Name: mammary transplantation of stromal cells and carcinoma cells in c57bl/6j mice
Uploaded: 2011-08-12T12:00:00
Description: Watch this Scientific Journal Video about Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice at JoVE.com

This project was funded through by NIH/NCI grant number R00 CA127357 and University of Kansas Cancer Center Endowment.

1. Isolation and extraction of donor collagen from C57BL/6J mice
<ol>
 <li>Sacrifice mature normal female C57BL/6J mice using approved IACUC methods.</li>
 <li>Harvest the tails and soak in 70% ethanol for 45 minutes to sterilize tissues. Dry the tails with tissue paper, wrap in aluminum foil. Store the tails in -20&deg;C until needed.</li>
 <li>Place the tails in a sterile environment such as a laminar flow hood. Using scissors, split the skin at the tail root and peel away from the tail. Remove 0.5 to 1 cm from...

<ol>
	<li>Hayward, S., Haughney, P. C., Rosen, M. A., Greulich, K. M., Weier, H. U., Dahiya, R., Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+adult+human+prostatic+epithelium+and+rat+urogenital+sinus+mesenchyme+in+a+tissue+recombination+model.">Interactions between adult human prostatic epithelium and rat urogenital sinus mesenchyme in a tissue recombination model.</a> Differentiation. 63, 131-131 (1998).</li><li>Cunha, G., Hom, Y. K., Young, P., Brody, J., Asch, B. B., Ip, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Mammary Gland Biology. , (2000).</li><li>Ethier, S. P., Ammerman, C. A., Dziubinski, M. L., Asch, B. B., Ip, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Mammary Gland Biology. , (2000).</li><li>Medina, D., Kittrell, F., Asch, B. B., Ip, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Mammary Gland Biology. , (2000).</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>DeNardo, D. G., Johansson, M., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cells+as+mediators+of+solid+tumor+metastasis.">Immune cells as mediators of solid tumor metastasis.</a> Cancer Metastasis. 27, 11-18 (2008).</li><li>Naito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+differentiation+and+function+in+health+and+disease.">Macrophage differentiation and function in health and disease.</a> Pathol Int. 58, 143-155 (2008).</li><li>Firestein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+T+cell+cometh:+interplay+between+adaptive+immunity+and+cytokine+networks+in+rheumatoid+arthritis.">The T cell cometh: interplay between adaptive immunity and cytokine networks in rheumatoid arthritis.</a> J Clin Invest. 114, 471-474 (2004).</li><li>Cheng, N., Chytil, A., Shyr, Y., Joly, A., 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=Enhanced+Hepatocyte+Growth+Factor+Signaling+by+Type+II+Transforming+Growth+Factor-{beta}+Receptor+Knockout+Fibroblasts+Promotes+Mammary+Tumorigenesis.">Enhanced Hepatocyte Growth Factor Signaling by Type II Transforming Growth Factor-{beta} Receptor Knockout Fibroblasts Promotes Mammary Tumorigenesis.</a> Cancer Res. 67, 4869-4877 (2007).</li><li>Qiu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+expression+profiling+identifies+signatures+of+tumor+virulence+in+MMTV-PyMT-transgenic+mice:+correlation+to+human+disease.">Global expression profiling identifies signatures of tumor virulence in MMTV-PyMT-transgenic mice: correlation to human disease.</a> Cancer Res. 64, 5973-5981 (2004).</li><li>Schaffhausen, B. S., Roberts, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+polyoma+middle+T+antigen+on+signaling+and+transformation:+A+DNA+tumor+virus+contribution+to+the+war+on+cancer.">Lessons from polyoma middle T antigen on signaling and transformation: A DNA tumor virus contribution to the war on cancer.</a> Virology. 384, 304-316 (2009).</li><li>Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+neural+cells+via+retrovirus-mediated+oncogene+transduction.">Immortalization of neural cells via retrovirus-mediated oncogene transduction.</a> Annu Rev Neurosci. 12, 47-65 (1989).</li><li>O'Hare, 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=Conditional+immortalization+of+freshly+isolated+human+mammary+fibroblasts+and+endothelial+cells.">Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.</a> Proc Natl Acad Sci U S A. 98, 646-651 (2001).</li><li>Shay, J. W., Wright, W. E., Werbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+molecular+mechanisms+of+human+cell+immortalization.">Defining the molecular mechanisms of human cell immortalization.</a> Biochim Biophys Acta. 1072, 1-7 (1991).</li><li>Gudjonsson, T., Villadsen, R., Ronnov-Jessen, L., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+protocols+used+in+cell+culture+models+of+human+breast+morphogenesis.">Immortalization protocols used in cell culture models of human breast morphogenesis.</a> Cell Mol Life Sci. 61, 2523-2534 (2004).</li><li>Raschke, W. C., Baird, S., Ralph, P., Nakoinz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+macrophage+cell+lines+transformed+by+Abelson+leukemia+virus.">Functional macrophage cell lines transformed by Abelson leukemia virus.</a> Cell. 15, 261-267 (1978).</li><li>Shen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+endothelial+cells+differentiated+from+mouse+embryonic+stem+cells.">Immortalization of endothelial cells differentiated from mouse embryonic stem cells.</a> Shi Yan Sheng Wu Xue Bao. 35, 218-228 (2002).</li><li>Wang, S. J., Greer, P., Auerbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+propagation+of+yolk-sac-derived+endothelial+cells+from+a+hypervascular+transgenic+mouse+expressing+a+gain-of-function+fps/fes+proto-oncogene.">Isolation and propagation of yolk-sac-derived endothelial cells from a hypervascular transgenic mouse expressing a gain-of-function fps/fes proto-oncogene.</a> In Vitro Cell Dev Biol Anim. 32, 292-299 (1996).</li><li>Tiede, B. J., Owens, L. A., Li, F., DeCoste, C., Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mouse+model+for+non-invasive+single+marker+tracking+of+mammary+stem+cells+in+vivo+reveals+stem+cell+dynamics+throughout+pregnancy.">A novel mouse model for non-invasive single marker tracking of mammary stem cells in vivo reveals stem cell dynamics throughout pregnancy.</a> PLoS One. 4, e8035-e8035 (2009).</li><li>Guzman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracarotid+injection+of+fluorescence+activated+cell-sorted+CD49d-positive+neural+stem+cells+improves+targeted+cell+delivery+and+behavior+after+stroke+in+a+mouse+stroke+model.">Intracarotid injection of fluorescence activated cell-sorted CD49d-positive neural stem cells improves targeted cell delivery and behavior after stroke in a mouse stroke model.</a> Stroke. 39, 1300-1306 (2008).</li><li>Duda, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+transplantability+of+tumor-associated+stromal+cells.">Differential transplantability of tumor-associated stromal cells.</a> Cancer Res. 64, 5920-5924 (2004).</li></ol>

The functional contribution of fibroblasts in tumor progression has been demonstrated through transplantation models, in which carcinoma associated fibroblasts co-transplanted with benign mammary epithelial cells results in increased tumor growth and invasiveness5. Conventional transplantation approaches have involved the use of SCID or nude mice to co-transplant stromal and epithelial cells from different genetic mouse backgrounds or different species. Immunocompromised mice lack functional T cells, which pl...

<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>C57BL/6N mice</td>
 <td>Harlan</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MMTV-PyVmT transgenic mice</td>
 <td>Jackson laboratories</td>
 <td>002374</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Fisher</td>
 <td>SH3039603PR</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>VWR</td>
 <td>10000113873</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin</td>
 <td>Fisher</td>
 <td>MT-30-001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>amphotericin</td>
 <td>fisher</td>
 <td>BP2645-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Amicon filtration columns ultracel 50k</td>
 <td>Millipore</td>
 <td>UFC905008</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tubes for Beckman TI rotor</td>
 <td>Beckman</td>
 <td>355618</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rat tail collagen</td>
 <td>Fisher</td>
 <td>CB 40236</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10x EBSS</td>
 <td>Sigma Aldrich</td>
 <td>E7510-100ML</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin 1X, 0.25% in HBSS w/o Calcium and Magnesium

</td>
 <td>Fisher</td>
 <td>MT-25-050-CI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glacial acetic acid</td>
 <td>Fisher</td>
 <td>A491-212</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Coomasie blue</td>
 <td>Fisher</td>
 <td>BP101 25</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Sigma Aldrich</td>
 <td>T3924-100ml</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase A</td>
 <td>Sigma AldrichC0130-50</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>hyalronidase</td>
 <td>Sigma Aldrich</td>
 <td>H3884</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>Sigma Aldrich</td>
 <td>D5025</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kaleidoscope Protein standard</td>
 <td>Biorad</td>
 <td>1610375</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass slides</td>
 <td>Fisher</td>
 <td>12545-78</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass coverslips</td>
 <td>VWR</td>
 <td>101400-042</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vimentin antibody S-20</td>
 <td>Santa Cruz Biotechnology</td>
 <td>SC-7558

</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&alpha;-smooth muscle actin antibody</td>
 <td>Abcam</td>
 <td>ab5694</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CK14 antibody</td>
 <td>Santa Cruz Biotechnology</td>
 <td>sc-53253</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CK18 antibody</td>
 <td>Abcam</td>
 <td>ab668</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Sigma Aldrich</td>
 <td>D9542</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse biotinylated</td>
 <td>Vector laboratories</td>
 <td>BA9200</td>
 <td>Distributed through Fisher</td>
 </tr>
 <tr>
 <td>Anti-mouse-alexa-568</td>
 <td>Invitrogen</td>
 <td>A10037</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse- alexa-488</td>
 <td>Invitrogen</td>
 <td>A11001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Streptavidin- alexa-488</td>
 <td>Invitrogen</td>
 <td>S11226</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Invitrogen</td>
 <td>D21490</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Prolong antifade</td>
 <td>Invitrogen</td>
 <td>P-36930</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Surgical scissors</td>
 <td>Fine Science Tools</td>
 <td>91400-12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fine spring scissors</td>
 <td>Fine Science Tools</td>
 <td>15000-02</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Blunt forceps</td>
 <td>Fine Science Tools</td>
 <td>11002-12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td># 5 fine forceps</td>
 <td>Fine Science Tools</td>
 <td>11251-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gut chromic suture</td>
 <td>Fisher</td>
 <td>NC9326254</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 <td>Glass Pasteur pipet</td>
 <td>Fisher</td>
 <td>22-042-815</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 <td>Ethanol</td>
 <td>Fisher</td>
 <td>A406P 4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>betadine</td>
 <td>fisher</td>
 <td>NC9386574</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Wound clips</td>
 <td>Fisher</td>
 <td>12032-07</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Wound staple</td>
 <td>Fisher</td>
 <td>12031-07</td>
 <td>&nbsp;</td>
 </tr> 
 </tbody>
</table>

mammary transplantation of stromal cells and carcinoma cells in c57bl/6j mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

Watch this Scientific Journal Video about Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice at JoVE.com

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Research

JoVE Journal

Medicine

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

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 ...

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

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 ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...