><p>b. Lab will include quality control data such as A<sub>260</sub>/A<sub>280</sub> ratios from quantifying DNA concentration.</p></list-item></list></list-item><list-item><p>Run the following PCR reactions:</p><list list-type="simple"><list-item><p>a. Experimental.<table-wrap id="tblu3" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Template</th><th>Forward primer</th><th>Reverse primer</th></tr></thead><tbody><tr><td><italic>E. coli</italic> NC101 gDNA</td><td>L1</td><td>L2</td></tr><tr><td><italic>E. coli</italic> NC101Δ<italic>pks</italic> gDNA</td><td>L1</td><td>L2</td></tr><tr><td>Water (no DNA control)</td><td>L1</td><td>L2</td></tr><tr><td><italic>E. coli</italic> NC101 gDNA</td><td>R1</td><td>R2</td></tr><tr><td><italic>E. coli</italic> NC101Δ<italic>pks</italic> gDNA</td><td>R1</td><td>R2</td></tr><tr><td>Water (no DNA control)</td><td>R1</td><td>R2</td></tr><tr><td><italic>E. coli</italic> NC101 gDNA</td><td>ClbB-F</td><td>ClbB-R</td></tr><tr><td><italic>E. coli</italic> NC101Δ<italic>pks</italic> gDNA</td><td>ClbB-F</td><td>ClbB-R</td></tr><tr><td>Water (no DNA control)</td><td>ClbB-F</td><td>ClbB-R</td></tr></tbody></table></table-wrap></p></list-item><list-item><p>b. Controls (additional control).<table-wrap id="tblu4" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Template</th><th>Forward primer</th><th>Reverse primer</th></tr></thead><tbody><tr><td><italic>E. coli</italic> NC101 gDNA</td><td>16S F</td><td>16S R</td></tr><tr><td><italic>E. coli</italic> NC101Δ<italic>pks</italic> gDNA</td><td>16S F</td><td>16S R</td></tr><tr><td>Water (no DNA control)</td><td>16S F</td><td>16S R</td></tr></tbody></table></table-wrap></p></list-item><list-item><p>c. Primers.<table-wrap id="tblu5" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Primer</th><th>Sequence</th><th>Expected band size</th><th>Comment</th></tr></thead><tbody><tr><td>L1</td><td>5′-AAT CAA CCC AGC TGC AAA TC-3′</td><td rowspan="2">1824 bp</td><td rowspan="2">L1 + L2 detect the 3′ end of the <italic>pks</italic></td></tr><tr><td>L2</td><td>5′-CAC CCC CAT CAT TAA AAA CG-3′</td></tr><tr><td>R1</td><td>5′-AGC CGT ATC CTG CTC AAA AC-3′</td><td rowspan="2">1413 bp</td><td rowspan="2">R1 + R2 detect the 5′ end of the <italic>pks</italic></td></tr><tr><td>R2</td><td>5′-TCG GTA TGT CCG GTT AAA GC-3′</td></tr><tr><td>ClbB-F</td><td>5′-GCG CAT CCT CAA GAG TAA ATA-3′</td><td rowspan="2">280 bp</td><td rowspan="2"/></tr><tr><td>ClbB-R</td><td>5′-GCG CTC TAT GCT CAT CAA CC-3′</td></tr><tr><td>16S F</td><td>5′-GTG STG CAY GGY TGT CGT CA-3′</td><td rowspan="2"/><td rowspan="2"/></tr><tr><td>16S R</td><td>5′-GTG STG CAY GGY TGT CGT CA-3′</td></tr></tbody></table></table-wrap></p></list-item><list-item><p>d. Reaction set-up.<table-wrap id="tblu6" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>10× buffer</th><th>5 µl</th></tr></thead><tbody><tr><td>5 µM dNTPs</td><td>0.5 µl</td></tr><tr><td>50 mM MgCl<sub>2</sub></td><td>1.5 µl</td></tr><tr><td>5 µM F primer</td><td>0.5 µl</td></tr><tr><td>5 µM R primer</td><td>0.5 µl</td></tr><tr><td>Invitrogen Taq polymerase</td><td>0.5 µl</td></tr><tr><td>Bacterial genomic DNA</td><td>2 µl</td></tr><tr><td>Water</td><td>Bring up to 50 µl</td></tr></tbody></table></table-wrap></p></list-item><list-item><p>e. Cycling parameters.</p><list list-type="simple"><list-item><p>i. Denature at 95°C for 5 min.</p></list-item><list-item><p>ii. 35 (up to 50) cycles of:</p><list list-type="simple"><list-item><p>■ 95°C for 45 s.</p></list-item><list-item><p>■ 56°C for 45 s.</p></list-item><list-item><p>■ 72°C for 45 s.</p></list-item></list></list-item><list-item><p>iii. 72°C for 10 min.</p></list-item><list-item><p>iv. Hold at 4°C forever.</p></list-item></list></list-item></list></list-item><list-item><p>Run out PCR amplicons on a 1.5% agarose gel alongside a size marker. Visualize with ethidium bromide.</p></list-item><list-item><p>Sequence <italic>pks</italic> amplicons by Sanger automated DNA sequencing.</p><list list-type="simple"><list-item><p>a. BLAST sequencing results against the <italic>E. coli</italic> Colibactin synthesis cluster (AM229678.1).</p></list-item></list></list-item><list-item><p>Sequence 16S amplicons to confirm identity of bacterial strains (additional control).</p></list-item></list></p></sec><sec id="s2-2-4"><title>Deliverables</title><p><list list-type="bullet"><list-item><p>Data to be collected:</p><list list-type="simple"><list-item><p>a. Full gel image of ethidium bromide stained gel showing PCR products for <italic>pks</italic>-L, <italic>pks</italic>-R and 16S amplicons.</p></list-item><list-item><p>b. Sequencing chromatograms.</p></list-item><list-item><p>c. BLAST comparison results.</p></list-item></list></list-item></list></p></sec><sec id="s2-2-5"><title>Confirmatory analysis plan</title><p><list list-type="bullet"><list-item><p>Statistical analysis of the replication data:</p><list list-type="simple"><list-item><p>a. No statistical test required.</p></list-item><list-item><p>b. Visually confirm presence of <italic>pks</italic> bands in <italic>E. coli</italic> NC101 and absence in <italic>E. coli</italic> NC101Δ<italic>pks</italic>.</p></list-item></list></list-item></list></p></sec><sec id="s2-2-6"><title>Known differences from the original study</title><p><list list-type="bullet"><list-item><p>Lab will use in-house bacterial genomic DNA extraction protocol.</p><list list-type="simple"><list-item><p>a. Original was unspecified.</p></list-item></list></list-item></list></p></sec><sec id="s2-2-7"><title>Provisions for quality control</title><p>All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (<ext-link ext-link-type="uri" xlink:href="https://osf.io/y4tvd/">https://osf.io/y4tvd/</ext-link>).<list list-type="bullet"><list-item><p>We will sequence the 16S rRNA of the bacteria to confirm the identity of the strain. The sample purity (A<sub>260</sub>/A<sub>280</sub> ratio) of the extracted DNA will be recorded.</p></list-item></list></p></sec></sec><sec id="s2-3"><title>Protocol 3: mono-associate mice with <italic>E. coli</italic> NC101 or NC101∆pks and analyze intestinal tumorigenesis and inflammation</title><p>This protocol describes the inoculation of germ-free <italic>Il10</italic><sup><italic>−/−</italic></sup> mice with <italic>E. coli</italic> as well as treatment with the carcinogen azoxymethane (AOM), as seen in Figure 4.</p><sec id="s2-3-1"><title>Sampling</title><p><list list-type="bullet"><list-item><p>This experiment will use at least 10 mice per group for a final power of 80–96%.</p><list list-type="simple"><list-item><p>a. See ‘Power calculations’ section for details.</p></list-item></list></list-item><list-item><p>The experiment consists of the following cohorts:</p><list list-type="simple"><list-item><p>a. Germ-free <italic>IL10</italic><sup><italic>−/−</italic></sup> mice treated with AOM and inoculated with <italic>E. coli</italic> NC101, harvested at 18 weeks post AOM.</p><list list-type="simple"><list-item><p>■ <italic>n</italic> = 14.</p><list list-type="simple"><list-item><p>i. Expected survival is 75% (as seen in Supplemental figure 10), thus 14 mice are required to predict 10 will survive.</p></list-item></list></list-item></list></list-item><list-item><p>b. Germ-free <italic>IL10</italic><sup><italic>−/−</italic></sup> mice treated with AOM and inoculated with <italic>E. coli</italic> NC101Δ<italic>pks</italic>, harvested at 18 weeks post AOM.</p><list list-type="simple"><list-item><p>■ <italic>n</italic> = 16.</p><list list-type="simple"><list-item><p>i. Expected survival is 62.5% (as seen in Supplemental figure 10), thus 16 mice are required to predict 10 will survive.</p></list-item></list></list-item></list></list-item></list></list-item></list></p></sec><sec id="s2-3-2"><title>Materials and reagents</title><p><list list-type="bullet"><list-item><p>Reagents that differ from those used originally are indicated with an *.</p></list-item></list><table-wrap id="tblu7" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Reagent</th><th>Type</th><th>Manufacturer</th><th>Catalog #</th><th>Comments</th></tr></thead><tbody><tr><td>IL10<sup>−/−</sup> mice</td><td>Mice</td><td>Original lab</td><td>n/a</td><td/></tr><tr><td>Wild-type mice</td><td>Mice</td><td>Original lab</td><td>n/a</td><td/></tr><tr><td>Azoxymethane (AOM)</td><td>reagent</td><td>Sigma</td><td>A5486</td><td/></tr><tr><td>Formalin*</td><td>Reagent</td><td>Sigma</td><td>HT501128</td><td>original unspecified</td></tr><tr><td>0.1 mm zirconium beads and bead beater</td><td>Equipment</td><td>BioSpec Products</td><td>1107900-101</td><td/></tr><tr><td>DNeasy kit</td><td>Reagent</td><td>Qiagen</td><td>69504</td><td/></tr><tr><td>16S F</td><td>Primer</td><td>At replicating lab's discretion</td><td>n/a</td><td/></tr><tr><td>16S R</td><td>Primer</td><td>At replicating lab's discretion</td><td>n/a</td><td/></tr></tbody></table></table-wrap></p></sec><sec id="s2-3-3"><title>Procedure</title><sec id="s2-3-3-1"><title>Notes</title><p><list list-type="bullet"><list-item><p>Experiment should be conducted by experimenters blinded to genotype and treatment group.</p></list-item><list-item><p>Azoxymethane (AOM) can show lot-to-lot variation in potency, and will lose potency and gain toxicity over time. In order to minimize these effects, a single lot of AOM will be used throughout the experiment. AOM will be aliquoted into 25 mg/ml aliquots and stored at −80°C. A fresh aliquot will be used each time AOM is needed to avoid repeated freeze-thaw cycles.</p><list list-type="order"><list-item><p>Breed and raise <italic>IL10</italic><sup><italic>−/−</italic></sup> mice in germ-free isolators.</p><list list-type="simple"><list-item><p>a. Use both male and female mice; house sexes separately.</p></list-item><list-item><p>b. House mice 2–4 mice per cage.</p></list-item><list-item><p>c. 12 hr/12 hr light/dark cycle.</p></list-item><list-item><p>d. Diet is Purine Lab Diet 3500.</p></list-item></list></list-item><list-item><p>At age 7–12 weeks initiate program to induce colitis/colorectal cancer.</p><list list-type="simple"><list-item><p>a. Maintain mice in gnotobiotic isolators.</p></list-item></list></list-item><list-item><p>Randomly assign Il10<sup>−/−</sup> mice to two groups. As each mouse becomes eligible for induction of colitis/colorectal cancer, randomly assign to a treatment group using the adaptive randomization approach with the gender of the mice as the covariate that is assessed as mice are sequentially assigned to a particular treatment group. Assignment will aim for a similar distribution of genders in each cohort while also taking into account the pre-determined size of each treatment group.</p><list list-type="simple"><list-item><p>a. Group 1: <italic>E. coli</italic> NC101; <italic>n</italic> = 14.</p></list-item><list-item><p>b. Group 2: <italic>E. coli</italic> NC101∆pks; <italic>n</italic> = 16.</p></list-item></list></list-item><list-item><p>Colonize mice by oral gavage and rectal swabbing (dip sterile Q-tip in culture and swab anus) with log phase growth bactera:</p><list list-type="simple"><list-item><p>a. Use 200 µl of an overnight bacterial culture with a concentration of 2 × 10<sup>9</sup> CFU/ml.</p><list list-type="simple"><list-item><p>i. Record OD600 of culture used.</p></list-item><list-item><p>ii. Perform serial dilution and plating of the culture used to swab for quantitative culturing. Record the actual CFUs of the culture used to colonize each mouse.</p></list-item></list></list-item><list-item><p>b. <italic>E. coli</italic> NC101 or <italic>E. coli</italic> NC101∆pks for <italic>Il10</italic><sup><italic>−/−</italic></sup> mice.</p></list-item><list-item><list list-type="simple"><list-item><p>i. Maintain in separate gnotobiotic isolators that will contain only the bacterium of interest throughout the study.</p></list-item></list></list-item><list-item><p>c. After 4 weeks, confirm colonization by stool culture. Note: Steps i through vi are derived from <xref ref-type="bibr" rid="bib8">Uronis et al. (2011)</xref>. Confirm bacterial strain by 16S RT-PCR:</p><list list-type="simple"><list-item><p>i. Collect up to 500 mg of fecal material.</p><list list-type="simple"><list-item><p>■ Pellets can be stored in an eppendorf tube at −80°C until processing.</p></list-item></list></list-item><list-item><p>ii. Resuspend in lysis buffer with 20 mg/ml lysozyme and incubate at 37°C for 30 min.</p></list-item><list-item><p>iii. Add 10% SDS and 350 µg/ml Proteinase K for further lysis.</p></list-item><list-item><p>iv. Homogenize samples with a bead beater and 0.1 mm zirconium beads.</p></list-item><list-item><p>v. Extract DNA with a DNeasy kit.</p></list-item><list-item><p>vi. Amplify the bacterial 16S ribosomal RNA gene.</p><list list-type="simple"><list-item><p>■ Forward primer: 5′-GTG STG CAY GGY TGT CGT CA-3′.</p></list-item><list-item><p>■ Reverse primer: 5′-ACG TCR TCC MCA CCT TCC TC-3′.</p></list-item><list-item><p>■ See Protocol 2, Procedure Step 2d and e for reaction and annealing conditions.</p></list-item></list></list-item><list-item><p>vii. *Sequence amplicons and BLAST sequencing results to confirm colonization with <italic>E. coli.</italic></p></list-item></list></list-item><list-item><p>d. Quantify level of colonization by serial dilution culture:</p><list list-type="simple"><list-item><p>i. Collect at least 200 mg of fecal material.</p></list-item><list-item><p>ii. Serially dilute 200 mg fecal material.</p></list-item><list-item><p>iii. Plate dilutions on LB agar plates.</p><list list-type="simple"><list-item><p>■ Dilute enough to resolve single colony forming units (CFUs).</p></list-item></list></list-item><list-item><p>iv. Calculate the total number of CFUs per 200 mg fecal matter.</p></list-item></list></list-item></list></list-item><list-item><p>At the same time as first stool culture, intraperitoneally inject with 10 mg/kg azoxymethane (AOM).</p></list-item><list-item><p>Repeat AOM injections every week for 5 more weeks (6 weeks total).</p></list-item><list-item><p>18 weeks after last AOM injection, sacrifice mice.</p><list list-type="simple"><list-item><p>a. #Mice are anesthetized with isofluorane in a drop jar. Once respiration has ceased, the mice are exsanguinated.</p></list-item><list-item><p>b. *Collect stool and quantify colonization as performed in Step 5b.</p></list-item></list></list-item><list-item><p>Macroscopically examine tumor formation:</p><list list-type="simple"><list-item><p>a. Remove colons from the cecum to the rectum, flush with PBS, and splay longitudinally.</p></list-item><list-item><p>b. Blindly count tumors per mouse and measure tumor diameter macroscopically. Image colon and tumors.</p></list-item></list></list-item><list-item><p>Prepare tissue for histological analysis:</p><list list-type="simple"><list-item><p>a. Collect distal colon tissue samples.</p></list-item><list-item><p>b. Swiss-roll colon tissue samples from the distal to the proximal end and fix overnight in 10% formalin.</p></list-item></list></list-item><list-item><p>Paraffin-embed tissues.</p><list list-type="simple"><list-item><p>a. #The replicating lab uses an automated embedding station:</p><list list-type="simple"><list-item><p>i. Samples are passed through a dehydration series consisting of 70%, 80%, 2 × 95% and 3 × 100% ethanol for 30 min each wash.</p></list-item><list-item><p>ii. Samples are washed into xylene; first wash is 30 min, the second is an hour.</p></list-item><list-item><p>iii. The samples are washed into 57°C Paraplast; four washes of 30 min each.</p></list-item><list-item><p>iv. Samples are mounted in mold and allowed to cool and harden.</p></list-item></list></list-item></list></list-item><list-item><p>Cut 6 µm sections and mount on slides.</p></list-item><list-item><p>Stain with hematoxylin and eosin for histologic analysis.</p><list list-type="simple"><list-item><p>a. Lab will record H&E staining protocol used.</p></list-item></list></list-item><list-item><p>Blindly score for inflammation, dysplasia, and invasion (scoring should by performed by an expert animal histopathologist).</p><list list-type="simple"><list-item><p>a. Score mucosal inflammation (0–4) by the degree of lamina propria mononuclear cell (LPMNC) infiltration, crypt hyperplasia, goblet cell depletion, and architectural distortion.</p><list list-type="simple"><list-item><p>i. See Table 2 in <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC507509/pdf/980945.pdf">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC507509/pdf/980945.pdf</ext-link>.</p></list-item></list></list-item><list-item><p>b. Score dysplasia as follows:</p><list list-type="simple"><list-item><p>i. 0 = no dysplasia.</p></list-item><list-item><p>ii. 1 = mild dysplasia characterized by aberrant crypt foci (ACF), +0.5 for small gastrointestinal neoplasia (GIN), or multiple ACF.</p></list-item><list-item><p>iii. 2 = moderate dysplasia with GIN, +0.5 for multiple occurrences or small adenoma.</p></list-item><list-item><p>iv. 3 = severe or high grade dysplasia restricted to the mucosa.</p></list-item><list-item><p>v. 3.5 = adenocarcinoma, invasion through the muscularis mucosa.</p></list-item><list-item><p>vi. 4 = adenocarcinoma, full invasion through the submucosa and into or through the muscularis propria.</p></list-item></list></list-item><list-item><p>c. Score invasion as follows:</p><list list-type="simple"><list-item><p>i. 0 = no invasion.</p></list-item><list-item><p>ii. 1 = 5–10% involvement.</p></list-item><list-item><p>iii. 2 = 10–15% involvement.</p></list-item><list-item><p>iv. 3 = 25–50% involvement.</p></list-item><list-item><p>v. 4 = >50% involvement.</p><list list-type="simple"><list-item><p>■ Multiply invasion score by 1 if invasion is through the muscularis mucosa, and by 2 if invasion is through the muscularis propria and serosa.</p></list-item></list></list-item></list></list-item></list></list-item></list></list-item></list></p></sec></sec><sec id="s2-3-4"><title>Deliverables</title><p><list list-type="bullet"><list-item><p>Data to be collected:</p><list list-type="simple"><list-item><p>a. Mouse records (gender used in each group, type of colonization procedure, health records, condition for early euthanasia, etc).</p></list-item><list-item><p>b. OD<sub>600</sub> of overnight bacterial cultures used in colonization.</p></list-item><list-item><p>c. (Compare to Supplemental figure 10): Raw data and Kaplan–Meier survival curve of mice for all conditions.</p></list-item><list-item><p>d. Sequencing chromatograms and gel image of amplicons from stool sample confirming colonization with <italic>E. coli</italic>.</p></list-item><list-item><p>e. (Compare to Figure 4D): Images, raw numbers and dot plot graph of macroscopic tumor number per mouse (multiplicity) at 18 weeks of colon tissue from mice for all conditions.</p></list-item><list-item><p>f. (Compare to Figure 4F): Micrographs of H&E histology for each mouse at 18 weeks for all conditions.</p></list-item><list-item><p>g. (Compare to Figure 4E): Raw numbers and dot plot graph of mean macroscopic tumor diameter in each mouse at 18 weeks of colon tissue from mice for all conditions.</p></list-item><list-item><p>h. (Compare to Figure 4A): Raw numbers and dot plot graph of inflammation scores at 18 weeks of colon tissue from mice for all conditions.</p></list-item><list-item><p>i. (Compare to Figure 4B): Raw numbers and dot plot graph of neoplasia scores at 18 weeks of colon tissue from mice for all conditions.</p></list-item><list-item><p>j. (Compare to Figure 4C): Raw numbers and dot plot graph of invasion scores at 18 weeks of colon tissue from mice for all conditions.</p></list-item><list-item><p>k. Counts of bacterial colonization from stool sample per mouse at 4 week time point and at sacrifice.</p></list-item></list></list-item></list></p></sec><sec id="s2-3-5"><title>Confirmatory analysis plan</title><p><list list-type="bullet"><list-item><p>Statistical analysis of the replication data:</p><list list-type="simple"><list-item><p>a. (As seen in Supplemental figure 10): Compare survival of AOM-treated <italic>Il10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101 relative to NC101∆<italic>pks.</italic></p><list list-type="simple"><list-item><p>■ Log-rank test (Mantel Cox).</p></list-item></list></list-item><list-item><p>b. (As seen in Figure 4A, right panel): Compare mean inflammation scores of AOM-treated <italic>Il10</italic><sup><italic>−/−</italic></sup> mice mono-associated with <italic>E. coli</italic> NC101 relative to NC101∆<italic>pks.</italic></p><list list-type="simple"><list-item><p>■ Unpaired two-tailed Student's <italic>t</italic>-test.</p></list-item></list></list-item><list-item><p>c. (As seen in Figure 4B): Compare mean neoplasia score of AOM-treated <italic>Il10</italic><sup><italic>−/−</italic></sup> mice mono-associated with <italic>E. coli</italic> NC101 relative to NC101∆<italic>pks.</italic></p><list list-type="simple"><list-item><p>■ Unpaired two-tailed Student's <italic>t</italic>-test.</p></list-item></list></list-item><list-item><p>d. (As seen in Figure 4C): Compare mean invasion score of AOM-treated <italic>Il10</italic><sup><italic>−/−</italic></sup> mice mono-associated with <italic>E. coli</italic> NC101 relative to NC101∆pks.</p><list list-type="simple"><list-item><p>■ Unpaired two-tailed Student's <italic>t</italic>-test.</p></list-item></list></list-item><list-item><p>e. (As seen in Figure 4D): Compare mean macroscopic tumor number (multiplicity) of AOM-treated <italic>Il10</italic><sup><italic>−/−</italic></sup> mice mono-associated with <italic>E. coli</italic> NC101 relative to NC101∆<italic>pks.</italic></p><list list-type="simple"><list-item><p>■ Unpaired two-tailed Student's <italic>t</italic>-test.</p></list-item></list></list-item><list-item><p>f. (As seen in Figure 4E): Mean macroscopic tumor diameter per mouse of AOM-treated <italic>Il10</italic><sup><italic>−/−</italic></sup> mice mono-associated with <italic>E. coli</italic> NC101 relative to NC101∆<italic>pks.</italic></p><list list-type="simple"><list-item><p>■ Unpaired two-tailed Student's <italic>t</italic>-test.</p></list-item></list></list-item><list-item><p>g. Compare mean number of CFUs per 200 mg stool pellet between <italic>E. coli</italic> NC1010-colonized and <italic>E. coli</italic> NC1010Δ<italic>pks</italic>-colonized mice at 4 weeks post-inoculation and at sacrifice.</p><list list-type="simple"><list-item><p>■ Two-way ANOVA.</p></list-item></list></list-item></list></list-item><list-item><p>Meta-analysis of original and replication attempt effect sizes:</p><list list-type="simple"><list-item><p>a. This replication attempt will perform the statistical analysis listed above, compute the effects sizes, compare them against the reported effect size in the original paper and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.</p></list-item></list></list-item></list></p></sec><sec id="s2-3-6"><title>Known differences from the original study</title><p><list list-type="bullet"><list-item><p>The replication attempt will be restricted to the 18 week time point.</p></list-item><list-item><p>The microscope used in the original lab was an Olympus CX41; the replicating lab will use an Olympus BX41.</p></list-item><list-item><p>We will be using a different lot of azoxymethane that used by the original authors. AOM is known to have lot-to-lot variation in efficacy that may affect the absolute numbers of tumors generated per mouse.</p></list-item><list-item><p>The original study collected data on 4 female and 8 male <italic>IL10<sup>-/-</sup></italic> mice inoculated with <italic>E. coli</italic> NC101 and 8 male <italic>IL10<sup>-/-</sup></italic> mice inoculated with <italic>E. coli</italic> NC101Δpks. While the gender of the mice in the replication is not currently known, the mice will be randomly assigned when they reach the age for inoculation with the aim of a similar gender distribution in each group. This will likely generate a different gender distribution than the original study.</p></list-item></list></p></sec><sec id="s2-3-7"><title>Provisions for quality control</title><p>All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (<ext-link ext-link-type="uri" xlink:href="https://osf.io/y4tvd/">https://osf.io/y4tvd/</ext-link>).<list list-type="bullet"><list-item><p>The experiment will be performed by a Science Exchange lab with expertise in germ free mouse studies.</p></list-item><list-item><p>Experimenters will be blinded to the genotype and treatment group. Mice will be randomly assigned to treatment groups.</p></list-item></list></p></sec></sec></sec><sec id="s3"><title>Power calculations</title><sec id="s3-1"><title>Protocol 1</title><p><list list-type="bullet"><list-item><p>Not applicable.</p></list-item></list></p></sec><sec id="s3-2"><title>Protocol 2</title><p><list list-type="bullet"><list-item><p>Not applicable.</p></list-item></list></p></sec><sec id="s3-3"><title>Protocol 3</title><sec id="s3-3-1"><title>Summary of original data</title><p>Figure 4A:<table-wrap id="tblu8" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>4A: Inflammation score at 18 weeks</th><th align="center">Mean</th><th align="center">SD</th><th align="center">N</th></tr></thead><tbody><tr><td><italic>IL10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101</td><td align="center">4</td><td align="center">0</td><td align="center">9</td></tr><tr><td><italic>IL10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101delta<italic>PKS</italic></td><td align="center">3.8</td><td align="center">0.45</td><td align="center">5</td></tr></tbody></table></table-wrap></p></sec><sec id="s3-3-2"><title>Test family</title><p><list list-type="bullet"><list-item><p>Two-tailed t-test, difference between two independent means, alpha error = 0.05.</p><list list-type="simple"><list-item><p>a. Sensitivity calculations were performed using G*power software (<xref ref-type="bibr" rid="bib6">Faul et al., 2007</xref>).</p></list-item></list></list-item></list></p></sec><sec id="s3-3-3"><title>Power calculations</title><p><list list-type="bullet"><list-item><p>Because the original data shows a non-significant effect, we will not be powering this replication to detect an effect. Based on the sample size of 10 mice per group derived from Figure 4C, with α of 0.05 we will be powered to 80% to detect a Cohen's <italic>d</italic> of 1.3249474.</p></list-item></list></p></sec><sec id="s3-3-4"><title>Summary of original data</title><p><list list-type="bullet"><list-item><p>Note: Raw data values obtained from scatterplot with confirmation of accuracy from the authors.<table-wrap id="tblu9" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>4B: Neoplasia score at 18 weeks with AOM treatment</th><th>Mean</th><th>SEM</th><th>SD</th><th>N</th></tr></thead><tbody><tr><td><italic>IL10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101</td><td>4.44</td><td>0.18</td><td>0.53</td><td>9</td></tr><tr><td><italic>IL10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101Δ<italic>pks</italic></td><td>3.60</td><td>0.24</td><td>0.55</td><td>5</td></tr></tbody></table></table-wrap></p></list-item></list><list list-type="bullet"><list-item><p>Stdev was calculated using formula SD = SEM*(SQRT n).</p></list-item></list></p></sec><sec id="s3-3-5"><title>Test family</title><p><list list-type="bullet"><list-item><p>Two-tailed t-test, difference between two independent means, alpha error = 0.05.</p><list list-type="simple"><list-item><p>a. Power calculations were performed for statistically significant effects reported in original study using G*power software (<xref ref-type="bibr" rid="bib6">Faul et al., 2007</xref>).</p></list-item></list></list-item></list></p></sec><sec id="s3-3-6"><title>Power calculations</title><p><table-wrap id="tblu10" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Group 1 vs</th><th>Group 2</th><th>Pooled SD</th><th>Effect size</th><th>A priori power</th><th>Group 1 sample size</th><th>Group 2 sample size</th></tr></thead><tbody><tr><td>NC101</td><td>NC101Δ<italic>pks</italic></td><td>0.534</td><td>1.573034</td><td>83.2%<xref ref-type="table-fn" rid="tblfn1">*</xref></td><td>8<xref ref-type="table-fn" rid="tblfn1">*</xref></td><td>8<xref ref-type="table-fn" rid="tblfn1">*</xref></td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>*</label><p>Based on the sample size required for Figure 4C, we will use 10 mice per group. This brings the a priori power to 91.3%.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="s3-3-7"><title>Summary of original data</title><p><list list-type="bullet"><list-item><p>Note: Raw data values obtained from scatterplot with confirmation of accuracy from the authors.</p></list-item></list><table-wrap id="tblu11" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>4C: Invasion score at 18 weeks with AOM treatment</th><th>Mean</th><th>SEM</th><th>SD</th><th>N</th></tr></thead><tbody><tr><td><italic>IL10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101</td><td>3</td><td>0.63</td><td>1.9</td><td>9</td></tr><tr><td><italic>IL10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101Δ<italic>pks</italic></td><td>0.8</td><td>0.37</td><td>0.84</td><td>5</td></tr></tbody></table></table-wrap><list list-type="bullet"><list-item><p>Stdev was calculated using formula SD = SEM*(SQRT n).</p></list-item></list></p></sec><sec id="s3-3-8"><title>Test family</title><p><list list-type="bullet"><list-item><p>Two-tailed t-test, difference between two independent means, alpha error = 0.05.</p><list list-type="simple"><list-item><p>a. Power calculations were performed for statistically significant effects reported in original study using G*power software (<xref ref-type="bibr" rid="bib6">Faul et al., 2007</xref>).</p></list-item></list></list-item></list></p></sec><sec id="s3-3-9"><title>Power calculations</title><p><table-wrap id="tblu12" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Group 1 vs</th><th>Group 2</th><th>Pooled SD</th><th>Effect size</th><th>A priori power</th><th>Group 1 sample size</th><th>Group 2 sample size</th></tr></thead><tbody><tr><td>NC101</td><td>NC101Δ<italic>pks</italic></td><td>4.410</td><td>1.814059<xref ref-type="table-fn" rid="tblfn2">*</xref></td><td>80.1%</td><td>6<xref ref-type="table-fn" rid="tblfn2">*</xref></td><td>6<xref ref-type="table-fn" rid="tblfn2">*</xref></td></tr></tbody></table><table-wrap-foot><fn id="tblfn2"><label>*</label><p>Based on the sample size required for 4C, we will use 10 mice per group. This brings the a priori power to 96.9%.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="s3-3-10"><title>Summary of original data</title><p><list list-type="bullet"><list-item><p>Note: Raw data values obtained from scatterplot with confirmation of accuracy from the authors.</p></list-item></list><table-wrap id="tblu13" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>4D: Tumor number per mouse at 18 weeks with AOM treatment</th><th>Mean</th><th>SEM</th><th>SD</th><th>N</th></tr></thead><tbody><tr><td><italic>IL10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101</td><td>10.6</td><td>1.8</td><td>5.3</td><td>9</td></tr><tr><td><italic>IL10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101Δ<italic>pks</italic></td><td>2.6</td><td>0.7</td><td>1.5</td><td>5</td></tr></tbody></table></table-wrap><list list-type="bullet"><list-item><p>Stdev was calculated using formula SD = SEM*(SQRT n).</p></list-item></list></p></sec><sec id="s3-3-11"><title>Test family</title><p><list list-type="bullet"><list-item><p>Two-tailed t-test, difference between two independent means, alpha error = 0.05.</p><list list-type="simple"><list-item><p>a. Power calculations were performed for statistically significant effects reported in original study using G*power software.</p></list-item></list></list-item></list></p></sec><sec id="s3-3-12"><title>Power calculations</title><p><table-wrap id="tblu14" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>Group 1 vs</th><th>Group 2</th><th>Pooled SD</th><th>Effect size</th><th>A priori power</th><th>Group 1 sample size</th><th>Group 2 sample size</th></tr></thead><tbody><tr><td>NC101</td><td>NC101Δ<italic>pks</italic></td><td>1.625</td><td>1.353846</td><td>81.7%</td><td>10</td><td>10</td></tr></tbody></table></table-wrap></p><p>Figure 4E:</p><table-wrap id="tblu15" position="anchor"><table frame="hsides" rules="groups"><thead><tr><th>4E: Mean macroscopic tumor diameter at 18 weeks</th><th align="center">Mean</th><th align="center">SD</th><th align="center">N</th></tr></thead><tbody><tr><td><italic>IL10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101</td><td align="center">3.02</td><td align="center">0.74</td><td align="center">9</td></tr><tr><td><italic>IL10</italic><sup><italic>−/−</italic></sup> mice inoculated with <italic>E. coli</italic> NC101delta<italic>PKS</italic></td><td align="center">3.66</td><td align="center">1.59</td><td align="center">5</td></tr></tbody></table></table-wrap></sec><sec id="s3-3-13"><title>Test family</title><p><list list-type="bullet"><list-item><p>Two-tailed t-test, difference between two independent means, alpha error = 0.05.</p><list list-type="simple"><list-item><p>a. Sensitivity calculations were performed using G*power software (<xref ref-type="bibr" rid="bib6">Faul et al., 2007</xref>).</p></list-item></list></list-item></list></p></sec><sec id="s3-3-14"><title>Power calculations</title><p><list list-type="bullet"><list-item><p>Because the original data shows a non-significant effect, we will not be powering this replication to detect an effect. Based on the sample size of 10 mice per group derived from Figure 4C, with α of 0.05 we will be powered to 80% to detect a Cohen's <italic>d</italic> of 1.3249474.</p></list-item></list></p><p>Supplemental figure 10:</p></sec><sec id="s3-3-15"><title>Test family</title><p><list list-type="bullet"><list-item><p>Two-tailed t-test, difference between two independent means, alpha error = 0.05.</p><list list-type="simple"><list-item><p>a. Sensitivity calculations were performed using G*power software (<xref ref-type="bibr" rid="bib6">Faul et al., 2007</xref>).</p></list-item></list></list-item></list></p></sec><sec id="s3-3-16"><title>Power calculations</title><p><list list-type="bullet"><list-item><p>Because the original data shows a non-significant effect, we will not be powering this replication to detect an effect. Based on the sample size of 10 mice per group derived from Figure 4C, with α of 0.05 we will be powered to 80% to detect a Cohen's <italic>d</italic> of 1.1453705.</p></list-item></list></p></sec></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>The Reproducibility Project: Cancer Biology core team would like to thank the original authors, in particular Janelle C Arthur and Christian Jobin, for generously sharing critical information as well as reagents to ensure the fidelity and quality of this replication attempt. We would also like to thanks the following companies for generously donating reagents to the Reproducibility Project: Cancer Biology; American Tissue Culture Collection (ATCC), BioLegend, Cell Signaling Technology, Charles River Laboratories, Corning Incorporated, DDC Medical, EMD Millipore, Harlan Laboratories, LI-COR Biosciences, Mirus Bio, Novus Biologicals, Sigma–Aldrich, and System Biosciences (SBI).</p></ack><sec sec-type="additional-information" id="s4"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>KE: The Germ Free Laboratory is a Science Exchange associated lab.</p></fn><fn fn-type="conflict" id="conf2"><p>RP:CB: We disclose that EI, FT, and JL are employed by and hold shares in Science Exchange Inc. The experiments presented in this manuscript will be conducted by KE at the Germ Free Laboratory, which is a Science Exchange lab.</p></fn><fn fn-type="conflict" id="conf3"><p>The other authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>KE, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>WY, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>RP:CB, Conception and design, Drafting or revising the article</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Arthur</surname><given-names>JC</given-names></name><name><surname>Gharaibeh</surname><given-names>RZ</given-names></name><name><surname>Mühlbauer</surname><given-names>M</given-names></name><name><surname>Perez-Chanona</surname><given-names>E</given-names></name><name><surname>Uronis</surname><given-names>JM</given-names></name><name><surname>McCafferty</surname><given-names>J</given-names></name><name><surname>Fodor</surname><given-names>AA</given-names></name><name><surname>Jobin</surname><given-names>C</given-names></name></person-group><year>2014</year><article-title>Microbial genomic analysis reveals the essential role of inflammation in bacteria-induced colorectal cancer</article-title><source>Nature Communications</source><volume>5</volume><fpage>1</fpage><lpage>11</lpage><pub-id pub-id-type="doi">10.1038/ncomms5724</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Arthur</surname><given-names>JC</given-names></name><name><surname>Perez-Chanona</surname><given-names>E</given-names></name><name><surname>Muhlbauer</surname><given-names>M</given-names></name><name><surname>Tomkovich</surname><given-names>S</given-names></name><name><surname>Uronis</surname><given-names>JM</given-names></name><name><surname>Fan</surname><given-names>TJ</given-names></name><name><surname>Campbell</surname><given-names>BJ</given-names></name><name><surname>Abujamel</surname><given-names>T</given-names></name><name><surname>Dogan</surname><given-names>B</given-names></name><name><surname>Rogers</surname><given-names>AB</given-names></name><name><surname>Rhodes</surname><given-names>JM</given-names></name><name><surname>Stintzi</surname><given-names>A</given-names></name><name><surname>Simpson</surname><given-names>KW</given-names></name><name><surname>Hansen</surname><given-names>JJ</given-names></name><name><surname>Keku</surname><given-names>TO</given-names></name><name><surname>Fodor</surname><given-names>AA</given-names></name><name><surname>Jobin</surname><given-names>C</given-names></name></person-group><year>2012</year><article-title>Intestinal inflammation targets cancer-inducing activity of the microbiota</article-title><source>Science</source><volume>338</volume><fpage>120</fpage><lpage>123</lpage><pub-id pub-id-type="doi">10.1126/science.1224820</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Buc</surname><given-names>E</given-names></name><name><surname>Dubois</surname><given-names>D</given-names></name><name><surname>Sauvanet</surname><given-names>P</given-names></name><name><surname>Raisch</surname><given-names>J</given-names></name><name><surname>Delmas</surname><given-names>J</given-names></name><name><surname>Darfeuille-Michaud</surname><given-names>A</given-names></name><name><surname>Pezet</surname><given-names>D</given-names></name><name><surname>Bonnet</surname><given-names>R</given-names></name></person-group><year>2013</year><article-title>High prevalence of mucosa-associated <italic>E. coli</italic> producing cyclomodulin and genotoxin in colon cancer</article-title><source>PLOS ONE</source><volume>8</volume><fpage>e56964</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0056964</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cougnoux</surname><given-names>A</given-names></name><name><surname>Dalmasso</surname><given-names>G</given-names></name><name><surname>Martinez</surname><given-names>R</given-names></name><name><surname>Buc</surname><given-names>E</given-names></name><name><surname>Delmas</surname><given-names>J</given-names></name><name><surname>Gibold</surname><given-names>L</given-names></name><name><surname>Sauvanet</surname><given-names>P</given-names></name><name><surname>Darcha</surname><given-names>C</given-names></name><name><surname>Dechelotte</surname><given-names>P</given-names></name><name><surname>Bonnet</surname><given-names>M</given-names></name><name><surname>Pezet</surname><given-names>D</given-names></name><name><surname>Wodrich</surname><given-names>H</given-names></name><name><surname>Darfeuille-Michaud</surname><given-names>A</given-names></name><name><surname>Bonnet</surname><given-names>R</given-names></name></person-group><year>2014</year><article-title>Bacterial genotoxin colibactin promotes colon tumour growth by inducing a senescence-associated secretory phenotype</article-title><source>Gut</source><volume>63</volume><fpage>1932</fpage><lpage>1942</lpage><pub-id pub-id-type="doi">10.1136/gutjnl-2013-305257</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cuevas-Ramos</surname><given-names>G</given-names></name><name><surname>Petit</surname><given-names>CR</given-names></name><name><surname>Marcq</surname><given-names>I</given-names></name><name><surname>Boury</surname><given-names>M</given-names></name><name><surname>Oswald</surname><given-names>E</given-names></name><name><surname>Nougayrède</surname><given-names>JP</given-names></name></person-group><year>2010</year><article-title><italic>Escherichia coli</italic> induces DNA damage in vivo and triggers genomic instability in mammalian cells</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>107</volume><fpage>11537</fpage><lpage>11542</lpage><pub-id pub-id-type="doi">10.1073/pnas.1001261107</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Faul</surname><given-names>F</given-names></name><name><surname>Erdfelder</surname><given-names>E</given-names></name><name><surname>Lang</surname><given-names>AG</given-names></name><name><surname>Buchner</surname><given-names>A</given-names></name></person-group><year>2007</year><article-title>G* Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences</article-title><source>Behavior Research Methods</source><volume>39</volume><fpage>175</fpage><lpage>191</lpage><pub-id pub-id-type="doi">10.3758/BF03193146</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nougayrede</surname><given-names>JP</given-names></name></person-group><year>2006</year><article-title><italic>Escherichia coli</italic> induces DNA double-strand breaks in Eukaryotic cells</article-title><source>Science</source><volume>313</volume><fpage>848</fpage><lpage>851</lpage><pub-id pub-id-type="doi">10.1126/science.1127059</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Uronis</surname><given-names>JM</given-names></name><name><surname>Arthur</surname><given-names>JC</given-names></name><name><surname>Keku</surname><given-names>T</given-names></name><name><surname>Fodor</surname><given-names>A</given-names></name><name><surname>Carroll</surname><given-names>IM</given-names></name><name><surname>Cruz</surname><given-names>ML</given-names></name><name><surname>Appleyard</surname><given-names>CB</given-names></name><name><surname>Jobin</surname><given-names>C</given-names></name></person-group><year>2011</year><article-title>Gut microbial diversity is reduced by the probiotic VSL#3 and correlates with decreased TNBS-induced colitis</article-title><source>Inflammatory Bowel Diseases</source><volume>17</volume><fpage>289</fpage><lpage>297</lpage><pub-id pub-id-type="doi">10.1002/ibd.21366</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04186.002</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Sears</surname><given-names>Cynthia</given-names></name><role>Reviewing editor</role><aff><institution>Johns Hopkins Univ SOMl</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Registered report: Intestinal inflammation targets cancer-inducing activity of the microbiota” for consideration at <italic>eLife</italic>. Your Registered report has been favorably evaluated by Richard Losick (Senior editor), a guest editor, 4 reviewers, and a biostatistician.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>The proposed experiments are largely appropriately designed although there are important points of clarification required.</p><p>1) The authors accurately summarize the literature directly related to the original paper, but should also be aware of two key manuscripts relevant to Colibactin: PMIDs 20534522 (PNAS 107:11537, 2010) and 16902142 (Science 313:848, 2006).</p><p>2) There are key concerns regarding the azoxymethane lot and its handling and documentation. First, the authors should note that each lot of azoxymethane may have differing potency, so one lot should be used throughout this replication experiment. Furthermore, this lot should first be tested for its ability to induce tumors in the AOM/DSS model. Using a different lot of azoxymethane from the original study could result in differences in tumorigenesis (i.e. penetrance, size, invasion, etc.) between the original study and the reproduced study. Importantly though, this should not affect differences in tumorigenesis between experimental groups but could lead to uninterpretable results if tumorigenesis is very low in the <italic>E. coli</italic> NC101, positive control group, associated with the lot of AOM. Second, the replication authors should describe better how they aliquot, store and use AOM, which soon after dilution becomes less stable and less carcinogenic, but more toxic and therefore can cause unexpected mortality that would hamper the experimental design.</p><p>3) While this project has been under review, another original paper has been published by the authors of the paper to be replicated: Arthur et al., Nature Communications, Sept 2014 (on-line). The <italic>E. coli</italic> NC101 tumorigenesis model is further explored in this manuscript. In consultation with the original authors, the replication project should clarify the contribution of AOM to the model. In Uronis et al. (PLOS ONE 4:e6026, 2009), it appears that <italic>IL10</italic><sup><italic>-/-</italic></sup> mice alone were not evaluated for tumorigenesis upon colonization with <italic>E. coli</italic> NC101. In this replication project, the text of the original Science paper suggests that <italic>IL10</italic><sup><italic>-/-</italic></sup> is insufficient to promote tumorigenesis upon colonization with NC101 whereas in the most recent Arthur publication noted above, <italic>IL10</italic><sup><italic>-/-</italic></sup> mice exhibit similar tumorigenesis, but not invasive extent of neoplasia, compared to the AOM + <italic>IL10</italic><sup><italic>-/-</italic></sup> model (Figure 4, Nature Communications, on-line as above). The authors of the replication project should comment on the impact of these recent data on their replication plan. We query whether the replication authors should consider including IL-10<sup>-/-</sup> mice colonized with <italic>E. coli</italic> NC101 without AOM treatment as a group into their experiments.</p><p>4) There is no explicit indication that the mice in the gnotobiotic study will be age-matched or sex-matched. It is only stated that cancer/colitis will be induced at 7-12 weeks and that male and female mice will be used. Although not explicitly stated, one assumes that male and female mice will be housed in separate isolators. Will the numbers of male and female mice used approximate that in the original experiments?</p><p>5) Because application of histopathologic criteria can be somewhat subjective and pathologist-dependent, the authors should consider whether histologic images should be evaluated independently by two pathologists and, if possible, by the animal histopathologist who evaluated the images in the original publication. The scoring system is specific about lamina propria mononuclear cell infiltration but polymorphonuclear infiltration should also be considered.</p><p>6) The method of inoculation allows for more than one approach although the original paper utilized gavage and rectal swabbing. While the authors suggest colonization with oral swab as an alternative, oral gavage is likely to yield more reproducible colonization results. Please clarify.</p><p>7) Please specify the typical OD600 or range considered acceptable to be used for the bacterial inoculation cultures. Confirmation of exact CFUs and culture purity by quantitative culturing of the inoculum should be performed. Confirmation that the inoculated strains are ClbB-positive and ClbB-negative should be done. Similarly, cultures to verify colonization with the specific strain inoculated at week 4 and at the end of the experiment, should be completed.</p><p>8) Sampling. It is not clear why the expected mortality for <italic>E. coli</italic> NC101Δpks is higher, when this strain induces less tumorigenesis than the parent strain <italic>E. coli</italic> NC101.</p><p>9) Can the authors clarify the approach/plan if the proposed experiments do not replicate the original data? Is there any back-up plan to adjust the experimental plan to adjust for potential technical concerns such as the potency of the azozymethane lot or if the experimental course differs in a different facility (e.g., more mouse death)?</p><p>Statistical comments:</p><p>1) Cross-study variation should be taken into account to determine power pre-data collection. This is hard to estimate, but papers by Giovanni Parmigiani and collaborators at the Dana Farber provide some estimates about cross-study variation that could be used for this purpose. The authors should budget some additional variability because of cross-study reproducibility, and increase the sample size on-the-fly, as they deem appropriate.</p><p>2) The final report on the replicated study should report the actual power of the tests, based on the standard deviations in the replicated study.</p><p>3) Please clarify the comment 'based on the sample size of 13 mice per group derived from Figure 4C'. The sample size for Figure 4C is stated as 10 to achieve 80% power, α = 0.05.</p><p>[Editors' note: further revisions were requested prior to acceptance, as described below.]</p><p>Thank you for resubmitting your article entitled “Registered report: Intestinal inflammation targets cancer-inducing activity of the microbiota” for further consideration at <italic>eLife</italic>. Your revised Registered Report has been favorably evaluated by Richard Losick (Senior editor), a guest Reviewing editor, three of the original reviewers, and a biostatistician.</p><p>There are two remaining issues that need to be addressed before acceptance, as outlined below:</p><p>1) Response to point 7: the reviewers feel strongly that quantitative cultures of the inoculum to prove purity and numbers of organisms inoculated should be performed. Relying on OD600 solely does not account for culture purity, nor potentially nonviable organisms in the culture.</p><p>2) Response to point 1 of the statistical review: one approach to incorporate putative extra variation in the proposed study would be to calculate sample size by assuming a range of the anticipated effect size. For example, the effect size for, say, Figure 4C used in the power calculation is 1.353846. So, one may examine sample size for smaller effect size: say, effect size of 1.2 and 1.1 and see how much the sample size would change in these two additional settings. When we do this calculation, we can see that the sample size is approximately 11 mice in each group if the effect size is 1.2, and approximately 13 mice in each group when the effect size is 1.1. The sample size does not increase drastically under these additional effect sizes. So, perhaps, the sample size of 10 is fine. Of course, if the observed effect size at the end of the study is less than 1.353846, then the result is likely going to be non-significant since the study was powered to detect an effect size of 1.353846 or more.</p><p>We would not recommend a post-hoc power calculation. But the investigators should consider saying upfront in the Methods section (when they publish their work) that they calculated the power at the beginning of the study using a two-sample t-test framework to detect a certain effect size.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04186.003</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The authors accurately summarize the literature directly related to the original paper, but should also be aware of two key manuscripts relevant to Colibactin: PMIDs 20534522 (PNAS 107:11537, 2010) and 16902142 (Science 313:848, 2006)</italic>.</p><p>Thank you for bringing these two papers to our attention. We have added references to these papers in the Introduction.</p><p><italic>2) There are key concerns regarding the azoxymethane lot and its handling and documentation. First, the authors should note that each lot of azoxymethane may have differing potency, so one lot should be used throughout this replication experiment. Furthermore, this lot should first be tested for its ability to induce tumors in the AOM/DSS model. Using a different lot of azoxymethane from the original study could result in differences in tumorigenesis (i.e. penetrance, size, invasion, etc.) between the original study and the reproduced study. Importantly though, this should not affect differences in tumorigenesis between experimental groups but could lead to uninterpretable results if tumorigenesis is very low in the</italic> E. coli <italic>NC101, positive control group, associated with the lot of AOM. Second, the replication authors should describe better how they aliquot, store and use AOM, which soon after dilution becomes less stable and less carcinogenic, but more toxic and therefore can cause unexpected mortality that would hamper the experimental design</italic>.</p><p>We would like to thank the reviewers for bringing these details to our attention. Unfortunately it is beyond the scope of this project to include a full pilot study testing the lot of AIM independently in addition to the experimental arms already proposed. We will clearly note, however, in the Replication Study that details the results of the replication experiment that lot-to-lot variability in the effectiveness of azoxymethane needs to be taken into consideration when comparing the original and replication data. When we are performing the comparative analysis of the replication and original data, we will assess both the quantitative numbers of tumors (which may be affected by differences in AOM lots), but also the trends between experimental groups, which should not be. Our replicating lab, which has previous experience working with AOM, will make a 25 mg/mL stock solution of AOM and store it in aliquots at -80°C. They will use a fresh aliquot each time they need AOM to prevent multiple freeze thaw cycles. Additionally, we will note in the “Known differences from the original study” section that we will be using a different lot of AOM, and that AOM is known to have lot-to-lot variation in efficacy. The manuscript has been updated to reflect this change.</p><p><italic>3) While this project has been under review, another original paper has been published by the authors of the paper to be replicated: Arthur et al., Nature Communications, Sept 2014 (on-line). The</italic> E coli <italic>NC101 tumorigenesis model is further explored in this manuscript. In consultation with the original authors, the replication project should clarify the contribution of AOM to the model. In Uronis et al. (PLOS ONE 4:e6026, 2009), it appears that</italic> IL10<sup>-/-</sup> <italic>mice alone were not evaluated for tumorigenesis upon colonization with E coli NC101. In this replication project, the text of the original Science paper suggests that</italic> IL10<sup>-/-</sup> <italic>is insufficient to promote tumorigenesis upon colonization with E coli NC101 whereas in the most recent Arthur publication noted above,</italic> IL10<sup>-/-</sup> <italic>mice exhibit similar tumorigenesis, but not invasive extent of neoplasia, compared to the AOM +</italic> IL10<sup>-/-</sup> <italic>model (Figure 4, Nature Communications, on-line as above). The authors of the replication project should comment on the impact of these recent data on their replication plan. We query whether the replication authors should consider including IL-10</italic><sup><italic>-/-</italic></sup> <italic>mice colonized with</italic> E. coli <italic>NC101 without AOM treatment as a group into their experiments</italic>.</p><p>We thank the reviewers for bringing this additional literature to our attention. From a purely intellectual perspective it is interesting to consider the contributions of each factor—AOM, genetic status and colonization with types of bacteria—to developing colorectal cancer. However, the aim of the Reproducibility Project: Cancer Biology is to replicate as faithfully as possible experiments, and not the conclusions and implications drawn from those experiments. As such, it is beyond the scope of this project to add an additional arm of mouse treatments that was not part of the original experiment based on data from a separate paper. We have included a discussion of the Arthur 2014 paper in the Introduction to describe the relevance of these newer findings to our replication study.</p><p><italic>4) There is no explicit indication that the mice in the gnotobiotic study will be age-matched or sex-matched. It is only stated that cancer/colitis will be induced at 7-12 weeks and that male and female mice will be used. Although not explicitly stated, one assumes that male and female mice will be housed in separate isolators</italic>. <italic>Will the numbers of male and female mice used approximate that in the original experiments?</italic></p><p>Our intention is indeed to use similar numbers of male and female mice as used in the original experiment. As of the writing of this letter, we are currently negotiating the MTA with the UNC Gnotobiotic core for our lab to receive the IL10 null mice. Due to the efforts involved in breeding and treating these mice, the replicating lab will approximate as closely as possible the same numbers of male and female mice used, based on the sexes they obtain in their breeding. We will record the sex of each mouse for later comparison to the original numbers of male and female mice used. The manuscript has been updated to include this detail.</p><p><italic>5) Because application of histopathologic criteria can be somewhat subjective and pathologist-dependent, the authors should consider whether histologic images should be evaluated independently by two pathologists and, if possible, by the animal histopathologist who evaluated the images in the original publication. The scoring system is specific about lamina propria mononuclear cell infiltration but polymorphonuclear infiltration should also be considered.</italic></p><p>This is a strong point to consider. However, the aim of the Reproducibility Project: Cancer Biology is to perform independent replications, and to assess if an effect is repeatable when performed by a separate group of people in a separate lab. Given that the person who scored the slides was Dr. Janelle Arthur, the original study’s first author (along with a second qualified colleague), we feel that involving her in the scoring of the replication data would affect the independence of the replication. To address the subjective nature of the histopathological scoring, Dr. Arthur has kindly provided detailed guidelines to the histopathologist who will score the replication data. In addition, the raw images of the stained tissues will be made publicly available through the Open Science Framework, allowing the public to see and evaluate the data directly and not be solely reliant upon each image’s score. This will allow examination of features of the stained tissues not included in the original criteria for scoring, such as polymorphonuclear infiltration.</p><p><italic>6) The method of inoculation allows for more than one approach although the original paper utilized gavage and rectal swabbing. While the authors suggest colonization with oral swab as an alternative, oral gavage is likely to yield more reproducible colonization results. Please clarify</italic>.</p><p>Thank you for clarifying the potential differences between the two methods. We will use oral gavage to colonize the mice. We have updated the manuscript to reflect this change.</p><p><italic>7) Please specify the typical OD600 or range considered acceptable to be used for the bacterial inoculation cultures. Confirmation of exact CFUs and culture purity by quantitative culturing of the inoculum should be performed. Confirmation that the inoculated strains are ClbB-positive and ClbB-negative should be done. Similarly, cultures to verify colonization with the specific strain inoculated at week 4 and at the end of the experiment, should be completed</italic>.</p><p>The <italic>E. coli</italic> strains being used in this project have been shared directly by the original authors. We will verify them by PCR for the <italic>pks</italic> genotoxic island, as outlined in Protocol 2. Steps to confirm colonization of the mice by stool culture are already present in the protocol. Please see Protocol 3 Step 4d for confirmation of colonization 4 weeks after inoculation and step 7b for the second confirmation of colonization at the end of the study. Based on direct communication from the first author, we are not aware of any exclusion criteria based on an OD600 reading for using an overnight culture to inoculate mice. We will be recording the OD600 of each culture used (see Protocol 3 Step 4a).</p><p><italic>8) Sampling. It is not clear why the expected mortality for</italic> E. coli <italic>NC101Δpks is higher, when this strain induces less tumorigenesis than the parent strain</italic> E. coli <italic>NC101</italic>.</p><p>Percent survival was calculated from the Kaplan-Meier curve in Supplemental Figure 10 of the original paper. The difference in percent survival was found by the original authors to not be statistically significant; however we wished to ensure that enough mice would survive the experiment in order to have highly powered statistical analysis.</p><p><italic>9) Can the authors clarify the approach/plan if the proposed experiments do not replicate the original data? Is there any back-up plan to adjust the experimental plan to adjust for potential technical concerns such as the potency of the azozymethane lot or if the experimental course differs in a different facility (e.g., more mouse death)?</italic></p><p>The overall aim of the Reproducibility Project: Cancer Biology is in fact to attempt to evaluate the replicability of key experiments from this paper. It will be an interesting result if the experiments do or do not replicate. There are many reasons besides inaccuracy of the original experiment that could result in non-replication; the reviewers themselves have highlighted some already. The Replication Study that will be published upon completion of data collection will include a thorough discussion of reasons the experiment could have succeeded or failed to replicate, including variation in the efficacy of AOM. Another point the reviewers explore is if we happen to have more deaths amongst our mice than was seen by the original authors. Because of the scope of this project, we would not be able to add additional mice in the event of experimental animals dying, which is why we have attempted to estimate a sufficient number of animals to ensure a fully powered dataset is achieved at the end of the experiment. However, even if unexpected deaths do reduce the number of animals in the replication dataset resulting in the replication being underpowered, we still believe there will be value in assessing the effect size determined from the remaining animals.</p><p><italic>Statistical comments</italic>:</p><p><italic>1) Cross-study variation should be taken into account to determine power pre-data collection. This is hard to estimate, but papers by Giovanni Parmigiani and collaborators at the Dana Farber provide some estimates about cross-study variation that could be used for this purpose. The authors should budget some additional variability because of cross-study reproducibility, and increase the sample size on-the-fly, as they deem appropriate</italic>.</p><p>We thank the reviewers for these suggestions. The cross-study variation, such as approaches that utilize the 95% confidence interval of the effect size, can be useful in conducting power calculations when planning adequate sample sizes for detecting the true population effect size, which requires a range of possible observed effect sizes. However, the Reproducibility Project: Cancer Biology is designed to conduct replications that have 80% power to detect the point estimate of the originally reported effect size. While this has the limitation of being underpowered to detect smaller effects than what is originally reported, this standardizes the approach across all studies to be designed to detect the originally reported effect size with at least 80% power. Also, while the minimum power guarantee is beneficial for observing a range of possible effect sizes, the experiments in this replication, and all experiments in the project, are designed to detect the originally reported effect size with a minimum power of 80%. Thus, performing power calculations during or after data collection is not necessary in this replication attempt as all studies included are already designed to meet a minimum power or are identified beforehand as being underpowered and thus are not included in the confirmatory analysis plan. The papers by Giovanni Parmigiani and collaborators highlight the importance of accounting for variability that can occur across different studies, specifically gene expression data. While it is possible for a difference in variance between the originally reported results and the replication data, this will be reflected in the presentation of the data and a possible reason for obtaining a different effect size estimate.</p><p><italic>2) The final report on the replicated study should report the actual power of the tests, based on the standard deviations in the replicated study</italic>.</p><p>We do not see the value in performing post-hoc power calculations on the obtained data. However, we do agree that reporting the actual power of the tests to detect the originally reported effect size estimate based on the sample size analyzed in the replication study is important and will be reported.</p><p><italic>3) Please clarify the comment 'based on the sample size of 13 mice per group derived from Figure 4C'. The sample size for Figure 4C is stated as 10 to achieve 80% power, α = 0.05.</italic></p><p>Thank you for identifying this typo. We have corrected it in the manuscript.</p><p>[Editors' note: further revisions were requested prior to acceptance, as described below.]</p><p><italic>There are two remaining issues that need to be addressed before acceptance, as outlined below</italic>:</p><p><italic>1) Response to point 7: the reviewers feel strongly that quantitative cultures of the inoculum to prove purity and numbers of organisms inoculated should be performed. Relying on OD600 solely does not account for culture purity, nor potentially nonviable organisms in the culture</italic>.</p><p>We agree with the reviewers that quantitative culturing is an important step. Based on our understanding, as well as that of the experts at the Germ Free Laboratory, the procedures we propose satisfy the terms of quantitative culturing to determine the exact CFUs of bacteria in the inoculum and also confirm living organisms were inoculated. We have included a letter from Dr. Kate Eaton, the director of the Germ Free Laboratory, detailing their standard procedures for quantitative culturing of inoculum and confirmation of colonization.</p><p><italic>2) Response to point 1 of the statistical review: one approach to incorporate putative extra variation in the proposed study would be to calculate sample size by assuming a range of the anticipated effect size. For example, the effect size for, say, Figure 4C used in the power calculation is 1.353846. So, one may examine sample size for smaller effect size: say, effect size of 1.2 and 1.1 and see how much the sample size would change in these two additional settings. When we do this calculation, we can see that the sample size is approximately 11 mice in each group if the effect size is 1.2, and approximately 13 mice in each group when the effect size is 1.1. The sample size does not increase drastically under these additional effect sizes. So, perhaps, the sample size of 10 is fine. Of course, if the observed effect size at the end of the study is less than 1.353846, then the result is likely going to be non-significant since the study was powered to detect an effect size of 1.353846 or more</italic>.</p><p><italic>We would not recommend a post-hoc power calculation. But the investigators should consider saying upfront in the Methods section (when they publish their work) that they calculated the power at the beginning of the study using a two-sample t-test framework to detect a certain effect size</italic>.</p><p>We agree with the reviewers and plan to be fully transparent concerning our choice of statistical analyses, both in the Registered Report as well as the follow-up Replication Study. Additional materials relating to the Registered Report, including details on power calculations, are posted on the study’s wiki page on the Open Science Framework, and are made public upon publication of the Registered Report.</p></body></sub-article></article> |