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 generøse støtte og tilsyn med dette arbejde og Mr. Kieran Mellody (University of Manchester, Manchester) for kritisk redigering af dette manuskript. Dette projekt blev støttet af Research Storbritannien (CR-britisk) indrømme nummer C147/A6058 (AO)

1. Isolering af primær dyrkede humane normale brystkirtler fibroblaster Eksperimentelle procedurer til at isolere primære dyrkede humane normale brystkirtler fibroblaster er skitseret i Fig. 1A. <ol><li> Forbered cellen dissociation buffer, som beskrevet tidligere 12: Dulbecco ændrede Eagle er Medium (DMEM) med 10% føtalt kalveserum (FCS), penicillin-streptomycin (200 enheder / ml), collagenase type I (1 mg / ml) og hyaluronidase ( 125 enheder / ml). </li><li> Vask brystvæv...

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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>

Manglen på CAF-specifikke markører og graden af ​​heterogenitet observeret blandt cafeer gøre karakteriseringen af ​​denne celle type, en udfordring i sig selv. At studere cafeer in vitro har også været hæmmet af den ekstra komplikation, at disse celler senesce og stoppe prolifererende når dyrkes i en længere periode. Vores tidligere forsøg på direkte at udødeliggøre primære cafeer ved hjælp af en retroviral hTERT cDNA konstruere mislykkedes. Derfor, for yderligere at undersøge tumorfremm...

<table border="1"><tr><td> Materialets navn </td><td> Type </td><td> Firma </td><td> Katalognummer </td><td> Kommentar </td></tr><tr><td> DMEM </td><td></td><td> Invitrogen </td><td> 61965-026 </td><td></td></tr><tr><td> Føtalt kalveserum </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) antistof </td><td></td><td> Novocastra Laboratories </td><td> NCL-L- VIM-V9 </td><td></td></tr><tr><td> Tenascin C (BC-8) antistof </td><td></td><td></td><td></td><td> en gave fra Dr. <strong&gt; Luciano Zardi </td></tr><tr><td> α-SMA-Cy3 (1A4) antistof </td><td></td><td> Sigma </td><td> C6198 </td><td></td></tr><tr><td> Prolyl-4-hydroxylase </td><td></td><td> Dako </td><td> M0877 </td><td></td></tr><tr><td> (5B5) antistof </td><td></td><td></td><td></td><td></td></tr><tr><td> Kollagen Type1 1A antistof </td><td></td><td> Sigma </td><td> HPA011795 </td><td></td></tr><tr><td> Pan-cytokeratin antistof </td><td></td><td> Sigma </td><td> C5992 </td><td></td></tr><tr><td> Fibronektin antistof </td><td></td><td> BD Biosciences </td><td> 610077 </td><td></td></tr><tr><td> S100A4/FSP-1 (fibroblast- specifikt protein-1) antistof </td><td></td><td> Dako </td><td> A5114 </td><td></td></tr><tr><td> Fibroblast overflade protein (Klon 1B10) antistof </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> Anmodning til forfatterne </td></tr><tr><td> pBabe-Puro konstruere </td><td></td><td></td><td></td><td> Køb 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 koniske rør </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> Kvinde NCR nøgen </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

Carcinomer er komplekse væv består af neoplastiske celler og en ikke-kræft rum benævnt &quot;stroma&quot;. Den stroma består af ekstracellulære matrix (ECM) og en række mesenchymale celler, inklusive fibroblaster, myofibroblasts, endotelceller, pericytes og leukocytter 1-3. Den tumor-associerede stroma er lydhør over for betydelige paracrine signaler, der frigives ved omkringliggende carcinom celler. Under sygdomsforløbet, ofte stroma bliver befolket af carcinoma-associerede fibroblaster (cafeer), herunder et stort antal myofibroblasts. Disse celler har tidligere været udvundet fra mange forskellige typer af mennesker karcinomer for deres in vitro kultur. En delpopulation af cafeer adskiller sig gennem deres op-regulering af α-glat muskulatur actin (α-SMA) udtryk 4,5. Disse celler er et adelsmærke for &quot;aktiverede fibroblaster &#39;, der deler lignende ejendomme med myofibroblasts commonly observeret i såret, og fibrøse væv 6. Tilstedeværelsen af ​​denne myofibroblastic CAF delmængde er stærkt relateret til høj kvalitet maligniteter og forbundet med dårlig prognose hos patienter. Mange laboratorier, herunder vores egen, har vist, at cafeer, når de injiceres med carcinom celler i nedsat immunforsvar mus, er i stand til væsentligt at fremme tumorgenese 7-10. Cafeer fremstillet af carcinoma patienter, dog ofte undergår ældning under formering i kultur begrænser hvor omfattende deres brug i hele løbende eksperimenter. For at løse dette problem, har vi udviklet en ny teknik til at eksperimentelt generere foreviget menneskelige brystkirtler CAF cellelinier (exp-cafeer) fra menneskelige brystkirtler fibroblaster, ved hjælp af en coimplantation bryst tumor xenograft model. For at skabe exp-cafeer, forældrenes menneskelige brystkirtler fibroblaster, fremstillet af en reduktion mammoplasty væv, blev først immortalised med hTERT, den katalytiske subunit af telomerase holoenzyme, og konstrueret til at udtrykke GFP og en puromycin resistensgen. Disse celler blev coimplanted med MCF-7 humane brystcancer celler, der udtrykker en aktiveret Ras oncogene (MCF-7-ras celler) ind i en mus xenograft. Efter en inkubationstid in vivo, var det oprindeligt injiceret menneskelige brystkirtler fibroblaster udvundet fra tumorxenoplantater på grundlag af deres puromycin modstand 11. Vi observerede, at beboeren menneskelige mælkekirtlen fibroblaster har differentieret, vedtage en myofibroblastic fænotype og erhvervet tumorfremmende egenskaber i løbet af tumorprogression. Det er vigtigt, disse celler, defineret som exp-cafeer, nøje efterligner den tumorfremmende myofibroblastic fænotype af cafeer isoleret fra bryst carcinomer dissekeret fra patienter. Vores tumor xenograft afledt exp-cafeer giver derfor en effektiv model til at studere biologi cafeer i human breast karcinomer. De beskrevne protokol kan også udvides til at generere og karakterisere forskellige CAF befolkninger stammer fra andre typer af menneskelig karcinomer.

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

Carcinoma-associeret fibroblaster (cafeer) rig på myofibroblasts stede i tumor stroma, spiller en vigtig rolle som drivkraft for tumorprogression. Vi udviklede en coimplantation tumor xengraft model for eksperimentelt at generere cafeer fra menneskelige brystkirtler fibroblaster. Protokollen beskriver, hvordan man etablerer CAF myofibroblasts, der erhverver en evne til at fremme tumorgenese.

Eksperimentel Generering af Carcinoma-associerede fibroblaster (cafeer) fra Human brystkirtler 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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