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 reduction mammoplasty (~0.5 gram) several times in phosphate buffered saline (PBS). Mince the tissue into small fragments (&lt;1.5 mm3) using sterile razor blades.</li>
	<li>Transfer the tissue fragments into a 15 ml conical tube containing an appropriate amount of the above cell dissociation buffer (10 ml per 0.5 gram of tissue) and vortex for 1 min at maximum speed.</li>
	<li>Digest the tissue fragments in the cell dissociation buffer for 12-18 h at 37&deg;C with slow agitation. *Note that there will be still many pieces of undigested tissue fragments remaining in the tube.</li>
	<li>Incubate the tube for 5 min at room temperature without agitation. Transfer the resulting stromal cell-enriched supernatant into a new conical tube using a 5 ml serological pipet.</li>
	<li>Centrifuge the stromal fraction for 5 min at 250 X g and resuspend the cell pellet in PBS. Centrifuge again for 5 min at 250 X g and resuspend the cell pellet in DMEM supplemented with 10% FCS. Culture the cells in DMEM containing 10% FCS in a 15 cm petri dish at 37&deg;C and 5% carbon dioxide.</li>
	<li>Propagate the cells until confluent. It will usually take 8-10 days. Store the cells at -80&deg;C using freezing medium (10 % dimethyl sulfoxide and 20% FCS in DMEM). Prepare 5 frozen vials as an original stock. Thaw one vial and expand the cells to prepare 5 vials for the secondary stock containing fibroblasts passaged within 5 population doublings (PDs) for the subsequent experiments. These procedures minimise clonal selection and culture stress that could occur during extended tissue culture. *Note that P1.1-7 can also be used for isolating CAFs from breast carcinomas obtained by a mastectomy12.</li>
</ol>
2. Generation of GFP-labelled, puromycin-resistant, immortalised human normal mammary fibroblasts
<ol>
	<li>To immortalise the primary human normal mammary fibroblasts isolated in P1.7), introduce a retroviral pMIG (MSCV-IRES-GFP) vector12, expressing both hTERT and GFP. Culture the cells for 4-5 days and then sort the GFP-positive cells using flow cytometry.</li>
	<li>Introduce a retroviral pBabe-puro construct encoding a puromycin resistance gene into the GFP-labelled immortalised fibroblasts. Culture the cells for 5-7 days in the presence of puromycin (final concentration: 1 &mu;g/ml) to isolate the GFP-positive (GFP+), puromycin-resistant (puroR) immortalised human mammary fibroblasts.</li>
	<li>Next, to examine whether these fibroblasts produce active viruses, which can lead to the horizontal transfer of genes encoding GFP and puromycin resistance, grow 2.5x105 GFP+puroR immortalized human mammary fibroblasts in a 6 cm Petri dish for 2 days.</li>
	<li>Filter medium conditioned by these fibroblasts using a syringe filter (pore size: 0.45 &mu;m) and add it onto 10T1/2 mouse fibroblastic cells for 12h in the presence of protamine sulfate (5 &mu;g/ml) which increases efficiency of the virus infection. The medium is then changed to 10% FCS-DMEM for additional 2 days.</li>
	<li>Examine the cells by a fluorescence microscopy. 10T1/2 cells infected by GFP virus will become GFP-positive. Treat also the cells with puromycin (1 &mu;g/ml) for 5-7 days and observe the plate if any puroR 10T1/2 cells are present and form colonies after the puromycin treatment.</li>
	<li>Note that the horizontal gene transfer by active viruses produced by the parental GFP+puroR human mammary fibroblasts might make the surrounding murine cells and/or carcinoma cells GFP+puroR within tumour xenografts. This may hinder the processes of isolating the initially injected GFP+puroR human mammary fibroblasts from the tumour xenografts.</li>
</ol>
3a. Coinjection of human mammary fibroblasts with breast carcinoma cells into an immunodeficient mouse
Experimental procedures for generating exp-CAFs are illustrated in Fig. 1Ba.
<ol>
	<li>Mix 1x106 MCF-7-ras human breast carcinoma cells and 3x106 GFP+puroR, immortalised human mammary fibroblasts generated in P2.2). Prepare the cells in 400 &mu;l of culture medium with 50% (v/v) Matrigel per injection.</li>
	<li>Inject the cell mixture subcutaneously into an immunodeficient nude mouse. When CAFs are extracted independently from different tumours, implant each mixture into right and left flanks of several mice.</li>
</ol>
3b. Injection of human mammary fibroblasts into an immunodeficient mouse
<ol>
	<li>To generate control fibroblasts against exp-CAFs, prepare 3x106 GFP+puroR immortalised human mammary fibroblasts generated in P2.2 in 400 &mu;l of culture medium with 50% (v/v) Matrigel per injection (Fig. 1Bb). DO NOT mix the fibroblasts with carcinoma cells.</li>
	<li>Implant the fibroblasts prepared in P3b.1 into subcutaneous sites of nude mice. </li>
</ol>
*Note that the inoculated fibroblasts will form the fibroblastic tissue but not tumour under the skin.
4a. Dissection of tumour xenograft
<ol>
	<li>Euthanise the mouse harbouring a subcutaneous human breast cancer xenograft 42 days post-implantation (Fig. 1Ba). *Note that when other carcinoma cells and/or fibroblasts are coimplanted, fibroblasts may need to be incubated for longer than 42 days within the tumour xenograft to initiate their myofibroblastic phenotype.</li>
	<li>Harvest the tumour xenograft from the flank of the mouse by blunt dissection using forceps and scissors. Immerse the tumour tissue in PBS.</li>
</ol>
4b. Dissection of fibroblast xenograft
<ol>
	<li>Euthanise the mouse harbouring the fibroblast xenograft 42 days post-implantation (Fig. 1Bb).</li>
	<li>Dissect the subcutaneous fibroblast xenograft using forceps and scissors. Place the fibroblastic tissue into PBS. *Note that a fibroblast xenograft, which appears as a tiny transparent tissue, may be difficult to find under the skin.</li>
</ol>
5. Preparation of primary cultured cells from xenografts
<ol>
	<li>Use a sterile biosafety cabinet to dissect the tumour (&tilde;0.5 gram) or fibroblastic tissue (&tilde;0.1 gram). Transfer the excised tissues onto the top of a 15 cm culture dish with 1 ml of PBS. Mince the tissues into small tissue fragments (&lt;1.5 mm3) using sterile razor blades. *Note that if the fibroblast xenograft weighs less than 0.1 gram, collect a few additional fibroblast xenografts and mix them together to increase the number of cells for isolation.</li>
	<li>Transfer the tissue fragments into a 15 ml conical tube containing the cell dissociation buffer (10 ml per 0.5 gram of tissue) and vortex for 1 min at maximum speed.</li>
	<li>Incubate the cell suspension for 3 h at 37&deg;C with continuous slow agitation.</li>
</ol>
6. Isolation of puromycin-resistant cells in culture
<ol>
	<li>Centrifuge the cell suspension dissociated either from tumour or fibroblast xenograft for 5 min at 250 X g and resuspend the cell pellet in PBS. Centrifuge again for 5 min at 250 X g. Resuspend the resulting cell pellet in 10% FCS-DMEM. Culture the cells in a 15 cm petri dish in the presence of puromycin (1 &mu;g/ml) at 37&deg;C and 5% carbon dioxide in order to eliminate any contaminating carcinoma cells and/or murine stromal cells. Propagate the cells until confluent (2-4 weeks). Store the cells at -80&deg;C using freezing medium. *Note that the resulting puroR cells, designated 42-day-old exp-CAF1 (Fig. 1Ba) or control fibroblast-1 cells (Fig. 1Bb), are immortal and positive for GFP. It is recommended to check if these cells produce active viruses using 10T1/2 cells, as described in P2.3-6.</li>
</ol>
7a. Isolation of exp-CAF2 cells in culture
To further boost the activated myofibroblastic phenotype of CAFs, mix the 42-day-old exp-CAF1 cells with MCF-7-ras cells and inject them again subcutaneously into a nude mouse for additional periods of 200 days. Dissect and dissociate the tumour xenograft into a single-cell suspension and culture the cells in a 15 cm petri dish with 10% FCS-DMEM in the presence of puromycin (1 &mu;g/ml), as indicated in P6.1. Propagate the cells until confluent (8-10 days). Store the cells at -80&deg;C using freezing medium. The resulting puroR cells are named 242-day-old exp-CAF2 cells (Fig. 1Ba).
7b. Extraction of control fibroblast-2 cells in culture
To generate control fibroblasts against exp-CAF2 cells, inject 42-day-old control fibroblast-1 cells alone without carcinoma cells subcutaneously into a nude mouse additionally for 200 days. Dissect and dissociate the fibroblastic tissue into a single cell suspension and culture the cells in a 15 cm petri dish with 10% FCS-DMEM in the presence of puromycin (1 &mu;g/ml) as indicated in P6.1. Propagate the cells until confluent (3-4 weeks). Store the cells at -80&deg;C using freezing medium. The resulting puroR cells are named control fibroblast-2 cells (Fig. 1Bb).
8. Representative Results:
Control fibroblast-2 and exp-CAF2 cells, which have been extracted from breast tumour xenografts, stained strongly positive for mesenchymal markers, including human-specific vimentin, prolyl-4-hydroxylase, collagen 1A, fibronectin, S100A4, and fibroblast surface protein (Fig. 2A)11, indicating human origin and mesenchymal nature of these cells. In contrast, the cytokeratin, a marker for epithelial cells, was not stained in these GFP+fibroblasts (Fig 2B). These findings therefore suggest that the extracted exp-CAF2 and control fibroblast-2 cells have originated from the parental human mammary fibroblasts initially introduced into mouse xenografts.
Importantly, a higher proportion of exp-CAF2 cells stained positive for &alpha;-SMA and extracellular matrix glycoprotein tenascin-C5, both of which are markers of myofibroblasts compared to 42-day-old exp-CAF1 and control fibroblast-2 cells (Fig. 2C, D)11. These data indicate that resident human mammary fibroblasts progressively evolve into CAF myofibroblasts within tumour xenografts.
<img alt="Figure 1" src="/files/ftp_upload/3201/3201fig1.jpg" /> 
Figure 1 Schematic representation of isolation of human mammary fibroblasts. A) The reduction mammoplasty tissue was minced using sterile razor blades (P1.2) and transferred into a 15 ml conical tube (P1.3). The small tissue fragments were digested in the cell dissociation buffer (P1.4) and prepared for culture in vitro (P1.5-7). To immortalise the isolated primary human mammary fibroblasts, a retroviral pMIG (MSCV-IRES-GFP) vector, expressing both hTERT and GFP, was introduced, and the resulting GFP-positive cells were sorted using flow cytometry (P2.1). A retroviral pBabe-puro vector encoding a puromycin resistance gene was then introduced into these cells. Upon the puromycin treatment, GFP-labelled (GFP+) puromycin-resistant (PuroR), immortalised human mammary fibroblasts were isolated (P2.2).
Ba) To generate exp-CAFs, GFP+puroR immortalised human mammary fibroblasts were coinjected with MCF-7-ras breast carcinoma cells subcutaneously into an immunodeficient nude mouse (P3a). The tumour xenograft was resected at 42 days after implantation (P4a) and dissociated into a single-cell suspension (P5). These cells were then cultured in vitro in the presence of puromycin to eliminate any contaminating carcinoma cells and mouse stromal cells (P6). The resulting puromycin-resistant cells were termed experimentally generated CAF1 (exp-CAF1) cells. These cells, resected 42 days post-implantation, were once again mixed with MCF-7-ras cells and implanted subcutaneously into a host mouse as before (P7a). The resulting tumour was allowed to grow for additional periods of 200 days, then dissected, dissociated, and cultured in the presence of puromycin. The isolated puromycin-resistant cells were termed exp-CAF2 cells (242-day-old).
Bb)To isolate control cells against exp-CAFs, GFP+puroR immortalized human mammary fibroblasts were injected subcutaneously into a nude mouse as pure cultures without MCF-7-ras cells (P3b). The fibroblastic tissue, dissected 42 days post-implantation (P4b), was dissociated into single-cell suspensions (P5) and puromycin-resistant cells, named control fibroblast-1 cells, were isolated as described earlier (P6). These fibroblasts were once again implanted subcutaneously into a nude mouse without MCF-7-ras cells additionally for 200 days (P7b). The fibroblastic tissue was dissected, dissociated, and cultured in the presence of puromycin. The isolated puromycin-resistant cells were termed control fibroblast-2 cells (242-day-old).
<img alt="Figure 2" src="/files/ftp_upload/3201/3201fig2.jpg" /> 
Figure 2 Exp-CAFs and the control fibroblasts originate from the parental human mammary fibroblasts. (A) Immunofluorescence analyses of control fibroblast-2 (control f.) and exp-CAF2 cells. The both cell types stain positive for mesenchymal markers (red), including human vimentin, prolyl-4-hydroxylase, collagen 1A, fibronectin, S100A4, and fibroblast surface protein. (B) In contrast, pan-cytokeratin, a marker for epithelial cells, is not detected in exp-CAF2 cells expressing GFP (green). Cell nuclei are stained with 4'-6-Diamidino-2-phenylindole (DAPI) (blue). Scale bar, 50 &mu;m (referred from Kojima et al.11)
(C) Immunofluorescence of exp-CAF2 cells and control fibroblast-2 cells (control f.) using antibodies against &alpha;-SMA (red) or tenascin-C (TN-C) (red). Cell nuclei are stained with DAPI (blue). Scale bar, 50 &mu;m. (D) 48% of exp-CAF2 cells stain positive for &alpha;-SMA, whereas 14% of 42-day-old exp-CAF1 and 2.5% of the control fibroblast-2 cell populations are positive for &alpha;-SMA. (referred from Kojima et al.11)

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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>

No conflicts of interest declared.

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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23.3K Views.  University of Manchester. 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.

Video: 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 protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

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

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

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

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

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

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

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

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

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 risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

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 ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

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

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

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

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...