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 both ends of the tail and divide the remaining tail up into three or four pieces. Dissect the tendons from the tails and separate the tendons into individual fibers using a scalpel or razor blade.</li>
 <li>Transfer the tendon fibers to a sterile container and wash with sterile distilled water. Transfer tendons from tail of 5-7 mice to a 50 ml conical tube containing 35 ml sterile de-ionized water containing 50 units/ml penicillin penicillin, 50 &mu;g/ml streptomycin and 250 ng/ml amphotericin, and 0.034 N acetic acid diluted from a stock solution of glacial acetic acid (17 N). Place on rocker at 4&deg;C for a week to extract the collagen.</li>
 <li>Spin down debris in a table top centrifuge at 3000 g for 15 minutes. Transfer supernatant, approximately 30 mls to two polycarbonate tubes adapted for the Beckman 50.2 Ti rotor and spin at 35,000 g (approximately 17000 rpm) for 1 hour.</li> 
 <li>The volume of the supernatant will need to be reduced to achieve a concentration of 1-2 mg/ml. Transfer the supernatant to an ultracel 50 k Amicon filtration unit and spin in a table top centrifuge at 3000 rpm for 15 at 4&deg;C. Transfer the filtrate to a sterile conical tube and save. Repeat the centrifugation until the volume is reduced to 5-6 ml.</li>
 <li>Read the protein concentration. We use a standard Bradford assay (Biorad), using BSA standards (Biorad) and undiluted samples. Measure a sample from your filtrate, as a negative control.</li>
 <li>Further check for purity of extraction by resolving a sample of purified collagen on a 6% SDS polyacrylamide gel, 10 lanes, 1.5 mm gel thickness. Prepare 20 &mu;g of sample in 30 &mu;l 1x SDS-PAGE loading buffer containing 63 mM Tris HCl 10% glycerol, 2% SDS 0.0025% bromophenol blue in 1.5 ml eppendorf tubes. Prepare an equivalent amount of protein from rat tail collagen type I as a positive control. In addition, prepare a set of BSA standards at 2.5, 5, 10, 20 &mu;g diluted in PBS and 1X loading buffer. Boil the samples at 95&deg;C for 5 minutes.</li>
 <li>Let the samples cool and briefly microfuge (maximum speed for 10 seconds) to spin down condensation.</li>
 <li>Load the samples samples along with a molecular weight marker. For example, load 5 &mu;l of Kaleidoscope Biorad protein standard. Electrophorese at 150 volts for approximately 1 hour or until the dye front reaches the bottom.</li>
 <li>Remove the gel and stain in coomassie blue buffer. Prepare this buffer by mixing 2 g of coomassie blue, 75 ml glacial acetic acid, 500 ml ethanol in a total volume of 1000 ml containing de-ionized water. Also prepare the same amount of destain buffer using the recipe except without coomassie blue reagent. Cover the gel with a sufficient volume for coomassie blue and place on a rocker.</li>
 <li>Rock gently for 1 hour at RT and decant the stain. Replace the destain solution when it turns dark blue. Detain the gel overnight or until the individual bands are resolved on the gel. Locate two collagen bands (molecular weights= 90 kda and 130 kda) and note the strength of the bands against the standards and controls. Image the gel using a gel doc system, if necessary.</li> 
</ol>
2. Isolation and culture of donor mammary carcinoma cells and fibroblasts from normal and PyVmT C57BL/6J mice
<ol>
 <li>Sacrifice age matched female normal or PyVmT transgenic mice after 10 weeks of age when tumors are evident and palpable in PyVmT transgenic mice using approved IACUC methods.</li>
 <li>Expose the mouse on its back on a flat surface and immobilize the limbs using tape adhesive.</li>
 <li>Make an upside down T incision between the nipples of the thoracic and inguinal mammary gland and pull the skin flap back to expose the mammary glands. Remove the mammary tissues and mince into small pieces using surgical scissors.</li>
 <li>Prepare 100 ml of an enzymatic digestion cocktail containing: 200 mg trypsin, 500 mg collagenase, 4 mg DNAse, 100,000 units hyaluronidase in sterile PBS and antibiotics overnight on ice and then for 3 h at 37&deg;C.</li>
 <li>Add 10 ml of PBS containing 10% fetal bovine serum (FBS) to each sample and centrifuge cell pellet in a table top centrifuge at 1500 rpm for 5 minutes at 4&deg;C.</li>
 <li>Gently aspirate the supernatant and resuspend the cell pellet in 5-7 ml of PBS/10% FBS. Repeat the spin and wash two more times.</li>
 <li>Resuspend the cell pellet in 10 ml of DMEM containing 10% FBS containing 50 units/ml penicillin , 50 &mu;g/ml streptomycin and 250 ng/ml amphotericin on 10 cm dishes coated with collagen type I. To coat plates with collagen, dilute the collagen 1 ml (using stock 1.5 mg/ml) into 39 ml of 0.02 N acetic acid diluted in sterile distilled water or PBS. Pipet 5 mls of collagen working solution onto 10 cm plates. Incubate at room temperature for a minimum for 10 minutes. Aspirate excess collagen. Parafilm unused plates and store at 4&deg;C for a week.</li>
 <li>Change the media 2- 3 times a week. Successful digestion of tissues will be characterized by the presence of epithelial foci surrounded by spindle shaped fibroblastic cells.</li> 
 <li>Separate the fibroblasts and epithelial cells by selective tryspinization. Aspirate the media and wash the cells with PBS once. Pipet 1 ml of 0.25% trypsin/0.54 mM EDTA onto cells and incubate at room temperature. Fibroblasts are more loosely adherent than epithelial cells. Check for fibroblast detachment under the microscope after 2 minutes; cells should be floating in suspension or rounded up while epithelial foci should still be adhered to the surface.</li> 
 <li>Lightly tap the dish to loosen the fibroblasts and gently pipet 10 mls of serum containing medium in the dish to remove the fibroblasts.</li>
 <li>Pipet the media containing fibroblasts in a 15 ml conical tube and centrifuge as described in step 5). Replate these cells onto collagen coated 10 cm dishes and repeat the selective trypsinization process until fibroblasts and epithelial cells are completely separated.</li>
 <li>Check for cell purity by immunofluorescence staining for epithelial markers such as CK14 and CK18 and fibroblast markers such as alpha smooth muscle actin (&alpha;-sma).</li>
</ol>
3. Immunofluorescence staining of cultured cells 
<ol>
 <li>Place glass coverslips in 6 cm dishes. Sterilize by UV exposure for 5 to 10 minutes in a laminar flow hood.</li>
 <li>Coat the coverslip with 1-2 ml of working stock collagen solution for 10 minutes at room temperature (RT). Aspirate excess.</li>
 <li>Trypsinize and plate 200,000 epithelial cells or fibroblasts per sample. Add 3-5 ml of complete media and incubate for 24 hours.</li> 
 <li>Aspirate the media, wash cells with PBS and fix in ice cold methanol at -20&deg;C for 7 minutes.</li>
 <li>Wash cells with PBS twice to remove ethanol, and block with 1-2 ml of PBS containing 1% FBS for 1 hour at RT.</li>
 <li>Using forceps, remove the coverslips and place in a large shallow container covered with parafilm or other hydrophobic surface.</li> 
 <li>Dilute the following primary antibodies 1:100 in blocking solution: anti-smooth muscle actin (&alpha;-sma, anti-CK14 (basal epithelial marker), anti-CK18 (luminal epithelial marker).</li>
 <li>Pipet 100 &mu;l of antibodies directly onto the coverslips for 1 hour at RT. Incubate a separate coverslip of cells in blocking solution; this sample will serve as the secondary antibody control.</li>
 <li>Aspirate antibody and pipet 1 ml of PBS directly on the coverslip to wash. Aspirate and repeat 3-5 times.</li>
 <li>Dilute the following secondary antibodies in blocking solution: anti-mouse-alexa 488 at 1:100, anti-mouse biotinylated at 1: 500, anti-mouse-alexa-568.</li> 
 <li>Wrap the container with the coverslips in aluminum foil. Pipet 100 &mu;l of appropriate secondary antibody directly on the coverslips: &alpha;-sma with anti-mouse-alexa 568, CK 14 staining with anti-mouse biotinylated and CK18 with anti-mouse-alexa 488. Cover the container to protect the samples from visible light.</li>
 <li>Aspirate antibody and wash with PBS as in step 9). Leave the samples in PBS, except for CK14 staining.</li> 
 <li>Dilute streptavidin conjugated to alexa 568 1:500 in PBS. Incubate the CK14 stained samples with strepatividin-alexa 568 for 30 minutes at RT in the dark. Wash in PBS as in step 9)</li>
 <li>Dilute DAPI 1: 500 in PBS. Aspirate the PBS from the samples and incubate with DAPI for 10 minutes RT in the dark.</li> 
 <li>Rinse samples with PBS. Pipet 100 &mu;l of Prolong anti-fade reagent onto glass slides, turn the coverslip over so that the cells are facing the glass slide. Tilt the coverslip to one angle to gently make contact with the mounting media.</li>
 <li>Image the samples using an immunofluorescence microscope. Keep the samples in the dark to protect the immunofluorescence signal, which will last approximately 2 weeks.</li> 
</ol>
4. Preparation of collagen embedded cells for grafting
<ol>
 <li>To embed cells in collagen, it is necessary to lower the pH of the collagen to achieve polymerization by mixing the collagen with a setting solution. 
 Prepare the setting solution by mixing 100 ml of 10X Earl's Balanced Salt Solution (EBSS) with 2.45 g of NaHCO3, 7.5 ml of 1 M NAOH and 42.5 ml of sterile distilled water. Further sterilize using a bottle top 0.22 micron filter unit attached to a sterile bottle.</li>
 <li>Mix the collagen (stock concentration 1 mg/ml) with setting solution at a starting 4: 1 ratio. For one graft, mix 100 &mu;l of collagen with 25 &mu;l of setting solution in an eppendorf tube. Vortex briefly or pipet the sample up and down to mix the sample thoroughly.</li> 
 <li>Add collagen or setting solution at 5-10 &mu;l increments and mix thoroughly until the phenol red dye in the mixture changes to a light pink to orange color, reflecting a neutral pH. A yellow color indicates acidic pH while dark pink reflects basic pH. Keep the mixture on ice to prevent polymerization.</li> 
 <li>Remove the fibroblasts and epithelial cells from the plate by trypsinization. First, aspirate the media and wash with 5 ml of PBS. Pipet 1 ml of 0.25% trypsin/0.54 mM EDTA in HBSS per plate. Incubate fibroblasts at RT for 2-5 minutes. Incubate epithelial cells at 37oC for 2-6 minutes. Tap the plates gently to loosen the cells. Check for detachment using a microscope.Quench the trypsin by pipetting 9 ml of complete medium on the plate and transfer the cells to a sterile 15 ml conical tube.</li> 
 <li>Remove 50 ul and count the cells by hemocytometer. Note the total volume of cells in each tube (approximately 10 ml).</li>
 <li>Pellet the cells at 1500 rpm for 5 min at RT. Aspirate the cell pellet and resuspend the cells to a concentration of 100,000 cells/100 &mu;l. For example, resuspend 500,000 cells in a total volume of 500 &mu;l of complete media.</li>
 <li>Mix 250,000 stromal cells (250 &mu;l) and 100,000 tumor cells (100 &mu;l) in a separate tube. Microfuge 10 sec 1000 rpm, remove supernatant by pipetting. Add 50 &mu;l of the adjusted collagen solution to cells. Pipet up and down to disperse the cells evenly. Pipet the 50 &mu;l in a sterile 6 cm tissue culture plate to form a circular drop. Incubate the graft at 37&deg;C for 10 minutes to polymerize.</li>
 <li>Gently add 5 mls of complete media in the plate by tilting the plate at an angle and slowly pipetting the media by one side of the plate. Tilt the plate around until the media covers the graft.</li>
 <li>Incubate for 24 hours at 37&deg;C. Transplant immediately.</li>
</ol>
5. Orthoptic transplantation of collagen embedded cells in C57BL/6J mice
<ol>
 <li>Fabricate a thin glass rod for the transplantation surgery. Heat a glass Pasteur pipet using a Bunsen burner. Using forceps, stretch the end of the pipet out to 2-3 mm in thickness. Let cool, and sterilize by 70% ethanol.</li>
 <li>Sterilize the following surgical instruments by autoclaving and 70% ethanol: fine
 surgical scissors, blunt surgical scissors, blunt forceps, thin forceps, wound clips, wound staple</li>
 <li>Anesthetize mouse using with 2-3% isoflurane using an isoflurane scavenging machine. The average O2 flow rate used is 1 L/ minute through the nose cone.</li>
 <li>Lay the mouse on its back and immobilize the limbs with tape or other removable adhesive. Remove the hair with chemical hair remover such as Nair. Sterilize the abdomen with cotton swabbing alternatively between 70% ethanol and betadine in a target-like fashion.</li>
 <li>Hold up a part of the skin by the mammary gland using blunt forceps with one hand. With the other hand, using blunt surgical scissors to make an upside down T- incision between the #9 and #10 nipples or #4 and #5 nipples of the inguinal mammary glands to expose the mammary glands.</li>
 <li>Make a pocket incision under the lymph node or mammary artery with small surgical spring scissors.</li>
 <li>Remove the collagen graft from the tissue culture dish using forceps. Remove excess liquid from the collagen graft by gently dabbing onto a sterile wipe and slide the collagen graft completely into the pocket using a thin glass rod.</li>
 <li>Suture the skin flap using #2 gut absorbable sutures or wound staples.</li>
 <li>Let the mouse recover in the cage.</li>
 <li>Monitor the mice twice weekly at a minimum, palpating for tumors. Sacrifice the mice when the tumors reach 1 cm in diameter. Harvest tissues for analysis. </li>
</ol>

6. Representative Results: 
Isolation and extraction of collagen from C57BL/6J mice
These procedures were adapted from1. Extraction of collagen protein from 5-7 mouse tails yields approximately 1-1.5 mg/ml in a 6 ml final volume, or 6- 9 mg of protein. By coommassie stain, bands corresponding to 90 kda and 130 kda are detected in the lanes loaded with samples extracted from mouse tails, indicating the presence of collagen type I and pro-collagen respectively (Figure 1).
Isolation and culture of mammary carcinoma cells and fibroblasts from normal and PyVmT C57BL/6J mice.
The procedures were adapted from2. PyVmT carcinoma cells and fibroblasts can be distinguished by differences in cell morphology and expression of specific epithelial and mesenchymal markers. PyVmT carcinoma cells are identified by cobblestone shape and co-express CK18, a luminal epithelial marker and CK14, a basal epithelial marker, but not express &alpha;-sma (Figure 2). Mammary fibroblasts are larger cells with a spindle shaped phenotype and express high levels of &alpha;-sma but do not express CK14 or CK 18 (Figure 2). These data indicate over 95% cell purity for fibroblasts and epithelial cells using the outlined procedures.
Orthoptic transplantation of collagen embedded cells in C57BL/6J mice
These procedures were adapted from3, 4. Transplantation recipient mice are sacrificed when tumors in either experimental group reach 1.0 cm in diameter, or approximately 60 days. While transplantation of PyVmT cells alone results in palpable tumors after 30- 40 days, reaching a mean tumor mass of 0.335 grams at 60 days, co-transplantation of PyVmT carcinoma cells with mammary fibroblasts results in a mean tumor mass of 0.630 grams, indicating enhancement of tumor growth by fibroblasts (Figure 3).
<img src="/files/ftp_upload/2716/2716fig1.jpg" alt="Figure 1"/> Figure 1. Coomassie stain analysis of collagen type I extraction from mouse tails. BSA (&tilde; 66 kda) is indicated by arrow. Commercial rat tail collagen (20 &mu;g protein), and purified mouse tail collagen (20 &mu;g protein) are indicated by box (&tilde;130 kda, &tilde; 90 kda proteins). Std= molecular weight standard.
<img src="/files/ftp_upload/2716/2716fig2.jpg" alt="Figure 2"/> Figure 2. Immunofluorescence staining of donor PyVmT mammary carcinoma cells and fibroblasts. Panels a and b represent PyVmT mammary carcinoma cells immunostained for antibodies to CK14 and CK18. Panel c represents fibroblasts stained with antibodies to &alpha;-sma. Images shown with DAPI overlay at 20x magnification.
<img src="/files/ftp_upload/2716/2716fig3.jpg" alt="Figure 3"/> Figure 3. Mammary tumor development in C57BL/6J mice in the presence or absence of fibroblasts. Mammary tumors were harvested from mice transplanted with PyVmT carcinoma cells in the presence or absence of mammary fibroblasts and weighed. Mean+standard error of the mean. N=6 per group.

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Animal experiments:
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the IACUC committee at the University of Kansas Medical Center.

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

Research

JoVE Journal

Medicine

17.6K 次观看.  University of Kansas Medical Center. 在这份报告中，我们展示了一个系统，以隔离和文化的供体细胞，从小鼠乳腺和原位移植受体小鼠，这些细胞分析基质上皮细胞的相互作用：在乳腺肿瘤的发展。

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

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.

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.

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

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 ...

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 pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

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 pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

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 particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

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 functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

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 cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

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 initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

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 this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

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

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

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

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

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

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Subtitles

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