This work was supported by grants from the National Health Research Institutes (05A1-CSPP16-014, H.J.L.) and from the Ministry of Science and Technology (MOST 103-2320-B-400-015-MY3, H.J.L).

All research involving animals complied with protocols approved by the Institutional Committee on Animal Care.
1. Extracellular Vesicle/exosome Isolation and Validation
<ol>
	<li>Culture mammary epithelial basal cells, NAMECs4, with fresh, serum-free medium made of 500 mL of MCDB 170, pH 7.4 + 500 mL of DMEM/F12 with sodium bicarbonate (0.2438%); EGF (5 ng/mL); hydrocoritisone (0.5 &#181;g/mL); insulin (5 &#181;g/mL); bovine pituitary extract (BPE; 35 &#181;g/mL); and GW627368X (1 &#181;g/mL) in 15 cm dishes.</li>
	<li>After counting the cells with a hemocytometer, seed 1.2 x 106 cells in 12 mL of medium per 15-cm dish on day 0 for 4 days4.</li>
	<li>After 4 days in culture, centrifuge the culture medium at 300 x g for 5 min using a table-top centrifuge. Transfer the supernatant to a conical tube (Figure 1).</li>
	<li>Centrifuge the supernatant at 2,000 x g for 20 min in a table-top centrifuge. Transfer the supernatant to an ultracentrifuge tube and leave the dead cells and cell debris (Figure 1).</li>
	<li>Centrifuge the supernatant at 10,000 x g for 30 min at 4 &#176;C. Transfer the supernatant to a new ultracentrifuge tube (Figure 1).</li>
	<li>Centrifuge the supernatant at 110,000 x g for 60 min at 4 &#176;C. Remove the supernatant and resuspend the EV/exosome pellet in PBS (Figure 1).</li>
	<li>Centrifuge the supernatant at 110,000 x g for 60 min at 4 &#176;C. Remove the supernatant. Resuspend the EV/exosome pellet in PBS (Figure 1). Resuspend the pellet isolated from 240-480 mL of NAMEC-conditioned medium in 100 &#181;L.</li>
	<li>Measure the protein concentration of the EV suspension with the BCA protein assay. Ensure that the concentration is around 20-40 &#181;g/100 &#181;L. Store at -20 &#176;C for further analysis.</li>
	<li>Measure the concentration and size of the EVs/exosomes by nanoparticle tracking analysis (NTA), as described previously by Gardiner et al.11. Dilute the EVs/exosomes (20 &#181;g/100 &#181;L) with PBS to 10,000-fold for NTA analysis. 
	NOTE: The result of NTA analysis reflects the number and size of the vesicles analyzed.</li>
	<li>Image the EVs/exosomes with transmissionelectron microscopy (TEM), as described previously by Lin et al4.</li>
</ol>
2. Exosome Purification Using a Density Gradient
<ol>
	<li>Resuspend the 110,000 g pellet from step 1.7 in 40% (w/v) iodixanol in PBS (2 mL). Overlay the mixture in sequence with aliquots of 30%, 20%, 10%, and 5% (w/v) iodixanol in PBS (2 mL each) to form a density gradient in an ultracentrifuge tube.</li>
	<li>Centrifuge the mixture at 200,000 x g for 8 h at 4 &#176;C.</li>
	<li>Collect each gradient fraction (10 fractions; 1 mL/fraction) with a pipette from the top of the tube.</li>
	<li>Analyze the presence of exosome markers (e.g., CD81, CD9, CD63, and Tsg101) in each fraction by SDS-PAGE12 and Western blot. Load 50-&#181;L suspensions of each fraction onto a 10% gel containing 0.1% (w/v) SDS and separate the proteins in fractions with gel electrophoresis.</li>
	<li>Transfer the proteins from a gel to a PVDF membrane and incubate the membrane with antibodies against the exosome markers (e.g., CD81, CD9, CD63, and Tsg101) and housekeeping protein GAPDH overnight (Table of Materials) at 4 &#176;C. 
	NOTE: The result identifies the fraction containing exosomes.</li>
</ol>
3. Extracellular Vesicle/Exosome Labeling
<ol>
	<li>Suspend the EVs/exosomes, obtained in step 1.7, in 10 &#181;M carboxyfluorescein succinimidyl diacetate ester (CFSE) at 20 &#181;g of exosomal protein/100 &#181;L. Prepare a parallel sample containing only CFSE and PBS, processed in the same manner, as a negative control for the later EV/exosome uptake assays. Leave the mixtures at 37 &#176;C for 30 min.</li>
	<li>Suspend the EVs/exosomes in 50x volume of PBS and centrifuge the suspension at 110,000 x g for 60 min at 4 &#176;C. Remove the supernatant and resuspend the EV/exosome pellet in PBS. Repeat step 3.2 once.</li>
	<li>Suspend the EVs/exosomes in PBS at a concentration of 20 &#181;g of exosomal protein/100 &#181;L and then filter the EVs/exosomes through 0.22-&#181;m membranes before adding the EVs/exosomes to the cells.</li>
</ol>
4. Extracellular Vesicle/Exosome Uptake Assay
<ol>
	<li>To make culture medium, mix 500 mL of MCDB 170, pH 7.4 + 500 mL of DMEM/F12 with sodium bicarbonate (0.2438%); EGF (5 ng/mL); hydrocoritisone (0.5 &#181;g/mL); insulin (5 &#181;g/mL); and BPE (35 &#181;g/mL)4. Plate the human mammary epithelial HMLE cells in 6-well dishes (1 x 106 cells/well) one day before EV/exosome treatment. Add 2 &#181;g/mL CFSE fluorescence-labeled EVs/exosome, obtained in step 3.3, to the culture medium of the HMLE cells for 2-6 h. Treat the HMLE cells of the negative control group with the parallel preparation, described in step 3.1.</li>
	<li>After the 2- to 6-h incubation, wash the cells twice with 4 mL of PBS at room temperature.</li>
	<li>Detach the cells with 0.25% trypsin for 10 min and re-suspend the cells in PBS containing 0.2% FBS. Measure the EV/exosome uptake from the fluorescence intensity in the cells using a fluorescence cell analyzer4. Image the cells treated with EVs/exosomes or the negative control using confocal microscopy. 
	NOTE: The green fluorescence in the cells is caused by the EV/exosome uptake. See the legend of Figure 6 for the microscope settings.</li>
</ol>
5. Isolation of Primary Mouse Mammary Epithelial Cells
<ol>
	<li>Prepare gelatin-coated dishes by adding 12 mL of 0.1% gelatin solution to 10 cm dishes. Place the plates in a 37 &#176;C incubator for 30 min. Remove the gelatin solution and leave the lids off the dishes in a laminar flow hood for 4 h until the gelatin coating has dried.</li>
	<li>Dissect the number 2, 3, 4, and 5 mammary glands (Figure 2) from 12-week-old virgin female C57BL/6 mice using scissors and cut the glands into small pieces (2 mm2) using a razor.</li>
	<li>Further dissociate the slurry of mammary glands (from 10 mice) for 60 min at 37 &#176;C, 120 rpm agitation with 50 mL of DMEM/F12 containing 0.2% collagenase type IV, 0.2% trypsin, 5% FBS, 5 &#181;g/mL gentamycin, and 1x pen-strep.</li>
	<li>Pellet down the epithelial organoids from the mixture by centrifugation at 350 x g for 10 min.</li>
	<li>Suspend the epithelial organoids in 4 mL of DMEM/F12 with 0.1 mg/mL DNase I for 5 min at room temperature. Add 6 mL of DMEM/F12 to a final volume of 10 mL.</li>
	<li>Centrifuge the suspension at 400 x g for 10 min at room temperature and discard the supernatant.</li>
	<li>Resuspend the pellet in 10 mL of DMEM/F12. Centrifuge the suspension but hit the brake when the speed reaches 400 x g. Discard the supernatant. 
	NOTE: By hitting the brake, epithelial organoids are quickly pelleted down, while fibroblasts, as single cells, still stay in the supernatant.</li>
	<li>Repeat step 5.6 and 5.7 5-7 times. Add a drop of the suspension to a hemocytometer and then check the clearance of fibroblasts from the organoid mixture under microscopy after each round of centrifugation (Figure 3).</li>
	<li>Plate the organoids in the gelatin-coated dish, obtained in step 5.1, for 48 h with DMEM/F12 containing 1x ITS, 5% FBS, 50 &#181;g/mL gentamycin, 10 ng/mL EGF, and 1x pen-strep.</li>
	<li>After 48 h, remove the floating cells in the culture by replacing the medium with fresh culture medium without FBS (DMEM/F12 containing 1x ITS, 50 &#181;g/mL gentamycin, 10 ng/mL EGF, and 1x pen-strep).</li>
	<li>Confirm that a monolayer of mammary epithelial cells migrates and grows out from the attached epithelial organoids in 3 days.</li>
</ol>
6. Separation of Primary Mouse Basal/Luminal Mammary Epithelial Cells
<ol>
	<li>After the 3-day culture, detach the mouse primary mammary cells with a natural enzyme mixture with proteolytic and collagenolytic enzyme activity for 20 min. Neutralize the enzyme activity with 5% FBS in PBS.</li>
	<li>Centrifuge the suspension at 450 x g for 10 min at room temperature and discard the supernatant. Resuspend the cell pellet in 5 mg/mL dispase for 20 min. Neutralize the enzyme activity with 5% FBS in PBS.</li>
	<li>Centrifuge the suspension at 450 x g for 10 min at room temperature and discard the supernatant.</li>
	<li>Re-suspended the cells in 100 &#181;L PBS with 0.2% FBS at a concentration of 107 cells/mL with the diluted anti-CD49f and anti-EpCAM antibodies on ice for 1 h in the dark.</li>
	<li>Wash the cells with PBS and then incubate the cells with fluorophore-conjugated secondary antibody on ice for 30 min in the dark.</li>
	<li>Wash and re-suspend the cells in PBS with 0.2% FBS.</li>
	<li>Sort the EpCAMhi/CD49flo luminal mammary epithelial cells on a cell sorter4.</li>
</ol>
7. Extracellular Vesicle/Exosome Treatment
<ol>
	<li>Seed the sorted EpCAMhi/CD49flo luminal mammary epithelial cells from step 6.7 on gelatin-coated 6-well dishes (2 x 105 cell/well) and then treat them with PBS or the 2 &#181;g/mL NAMEC-derived EVs/exosomes obtained in step 1.7.</li>
	<li>To ensure the biological efficacy of the EVs/exosomes in long-term culture treatment, replace the cell culture medium with fresh medium containing PBS or 2 &#181;g/mL NAMEC-derived EVs/exosomes every two days. Do not split the mouse primary luminal cells during the 10-day treatment.</li>
</ol>
8. Fat Pad Injection of Mammary Epithelial Cells
<ol>
	<li>Anesthetize a 3-week-old female C57BL/6 mice with isofluorane 2-3% inhalant.</li>
	<li>Place the anesthetized mouse on its back. Remove the fur on the mid-abdomen with a razor/hair cream and clean the surgery site with three alternating cycles of 70% alcohol and povidone-iodine.</li>
	<li>Make a 1.5 cm vertical incision through the skin along the ventral thoracic-inguinal region with scissors and then alternately expose the right and left 4th mammary fat pads.</li>
	<li>Clear each fat pad by removing gland parenchyma with scissors. Locate the lymph node in the fat pad and then remove the whole gland parenchyma below the lymph node. 
	NOTE: Two-thirds of the fat pad should remain in place.</li>
	<li>Detach the cells obtained from step 7.2 with a natural enzyme mixture (see the Table of Materials) with proteolytic and collagenolytic enzyme activity for 10 min. Neutralize the enzyme activity with 5% FBS in PBS.</li>
	<li>Centrifuge the suspension at 450 x g for 10 min at room temperature and discard the supernatant. Count the cells with a hemocytometer and suspend the cells in PBS at a concentration such that 15 &#181;L contains the desired cell dose (104-102 cell/pad).</li>
	<li>Inject 15 &#181;L of mammary epithelial cell suspension into a fat pad using a 100 &#181;L glass syringe attached to a 27G needle.</li>
	<li>Repeat steps 8.4-8.7 for the fat pad on the other side.</li>
	<li>Close the skin with wound clips.</li>
</ol>
9. Mammary Gland Whole Mount
<ol>
	<li>Euthanize the mouse with CO2 plus cervical dislocation at 8 weeks after the cell injection (Step 8.7).</li>
	<li>Make a vertical incision through the skin layer from the thoracic region to the inguinal region using scissors and then expose both the right and left 4th mammary fat pads. Remove the 4th mammary glands (Figure 2).</li>
	<li>Spread the fat pads on glass microscope slides and fix the fat pads with Kahle&#39;s fixative (4% formaldehyde, 30% EtOH, and 2% glacial acetic acid) at room temperature overnight. 
	Caution: Kahle&#39;s fixative is an irritant. Perform this step in a chemical hood.</li>
	<li>Wash the fat pads in 250 mL of 70% EtOH for 15 min and then in 250 mL of dH2O for 5 min. Stain the fat pads with carmine alum (1 g of carmine and 2.5 g of aluminum potassium sulfate in 500 mL of dH2O) at room temperature overnight.</li>
	<li>Wash the fat pads with 250 mL of 70% EtOH for 15 min, 250 mL of 95% EtOH for 15 min, and 250 mL of 100% EtOH for 15 min.</li>
	<li>Clean the fat pads in xylene for days and stop the xylene incubation when the fat pads become transparent. 
	Caution: Xylene is an irritant. Perform this step in a chemical hood.</li>
	<li>Mount the slides with mounting medium and take images (2,400 dpi) of the fat pads using a digital slide scanner.</li>
</ol>

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The authors have nothing to disclose.

Exosomes often carry characteristics of the cells that released them, and the amount of released exosomes can be induced by stimuli4. The culture medium of cells can be collected and subjected to differential ultracentrifugation for EV/exosome collection (Figure 1). There is currently no general agreement on an ideal method to isolate EVs/exosomes. The optimal method used here has been determined by the downstream application14. Ultracentrifuga...

Exosomes can mediate cellular communication by transferring membrane and cytosolic proteins, lipids, and RNAs between cells1. Exosome-mediated communication has been demonstrated to be involved in many physiological and pathological processes (i.e., antigen presentation, development of tolerance2, and tumor progression3). Exosomes often have contents similar to those of the source cells releasing them. Thus, the exosomes can carry specific cell properties from the source cells and transfer these properties to the cells ingesting them4.
Exosomes are 50- to 150-nm double-layer membrane vesicles and present specific markers (e.g., CD9, CD81, CD63, HSP70, Alix, and TSG101). Thus, exosomes must be characterized by various methods for different aspects. Transmission electron microscopy can be used to visualize membrane vesicles such as exosomes4,5. Nanoparticle tracking analysis (NTA) and dynamic light scattering analysis (DLS) are used for measuring the size and number of purified exosomes4. The lipid membrane content of exosomes can be verified by density gradient. Exosomal markers, such as CD9, CD81, CD63, HSP70, Alix, and TSG1016,7, can be measured by Western blotting.
Mammary basal cells have the ability to generate mammary glands when implanted into fat pads, while luminal cells cannot8,9,10. Thus, mammary basal cells are also referred to as mammary repopulating units. By using the model of mammary basal and luminal cells, the ability of EVs/exosomes to transfer cell characteristics between different cell populations can be examined. This work demonstrates the method of transferring gland-forming ability from mammary basal epithelial cells to mammary luminal epithelial cells by using EVs/exosomes derived from mammary basal epithelial cells. Luminal mammary epithelial cells acquired basal cell properties following the ingestion of EVs/exosomes secreted from basal cells and can then form mammary glands4.

<table><tbody><tr><td>MCDB 170&amp;nbsp;</td><td>USBiological</td><td>M2162</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo</td><td>1250062</td><td></td></tr><tr><td>Optima L-100K ultracentrifuge</td><td>Beckman</td><td>393253</td><td></td></tr><tr><td>SW28 Ti Rotor</td><td>Beckman</td><td>342204</td><td></td></tr><tr><td>SW41 Rotor</td><td>Beckman</td><td>331306</td><td></td></tr><tr><td>NANOSIGHT LM10</td><td>Malvern</td><td>NANOSIGHT LM10</td><td>for nanoparticle tracking analysis (NTA)</td></tr><tr><td>Optiprep&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D1556</td><td>60% (w/v) solution of iodixanol in water (sterile).</td></tr><tr><td>CD81 antibody</td><td>GeneTex</td><td>GTX101766</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CD9 antibody</td><td>GeneTex</td><td>GTX100912</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CD63 antibody</td><td>Abcam</td><td>Ab59479</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>TSG101 antibody</td><td>GeneTex</td><td>GTX118736</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>GAPDH</td><td>GeneTex</td><td>GTX100118</td><td>1:6,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CFSE (carboxyfluorescein succinimidyl diacetate ester)</td><td>Thermo</td><td>V12883</td><td></td></tr><tr><td>FACSCalibur</td><td>BD Biosciences</td><td></td><td>fluorescence cell analyzer</td></tr><tr><td>collagenase Type IV&amp;nbsp;</td><td>Thermo</td><td>17104019</td><td></td></tr><tr><td>trypsin</td><td>Thermo</td><td>27250018</td><td></td></tr><tr><td>&amp;nbsp;ITS</td><td>Sigma-Aldrich</td><td>I3146</td><td>a mixture of recombinant human insulin, human transferrin, and sodium selenite</td></tr><tr><td>accutase</td><td>ebioscience</td><td>00-4555-56</td><td>a natural enzyme mixture with proteolytic and collagenolytic enzyme activity</td></tr><tr><td>dispase&amp;nbsp;</td><td>STEMCELL</td><td>7913</td><td>5 mg/mL = 5 U/mL</td></tr><tr><td>anti-CD49f antibody</td><td>Biolegend</td><td>313611</td><td>1:50</td></tr><tr><td>anti-EpCAM antibody</td><td>Biolegend</td><td>118213</td><td>1:200</td></tr><tr><td>FACSAria</td><td>BD Biosciences</td><td></td><td>cell sorter</td></tr><tr><td>carmine alum</td><td>Sigma-Aldrich</td><td>C1022</td><td></td></tr><tr><td>human mammary epithelial cells (HMLE cells, NAMECs)</td><td></td><td></td><td>gifts from Dr. Robert Weinberg</td></tr><tr><td>permount</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>SP15-500</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Zymeset&amp;nbsp;</td><td>BSB101</td><td></td></tr><tr><td>EGF</td><td>Peprotech</td><td>AF-100-015</td><td></td></tr><tr><td>Hydrocoritisone</td><td>Sigma-Aldrich</td><td>SI-H0888</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SI-I9278</td><td></td></tr><tr><td>BPE (bovine pituitary extract)</td><td>Hammod Cell Tech&amp;nbsp;</td><td>1078-NZ</td><td></td></tr><tr><td>GW627368X&amp;nbsp;</td><td>Cayman</td><td>10009162</td><td></td></tr><tr><td>15 cm culture dish</td><td>Falcon&amp;nbsp;</td><td>353025</td><td></td></tr><tr><td>table-top centrifuge</td><td>Eppendrof&amp;nbsp;</td><td>Centrifuge 3415R</td><td></td></tr><tr><td>ultracentrifuge tube</td><td>Beckman</td><td>344058</td><td></td></tr><tr><td>PBS (Phosphate-buffered saline)&amp;nbsp;</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>BCA Protein Assay</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>23228</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>Hitachi</td><td>HT7700</td><td></td></tr><tr><td>gelatin&amp;nbsp;</td><td>STEMCELL</td><td>7903</td><td></td></tr><tr><td>10 cm culture dish</td><td>Falcon&amp;nbsp;</td><td>353003</td><td></td></tr><tr><td>6-well culture dish</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>female C57BL/6 mice</td><td>NLAC (National Laboratory Animal Center</td><td></td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>BioWest&amp;nbsp;</td><td>S01520</td><td></td></tr><tr><td>gentamycin</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>15710072</td><td></td></tr><tr><td>Pen/Strep</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>DNase I</td><td>5PRIMER</td><td>2500120</td><td></td></tr><tr><td>isofluorane&amp;nbsp;</td><td>Halocarbon</td><td>NPC12164-002-25</td><td></td></tr><tr><td>formaldehyde</td><td>MACRON</td><td>H121-08</td><td></td></tr><tr><td>EtOH (Ethanol)</td><td>J.T. Baker</td><td>800605</td><td></td></tr><tr><td>glacial acetic acid</td><td>Panreac</td><td>131008.1611</td><td></td></tr><tr><td>aluminum potassium sulfate</td><td>Sigma-Aldrich</td><td>12625</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Leica</td><td>3803665</td><td></td></tr><tr><td>0.22 &amp;mu;m membranes</td><td>Merck Millipore</td><td>Millex-GP</td><td></td></tr><tr><td>AUTOCLIP Wound Clips, 9 mm</td><td>BD Biosciences</td><td>427631</td><td></td></tr><tr><td>AUTOCLIP Wound Clip Applier</td><td>BD Biosciences</td><td>427630</td><td></td></tr><tr><td>CellMask&amp;trade; Deep Red</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>C10046</td><td>plasma membrane stain</td></tr></tbody></table>

transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

Since it has been shown that blocking PGE2/EP4 signaling triggers EV/exosome release from mammary basal-like stem cells4, this work presents a method of isolating the induced EVs/exosomes from mammary epithelial basal cell (NAMEC) culture. Since NAMECs are cultured in serum-free medium, there are no pre-existing EVs/exosomes derived from serum13. For cells cultured in serum-containing medium, pre-existing exosomes in th...

Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

transfer-mammary-gland-forming-ability-between-mammary-basal

Research

JoVE Journal

Developmental Biology

11.6K Views.  National Health Research Institutes. This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Video: Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

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.

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 ...

Medicine

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Cancer Research

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Biology

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Summary

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Chapters in this video

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

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Mammary Epithelial Transplant Procedure

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Evaluation of Mammary Gland Development and Function in Mouse Models

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology