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

Мы благодарим д-р Роберт А. Вайнберга (Уайтхед Институт медико-биологических исследований, Кембридж) за щедрую поддержку и надзор за этой работой и г-н Киран Mellody (Манчестерский университет, Манчестер) для критических редактирование этой рукописи. Этот проект был поддержан исследований Великобритании (CR-Великобритания) номер гранта C147/A6058 (АО)

1. Изоляция первичной культивированных человеческих фибробластов нормальной молочной Экспериментальные процедуры выделения первичных культивированных человеческих фибробластов нормальной молочной приводятся на рис. 1А. <ol><li> Подготовка буфера клеточной диссоциаци...

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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>

Отсутствие CAF-специфических маркеров и уровень неоднородности наблюдается среди CAFS оказывают характеристики этого типа клеток задача сама по себе. Изучение CAFS в пробирке была также затруднена дополнительные сложности, что эти клетки стареть и остановить пролиферирующих когда к?...

<table border="1"><tr><td> Материал Имя </td><td> Тип </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарий </td></tr><tr><td> DMEM </td><td></td><td> Invitrogen </td><td> 61965-026 </td><td></td></tr><tr><td> Фетальной телячьей сыворотки </td><td></td><td> GIBCO </td><td> 10270 </td><td></td></tr><tr><td> Пенициллин-стрептомицин </td><td></td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Коллагеназы типа I. </td><td></td><td> Сигма </td><td> C0130-1G </td><td></td></tr><tr><td> гиалуронидазы </td><td></td><td> Сигма </td><td> H4272 </td><td></td></tr><tr><td> Виментин (V9) антител </td><td></td><td> Novocastra лаборатории </td><td> NCL-L- ВИМ-V9 </td><td></td></tr><tr><td> Tenascin C (БК-8) антител </td><td></td><td></td><td></td><td> подарок от Доктор <stroнг&gt; Лучано Zardi </td></tr><tr><td> α-SMA-Cy3 (1А4) антител </td><td></td><td> Сигма </td><td> C6198 </td><td></td></tr><tr><td> Пролил-4-гидроксилазы </td><td></td><td> Dako </td><td> M0877 </td><td></td></tr><tr><td> (5B5) антител </td><td></td><td></td><td></td><td></td></tr><tr><td> Коллаген type1 1A антител </td><td></td><td> Сигма </td><td> HPA011795 </td><td></td></tr><tr><td> Пан-цитокератин антител </td><td></td><td> Сигма </td><td> C5992 </td><td></td></tr><tr><td> Фибронектин антител </td><td></td><td> BD Biosciences </td><td> 610077 </td><td></td></tr><tr><td> S100A4/FSP-1 (фибробластов- специфического белка-1) антител </td><td></td><td> Dako </td><td> A5114 </td><td></td></tr><tr><td> Белок фибробластов поверхности (Клон 1B10) антител </td><td></td><td> Abcam </td><td> ab11333 </td><td></td> &lt;/ TR&gt; <tr><td> MSCV-IRES-GFP построить </td><td></td><td></td><td></td><td> Просьба к авторам </td></tr><tr><td> pBabe-Пуро построить </td><td></td><td></td><td></td><td> Покупка от Addgene </td></tr><tr><td> Пуромицин </td><td></td><td> Сигма </td><td> P8833 </td><td></td></tr><tr><td> DAPI </td><td></td><td> Сигма </td><td> D9564 </td><td></td></tr><tr><td> 15 мл коническую трубку </td><td></td><td> Гранулирование </td><td> 430766 </td><td></td></tr><tr><td> Обнаженная мыши </td><td></td><td> Taconic </td><td> NCRNU-F </td><td> Женский NCr обнаженной </td></tr><tr><td> C3H/10T1/2 клетки </td><td></td><td> АТСС </td><td></td><td> CCL-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

Карцинома связанных фибробластов (CAFS) богаты миофибробластов присутствует в опухоли стромы, играют важную роль в продвижении опухолевой прогрессии. Мы разработали xengraft coimplantation опухоли модель экспериментально генерации CAFS из молочной железы человека фибробластов. Протокол описывает, как установить CAF миофибробластов, которые приобретают способность продвигать tumourigenesis.

Экспериментальные Генерация Карцинома-Associated Фибробласты (CAFS) из молочной железы человека Фибробласты

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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Medicine

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

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

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

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Экспериментальные человека перевозке пневмококковой

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Выделение и функциональная характеристика человека желудочка кардиомиоцитов из свежих хирургических образцов

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

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Молекулярная профилирования из инвазивным опухоли микроокружения в 3-мерной модели клеток колоректального рака и<em&gt; Экс естественных</em&gt; Фибробласты

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The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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