Name: a preclinical mouse model of osteosarcoma to define the extracellular vesicle-mediated communication between tumor and mesenchymal stem cells
Uploaded: 2018-05-06T12:30:00
Description: Watch this Scientific Journal Video about A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells at JoVE.com

Løping Baglio ble støttet av et fellesskap av Associazione Italiana per la Ricerca sul Cancro (AIRC) delfinansiert av EU. I tillegg har dette prosjektet mottatt støtte fra EUs horisonten 2020 forskning og innovasjon programmet under Marie Sklodowska-Curie grant avtalen ingen 660200 (til SR Baglio).

Menneskelig liggende under adipose vev mesenchymal stamceller isolering var Hentet fra department of Plastisk kirurgi i Tergooi sykehus (Hilversum, Nederland) etter godkjenning av institusjonelle etiske komiteen og skriftlig samtykke. GFP-positive adipose MSCs ble innhentet fra Institutt for medisinsk og kirurgisk Sciences for barn og voksne (Universitetet i Modena og Reggio Emilia).
Dyreforsøk ble utført i henhold til nederlandsk dyr eksperimentering, og protokollen ble godkjent av komiteen...

<ol>
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S., Peinado, H., Lyden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+in+cancer:+Cell-to-cell+mediators+of+metastasis.">Extracellular vesicles in cancer: Cell-to-cell mediators of metastasis.</a> Cancer Cell. 30 (6), 836-848 (2016).</li><li>Skog, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+microvesicles+transport+RNA+and+protein+that+promote+tumor+growth+and+provide+diagnostic+biomarkers.">Glioblastoma microvesicles transport RNA and protein that promote tumor growth and provide diagnostic biomarkers.</a> Nat Cell Biol. 10 (12), 1470-1476 (2008).</li><li>Peinado, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanoma+exosomes+educate+bone+marrow+progenitor+cells+toward+a+pro-metastatic+phenotype+through+MET.">Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.</a> Nat Med. 18 (6), 883-891 (2012).</li><li>Zomer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+reveals+extracellular+vesicle-mediated+phenocopying+of+metastatic+behavior.">In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior.</a> Cell. 161 (5), 1046-1057 (2015).</li><li>Webber, J., Steadman, R., Mason, M. D., Tabi, Z., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+exosomes+trigger+fibroblast+to+myofibroblast+differentiation.">Cancer exosomes trigger fibroblast to myofibroblast differentiation.</a> Cancer Res. 70 (23), 9621-9630 (2010).</li><li>Webber, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+tumour-promoting+stromal+myofibroblasts+by+cancer+exosomes.">Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes.</a> Oncogene. 34 (3), 290-302 (2015).</li><li>Zhou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-secreted+miR-105+destroys+vascular+endothelial+barriers+to+promote+metastasis.">Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis.</a> Cancer Cell. 25 (4), 501-515 (2014).</li><li>Whiteside, T., Anastasopoulou, E., Voutsas, I., Papamichail, M., Perez, S., Nunes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+tumor-mediated+immune+suppression.">Exosomes and tumor-mediated immune suppression.</a> Expert Rev Mol Diagn. 15 (10), 1293-1310 (2016).</li><li>Verweij, F. J., Van Eijndhoven, M. A. J., Middeldorp, J., Pegtel, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+viral+microRNA+exchange+via+exosomes+in+vitro+and+in+vivo.">Analysis of viral microRNA exchange via exosomes in vitro and in vivo.</a> Methods Mol Biol. 1024, 53-68 (2013).</li><li>Baglio, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+marrow-+and+adipose-mesenchymal+stem+cells+secrete+exosomes+enriched+in+distinctive+miRNA+and+tRNA+species.">Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species.</a> Stem Cell Res Ther. 6 (1), 127 (2015).</li><li>Naaijkens, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+as+a+fetal+bovine+serum+substitute+improves+human+adipose-derived+stromal+cell+culture+for+future+cardiac+repair+applications.">Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications.</a> Cell Tissue Res. 348 (1), 119-130 (2012).</li><li>Grisendi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+mesenchymal+stem+cells+as+stable+source+of+tumor+necrosis+factor-related+apoptosis-inducing+ligand+delivery+for+cancer+therapy.">Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy.</a> Cancer Res. 70 (9), 3718-3729 (2010).</li><li>Cosette, J., Abdelwahed, R. B., Donnou-Triffault, S., Saut&#232;s-Fridman, C., Flaud, P., Fisson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence-based+tumor+quantification+method+for+monitoring+tumor+progression+and+treatment+effects+in+mouse+lymphoma+models.">Bioluminescence-based tumor quantification method for monitoring tumor progression and treatment effects in mouse lymphoma models.</a> J Vis Exp. (113), (2016).</li><li>Carbone, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+cervical+dislocation+as+a+humane+euthanasia+method+in+mice.">Assessing cervical dislocation as a humane euthanasia method in mice.</a> J Am Assoc Lab Anim Sci. 51 (3), 352-356 (2012).</li><li>Costa-Silva, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer+exosomes+initiate+pre-metastatic+niche+formation+in+the+liver.">Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.</a> Nat Cell Biol. 17 (6), 816-826 (2015).</li><li>Hoshino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+exosome+integrins+determine+organotropic+metastasis.">Tumour exosome integrins determine organotropic metastasis.</a> Nature. 527 (7578), 329-335 (2015).</li><li>Clayton, A., Mitchell, J. P., Court, J., Mason, M. D., Tabi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tumor-derived+exosomes+selectively+impair+lymphocyte+responses+to+interleukin-2.">Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2.</a> Cancer Res. 67 (15), 7458-7466 (2007).</li><li>Wieckowski, E. U., Visus, C., Szajnik, M., Szczepanski, M. J., Storkus, W. J., Whiteside, T. 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Svulst-utskilles ekstracellulære blemmer (EVs) kan endre fysiologi av lokale og fjerntliggende mesenchymal celler til å generere en svulst-støttende miljø. Her beskriver vi generering av en prekliniske musemodell osteosarcoma som lar Disseksjon av EV-mediert samspillet mellom kreftceller og mesenchymal stamceller (MSCs) i vivo. Vi viser at systemisk injeksjon av menneskelig svulst EV-utdannede MSCs i mus med osteosarcoma xenografts sterkt fremmer kreft vekst og metastasering dannelse av IL-6/STAT3 signalnett...

Svulst microenvironment deltar aktivt i de fleste, om ikke alle aspekter av tumorigenesis og kreft progresjon, inkludert metastasering formasjon og utvikling av resistens mot therapeutics1. Dette understreker behovet for prekliniske orthotopic kreft musen modeller at Disseksjon av komplekse svulst-stroma samhandlinger som oppstår i svulst nisje.
I mange mobilnettet komponentene i svulst microenvironment bidra stamceller (MSCs) sterkt til kreft progresjon i flere typer kreft som brystkreft, prostatakreft, hjernesvulster, myelom og osteosarcoma2 ,,3,,4,,5,,6,,7. MSCs er multipotent stamceller som finnes i ulike voksne og fosterets vev, inkludert beinmargen, fettvev, morkaken, navlestrengsblod og andre8,9. Svar på kreft-generert inflammatorisk signaler, MSCs migrere mot svulst nettsteder, innlemme i svulst microenvironment og til slutt skille ut støtte for kreft celler10. Disse kreft-assosiert MSCs gir viktige faktorer (dvs. vekstfaktorer, chemokines, cytokiner og suppressive meglere) for svulst progresjon opptrer både kreftceller og de omkringliggende stroma2, 3 , 11 , 12 , 13. mens svulst-fremme effekter av kreft-assosiert MSCs har vært undersøkt i mange kreft modeller, mekanismer som kreftceller omprogrammere MSCs å forme en kreft-fremme nisje er dårlig forstått. Her beskriver vi generering av en orthotopic xenograft modell som tillater studiet av pro-tumorigenic samspillet mellom bein kreftceller og MSCs via ekstracellulære blemmer (EVs).
EVs er avgjørende meglere intercellulære kommunikasjon mellom svulst og stromal celler14. EVs bære funksjonelle biomolecules av cellen for opprinnelse, inkludert proteiner og lipider forskrifter RNAs. Når utgitt i ekstracellulære plass, disse vesikler kan tas av omkringliggende celler eller gjennomført til fjerntliggende områder via blodet eller lymfesystemet sirkulasjon, og kan dypt påvirker målet cellen atferd. 15 , 16 , 17 For eksempel opptak av kreft EVs av stromal fibroblaster kan resultere i myofibroblast differensiering støtte angiogenese og akselererende tumor vekst i vivo18,19, internalisering av endothelial cellene kan stimulere svulst angiogenese og øke vaskulær permeabilitet16,20, og interaksjon med immunceller kan føre til undertrykkelse av antitumor immunrespons21.
Vi sist viste, med en bioluminescent orthotopic xenograft musemodell av osteosarcoma, at kreftceller løslate store mengder EVs som ber MSCs til å erverve en pro-tumorigenic og pro-metastatisk fenotypen. Denne effekten av en dramatisk endring i MSC cytokin profilen for expression (referert til som "MSC utdanning"), og kan forebygges ved administrasjonen av en terapeutisk interleukin-6 reseptor (IL-6R) antistoff7. Vårt arbeid har vist at kreft EVs er avgjørende modulatorer MSC atferd, gir en begrunnelse for microenvironment målrettede tilnærminger til stoppe osteosarcoma progresjon. Her beskriver vi en trinnvis protokoll for å undersøke den EV-mediert svulst-MSC samhandling i vivo. Denne modellen er beregnet til: 1) spesielt definere kreft EV-induserte endringer av MSC i svulst microenvironment, 2) vurdere hvordan denne samhandlingen bidrar til bone tumor vekst og metastasering dannelse, og 3) studie om forstyrrer EV-mediert crosstalk i vivo hindrer kreft progresjon.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Equipment</td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Ultra Centrifuge</td>
			<td >Beckman</td>
			<td >Optima L-90K</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Rotor SW32Ti</td>
			<td >Beckman</td>
			<td >369650</td>
			<td colspan="4">Referred to in the manuscript as ultra-swinging bucket rotor</td>
			<td></td>
		</tr>
		<tr >
			<td >Transmission electron microscope</td>
			<td >Zeiss</td>
			<td >EM109</td>
			<td>Or similar TEM</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Digital camera</td>
			<td >Nikon</td>
			<td >DMX 1200F</td>
			<td>Or similar camera</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Imaging software TEM&#160;</td>
			<td >Nikon</td>
			<td >ACT-1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescence microscope</td>
			<td >Zeiss</td>
			<td >Imager.D2</td>
			<td colspan="2">Or similar Fluorescence microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Imaging software FM</td>
			<td>Zeiss</td>
			<td >ZEN Blue</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Incubator</td>
			<td >Nuaire</td>
			<td >4750E</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Hettick</td>
			<td >ROTANTA 460R</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >-80 Freezer</td>
			<td >Thermo electro corporation</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >FACS</td>
			<td >BD</td>
			<td >BD FACScalibur</td>
			<td>Or similar flow cytometer</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Drill</td>
			<td >Ferm</td>
			<td >FCT-300</td>
			<td>With 0.8 mm drill</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >HSS micro twist drills, 0.8 mm</td>
			<td >Proxxon</td>
			<td >28 852</td>
			<td>0.8 mm drill</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >IVIS camera</td>
			<td >Xenogen</td>
			<td >Ivis Lumina</td>
			<td colspan="5">Referred to in the manuscript as bioluminescence camera. Xenogen is now part of Perkin Elmer</td>
		</tr>
		<tr >
			<td >Living image software2.60</td>
			<td >Xenogen / Igor Por</td>
			<td >n.a</td>
			<td colspan="2">Xenogen is now part of Perkin Elmer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >10 &#181;L Syringe</td>
			<td >Hamilton</td>
			<td >Neuros Model 1701 RN</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Needle: Hamilton RN Needle for Syringe, 26 Gauge, Pointstyle AS, custom length 2 cm</td>
			<td >Hamilton</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Caliper</td>
			<td >Mitutoyo</td>
			<td >G08004463</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Autoclave</td>
			<td >Astell</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Heat Lamp</td>
			<td >Philips</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Culture media</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >Hyclone</td>
			<td >RYG35912</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Platelet Lysate</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >IMDM medium</td>
			<td >Lonza</td>
			<td >BE12-722F</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >alpha-MEM medium</td>
			<td >Lonza</td>
			<td >BE02-002F</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >DMEM medium</td>
			<td >Lonza</td>
			<td>BE12-614F</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >pen/strep/glutamine</td>
			<td >GIBCO</td>
			<td >10378-016</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >heparin</td>
			<td >LEO</td>
			<td >012866-08</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin/EDTA (10x)</td>
			<td >GIBCO</td>
			<td >15400-054</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Cells</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >adipose deriverd MSCs</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >GFP-positive MSCs</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >human fibroblasts</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >143B cells</td>
			<td >ATCC</td>
			<td >CRL-8303</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >FLUC-143B cells</td>
			<td >ATCC</td>
			<td >CRL-8303</td>
			<td>Transduced</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Disposables</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Culture flasks 175 cm2</td>
			<td >CELLSTAR</td>
			<td >660175</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >50 mL tubes</td>
			<td >Greiner bio-one</td>
			<td >210261</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Freeze tubes</td>
			<td >Thermoscientific</td>
			<td >377224</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Ultra-Clear tubes</td>
			<td >Beckman</td>
			<td >344058</td>
			<td colspan="4">Referred to in the manuscript as ultra-centrifuge tubes</td>
			<td></td>
		</tr>
		<tr >
			<td >0,22 &#181;m filter</td>
			<td >Millex</td>
			<td >SLGV033RS</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >200 mesh Formvar-carbon-coated nickel grids</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >0.5 mL insulin syringes with 29G Needle</td>
			<td >Terumo</td>
			<td >U-100&#160;</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td >Sigma - Aldrich</td>
			<td >P7612</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Filter paper&#160;</td>
			<td >Thermo fisher Scientific</td>
			<td >50363215</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Reagents / kits</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >paraformaldehyde</td>
			<td >Alfa Aeser</td>
			<td >43368.9M</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Braun</td>
			<td >220/12257974/110</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >glutaraldehyde</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >16300</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >uranyl oxalate</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >22510</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >urany acetate</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >22400</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >methyl cellulose</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >1560</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >PKH67</td>
			<td >Sigma</td>
			<td >mini67-1kt</td>
			<td colspan="2">Referred to in the manuscript as GFLD</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >BSA</td>
			<td >Sigma</td>
			<td >A8412</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >CBA - human inflammatory cytokine kit</td>
			<td >BD</td>
			<td >551811</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Formaldehyde 37%</td>
			<td >VWR</td>
			<td >104003100</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Carbon Steel surgical blades</td>
			<td >Swann-Morton</td>
			<td >206</td>
			<td colspan="3">Referred to in the manuscript as surgical knife</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >anti-human vimentin antibody</td>
			<td >Santa Cruz</td>
			<td >sc-6260</td>
			<td>Clone V9</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Antibody diluent</td>
			<td >DAKO</td>
			<td >S0809</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >HRP-labeled anti mouse IgG antibody</td>
			<td >Life Technologies</td>
			<td >32230</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >DAB-kit</td>
			<td >DAKO</td>
			<td >K500711</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >hematoxyllin</td>
			<td >Sigma</td>
			<td >GHS232</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >EDTA-buffer</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Citrate buffer</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >rabbit polyclonal anti-GFP antibody</td>
			<td >Abcam</td>
			<td >n.a.</td>
			<td>Ab290</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI&#160;</td>
			<td >Life Technologies</td>
			<td >D1306</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Paracetamol, 120 mg / 5 ml syrup</td>
			<td >Bayer</td>
			<td >n.a.</td>
			<td colspan="2">Sinaspril, paracetamol solution for kids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane 1000 mg/g</td>
			<td >Vumc pharmacy</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >buprenofine hydrochloride, 0.3 mg/ml</td>
			<td >Indivior UK Limited</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >lidocaine-HCL 2%</td>
			<td >Vumc pharmacy</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >70% ethanol</td>
			<td >VWR</td>
			<td >93003.1006</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Tissue glue</td>
			<td >Derma+Flex, formulated medical cyanoacrylate</td>
			<td >Vygon</td>
			<td>LB604060</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Eyedrops: Vidisec Carbogel, 2 mg/ml</td>
			<td >Bausch+Lomb</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >D-luciferin, potassium salt</td>
			<td >Gold Biotechnology</td>
			<td >LUCK-1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Glass slides</td>
			<td >Thermo scientific</td>
			<td >630-0954</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Stainless steel loops&#160;</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Mice experiments</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Mice, Hsd:Athymic Nude-Foxn1nu,&#160; female, 6 weeks at arrival, bacterial status conform FELASA</td>
			<td >ENVIGO</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Paper-pulp smart home (cage enrichment)</td>
			<td >Bio Services</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Alpha-dri bedding material</td>
			<td >Shepperd Speciality Papers</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse food: Teklad global 18% protein rodent diet</td>
			<td >ENVIGO</td>
			<td >2918-11416M</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Sutures</td>
			<td >Ethicon</td>
			<td >V926H</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td >Sigma-Aldrich</td>
			<td >S3146-1EA</td>
			<td>(or similar)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Tweezers</td>
			<td >Sigma-Aldrich</td>
			<td >F4142-1EA</td>
			<td>(or similar)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a preclinical mouse model of osteosarcoma to define the extracellular vesicle-mediated communication between tumor and mesenchymal stem cells

I svulst microenvironment bidra bosatt eller rekruttert stamceller (MSCs) til ondartede progresjon i flere typer kreft. Under påvirkning av bestemte Miljøsignaler, kan disse voksen stamceller frigi paracrine meglere fører til akselerert tumor vekst og metastasering. Definere crosstalk mellom svulst og MSCs er av primær betydning å forstå mekanismene bak kreft progresjon og identifisere romanen mål for terapeutisk intervensjon.
Kreftceller produserer store mengder ekstracellulære blemmer (EVs), som kan dypt påvirke atferden til målcellene i svulst microenvironment eller i fjerntliggende områder. Svulst EVs setter funksjonelle biomolecules, inkludert inflammatorisk RNAs og (onco) proteiner, som kan utdanne stromal celler å forbedre metastatisk virkemåten av kreftceller eller delta i pre metastatisk nisje-formasjonen. I denne artikkelen beskrive vi utviklingen av en prekliniske kreft musemodell som aktiverer bestemte evaluering av EV-mediert crosstalk mellom svulst og mesenchymal stamceller. Først beskriver vi rensing og karakterisering av svulst-utskilles EVs og vurdering av EV internalisering av MSCs. Vi da gjøre bruk av en multiplex perle-baserte immunanalyse å vurdere endring av MSC cytokin uttrykk profilen indusert av kreft EVs. Til slutt, vi illustrere generering av en bioluminescent orthotopic xenograft musemodell av osteosarcoma som viser svulst-MSC samspillet og Vis bidrag av EV-utdannede MSCs tumor vekst og metastasering formasjonen.
Vår modell gir mulighet til å definere hvordan kreft EVs forme en svulst-støtte miljø, og vurdere om blokaden av EV-mediert kommunikasjon mellom svulst og MSCs hindrer kreft progresjon.

I denne studien utforsket vi evne til osteosarcoma-utskilles EVs å utdanne MSCs til et pro-tumorigenic og pro-metastatisk fenotypen. Vi viser at osteosarcoma celler slipper exosome-lignende EVs som er internalisert MSCs. Vi målt endring av MSC cytokin profilen for expression av kreft EVs og vurdert effekten av EV-utdannede MSCs på tumor vekst og metastasering formasjonen. Den generelle representasjonen av studien design er illustrert i figur 1.
<p clas...

Watch this Scientific Journal Video about A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells at JoVE.com

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

En prekliniske musemodell Osteosarcoma definere den ekstracellulære Vesicle-mediert kommunikasjonen mellom svulst og Mesenchymal stamceller

Direkte injeksjon av kreft-avledet ekstracellulære blemmer (EVs) fører til omprogrammering av benmarg støtte svulst progresjon; men er hvilke celler som megle denne effekten uklart. Her beskriver vi en trinnvis protokoll for å undersøke EV-mediert svulst-mesenchymal stamceller (MSC) interaksjoner i vivo, avslører en avgjørende rolle for EV-utdannede MSCs i metastasering.

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

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Cancer Research

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Utvikling og vedlikehold av en Preklinisk Pasient Avledet Tumor Xenotransplantat Modell for Undersøkelse av Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

En Mimic av svulstens mikromiljø: En enkel metode for å generere beriket celle populasjoner og Gransker inter Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

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

Colon-26 karsinom Tumor-bærende mus som en modell for studier av kreft kakeksi

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

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

Isolering av sirkulerende tumorceller i en ortotopisk musemodell av kolorektal kreft

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

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields)

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields TTFields

Bestemme Optimal Hemmende Frekvens for kreftceller Bruke Tumor Behandling av Fields (TTFields)

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), ...

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Analysere kommunikasjonen mellom monocytter og primære brystkreft celler i en ekstracellulær Matrix ekstrakt (ECME)-basert tredimensjonale System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

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

En metode for å definere effekten av miljømessig berikelse på kolon Microbiome biologisk mangfold i en mus kolon svulst modell

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

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Modellering Osteosarcoma bruker Li-Fraumeni syndrom pasient-avledede Induced Pluripotent stamceller

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Tredimensjonale bein ekstracellulær Matrix modell for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Fange små molekyl kommunikasjon mellom vev og celler ved hjelp av Imaging massespektrometri

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...