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

Summary

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.

Abstract

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 EpCAM^hi/CD49f^lo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAM^lo/CD49f^hi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAM^hi/CD49f^lo 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 EpCAM^hi/CD49f^lo luminal mammary epithelial cells.

Introduction

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 exosomes^4,5. 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 TSG101^6,7, can be measured by Western blotting.

Mammary basal cells have the ability to generate mammary glands when implanted into fat pads, while luminal cells cannot^8,9,10. 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.

Protocol

All research involving animals complied with protocols approved by the Institutional Committee on Animal Care.

1. Extracellular Vesicle/exosome Isolation and Validation

1. 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 µg/mL); insulin (5 µg/mL); bovine pituitary extract (BPE; 35 µg/mL); and GW627368X (1 µg/mL) in 15 cm dishes.
2. 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.
3. 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).
4. 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).
5. Centrifuge the supernatant at 10,000 x g for 30 min at 4 °C. Transfer the supernatant to a new ultracentrifuge tube (Figure 1).
6. Centrifuge the supernatant at 110,000 x g for 60 min at 4 °C. Remove the supernatant and resuspend the EV/exosome pellet in PBS (Figure 1).
7. Centrifuge the supernatant at 110,000 x g for 60 min at 4 °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 µL.
8. Measure the protein concentration of the EV suspension with the BCA protein assay. Ensure that the concentration is around 20-40 µg/100 µL. Store at -20 °C for further analysis.
9. 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 µg/100 µL) with PBS to 10,000-fold for NTA analysis.
   NOTE: The result of NTA analysis reflects the number and size of the vesicles analyzed.
10. Image the EVs/exosomes with transmissionelectron microscopy (TEM), as described previously by Lin et al⁴.

2. Exosome Purification Using a Density Gradient

1. 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.
2. Centrifuge the mixture at 200,000 x g for 8 h at 4 °C.
3. Collect each gradient fraction (10 fractions; 1 mL/fraction) with a pipette from the top of the tube.
4. Analyze the presence of exosome markers (e.g., CD81, CD9, CD63, and Tsg101) in each fraction by SDS-PAGE¹² and Western blot. Load 50-µL suspensions of each fraction onto a 10% gel containing 0.1% (w/v) SDS and separate the proteins in fractions with gel electrophoresis.
5. 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 °C.
   NOTE: The result identifies the fraction containing exosomes.

3. Extracellular Vesicle/Exosome Labeling

1. Suspend the EVs/exosomes, obtained in step 1.7, in 10 µM carboxyfluorescein succinimidyl diacetate ester (CFSE) at 20 µg of exosomal protein/100 µL. 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 °C for 30 min.
2. Suspend the EVs/exosomes in 50x volume of PBS and centrifuge the suspension at 110,000 x g for 60 min at 4 °C. Remove the supernatant and resuspend the EV/exosome pellet in PBS. Repeat step 3.2 once.
3. Suspend the EVs/exosomes in PBS at a concentration of 20 µg of exosomal protein/100 µL and then filter the EVs/exosomes through 0.22-µm membranes before adding the EVs/exosomes to the cells.

4. Extracellular Vesicle/Exosome Uptake Assay

1. 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 µg/mL); insulin (5 µg/mL); and BPE (35 µg/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 µg/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.
2. After the 2- to 6-h incubation, wash the cells twice with 4 mL of PBS at room temperature.
3. 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.

5. Isolation of Primary Mouse Mammary Epithelial Cells

1. Prepare gelatin-coated dishes by adding 12 mL of 0.1% gelatin solution to 10 cm dishes. Place the plates in a 37 °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.
2. 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.
3. Further dissociate the slurry of mammary glands (from 10 mice) for 60 min at 37 °C, 120 rpm agitation with 50 mL of DMEM/F12 containing 0.2% collagenase type IV, 0.2% trypsin, 5% FBS, 5 µg/mL gentamycin, and 1x pen-strep.
4. Pellet down the epithelial organoids from the mixture by centrifugation at 350 x g for 10 min.
5. 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.
6. Centrifuge the suspension at 400 x g for 10 min at room temperature and discard the supernatant.
7. 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.
8. 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).
9. 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 µg/mL gentamycin, 10 ng/mL EGF, and 1x pen-strep.
10. 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 µg/mL gentamycin, 10 ng/mL EGF, and 1x pen-strep).
11. Confirm that a monolayer of mammary epithelial cells migrates and grows out from the attached epithelial organoids in 3 days.

6. Separation of Primary Mouse Basal/Luminal Mammary Epithelial Cells

1. 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.
2. 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.
3. Centrifuge the suspension at 450 x g for 10 min at room temperature and discard the supernatant.
4. Re-suspended the cells in 100 µL 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.
5. Wash the cells with PBS and then incubate the cells with fluorophore-conjugated secondary antibody on ice for 30 min in the dark.
6. Wash and re-suspend the cells in PBS with 0.2% FBS.
7. Sort the EpCAM^hi/CD49f^lo luminal mammary epithelial cells on a cell sorter⁴.

7. Extracellular Vesicle/Exosome Treatment

1. Seed the sorted EpCAM^hi/CD49f^lo 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 µg/mL NAMEC-derived EVs/exosomes obtained in step 1.7.
2. 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 µg/mL NAMEC-derived EVs/exosomes every two days. Do not split the mouse primary luminal cells during the 10-day treatment.

8. Fat Pad Injection of Mammary Epithelial Cells

1. Anesthetize a 3-week-old female C57BL/6 mice with isofluorane 2-3% inhalant.
2. 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.
3. 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 4^th mammary fat pads.
4. 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.
5. 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.
6. 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 µL contains the desired cell dose (10⁴-10² cell/pad).
7. Inject 15 µL of mammary epithelial cell suspension into a fat pad using a 100 µL glass syringe attached to a 27G needle.
8. Repeat steps 8.4-8.7 for the fat pad on the other side.
9. Close the skin with wound clips.

9. Mammary Gland Whole Mount

1. Euthanize the mouse with CO₂ plus cervical dislocation at 8 weeks after the cell injection (Step 8.7).
2. 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 4^th mammary fat pads. Remove the 4^th mammary glands (Figure 2).
3. Spread the fat pads on glass microscope slides and fix the fat pads with Kahle's fixative (4% formaldehyde, 30% EtOH, and 2% glacial acetic acid) at room temperature overnight.
   Caution: Kahle's fixative is an irritant. Perform this step in a chemical hood.
4. 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.
5. 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.
6. 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.
7. Mount the slides with mounting medium and take images (2,400 dpi) of the fat pads using a digital slide scanner.

Representative Results

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 the medium must be pre-cleaned by ultracentrifugation at 110,000 x g before the medium is used to culture the source cells for the collection of EVs/exosomes⁵. EVs/exosomes from the 4 day induced NAMEC-conditioned medium can be isolated from the 110,000 x g pellet by differential ultracentrifugation, as illustrated in Figure 1. The number and size of the isolated vesicles in the 110,000 x g pellet can be measured using nanoparticle tracking analysis (NTA). The 110,000-g pellet fraction isolated by differential ultracentrifugation mainly contains ~100 nm vesicles (Figure 4A); this corresponds with the size of exosomes (50 - 150 nm) reported in the literature. In addition, TEM analysis showed that 110,000 g fractions of NAMEC-conditioned medium contain abundant membrane vesicles (Figure 4B). Although the differential ultracentrifugation can generate reasonably pure exosomes, the following purification step using a density gradient further eliminates contaminants (e.g., protein aggregates)⁵. In the density gradient, exosomes float in the gradient because of the lipid content in the vesicle, while protein aggregates, if any, stay at the bottom of the gradient. Each fraction of the gradient is collected for the detection of exosome makers (e.g., CD81, CD63, CD9, and TSG101^6,7) by Western blotting. Exosome markers can be detected in the fraction with ~20% iodixanol (Figure 5). The fraction containing exosomes can be diluted with PBS and subjected to 200,000 x g ultracentrifugation to isolate the exosomes. The isolated exosome pellet is washed once in PBS and then is resuspended in PBS and stored at -20 °C for further analysis.

Before measuring the uptake of NAMEC-derived EVs/exosomes by the non-stem counterpart of mammary epithelial cells-HMLE cells-the EVs/exosomes must be labeled with a fluorescent dye (e.g., carboxyfluorescein succinimidyl ester (CFSE)). A parallel sample containing only CFSE but no EV/exosome is processed in the same labeling procedure. This sample is a negative control used in the following EV/exosome uptake assay to reflect the effect of a trace amount of residual free CFSE dye. HMLE cells are cultured with CFSE-labeled, NAMEC-derived EVs/exosomes or the negative control for 2-6 h and are then subjected to flow cytometry. Compared to the untreated HMLE cells, the HMLE cells cultured with the negative control express a slightly higher level of CFSE signal (Figure 6A, red line versus orange line), which reflects the background level of CFSE signaling caused by residual free CFSE left from the EV/exosome labeling process. Furthermore, compared to the negative control-treated HMLE cells, the HMLE cells cultured with CFSE-labeled, NAMEC-derived exosomes express a 10-fold higher CFSE signal (Figure 6A, blue line versus red line), which results from the specific uptake of CFSE-labeled EVs/exosomes. The uptake of CFSE-labeled, NAMEC-derived EVs/exosomes by HMLE cells can also be observed by confocal microscopy. While the negative control-treated HMLE cells do not exhibit a CFSE signal, the uptake of NAMEC-derived EVs/exosomes by HMLE cells can be observed with the CFSE signal under the confocal microscope in CFSE-labeled EV/exosome-treated cells (Figure 6B).

To evaluate whether NAMEC-derived EVs/exosomes can transfer mammary gland-forming ability from stem-like mammary basal cells to mammary luminal cells, mouse mammary luminal cells are first isolated to allow for the analysis of mammary gland formation in mice. Mouse mammary epithelial cells are isolated from 12 week-old mice. The mammary glands are cut into small pieces and are further dissociated with collagenase and trypsin. The dissociated epithelial organoids and fibroblasts can be separated by differential centrifugation, as described in steps 5.7 and 5.8. In each round of centrifugation, the pellet at the bottom of the tubes should contain mainly epithelial organoids, and the fibroblasts and single cells should float in the supernatant. Compared to the mixture containing both epithelial organoids and fibroblasts before the differential centrifugation (Figure 3, upper panels), the six rounds of centrifugation clear out most fibroblasts and single cells in the mixture (Figure 3, bottom panel).

The mammary epithelial cells (Figure 7) of the epithelial organoids are further dissociated using a natural enzyme mixture with proteolytic and collagenolytic enzyme activity and dispase to generate single cells in suspension. Sorting the single-cell suspension by the expression of cell surface CD49f and EpCAM can separate mammary luminal cells (EpCAM^hi/CD49f^lo), mammary basal cells (EpCAM^lo/CD49f^hi), and non-epithelial cells (EpCAM^-) (Figure 8).

The isolated EpCAM^hi/CD49f^lo luminal mammary epithelial cells are cultured with NAMEC-derived EVs/exosomes for 10 days, and the fresh EVs/exosomes and medium are replaced every two days. After EV/exosome treatment, the mammary luminal cells are implanted into the 4^th mammary fat pads (Figure 2) of mice. After 8 weeks, the fat pads are isolated and stained for the analysis of mammary gland formation (Figure 9). The treatment with induced NAMEC-derived EVs/exosomes allows luminal cells to acquire mammary gland-forming ability⁴. The NAMEC-derived, EV/exosome-treated mammary luminal cells form mammary glands in mouse fat pads (Figure 9).

Figure 1: Illustration of the Extracellular Vesicle/Exosome Purification Method from Cell Culture Medium by Differential Ultracentrifugation. The speed and length of each centrifugation are indicated. After each of the first three centrifugations, the supernatant is kept for the next step. After the 110,000 × g centrifugations, the pellets are kept and the supernatants discarded. Please click here to view a larger version of this figure.

Figure 2: Mouse Mammary Gland Anatomy. Mice have five pairs of mammary glands, indicated by the numbers 1-5, located in the fat pads (red) directly underneath the skin. Please click here to view a larger version of this figure.

Figure 3: Images of a mixture of epithelial organoids and cells of fat pads before and after differential centrifugation. Bright-field images of the isolated fat-pad cell mixtures before and after differential centrifugation, taken from the hemocytometer. Arrowhead: epithelial organoids. Arrow: fibroblasts and single cells. Scale bar = 0.5 mm. Please click here to view a larger version of this figure.

Figure 4: Vesicle Size and Concentration Analysis of NAMEC110,000 x g Pellet Fraction. The 110,000 g pellet fraction of the NAMEC medium is collected and subjected to (A) nano- particle tracking analysis (NTA) and (B) transmissionelectron microscopy (TEM). Scale bar = 1 µm. Please click here to view a larger version of this figure.

Figure 5: Detecting Exosomes in Fractions of the Density Gradient. Western blot analysis of exosome markers CD81, CD9, CD63, and Tsg101 and housekeeping gene GAPDH reveals the 20% iodixanol fraction containing the exosomes. Please click here to view a larger version of this figure.

Figure 6: Detecting Extracellular Vesicle/Exosome Uptake by Flow Cytometry and Confocal Microscopy. EV/exosome uptake was measured in HMLE cells. CFSE-labeled, NAMEC-derived EVs/exosomes and negative control are added to the indicated cultures for 6 h. After the incubation, the cells are subjected to (A) flow cytometry and (B) confocal microscopy. The CFSE (green; excitation/emission (nm): 492/517; microscope laser line: 488) fluorescence intensities reflect the EV/exosome uptake. Cell nuclei are stained with DAPI (blue; excitation/emission (nm): 358/461; microscope laser line: 405) and the plasma membranes are stained with plasma membrane stain (red; excitation/emission (nm): 649/666; microscope laser line: 633; see the Table of Materials). Confocal objective lens: HCX PL APO 63x/1.40-0.60 Oil. Scale bar = 20 µm. Please click here to view a larger version of this figure.

Figure 7: Images of Attached Epithelial Organoids. Bright-field images of mammary epithelial cells formed by the cells migrating and growing out of the attached epithelial organoids. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 8: Sorting of Primary Mouse Mammary Epithelial Cells by Surface EpCAM and CD49f. Mouse mammary epithelial cells isolated from fat pads of 12-week-old mice are subjected to cell sorting. Mouse mammary luminal cells are enriched in the EpCAM^hi/CD49f^lo population, marked with the blue circle; basal cells were enriched in the EpCAM^lo/ CD49f^hi population, marked with the red circle. Non-epithelial cells are marked with the black box in the plot. Please click here to view a larger version of this figure.

Figure 9: Mammary Gland Formation by Primary Mouse Luminal Mammary Cells. The primary mouse EpCAM^hi/CD49f^lo luminal cells are treated with PBS or the NAMEC-derived EVs/exosomes in cell culture for 10 days and implanted into the cleared fat pads of 3-week-old mice. The mice are euthanized and necropsied after 8 weeks to analyze mammary gland formation. Scale bar = 0.75 cm.  Please click here to view a larger version of this figure.

	Percentage	MFI of EpCAM	MFI of CD49f
mammary luminal cell	0.437	4.57 x 104	8183
(EpCAMhi/CD49flo)
mammary basal cell	0.09	5452	2.23 x 104
(EpCAMlo/CD49fhi)
Non-epithelial cell (EpCAM-)	0.309	53	4619

Table 1: Percentage and Mean Fluorescence Intensity (MFI) of the Populations Described in Figure 8.

Discussion

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. Ultracentrifugation is a relatively fast method for the isolation of EVs/exosomes, which can preserve the biological activity of the EVs/exosomes. However, the vesicles isolated by ultracentrifugation generally contain a mixture of EVs, which contain exosomes produced via endosomal compartments and/or vesicles produced via budding from the cell membrane.

By analyzing the sizes of vesicles with NTA (Figure 4A), the vesicles found to purify at 110,000 x g during the differential ultracentrifugation were mainly 50-150 nm, which corresponds to the size of exosomes. The NTA data suggests that the NAMEC-derived exosomes can be isolated from the culture medium at 110,000 x g during differential ultracentrifugation. However, the NTA and TEM introduced here only allow for the visualization of vesicles or the measurement of the size and number of the whole population of EVs. These techniques cannot measure the heterogeneity of the EV populations or even sort the heterogeneous EV populations. Scientists have started to develop methods of using flow cytometers to analyze and sorting EV populations¹⁵.

Although differential ultracentrifugation can be used to purify exosomes, a density gradient should be used to further remove the contamination of protein aggregates from the exosome vesicles in the 110,000 x g isolated fraction⁵. Exosomes contain lipid membranes. The lipid in the exosome membranes makes the exosomes float in a density gradient, while protein aggregates fall to the bottom of the density gradient. Exosomes express specific markers (e.g., CD81, CD63, CD9, and TSG101^6,7). By analyzing the presence of exosome markers in the fractions of the density gradient, it is possible to identify exosomes in the fraction with ~20% iodixanol (Figure 5). Multiple exosome markers should be examined to identify exosomes in the density gradient, since each population of exosomes may express different exosome markers¹⁶.

The uptake of EVs/exosomes by cells can be measured using fluorescent dye-labeled EVs/exosomes. The number of cells ingesting labeled EVs/exosomes can be measured by flow cytometry (Figure 6A). To confirm that the fluorescence from the cells result from the uptake of labeled EVs/exosomes but not from free dye, the pattern of fluorescence was examined in the cells using microscopy. Confocal microscopy shows that the pattern of fluorescence in the EV/exosome-treated cells is punctate (Figure 6B right panel). The punctate signals likely result from labeled EVs/exosomes, not from free fluorescence. The punctate signals in the cells can be further analyzed with structured illumination super-resolution microscopy, which has the highest resolution at 85 nm^4,17. Super-resolution microscopy can confirm that the punctate signals are from ~100-nm hollow vesicles, which resemble exosomes⁴. These results suggest that NAMEC-derived exosomes can be ingested by mammary epithelial cells in culture.

NAMEC-derived EVs/exosomes often carry molecules (e.g., proteins and miRNAs) essential for the characteristics of certain cells¹. This suggests that NAMEC-derived EVs/exosomes can transfer the properties of NAMECs (e.g. mammary gland-forming ability) to their epithelial cell counterparts. Since human mammary epithelial cells cannot form mammary glands xenogeneically in mouse fat pads^18,19, the transfer of gland-forming ability can be examined using mouse primary mammary epithelial cells. Mouse primary mammary EpCAM^hi/CD49f^lo luminal cells, which do not form mammary glands, can be isolated from 6-week-old mice (Figure 7 and Figure 8). The isolated cells from mouse fat pads can be divided into three groups (i.e., EpCAM^hi/CD49f^lo luminal cells, EpCAM^lo/CD49f^hi basal cells, and EpCAM^- non-epithelial cells) by examining the levels of surface EpCAM and CD49f (Figure 8). EpCAM^lo/CD49f^hi basal cells can form mammary glands in fat fads when transplanted into fat pads, but EpCAM^hi/CD49f^lo luminal cells cannot⁴. Thus, the EpCAM^hi/CD49f^lo luminal cell population can be used to examine the ability of induced NAMEC-derived EVs/exosomes to transfer mammary gland-forming ability. The isolated EpCAM^hi/CD49f^lo luminal cells can be kept in culture for 7-10 days for the EV/exosome treatment⁴. It should be noted that keeping primary mammary epithelial cells in vitro for longer can attenuate the viability of the cells.

The EV/exosome-treated mammary luminal cells can be implanted into cleared fat pads to analyze the mammary gland-forming ability. The fat pads of mice used for implantation must be cleared at 3 weeks of age. At 3 weeks of age, mammary epithelial cells are confined to the region between the nipple and the lymph of a fat pad. Mammary epithelium in a fat pad can be cleared by removing the region between the nipple and the lymph at 3 weeks of age. Mammary luminal cells are implanted right after clearing the fat pad, and the gland formation by the implanted cells can be analyzed at 8 weeks after the implantation. The effect of NAMEC-derived EVs/exosomes on transferring mammary gland-forming ability to mammary luminal cells can be evaluated by the gland formation of luminal cells and EV/exosome-treated luminal cells (Figure 9). The induced EVs/exosomes from NAMECs carry the property of mammary basal epithelial cells – gland-forming ability – and the luminal cells that ingest the induced EVs/exosomes from NAMECs can acquire the property from NAMECs via EVs/exosomes. This work demonstrates evidence showing that molecules responsible for the EV/exosome-mediated transfer of mammary gland forming ability are present in lipid rafts of EVs/exosomes⁴.

Materials

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