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.

E....

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....

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 th.......

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. Ultracentrifuga.......

Materials

List of materials used in this article

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