Name: experimental generation of carcinoma-associated fibroblasts (cafs) from human mammary fibroblasts
Uploaded: 2011-10-25T12:00:00
Description: Watch this Scientific Journal Video about Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts at JoVE.com

Vi takker Dr. Robert A. Weinberg (Whitehead Institute for Biomedical Research, Cambridge) for generøs støtte og oppfølging av dette arbeidet og Mr. Kieran Mellody (University of Manchester, Manchester) for kritisk redigering av dette manuskriptet. Dette prosjektet ble støttet av Forskning Storbritannia (CR-britiske) stipend nummer C147/A6058 (AO)

1. Isolering av primære cultured human normal mammary fibroblaster Eksperimentelle prosedyrer for å isolere primære dyrkede humane normale mammary fibroblaster er skissert i fig. 1A. <ol><li> Forbered cellen dissosiasjon buffer, som beskrevet tidligere 12: Dulbecco modifiserte Eagle er Medium (DMEM) med 10% kalvefoster serum (FCS), penicillin-streptomycin (200 enheter / ml), collagenase type I (1 mg / ml) og Hyaluronidase ( 125 enheter / ml). </li><li> Vask brystvev dissekert...

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	<li>Ronnov-Jessen, L., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+by+proxy:+can+the+microenvironment+be+both+the+cause+and+consequence?.">Breast cancer by proxy: can the microenvironment be both the cause and consequence?.</a> Trends Mol. Med. 15, 5-13 (2009).</li><li>Mueller, M. M., Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friends+or+foes+-+bipolar+effects+of+the+tumour+stroma+in+cancer.">Friends or foes - bipolar effects of the tumour stroma in cancer.</a> Nat. Rev. Cancer. 4, 839-849 (2004).</li><li>Bhowmick, N. A., Neilson, E. G., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer+initiation+and+progression.">Stromal fibroblasts in cancer initiation and progression.</a> Nature. 432, 332-337 (2004).</li><li>Kalluri, R., Zeisberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+cancer.">Fibroblasts in cancer.</a> Nat. Rev. Cancer. 6, 392-401 (2006).</li><li>De Wever, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenascin-C+and+SF/HGF+produced+by+myofibroblasts+in+vitro+provide+convergent+pro-invasive+signals+to+human+colon+cancer+cells+through+RhoA+and+Rac.">Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac.</a> 18, 1016-1018 (2004).</li><li>Serini, G., Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+myofibroblast+activity+and+phenotypic+modulation.">Mechanisms of myofibroblast activity and phenotypic modulation.</a> Exp. Cell. Res. 250, 273-283 (1999).</li><li>Shimoda, M., Mellody, K. T., Orimo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinoma-associated+fibroblasts+are+a+rate-limiting+determinant+for+tumour+progression.">Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression.</a> Semin. Cell. Dev. Biol. 21, 19-25 (2010).</li><li>Pietras, K., Ostman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+interactions+with+the+tumor+stroma.">Hallmarks of cancer: interactions with the tumor stroma.</a> Exp. Cell. Res. 316, 1324-1331 (2010).</li><li>Polyak, K., Haviv, I., Campbell, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-evolution+of+tumor+cells+and+their+microenvironment.">Co-evolution of tumor cells and their microenvironment.</a> Trends in Genetics. 25, 30-38 (2009).</li><li>Franco, O. E., Shaw, A. K., Strand, D. W., Hayward, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+associated+fibroblasts+in+cancer+pathogenesis.">Cancer associated fibroblasts in cancer pathogenesis.</a> Semin. Cell. Dev. Biol. 21, 33-39 (2010).</li><li>Kojima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+TGF-beta+and+stromal+cell-derived+factor-1+(SDF-1)+signaling+drives+the+evolution+of+tumor-promoting+mammary+stromal+myofibroblasts.">Autocrine TGF-beta and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 20009-20014 (2010).</li><li>Orimo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+present+in+invasive+human+breast+carcinomas+promote+tumor+growth+and+angiogenesis+through+elevated+SDF-1/CXCL12+secretion.">Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.</a> Cell. 121, 335-348 (2005).</li></ol>

Mangelen på CAF-spesifikke markører og graden av heterogenitet observert blant kafeer gjengi karakterisering av denne cellen typen en utfordring i seg selv. Studerer kafeer in vitro har også blitt hindret av den ekstra komplikasjon som disse cellene senesce og stoppe proliferating når kultivert for en lang periode. Våre tidligere forsøk på å direkte immortalise primære kafeer bruke en retrovirale hTERT cDNA konstruere var mislykket. Derfor, for å ytterligere undersøke svulsten fremme egenskapene til ...

<table border="1"><tr><td> Material Name </td><td> Type </td><td> Firma </td><td> Katalog nummer </td><td> Kommentar </td></tr><tr><td> DMEM </td><td></td><td> Invitrogen </td><td> 61965-026 </td><td></td></tr><tr><td> Kalvefoster serum </td><td></td><td> GIBCO </td><td> 10270 </td><td></td></tr><tr><td> Penicillin-streptomycin </td><td></td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Collagenase type I </td><td></td><td> Sigma </td><td> C0130-1G </td><td></td></tr><tr><td> Hyaluronidase </td><td></td><td> Sigma </td><td> H4272 </td><td></td></tr><tr><td> Vimentin (V9) antistoff </td><td></td><td> Novocastra Laboratories </td><td> NCL-L- VIM-V9 </td><td></td></tr><tr><td> Tenascin C (BC-8) antistoff </td><td></td><td></td><td></td><td> en gave fra Dr. <strong&gt; Luciano Zardi </td></tr><tr><td> α-SMA-Cy3 (1A4) antistoff </td><td></td><td> Sigma </td><td> C6198 </td><td></td></tr><tr><td> Prolyl-4-hydroksylase </td><td></td><td> Dako </td><td> M0877 </td><td></td></tr><tr><td> (5B5) antistoff </td><td></td><td></td><td></td><td></td></tr><tr><td> Kollagen type1 1A antistoff </td><td></td><td> Sigma </td><td> HPA011795 </td><td></td></tr><tr><td> Pan-cytokeratin antistoff </td><td></td><td> Sigma </td><td> C5992 </td><td></td></tr><tr><td> Fibronektin antistoff </td><td></td><td> BD Biosciences </td><td> 610077 </td><td></td></tr><tr><td> S100A4/FSP-1 (fibroblast- spesifikke protein-1) antistoff </td><td></td><td> Dako </td><td> A5114 </td><td></td></tr><tr><td> Fibroblast overflate protein (Klone 1B10) antistoff </td><td></td><td> Abcam </td><td> ab11333 </td><td></td> &lt;/ Tr&gt; <tr><td> MSCV-IRES-GFP konstruere </td><td></td><td></td><td></td><td> Henvendelse til forfatterne </td></tr><tr><td> pBabe-Puro konstruere </td><td></td><td></td><td></td><td> Kjøp fra Addgene </td></tr><tr><td> Puromycin </td><td></td><td> Sigma </td><td> P8833 </td><td></td></tr><tr><td> DAPI </td><td></td><td> Sigma </td><td> D9564 </td><td></td></tr><tr><td> 15 ml konisk tube </td><td></td><td> Corning </td><td> 430766 </td><td></td></tr><tr><td> Nude mus </td><td></td><td> Taconic </td><td> NCRNU-F </td><td> Kvinne NCR nude </td></tr><tr><td> C3H/10T1/2 celler </td><td></td><td> ATCC </td><td></td><td> CCL-226 </td></tr></table>

experimental generation of carcinoma-associated fibroblasts (cafs) from human mammary fibroblasts

Karsinomer er komplekse vev består av neoplastiske celler og en ikke-cancerous kupé referert til som &quot;stroma &#39;. Den stroma består av ekstracellulære matrix (ECM) og en rekke mesenchymalceller, inkludert fibroblaster, myofibroblasts, endotelceller, pericytes og leukocytter 1-3. Svulsten-assosiert stroma er lydhør overfor betydelige parakrine signaler utgitt av nabolandet carcinoma celler. Under sykdommen prosessen, stroma ofte blir befolket av carcinoma-assosiert fibroblaster (kafeer), inkludert et stort antall myofibroblasts. Disse cellene har tidligere vært hentet fra mange forskjellige typer av humant karsinom for sine in vitro kultur. En undergruppe kafeer er gjenkjennelig gjennom sine oppregulering av α-glatt muskel aktin (α-SMA) uttrykk 4,5. Disse cellene er et kjennetegn på &#39;aktivert fibroblaster&#39; som deler lignende eiendommer med myofibroblasts commonly observert i skadet og fibrotiske vev 6. Tilstedeværelsen av denne myofibroblastic CAF subset er sterkt knyttet til høyverdig malignitet og assosiert med dårlig prognose hos pasienter. Mange laboratorier, inkludert vår egen, har vist at kafeer, når injisert med carcinoma celler i immunodeficient mus, er i stand til vesentlig å fremme tumourigenesis 7-10. Kafeer forberedt fra carcinoma pasienter imidlertid ofte gjennomgå senescence under forplantning i kultur begrense utbredelsen av bruken gjennom pågående eksperimentering. For å overvinne denne vanskeligheten, har vi utviklet en ny teknikk for å eksperimentelt generere udødeliggjort menneskelige mammary CAF cellelinjer (exp-kafeer) fra human mammary fibroblaster, ved hjelp av en coimplantation brystsvulst xenograft modell. For å generere exp-kafeer, foreldrenes menneskelige mammary fibroblaster, hentet fra reduksjonen mammoplasty vev, ble først immortaliSED med hTERT, den katalytiske subenhet av telomerase holoenzyme, og konstruert for å uttrykke GFP og en puromycin motstand genet. Disse cellene ble coimplanted med MCF-7 menneskelige brystkreft celler uttrykker en aktivert ras onkogen (MCF-7-ras celler) inn i en mus xenograft. Etter en periode med inkubasjon in vivo ble først injisert menneskelige mammary fibroblaster hentet fra svulsten xenografts på grunnlag av deres puromycin motstand 11. Vi observerte at beboeren menneskelige mammary fibroblaster har differensiert, vedta en myofibroblastic fenotype og ervervet tumor-fremme egenskaper i løpet av tumor progresjon. Viktigere, disse cellene, definert som exp-kafeer, tett etterligner tumor-fremme myofibroblastic fenotype av kafeer isolert fra brystcarsinomer dissekert fra pasienter. Våre tumor xenograft-avledet exp-kafeer derfor gi en effektiv modell for å studere biologi kafeer i menneskelig breast karsinomer. De beskrevne Protokollen kan også utvides for å generere og karakterisere ulike CAF populasjoner avledet fra andre typer av humant karsinomer.

Watch this Scientific Journal Video about Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts at JoVE.com

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Karsinom-assosiert fibroblaster (kafeer) rik på myofibroblasts stede i tumor stroma, spille en viktig rolle i å drive tumor progresjon. Vi utviklet en coimplantation svulst xengraft modell for eksperimentelt generere kafeer fra menneskelige mammary fibroblaster. Protokollen beskriver hvordan å etablere CAF myofibroblasts at få en evne til å fremme tumourigenesis.

Eksperimentell Generation of Karsinom-Associated Fibroblaster (kafeer) fra Human mammary Fibroblaster

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

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Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

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