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>
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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 ...

mammary-transplantation-stromal-cells-carcinoma-cells-c57bl6j

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Medicine

17.5K ビュー.  University of Kansas Medical Center. このレポートでは、我々はマウス乳腺から分離し、培養ドナー細胞にシステムのデモンストレーションを、と同所間質分析するためにレシピエントマウスにこれらの細胞を移植：乳腺腫瘍の開発中に上皮との相互作用を。