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

हम उदार और इस काम और इस पांडुलिपि के महत्वपूर्ण संपादन के लिए श्री कीरन Mellody (मैनचेस्टर, मैनचेस्टर विश्वविद्यालय) के समर्थन के पर्यवेक्षण के लिए डॉ. रॉबर्ट ए Weinberg (व्हाइटहेड जैव चिकित्सा अनुसंधान, कैम्ब्रिज के लिए संस्थान) धन्यवाद. इस परियोजना अनुसंधान (सीआर ब्रिटेन) अनुदान C147/A6058 संख्या (एओ) ब्रिटेन द्वारा समर्थित किया गया

1. प्राथमिक सभ्य मानव सामान्य स्तन fibroblasts के अलगाव प्राथमिक सभ्य मानव सामान्य स्तन fibroblasts को अलग करने के लिए प्रायोगिक प्रक्रियाओं छवि में रेखांकित कर रहे हैं. 1A. <ol><li> 10% भ्रूण बछड़ा (FCS) सीरम, (200 / इकाइ?...

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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>

CAF विशेष मार्करों और विविधता के स्तर की कमी मनाया बीच CAFs इस सेल प्रकार अपने आप में एक चुनौती के लक्षण वर्णन सौंपनेवाला. इन विट्रो में अध्ययन CAFs भी अतिरिक्त जटिलता द्वारा निस्र्द्ध गया है कि इन कोशिकाओ?...

<table border="1"><tr><td> सामग्री नाम </td><td> टाइप </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणी </td></tr><tr><td> DMEM </td><td></td><td> Invitrogen </td><td> 61965-026 </td><td></td></tr><tr><td> भ्रूण बछड़ा सीरम </td><td></td><td> GIBCO </td><td> 10270 </td><td></td></tr><tr><td> पेनिसिलिन - स्ट्रेप्टोमाइसिन </td><td></td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Collagenase प्रकार मैं </td><td></td><td> सिग्मा </td><td> C0130-1G </td><td></td></tr><tr><td> hyaluronidase </td><td></td><td> सिग्मा </td><td> H4272 </td><td></td></tr><tr><td> Vimentin एंटीबॉडी (V9) </td><td></td><td> Novocastra प्रयोगशालाओं </td><td> एनसीएल - एल - वीआइएम-V9 </td><td></td></tr><tr><td> Tenascin सी एंटीबॉडी (ई.पू. 8) </td><td></td><td></td><td></td><td> से एक उपहार डॉ. <stroएनजी&gt; लुसियानो Zardi </td></tr><tr><td> α-SMA Cy3 एंटीबॉडी (1A4) </td><td></td><td> सिग्मा </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> एंटीबॉडी (5B5) </td><td></td><td></td><td></td><td></td></tr><tr><td> कोलेजन type1 1A एंटीबॉडी </td><td></td><td> सिग्मा </td><td> HPA011795 </td><td></td></tr><tr><td> पान - cytokeratin एंटीबॉडी </td><td></td><td> सिग्मा </td><td> C5992 </td><td></td></tr><tr><td> Fibronectin एंटीबॉडी </td><td></td><td> बी.डी. बायोसाइंसेज </td><td> 610077 </td><td></td></tr><tr><td> S100A4/FSP-1 (fibroblast - विशिष्ट प्रोटीन-1) एंटीबॉडी </td><td></td><td> Dako </td><td> A5114 </td><td></td></tr><tr><td> Fibroblast सतह प्रोटीन (1B10 क्लोन) एंटीबॉडी </td><td></td><td> Abcam </td><td> ab11333 </td><td></td> &lt;/ Tr&gt; <tr><td> MSCV IRES - GFP निर्माण </td><td></td><td></td><td></td><td> लेखकों के लिए अनुरोध करें </td></tr><tr><td> pBabe - puro निर्माण </td><td></td><td></td><td></td><td> क्रय Addgene से </td></tr><tr><td> Puromycin </td><td></td><td> सिग्मा </td><td> P8833 </td><td></td></tr><tr><td> DAPI </td><td></td><td> सिग्मा </td><td> D9564 </td><td></td></tr><tr><td> 15 मिलीलीटर शंक्वाकार ट्यूब </td><td></td><td> कोर्निंग </td><td> 430766 </td><td></td></tr><tr><td> नंगा माउस </td><td></td><td> टैकॉनिक </td><td> NCRNU - एफ </td><td> स्त्री एनसीआर नग्न </td></tr><tr><td> C3H/10T1/2 कोशिकाओं </td><td></td><td> ATCC </td><td></td><td> सीसीएल - 226 </td></tr></table>

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

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

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

कार्सिनोमा - जुड़े fibroblasts (CAFs) ट्यूमर stroma भीतर मौजूद myofibroblasts में अमीर, ट्यूमर प्रगति ड्राइविंग में एक प्रमुख भूमिका निभाते हैं. हम प्रयोगात्मक मानव स्तन fibroblasts से CAFs पैदा करने के लिए एक coimplantation ट्यूमर xengraft मॉडल विकसित किया है. प्रोटोकॉल का वर्णन कैसे स्थापित करने के लिए CAF myofibroblasts कि tumourigenesis को बढ़ावा देने की क्षमता हासिल है.

कार्सिनोमा - एसोसिएटेड Fibroblasts के प्रायोगिक मानव स्तन Fibroblasts से जनरेशन (CAFs)

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

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

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Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

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Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

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Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

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पार्किंसंस रोग के क्षेत्र में Mitochondrial phenotypes की निगरानी के लिए प्राथमिक मानव fibroblasts का प्रयोग करें

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

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प्रायोगिक मानव न्यूमोकोकल कैरिज

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Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

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आण्विक कोलोरेक्टल कैंसर की कोशिकाओं का एक 3 आयामी मॉडल में आक्रामक ट्यूमर microenvironment की रूपरेखा और<em&gt; पूर्व vivo</em&gt; Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

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