We thank Dr. Robert A. Weinberg (Whitehead Institute for Biomedical Research, Cambridge) for generous support and supervision of this work and Mr. Kieran Mellody (University of Manchester, Manchester) for critical editing of this manuscript. This project was supported by Research UK (CR-UK) grant number C147/A6058 (A.O.).

1. Isolation of primary cultured human normal mammary fibroblasts
Experimental procedures for isolating primary cultured human normal mammary fibroblasts are outlined in Fig. 1A.
<ol>
	<li>Prepare the cell dissociation buffer, as described previously12: Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum (FCS), penicillin-streptomycin (200 units/ml), collagenase type I (1 mg/ml) and hyaluronidase (125 units/ml).</li>
	<li>Wash the breast tissue dissected from a red...

<ol>
	<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>

The lack of CAF-specific markers and the level of heterogeneity observed amongst CAFs render the characterisation of this cell type a challenge in itself. Studying CAFs in vitro has also been hindered by the additional complication that these cells senesce and stop proliferating when cultured for a long period. Our previous attempt to directly immortalise primary CAFs using a retroviral hTERT cDNA construct was unsuccessful. Therefore, to further investigate the tumour promoting properties of these cells, we dev...

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comment</td>
</tr>
<tr>
<td>DMEM </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>61965-026 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fetal calf serum </td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>10270</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Penicillin-streptomycin </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15140-122 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Collagenase type I </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C0130-1G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4272 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Vimentin (V9) antibody</td>
<td>&nbsp;</td>
<td>Novocastra Laboratories</td>
<td>NCL-L-
VIM-V9</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tenascin C (BC-8) antibody</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>a gift from 
&nbsp;Dr. Luciano 
&nbsp;Zardi</td>
</tr>
<tr>
<td>&alpha;-SMA-Cy3 (1A4) antibody</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C6198</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Prolyl-4-hydroxylase </td>
<td>&nbsp;</td>
<td>Dako</td>
<td>M0877</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>(5B5) antibody</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Collagen type1 1A antibody</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>HPA011795</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Pan-cytokeratin antibody</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C5992</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fibronectin antibody</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>610077</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>S100A4/FSP-1 (fibroblast-
specific protein-1) antibody</td>
<td>&nbsp;</td>
<td>Dako</td>
<td>A5114</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fibroblast surface protein 
(clone 1B10) antibody</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab11333</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MSCV-IRES-GFP construct</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Request to the the authors</td>
</tr>
<tr>
<td>pBabe-puro construct</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Purchase 
from Addgene</td>
</tr>
<tr>
<td>Puromycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8833</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DAPI</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D9564</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>15 ml conical tube</td>
<td>&nbsp;</td>
<td>Corning </td>
<td>430766</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nude mouse</td>
<td>&nbsp;</td>
<td>Taconic</td>
<td>NCRNU-F</td>
<td>Female NCr nude</td>
</tr>
<tr>
<td>C3H/10T1/2 cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</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

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

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 ...

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23.2K vistas.  University of Manchester. Carcinoma asociados fibroblastos (CAF), rica en miofibroblastos presentes en el estroma tumoral, juegan un papel importante en la conducción de la progresión del tumor. Hemos desarrollado un modelo de tumor coimplantation xengraft para generar experimentalmente CAF a partir de fibroblastos humanos mamarios. El protocolo describe cómo establecer miofibroblastos CAF que adquirir la capacidad de promover la génesis tumoral.