The gut microbiota\u27s function in regulating health has seen it linked to disease progression in several cancers. However, there is limited research detailing its influence in breast cancer (BrCa). This study found that antibiotic-induced perturbation of the gut microbiota significantly increases tumor progression in multiple BrCa mouse models. Metagenomics highlights the common loss of several bacterial species following antibiotic administration. One such bacteria

et al,

McKee, Alastair M

Weilbaecher, Katherine N

English

Digital Commons@Becker

iScience, Volume 24Supplemental informationAntibiotic-induced disturbancesof the gut microbiota result in acceleratedbreast tumor growthAlastair M. McKee, Benjamin M. Kirkup, Matthew Madgwick, Wesley J.Fowler, Christopher A. Price, Sally A. Dreger, Rebecca Ansorge, Kate A.Makin, Shabhonam Caim, Gwenaelle Le Gall, Jack Paveley, Charlotte Leclaire, MatthewDalby, Cristina Alcon-Giner, Anna Andrusaite, Tzu-Yu Feng, Martina Di Modica, TizianaTriulzi, Elda Tagliabue, Simon W.F. Milling, Katherine N. Weilbaecher, Melanie R.Rutkowski, Tamás Korcsmáros, Lindsay J. Hall, and Stephen D. RobinsonSupplementary Figure Legends   Figure S1. VNMAA treatment does not influence tumour histopathology but does alter caecal bacterial composition, related to Figure 1 and Figure 2. A) Representative photomicrographs of H&E stained tumours (left) from VNMAA and Control treated animals (Scale bar = 100 µm) and Representative photomicrographs of endomucin stained tumours (right) from VNMAA and Control treated animals (Scale bar = 100 µm); arrows demarcate Endomucin positive blood vessels. B) Bar graph showing mean (±s.e.m.) relative vessel counts in tumours from VNMAA treated and Control animals (N=1, n=5). C) stacked bar graph of the relative abundances of phyla in all individual samples obtained through shotgun metagenomics.  D) Principle component analysis (caecal metabolites) representing the relatedness of the Control and VNMAA samples shown in Figure 2B. Control samples (yellow circles) cluster separately from VNMAA samples (blue circles).  Figure S2. Immune cell populations are not altered by VNMAA administration, related to Figure 3. A) Examples of the hierarchical gating strategies used for myeloid and lymphoid flow-cytometry. B) Median Fluorescence Intensity analysis of CD206 and MHCII in intra-tumoural macrophages. Flow-cytometric analysis of C) spleen and D) mesenteric lymph nodes. Graphs in B, C and D are representative of 3 independent experiments (n=3-5 animals per condition per experiment).  Figure S3. Tumour infiltrating leucocytes are not influenced by VNMAA treatment in additional BrCa models, related to Figure 3. A) Flow cytometric population differences in tumour infiltrating myeloid (left) and lymphoid (right) leucocytes from VNMAA experiments using EO771 (top) and BRPKp110 (bottom) BrCa tumour models expressed as mean (± s.e.m.) percentage of total viable cells. The EO771 graphs are representative of two independent experiments (n=3-5 animals per condition per experiment) while the BRPKp110 graphs are of one experiment (n=5 animals per condition). B) Flow cytometric population differences in tumour infiltrating lymphocytes and B-cells expressed as mean (± s.e.m.) percentage of total viable cells (representative of 2 independent experiments; n=3-5 animals per condition).  Figure S4. Further examination of tumour stroma and collagen deposition, related to Figure 5. A) Heatmap showing the Log2 fold change in the expression of established marker genes used to identify the cell type clusters specified in Figure 5A and 5B. The stromal population containing mast cells is labelled in red. B) Picro-Sirius Red staining of tumours from VNMAA and control treated animals showing collagen deposition across whole sections. Scale bars = 100µm. Bar blot shows mean (± s.e.m.) percent area stained for collagen (n=5 tumours per condition).  Figure S5. Effect of VNMAA and Cephalexin treatment on caecal metabolite abundances, related to Figure 6. Heatmaps showing significantly different metabolite abundances in caecal samples following a multigroup comparison test (p=0.025, q=0.05) in animals treated with either VNMAA or Cephalexin compared to control. Colour ratio is shown according to Log2 fold change.  Figure S6. Differential abundances of metabolic pathways between Cephalexin treatment and controls, related to Figure 6. Plot displaying differences in median pathway abundances (symbols) and their confidence intervals (lines) as obtained by the Wilcoxon-Mann-Whitney test. The deviation from an estimate of zero (dotted grey line) indicates direction of change with pathways enriched in Cephalexin treatments to the right and pathways enriched in controls to the left side. Colours correspond to the Benjamini-Hochberg adjusted p-value. Dot symbols represent significant p-values (< 0.05) and triangles represent non-significant p-values (> 0.05). ControlVNMAAH&E EndomucinFigure S1A B05-5-10 -5 0 5 10PC 5 (5.2%)PC 1 (41.9%)ControlVNMAACDFigure S2% Total viable cells010203040nsnsnsControlVNMAASplenic lymphoid cellsCD3 CD4 CD8Lymph node lymphoid cells% Total viable cells020406080nsns nsCD3 CD4 CD8ControlVNMAA102345RatioM1:M2 Macrophage MFIControlVNMAAnsCBDACD45+Singlet-liveSSC-ACD45CD11b Ly6G F4/80SSC-ASSC-ALy6CCD11b+ Ly6g+ F4/80+CD4 FOXP3CD3CD19SSC-ACD8CD4CD3CD3+ CD8+ CD4+FOXP3+CD19+Figure S3Intra-tumoural myeloid cells Intra-tumoural lymphoid cellsnsnsnsnsControlVNMAAnsnsnsnsnsnsnsnsnsnsEO771BRPKp110Ly6GABControlVNMAACD45 CD3 CD4 CD8 CD19nsnsns nsns% Total viable cellsFigure S4Slc12a2Wfdc2PtnIgfbp5Wfdc18EpcamLcn2CkbAcp5Slc37a2Mmp9CtskAtp6v0d2Slc9b2Mt3Epsti1Fabp5Serpinb6bCcr7Tbc1d4Ccl5Ccl22Mgl2Cd74Il1r2H2−Ab1H2−AaH2−Eb1Plbd1NapsaIbspGng11MgpLgals7Rbp1Sparcl1Serpinf1Grem1Trbc1LckLef1Cd3eMs4a4bTracCd3dCd3gTrbc2Hspb1MtapAnkCol18a1Mxra8AceSdc2Nupr1Cd37FcmrCd79bIghdEbf1Iglc2Ly6dIghmCd79aIgkcIer3Cxcl2Arg1Cd14Il1bThbs1Ccl6Clec4eSrgnMafF13a1Ccl12Mrc1Ccl2Ccl7FcrlsPf4Stab1Prc1Tubb6Cdk1AnlnBirc5Tubb4bCenpfTop2aHist1h2apUbe2cCol6a1Col3a1Col6a2Igfbp7BgnSerpinh1Col1a1Col1a2PostnSparcHilpdaCcl24TrfC1qbC1qcC1qaMmp13Rgs1Ifitm1ApoeS100a6Plet1S100a10Prl2c3Lamc2Krt8Krt18CluLuminal cell1  Macrophage  Stromal cell  Proliferating cell  Macrophage C1qc high  Macrophage Lyz1 high B cell  Fibroblast  T cell  Stromal cell Col3a1 high  Dendritic cell Cst3 high  Dendritic cell Fscn1 high Stressed cell  Luminal 2cellControl VNMAAABValineLeucineIsoleucineTryptophanGlutamineAlanineAsparagineSucroseRaffinoseGlucose-1-phosphateAspartateNiacinamideStachyoseMaltoseDeoxy cytidine monoPArabinose2’-DeoxyadenosineDeoxythymidine triPGlucoseABX system like citrateDeoxyuridineDimethylamineUrocanateCholineTaurineFumarateValerateUracilHypoxanthineNicotinatePyruvate2-OxoisocaproateRiboseXanthine3-HydroxyphenylacetateAcetateButryatePropionate3-PhenylpropionateTyramineMethylsuccinateMethylaminePutrescineTrimethylamineOrnithine5-AminopentanoateGlutamateCaprylateAdenosine monoP2-MethylbutyrateControlCephalexinVNMAA 0.0-2.0-1.0 1.0 2.0Figure S5water cephalexinP124−PWY: Bifidobacterium shuntMETSYN−PWY: L−homoserine and L−methionine biosynthesisMET−SAM−PWY: superpathway of S−adenosyl−L−methionine biosynthesisPWY66−389: phytol degradationPWY−4321: L−glutamate degradation IVPWY0−162: superpathway of pyrimidine ribonucleotides de novo biosynthesisHOMOSER−METSYN−PWY: L−methionine biosynthesis IGOLPDLCAT−PWY: superpathway of glycerol degradation to 1,3−propanediolPWY−7209: superpathway of pyrimidine ribonucleosides degradationGLUDEG−I−PWY: GABA shuntPWY−6859: all−trans−farnesol biosynthesisLACTOSECAT−PWY: lactose and galactose degradation IPWY−241: C4 photosynthetic carbon assimilation cycle, NADP−ME typePWY−922: mevalonate pathway IPWY−5910: superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate)PWY0−1241: ADP−L−glycero−&beta;−D−manno−heptose biosynthesisCITRULBIO−PWY: L−citrulline biosynthesisPWY0−1061: superpathway of L−alanine biosynthesisHEME−BIOSYNTHESIS−II: heme biosynthesis I (aerobic)PWY−5189: tetrapyrrole biosynthesis II (from glycine)PWY−5840: superpathway of menaquinol−7 biosynthesisPWY−6519: 8−amino−7−oxononanoate biosynthesis IPWY−7664: oleate biosynthesis IV (anaerobic)PWY0−862: (5Z)−dodec−5−enoate biosynthesisPWY−5838: superpathway of menaquinol−8 biosynthesis IPWY−5863: superpathway of phylloquinol biosynthesisPWY−7115: C4 photosynthetic carbon assimilation cycle, NAD−ME typeFASYN−ELONG−PWY: fatty acid elongation −− saturatedP23−PWY: reductive TCA cycle IPWY−6531: mannitol cyclePWY−7210: pyrimidine deoxyribonucleotides biosynthesis from CTPPWY−5897: superpathway of menaquinol−11 biosynthesisPWY−5898: superpathway of menaquinol−12 biosynthesisPWY−5899: superpathway of menaquinol−13 biosynthesisPROPFERM−PWY: L−alanine fermentation to propanoate and acetatePWY−6895: superpathway of thiamin diphosphate biosynthesis IIPWY−6263: superpathway of menaquinol−8 biosynthesis IIPWY−7316: dTDP−N−acetylviosamine biosynthesisALLANTOINDEG−PWY: superpathway of allantoin degradation in yeastPWY−5861: superpathway of demethylmenaquinol−8 biosynthesisPWY−5918: superpathay of heme biosynthesis from glutamatePWY−7388: octanoyl−[acyl−carrier protein] biosynthesis (mitochondria, yeast)HEMESYN2−PWY: heme biosynthesis II (anaerobic)PWY−6470: peptidoglycan biosynthesis V (&beta;−lactam resistance)P4−PWY: superpathway of L−lysine, L−threonine and L−methionine biosynthesis IPWYG−321: mycolate biosynthesisPWY−5791: 1,4−dihydroxy−2−naphthoate biosynthesis II (plants)PWY−5837: 1,4−dihydroxy−2−naphthoate biosynthesis IPWY−6282: palmitoleate biosynthesis I (from (5Z)−dodec−5−enoate)PWY0−781: aspartate superpathwayP185−PWY: formaldehyde assimilation III (dihydroxyacetone cycle)REDCITCYC: TCA cycle VIII (helicobacter)PWY66−367: ketogenesisPWY−6891: thiazole biosynthesis II (Bacillus)FASYN−INITIAL−PWY: superpathway of fatty acid biosynthesis initiation (E. coli)BIOTIN−BIOSYNTHESIS−PWY: biotin biosynthesis IPWY−5265: peptidoglycan biosynthesis II (staphylococci)CRNFORCAT−PWY: creatinine degradation IPWY−5989: stearate biosynthesis II (bacteria and plants)PWY0−1415: superpathway of heme biosynthesis from uroporphyrinogen−IIIFOLSYN−PWY: superpathway of tetrahydrofolate biosynthesis and salvagePWY−5723: Rubisco shuntPOLYAMSYN−PWY: superpathway of polyamine biosynthesis IARG+POLYAMINE−SYN: superpathway of arginine and polyamine biosynthesisPWY66−409: superpathway of purine nucleotide salvagePWY−6612: superpathway of tetrahydrofolate biosynthesisPWY−7196: superpathway of pyrimidine ribonucleosides salvagePRPP−PWY: superpathway of histidine, purine, and pyrimidine biosynthesisMETHGLYUT−PWY: superpathway of methylglyoxal degradationPOLYAMINSYN3−PWY: superpathway of polyamine biosynthesis IIPWY−7200: superpathway of pyrimidine deoxyribonucleoside salvagePWY−7254: TCA cycle VII (acetate−producers)PWY−4984: urea cycleFUCCAT−PWY: fucose degradationPWY−7090: UDP−2,3−diacetamido−2,3−dideoxy−&alpha;−D−mannuronate biosynthesisPWY−6588: pyruvate fermentation to acetonePWY−7288: fatty acid &beta;−oxidation (peroxisome, yeast)PWY66−391: fatty acid &beta;−oxidation VI (peroxisome)PWY−7332: superpathway of UDP−N−acetylglucosamine−derived O−antigen building blocks biosynthesisPWY66−398: TCA cycle III (animals)PWY−6471: peptidoglycan biosynthesis IV (Enterococcus faecium)P161−PWY: acetylene degradationPWY0−1298: superpathway of pyrimidine deoxyribonucleosides degradationPWY4LZ−257: superpathway of fermentation (Chlamydomonas reinhardtii)PWY−5138: unsaturated, even numbered fatty acid &beta;−oxidationPWY0−1297: superpathway of purine deoxyribonucleosides degradationPWY0−1479: tRNA processingPWY−6953: dTDP−3−acetamido−3,6−dideoxy−&alpha;−D−galactose biosynthesisPWY−5005: biotin biosynthesis IIPWY−621: sucrose degradation III (sucrose invertase)PWY−5690: TCA cycle II (plants and fungi)P441−PWY: superpathway of N−acetylneuraminate degradationPWY−6749: CMP−legionaminate biosynthesis I−1e−05 0e+00 1e−05EstimatePathway0.000.250.500.75p_value.wilcoxon.adjustedsignificance<0.05>0.05C l iControlFigure S6

Antibiotic-induced disturbances of the gut microbiota result in accelerated breast tumor growth

https://digitalcommons.wustl.edu/cgi/viewcontent.cgi?filename=0&article=12385&context=open_access_pubs&type=additional

Antibiotic-induced disturbances of the gut microbiota result in accelerated breast tumor growth

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