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[Cancer Research 65, 10952-10958, December 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Novel Extracellular Vesicles Mediate an ABCG2-Dependent Anticancer Drug Sequestration and Resistance

Ilan Ifergan1, George L. Scheffer2 and Yehuda G. Assaraf1

1 Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel and 2 Department of Pathology, VU Medical Center, MB Amsterdam, the Netherlands

Requests for reprints: Yehuda G. Assaraf, Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel. Phone: 972-4-829-3744; E-mail: assaraf{at}tx.technion.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Overexpression of the multidrug efflux transporter ABCG2 in the plasma membrane of cancer cells confers resistance to various anticancer drugs, including mitoxantrone. Here, we explored the mechanism underlying drug resistance in the MCF-7 breast cancer sublines MCF-7/MR and MCF-7/FLV1000 cells in which wild-type (R482) ABCG2 overexpression is highly confined to cell-cell attachment zones. The latter comprised the membrane of novel extracellular vesicles in which mitoxantrone was rapidly and dramatically sequestered. After 12 hours of incubation with mitoxantrone, the estimated intravesicular drug concentration was ~1,000-fold higher than in the culture medium. This drug compartmentalization was prevented by the specific and potent ABCG2 transport inhibitors Ko143 and fumitremorgin C, thereby resulting in restoration of drug sensitivity. Consistently, this intravesicular drug concentration was abrogated by energy deprivation and was restored upon provision of energy substrates. Fine-structure studies corroborated the presence of numerous large extracellular vesicles that were highly confined to cell-cell attachment zones between neighbor cells. Furthermore, high-resolution electron microscopy revealed that the membrane of these extracellular vesicles contained microvilli-like invaginations protruding into the intravesicular lumen. It is likely that these microvilli-like projections increase the vesicular membrane surface, thereby allowing for a more efficient ABCG2-dependent intravesicular anticancer drug concentration. Hence, these novel extracellular vesicles mediate the ABCG2-dependent extraction of intracellular drug, thereby serving as cytotoxic drug disposal chambers shared by multiple neighbor cancer cells. This constitutes a novel modality of anticancer drug resistance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chemotherapeutic agents constitute a key component of the treatment of various human malignancies. However, the frequent emergence of cancer cells with a simultaneous resistance to multiple anticancer drugs, a trait known as multidrug resistance, poses a major obstacle towards curative cancer chemotherapy (1–6). Hence, the elucidation of the molecular mechanisms underlying inherent and acquired multidrug resistance is a prerequisite to the overcoming of anticancer drug resistance. Several transporters of the ATP-binding cassette (ABC) superfamily have the facility to actively translocate an extraordinarily diverse array of structurally dissimilar endogenous and exogenous substrates and their metabolites across cell membranes (reviewed in refs. 1, 2). Among these are three important anticancer drug efflux transporters, including P-glycoprotein (Pgp/ABCB1; reviewed in refs. 1, 2, 5, 6), the multidrug-resistant protein 1 (MRP1/ABCC1; ref. 3), and breast cancer resistance protein (BCRP/ABCG2; ref. 4). Overexpression of these multidrug-resistant efflux transporters in the plasma membrane of various malignant cells results in an ATP-dependent decrease in cellular drug accumulation (1, 2, 4–6) and acquisition of multidrug resistance to a multitude of anticancer drugs. However, little is known about the role of the overexpression of multidrug-resistant transporters in alternative localizations that are distinct from the plasma membrane (7). As a step toward this end, we here explored the mechanism underlying mitoxantrone resistance in breast cancer cells in which ABCG2 overexpression is highly confined to cell-cell attachment zones. We report on the identification of a novel mechanism of drug resistance that is based upon a dramatic ABCG2-dependent drug concentration in novel extracellular vesicles shared by neighbor breast cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals. Mitoxantrone hydrochloride was from Cyanamid of Great Britain Ltd. (Gosport, Hampshire, United Kingdom). Ko143 was generously provided by Dr. A.H. Schinkel (The Netherlands Cancer Institute, Amsterdam, the Netherlands), whereas fumitremorgin C and flavopiridol were kindly provided by Dr. S.E. Bates (National Cancer Institute, Bethesda, MD). Sodium azide and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were from Sigma (St. Louis, MO).

Tissue culture and growth inhibition with mitoxantrone. MCF-7 human breast cancer cells and their mitoxantrone-resistant MCF-7/MR (8) and flavopiridol-resistant MCF-7/FLV1000 (9) sublines kindly provided by Dr. S.E. Bates were grown as monolayers in RPMI 1640 as previously described (7, 10). It should be emphasized that in a previous study we found that MCF-7/MR cells overexpressed the wild-type R482 ABCG2; thus, no ABCG2 mutations were present in MCF-7/MR (10) and MCF-7/FLV1000 cells (11). Specifically, MCF-7/MR cells were pulsed with 100 nmol/L mitoxantrone every 2 weeks for 3 days, whereas MCF-7/FLV1000 cells were continuously grown in the presence of 1 µmol/L flavopiridol. All subsequent experiments were initiated after 4 days of incubation with mitoxantrone-free or flavopiridol-free medium. Cells (5 x 103 per well) were seeded in 24-well plates in growth medium (2 mL/well) and incubated for 48 hours at 37°C. Then, the medium of MCF-7 and MCF-7/MR cells was replaced with a fresh one lacking or containing Ko143 (0.4 µmol/L). After 2 hours of incubation, mitoxantrone was added at various concentrations. Then, the cells were exposed to this drug for 5 hours at 37°C, following which the drug-containing medium was aspirated, and three successive washes (each of 10 minutes) in RPMI 1640 containing 10% dialyzed fetal bovine serum (FBS) were done at 37°C. Drug-free medium was then added (2 mL/well), and cultures were incubated for four additional days at 37°C. Finally, cells were detached by trypsinization, and the number of viable cells was determined microscopically using trypan blue exclusion.

Western blot analysis of ABCG2. ABCG2 levels as normalized to ß-tubulin were determined by Western blots using monoclonal antibodies BXP-53 and clone 2-28-33, respectively, as previously described (7, 10).

Mitoxantrone accumulation and immunohistochemical localization of ABCG2 in specific colonies of MCF-7/MR and MCF-7/FLV1000 cells. Cells (5 x 104) were seeded in 25-mm tissue culture flasks (5-mL medium) and incubated for 4 days. Then, the growth medium was replaced with a fresh one containing 20 µmol/L mitoxantrone. After 12 hours of incubation at 37°C, monolayer cells were washed thrice with medium containing 10% dialyzed FBS. Then, 1 mL of medium was added to each flask, and random colonies were examined using a LEICA microscope at a bright-field mode. For immunohistochemical staining of ABCG2, monolayer cells were then processed as previously described (7, 10). The immunolocalization results obtained with BXP-53 were fully corroborated with other monoclonal antibodies to ABCG2, including BXP-21 and BXP-43.

Determination of the number of light-refracting extracellular vesicles. MCF-7 and MCF-7/MR cells (5 x 104) were seeded in 25-mm tissue culture flasks (5-mL medium per flask) and incubated for 4 days at 37°C, following which the medium was replaced with a fresh one (1 mL/flask). Random colonies were examined for visible, light-refracting extracellular vesicles using a LEICA microscope at a bright-field mode. Three independent experiments were done using ~200 cells in each determination for each cell line.

Inhibition of mitoxantrone accumulation with ABCG2 transport inhibitors and ATP-depleting agents. Cells were seeded in 24-well plates (104 per well) and incubated for 4 days at 37°C. Then, the medium of a control well was replaced with a fresh one. In contrast, the medium of the three remaining wells was replaced with one containing either 0.4 µmol/L Ko143, 5 µmol/L fumitremorgin C, or the combination of the metabolic energy inhibitors 5 µmol/L FCCP and 5 mmol/L sodium azide. Following 1 hour of incubation at 37°C, mitoxantrone was added to a final concentration of 20 µmol/L. After 6 hours of incubation at 37°C, the growth medium was aspirated and a wash step with medium containing 10% dialyzed FBS was done. Then, fresh medium was added (0.3 mL/well), and random colonies were rapidly examined for their mitoxantrone blue staining using a LEICA microscope at a bright-field mode. To remove the ABCG2 transport and metabolic energy inhibitors, the cells were incubated twice (each for 7 minutes) in fresh growth medium at 37°C followed by aspiration of the medium. Fresh medium containing 20 µmol/L mitoxantrone was then added; cells previously incubated with FCCP and azide were also supplemented with the ATP-restoring agents sodium pyruvate (1 mmol/L; Life Technologies Bethesda Research Laboratories, Gaithersburg, MD) and D-glucose (5 mmol/L; Sigma). After 6 hours of incubation at 37°C, the medium was discarded, and cells were washed once with medium containing 10% dialyzed FBS. Then, fresh medium was added (0.3 mL/well), and random colonies were examined microscopically for mitoxantrone accumulation.

Estimation of the intravesicular concentration of mitoxantrone. To explore the time dependence of mitoxantrone accumulation in the intravesicular lumen, we incubated cells with 20 µmol/L mitoxantrone for 3 to 12 hours at 37°C. Photographs of random colonies were taken using a LEICA microscope at a bright-field mode. To generate a calibration curve, 10-µL aliquots of standard solutions containing increasing mitoxantrone concentrations were dispensed onto glass slides, which were then covered by glass coverslips. Photographs were taken at random locations using a bright-field mode. Photographs were then transformed to a gray-scale format and analyzed individually by scanning densitometry using the program "TINA" (version 2.10g). The densitometric background levels of the calibration curve (i.e., at a zero mitoxantrone concentration) and the monolayer cell culture with unstained extracellular vesicles (t = 0) were numerically normalized. The experiment was done thrice using ~150 extracellular vesicles in each experiment for each incubation time with mitoxantrone.

Autofluorescence detection with viable cells. Cells (4 x 103) were seeded in 24-well plates (2-mL phenol red–free medium per well) for 4 days at 37°C in the presence or absence of 0.4 µmol/L Ko143. Then, 1.5 mL of the growth medium was removed, and random colonies were examined for their autofluorescence using a fluorescence microscope at an FITC-like mode; a bright-field mode examination was also used here.

Confocal microscopy of ABCG2 confinement to cell-cell attachment zones. Cells (4 x 103) were seeded in 24-well plates (1-mL medium per well) on sterile glass coverslips and incubated for 4 days at 37°C. Cells were then washed, reacted with the ABCG2-specific monoclonal antibody BXP-53 followed by a secondary goat anti-rat IgG and a third FITC-conjugated rabbit-anti-goat IgG as described recently (7). The fluorescent cells were then examined for cross-section and perpendicular section using a Bio-Rad (Richmond, CA) MRC1024 confocal microscope.

Confocal microscopy studies of the accessibility of the culture medium to the extracellular vesicles. MCF-F/MR cells (4 x 103 per well) were seeded in 24-well plates (2-mL medium per well) on sterile glass coverslips and incubated for 3 days at 37°C. The growth medium was removed, and monolayer cells were washed twice with a fresh medium. Then, a PBS solution containing 10% FCS, 15% tetramethylrhodamine isothiocyanate (TRITC)/goat anti-rabbit IgG (Sigma, T5268) was placed between the coverslips and the glass slides. The slides were then examined using a Bio-Rad MRC1024 confocal microscope for cross-section and perpendicular section. The FITC-like mode was used to follow the green autofluorescence of the vesicles and the red fluorescence of the culture medium was detected by a Kr/Ar laser (excitation at 568 nm and emission at 585 nm).

Electron microscopy studies. The presence of extracellular vesicles and their fine structure were studied by first seeding MCF-7/MR cells on glass slides mounted on eight-well tissue culture chambers (Lab-Tek, Nunc, Naperville, IL). The cells were grown for 4 days until confluence was achieved; an examination under a light microscope revealed the presence of numerous large vesicles. Slides containing monolayer cells were fixed using an overnight incubation in 2% glutardialdehyde in phosphate buffer and an additional 30-minute incubation in osmium tetroxide/collidine (2:1) buffer for 30 minutes. Slides were dehydrated using solutions containing increasing concentrations of ethanol (70-100%). Then, the chambers were discarded, and the slides containing monolayer cells were impregnated for 1.5 hours in a solution of Epon/propylene oxide/DMP-30 (1:1:0.02). Cells were embedded by placing on top of them open-end capsules that were filled with embedding fluid (Epon/DMP-30, 1:0.015), following which polymerization was allowed overnight at 70°C. After polymerization, the glass slides were removed by snap-freezing in liquid nitrogen and thawing, thereby resulting in the entrapment of the monolayer cells in the polymerized resin. Then, ultrathin sections (60-70 nm) were cut using a Diatome diamant knife and an LKB Ultrotome III and collected on support film-coated (1.5% Formvar in dichloroethane) Cu grids. The sections were then counterstained with 2% uranyl acetate for 20 minutes and lead nitrate/sodium tricitrate for 20 minutes and then examined with a Jeol 1200EX electron microscope. Photographs were finally printed using a Leitz Focomat IIc.

Statistical analysis. We used a Student's t test to examine the significance of the difference between two populations for a certain variable. A difference between the averages of two populations was considered significant if the obtained P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression and immunolocalization of ABCG2 to extracellular vesicles in mitoxantrone-resistant MCF-7/MR cells. MCF-7/MR breast cancer cells overexpress ABCG2 (7, 8) and consequently display 20-fold resistance to the established ABCG2 transport substrate mitoxantrone, relative to their parental MCF-7 cells (Fig. 1). Sensitivity to this anticancer drug was restored with Ko143, a potent and specific ABCG2 transport inhibitor (12). Previously, we have shown that ABCG2 was highly confined to cell-cell attachment zones in monolayers of MCF-7/MR cells (7). Microscopic examination of immunohistochemical staining with anti-BCRP as well as after hematoxylin staining of monolayers of MCF-7 (Fig. 2A-C) and MCF-7/MR cells (Fig. 2D-F) revealed numerous extracellular vesicle-like structures that were highly confined to cell-cell attachment zones between multiple neighbor cells. Immunohistochemical analysis of MCF-7/MR cells with a monoclonal antibody to ABCG2 (BXP-53) revealed that ABCG2 was highly confined to the vesicular membrane contacting the surrounding cells (Fig. 2D-E, continuous arrows) as well as to cell-cell attachment zones (Fig. 2E, dashed arrow); no staining was observed in the absence of BXP-53 antibodies (Fig. 2F). In contrast, parental MCF-7 cells which poorly express ABCG2 did not show any detectable staining of the vesicular membrane whether the BXP-53 antibody was present (Fig. 2A-B) or absent (Fig. 2C). These immunolocalization results with the BXP-53 antibody were recapitulated with additional monoclonal antibodies to ABCG2, including BXP-21 and BXP-34 (data not shown).



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  		Figure 1. Cellular growth inhibition with mitoxantrone. Parental MCF-7 and MCF-7/MR cells were exposed to various concentrations of mitoxantrone for 5 hours in the absence or presence of the ABCG2 inhibitor Ko143 (0.4 µmol/L). After 4 days of incubation in drug-free medium, the number of viable cells was determined. Points, means of three independent experiments; bars, SD. Inset, Western blot analysis of ABCG2 expression as normalized to ß-tubulin.

 


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  		Figure 2. Immunohistochemical localization of ABCG2 in parental MCF-7 cells and their MCF-7/MR subline. MCF-7 (A-C) and MCF-7/MR cells (D-F) grown in 24-well culture plates were fixed with formaldehyde and reacted with (A, B, D, and E) or without (C and F) the anti-BCRP monoclonal antibody BXP-53 followed by the addition of horseradish peroxidase–conjugated rabbit anti-mouse IgG as the second antibody. Color development was carried out using the chromogen 3,3'-diaminobenzidine (brown). Cells were then counterstained with hematoxylin (violet) and examined with a light microscope at a x200 magnification. Continuous arrows, extracellular vesicles (A-F). Note that ABCG2 precisely localizes to the membrane surrounding the extracellular vesicles (D and E, continuous arrows) and to cell-cell attachment zones in MCF-7/MR cells (E, dashed arrow). These immunolocalization results with BXP-53 were also corroborated with additional monoclonal antibodies to ABCG2, including BXP-21 and BXP-34 (data not shown).

 
Fine-structure studies corroborated the presence of multiple extracellular vesicles in MCF-7/MR cells (Fig. 3A-B, continuous arrows), particularly between multiple neighbor cells (Fig. 3B, dashed arrows). High-resolution electron microscopy revealed that the vesicular membrane had a typical lipid bilayer structure (Fig. 3C, continuous arrow). Moreover, these extracellular vesicles contained microvilli-like invaginations protruding into the vesicular lumen (Fig. 3C, dashed arrow).



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  		Figure 3. Transmission electron microscopy analysis of the extracellular vesicles in monolayers of MCF-7/MR cells. MCF-7 MR cells were grown on glass slides until confluence was achieved. Slides containing monolayer cells were then fixed, dehydrated with ethanol, embedded in an Epon/DMP-30 resin, cut with an ultrotome, and analyzed with an electron microscope as in Materials and Methods. A and C, continuous arrows, membrane of the extracellular vesicles. Magnification, x4500 (A) and x18,000 (C). B, dashed arrows, plasma membrane of neighbor cells surrounding the vesicle. Magnification, x9,000. C, furthermore, high-resolution electron microscopy revealed that these vesicles contained a lipid bilayer membrane (continuous arrow) with multiple microvilli-like invaginations protruding into the intravesicular lumen (dashed arrow).

 
Intravesicular concentration of mitoxantrone in an ATP-dependent and ABCG2-dependent manner. We explored the mechanism underlying mitoxantrone resistance in breast cancer MCF-7/MR cells in which ABCG2 overexpression is highly confined to these extracellular vesicles in cell-cell attachment zones. The intense blue color of mitoxantrone rendered drug accumulation readily discernible by light microscopy. Pulse exposure of MCF-7/MR breast cancer cells to 20 µmol/L mitoxantrone for 6 hours resulted in a dramatic sequestration of this blue drug in the lumen of these extracellular vesicles that were confined to cell-cell attachment zones (Fig. 4A). These extracellular vesicles residing in between neighbor cells refracted light; this was used to estimate the number of extracellular vesicles. The number of light-refracting extracellular vesicles per 100 cells was estimated to be 23.3 ± 2.5 and 3.2 ± 0.5 in drug-resistant MCF-7/MR and parental MCF-7 cells, respectively. Furthermore, the number of mitoxantrone-concentrating extracellular compartments was 44.1 ± 6.5 per 100 MCF-7/MR cells and none in parental cells. In agreement with the above results, immunohistochemical analysis revealed that ABCG2 staining formed a circumferential ring in the membrane of the extracellular vesicles (Fig. 4B), thereby establishing that ABCG2 is highly confined to the vesicular membrane contacting the surrounding cells. In contrast, ABCG2 was barely detectable in the apical and basal membranes of these vesicles (Fig. 4B). The intense blue color of the sequestered mitoxantrone in these extracellular vesicles allowed for the quantification of the intravesicular concentration of the drug. Based on a calibration curve of known mitoxantrone concentrations (Fig. 4C), the intravesicular concentration of the drug was estimated to be as high as 12.8 ± 3.5 mmol/L after 6 hours of incubation with 20 µmol/L mitoxantrone and further increased in a time-dependent manner to ~20 mmol/L after 12 hours (Fig. 4D). Hence, the intravesicular concentration of mitoxantrone after 12 hours of incubation with this drug was ~1,000-fold higher than in the culture medium (P = 5.5 x 10–12). Similarly, the intravesicular concentration of mitoxantrone was also explored in flavopiridol-resistant MCF-7/FLV1000 cells with ABCG2 overexpression. Consistently, MCF-7/FLV1000 cells, which also contained extracellular vesicles, albeit at a lower frequency than MCF-7/MR cells, displayed a robust intravesicular concentration of mitoxantrone (data not shown). This latter result suggests that the extracellular vesicles and their ability to sequester mitoxantrone in an ABCG2-dependent manner is not limited to mitoxantrone-resistant MCF-7/MR cells.



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  		Figure 4. Mitoxantrone accumulation in extracellular vesicles and immunohistochemical localization of ABCG2 in MCF-7/MR cells. Cells were incubated in growth medium containing 20 µmol/L mitoxantrone for 12 hours at 37°C. Then, cells were washed thrice, fresh medium was added, and random colonies were examined by light microscopy at a bright-field mode. A, note that mitoxantrone accumulated in extracellular vesicles (blue extracellular vesicles). B, cells were then processed for immunohistochemical localization of ABCG2 (brown); colonies were examined by light microscopy after counterstaining of nuclei with hematoxylin (violet). Note that ABCG2 precisely localizes to the membrane surrounding the extracellular vesicles that have previously accumulated mitoxantrone (A). The intravesicular concentration of mitoxantrone was estimated using a mitoxantrone calibration curve (C) revealing drug concentrations of ~20 mmol/L after 12 hours of incubation (D). Columns, means of three independent experiments using ~150 extracellular vesicles in each experiment for each incubation time; bars, SD. The estimated intravesicular concentration of mitoxantrone at 6 and 12 hours of incubation was significantly higher than that at 3 hours (P = 4.9 x 10–16 and 5.5 x 10–12, respectively).

 
The intravesicular concentration of mitoxantrone in MCF-7/MR cells (Fig. 5A) was prevented by the specific and potent ABCG2 drug efflux inhibitors Ko143 (Fig. 5B) and fumitremorgin C (Fig. 5C) as well as by energy deprivation achieved by treatment with the respiration inhibitor sodium azide and the uncoupler FCCP (Fig. 5D). To confirm that the high concentration of mitoxantrone did not impair the ability of the vesicles to concentrate this drug, we did an experiment in which the ABCG2 transport inhibitor and metabolic energy inhibitors were first washed out followed by mitoxantrone reaccumulation. Hence, washing out the drug efflux inhibitors followed by further incubation with mitoxantrone restored the intravesicular concentration of this drug (Fig. 5F-G) at a level that was comparable with untreated cells (Fig. 5E). Likewise, washing out the metabolic energy inhibitors followed by provision of the energy substrates glucose and pyruvate in the presence of mitoxantrone restored the intravesicular sequestration of the drug (Fig. 5H).



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  		Figure 5. Prevention of intravesicular mitoxantrone accumulation by ABCG2 transport inhibitors and metabolic energy deprivation: MCF-7/MR cells were incubated for 1 hour at 37°C in medium (A) lacking or containing either (B) 0.4 µmol/L Ko143, (C) 5 µmol/L fumitremorgin C, or a (D) combination of the metabolic energy inhibitors FCCP (5 µmol/L) and azide (5 mmol/L). Mitoxantrone was added at 20 µmol/L, and cells were incubated for six additional hours at 37°C. Random colonies were then rapidly examined for the intravesicular accumulation of mitoxantrone. After ridding off the various ABCG2 and metabolic energy inhibitors (F-H), fresh medium containing 20 µmol/L mitoxantrone was added, and cells that were previously incubated with FCCP and azide were supplemented with the ATP-restoring substrates sodium pyruvate (1 mmol/L) and D-glucose (5 mmol/L) along with mitoxantrone. After 6 hours of incubation at 37°C, the growth medium was removed, and cells were washed once with medium containing 10% dialyzed FBS. Random colonies were finally examined for the intravesicular accumulation of mitoxantrone using a light microscope at a bright-field mode (E-H).

 
Intravesicular concentration of an endogenous green fluorescent chromophore. Under normal growth in mitoxantrone-free medium, the extracellular vesicles were easily identifiable by an intense endogenous green fluorescence (Fig. 6A-C). This autofluorescence was retained in phenol red–free medium, thereby excluding the possibility that this common pH indicator is the endogenous fluorescent compound. However, this intravesicular fluorescence was completely lost upon cellular growth in the presence of Ko143 (Fig. 6D-F). This result indicated that ABCG2 mediated the intravesicular concentration of some endogenous fluorescent compound(s). Hence, ABCG2 mediates the intravesicular concentration of both mitoxantrone and the endogenous fluorescent compound(s). Taking advantage of this autofluorescence, the structural and functional characteristics of these vesicles were explored. The accessibility of the culture medium to the extracellular vesicles was first examined; a cell-impermeable red-fluorescence TRITC-IgG conjugate was used to label the extracellular milieu of MCF-7/MR monolayers (Fig. 6G and J). Whereas the extracellular vesicles were readily discernible by their endogenous green fluorescence (Fig. 6H and K), confocal laser microscopy of cross-sections of monolayer MCF-7/MR cells incubated in medium containing TRITC-IgG revealed that this red fluorescence chromophore was inaccessible to the green fluorescent extracellular vesicle from the cytosol (Fig. 6I). In contrast, a section perpendicular to the monolayer plane showed that the apical side of the extracellular vesicle was the only surface accessible to the TRITC-IgG-containing culture medium (Fig. 6J-L). The confinement of ABCG2 to the vesicular membrane of this cylindrical extracellular compartment was also corroborated by confocal laser microscopy after staining with a green fluorescent–labeled antibody to ABCG2 (Fig. 6M-O). Consistent with the above immunohistochemistry results (Fig. 2D-E and Fig. 4B), confocal analysis of a cross-section revealed that ABCG2 staining formed a circumferential ring (Fig. 6M). This further confirmed that ABCG2 was highly confined to the vesicular membrane contacting the surrounding cells but was barely detectable in the intravesicular lumen. Consistent with the cross-section, a section perpendicular to the plane of the monolayer established the confinement of ABCG2 to the walls lining the extracellular vesicle (Fig. 6N and O). In contrast, the apical membrane of the extracellular vesicle that faces the culture medium was devoid of ABCG2 (Fig. 6N). These results are in accord with the immunohistochemistry findings, which show that ABCG2 was barely detectable in the apical membrane of the extracellular vesicle. These analyses (Fig. 6) allowed for the estimation of the average volume of the cylindrical extracellular vesicle, which was found to be 190 ± 64 fL. These results establish that ABCG2, which is highly confined to the membrane walls lining the extracellular vesicles, mediates the ATP-driven transport of mitoxantrone from the cytosol into the intravesicular lumen of these extracellular compartments.



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  		Figure 6. Detection of intravesicular green autofluorescence in viable MCF-7/MR cells. MCF-7/MR monolayer cells were cultured in medium lacking (A-C) or containing 0.4 µmol/L Ko143 (D-F). Then, random colonies were examined with a fluorescence microscope for their green autofluorescence (B and E); microscopic examination using a bright-field mode revealed the intercellular localization of the extracellular vesicles (A and D, black arrows). The green autofluorescence merged perfectly with the extracellular vesicles (C), whereas this autofluorescence was absent from the extracellular vesicles in monolayer cells growing in the presence of Ko143 (E and F). The accessibility of the growth medium to the extracellular vesicles was examined by confocal microscopy (G-L). Cells grown on sterile glass coverslips were incubated in a buffer solution containing a TRITC-IgG conjugate (red fluorescence). Confocal analysis of cross-section (G-I) and perpendicular section (J-L) of the green and red fluorescence was done with viable monolayer cells; the FITC-like mode was used to detect the extracellular vesicles' green autofluorescence (H and K), whereas the culture medium red fluorescence of the cell-impermeable TRITC-IgG conjugate was detected using a Kr/Ar laser (G and J). Merging the green fluorescence of the extracellular vesicles and the red fluorescence is shown for both the cross-section (I) and perpendicular section (L) analyses. Note that the accessibility of the extracellular vesicles (green fluorescence) to the culture medium (red fluorescence) and not to the cytosol is restricted to its apical side (I and L). The confinement of ABCG2 to the circumferential membrane of the extracellular vesicles was revealed by cross-section (M) and perpendicular section (N) confocal microscopy after immunofluorescent staining with anti-ABCG2 antibodies. Cells grown on glass coverslips were fixed with methanol and reacted with a monoclonal antibody to ABCG2 followed by a second FITC-conjugated antibody. The ABCG2 green fluorescence was confined to the circumferential membrane of the extracellular vesicles upon a cross-section (M) and to the membrane walls lining the extracellular vesicles upon a perpendicular section (N). Merging the cross-section and perpendicular section is shown as well (O).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several lines of evidence establish that the intravesicular concentration of mitoxantrone is mediated by ABCG2. First, inhibition of ABCG2 transport activity by Ko143 and fumitremorgin C prevented the intravesicular concentration of mitoxantrone. Furthermore, washing out these drug transport inhibitors resulted in restoration of mitoxantrone compartmentalization. These results are in accord with the finding that Ko143 induced a near-complete reversal of mitoxantrone resistance in MCF-7/MR cells. Second, depletion of cellular ATP pools by the respiration inhibitor sodium azide (13) and the uncoupler FCCP (14) prevented the intravesicular concentration of mitoxantrone. Consistently, removal of metabolic energy inhibitors followed by restoration of cellular energy resources by provision of glucose and pyruvate in the presence of mitoxantrone resulted in the resumption of intravesicular drug concentration. These findings are in agreement with the tight coupling of ABCG2 drug transport to ATP hydrolysis and consequent intravesicular drug concentration.

The concentration of mitoxantrone in these extracellular vesicles by ABCG2 was energy and time dependent and reached a ~20 mmol/L concentration after 12 hours of incubation with 20 µmol/L drug. This 1,000-fold concentrative ability of ABCG2 gained further support by the dramatic intravesicular sequestration of a green fluorescent compound(s). Whereas this chromophore(s) was not fluorescently visible in the cytosol of neighbor cells surrounding the extracellular vesicle, it was intensely fluorescent in the lumen of this compartment in both MCF-7/MR and MCF-7/FLV1000 cells with ABCG2 overexpression. This intravesicular fluorescence was completely absent after 4 days of cellular growth in the presence of the specific ABCG2 transport inhibitor Ko143. Furthermore, the presence of the light-refracting extracellular vesicles in parental MCF-7 cells was less frequent. When present, these extracellular vesicles in parental MCF-7 cells with low-level R482 ABCG2 expression were completely devoid of the green autofluorescence that is characteristic of the vesicles in drug-resistant MCF-7/MR and MCF-7/FLV1000 cells, which overexpress the wild-type R482 ABCG2. This lack of intravesicular autofluorescence in drug-sensitive cells that poorly express ABCG2 is consistent with the finding that the intravesicular concentration of the green fluorescent compound(s) in MCF-7/MR and MCF-7/FLV1000 cells is mediated by ABCG2. Hence, the energy and time dependence and the ~1,000-fold concentrative capacity of mitoxantrone and that of the autofluorescent compound(s) by ABCG2 are consistent with the highly concentrative transport of various ABC transporters. First, lysosomal and vacuolar membranes contain V-type ATP-driven proton pumps that maintain a >100-fold proton gradient across the acidic lumen of the lysosome (pH ~4.5-5) and the neutral cytosol (pH ~7.0; ref. 15). Second, because an increase in the concentration of Ca2+ ions in the cytosol of mammalian cells (e.g., erythrocytes) is an important regulatory signal, the plasma membrane P-class Ca2+ ATPase efficiently transports Ca2+ out of the cell; consequently, the extracellular (i.e., blood) concentration of Ca2+ is as high as 3,600-fold (1.8 mmol/L) than in the cytosol of the erythrocyte (0.5 µmol/L; ref. 16). Similarly, Ca2+ ATPase from the sarcoplasmic reticulum of muscle cells efficiently pumps Ca2+ ions from the cytosol (0.1-1 µmol/L) into the lumen of the sarcoplasmic reticulum (10 mmol/L), thereby resulting in at least 10,000-fold concentrative transport (17). The third example concerns the H+,K+ ATPase present in the plasma membrane of acid-secreting parietal gastric cells. This P-type H+,K+ ATPase maintains an extremely acidic pH in the gastric fluid, whereas the cytosolic pH of these cells is neutral (pH 7.0). Thus, this H+,K+ ATPase concentrates protons by a factor of 100,000 (18).

Upon cross-section confocal microscopy experiments with a cell-impermeable TRITC-IgG conjugate, there was no accessibility of the culture medium containing this red chromophore to the extracellular vesicles. Whereas, a section that is perpendicular to the plane of the monolayer revealed that the only contact of these vesicles with the fluorochrome-labeled medium was from the apical side of this extracellular compartment. Furthermore, confocal microscopy and immunohistochemistry revealed that ABCG2 was primarily localized at the circumference of the extracellular vesicle but was absent from its apical side that faces the culture medium; ABCG2 was therefore localized at the walls lining this vesicle but was absent from its apical side. Thus, the ATP-binding fold and the substrate-binding site of ABCG2 must face the cytoplasm of the cells surrounding this extracellular vesicle. As such, ABCG2 presumably extracts mitoxantrone from the cytosol of the surrounding cells and highly concentrates it in the lumen of these extracellular vesicles.

Fine-structure studies corroborated the presence of numerous large extracellular vesicles emerging from cell-cell attachment zones. Furthermore, high-resolution electron microscopy revealed that these vesicles contained a lipid bilayer membrane with multiple microvilli-like invaginations protruding into the intravesicular lumen. Hence, these fine-structure projections are reminiscent of the microvilli invaginations of both the gastrointestinal mucosa and the placental epithelium. Given the ATP-driven ABCG2-dependent trans-vesicular transport of mitoxantrone into the intravesicular lumen, it is likely that these microvilli-like invaginations increase the vesicular membrane surface, thereby allowing for a more efficient intravesicular drug concentration. Similarly, the surface of the syncytial trophoblast of the human placenta is covered by a microvillus (i.e., brush) border that is in direct contact with maternal blood; this location is the site of a variety of transport and receptor activities. For example, endocytosis of gold-labeled LDL into primary human placental cells involved uncoated plasmalemmal invaginations, which subsequently became clathrin-uncoated endosome vesicles (19).

The encouraging results of the current study with anticancer drug-resistant breast cancer cell lines warrant further clinical evaluation of the presence of such drug-sequestering extracellular vesicles in tumor-derived specimens. The possible future finding of such extracellular vesicles, which could efficiently concentrate anticancer drugs, may have potential implications for cancer chemotherapy. First, inclusion of specific, potent, and nontoxic ABCG2 transport inhibitors, such as Ko143 (12) and GF120918 (20), which reverse anticancer drug resistance, may potentially prove effective in the prevention of drug compartmentalization in tumors, thereby resulting in reversal of drug resistance. Moreover, various approved cytotoxic drugs were recently found to be efficient inhibitors of ABCG2 efflux activity, including Iressa (ZD1839, Gefitinib), a selective epidermal growth factor receptor tyrosine kinase inhibitor (21, 22); Imatinib mesylate (Gleevec, STI571), a tyrosine kinase inhibitor selective for Bcr-Abl (23); and CI-1033, a HER tyrosine kinase inhibitor (24). In addition, phytoestrogens and flavonoids were also shown to efficiently reverse drug resistance mediated by ABCG2 (25). Clearly, these ABCG2 efflux inhibitors may prove effective reversal agents of drug resistance mediated by ABCG2 overexpression, including when the latter is highly confined to the vesicular membrane. Second, compounds that may interfere with the formation of these novel extracellular vesicles and/or with the sorting of ABCG2 to the vesicular membrane should render cells sensitive to anticancer drugs like mitoxantrone. For example, a recent article (26) reported on the rapid translocation of ABCG2 from the plasma membrane to the cytoplasmic compartment (endoplasmic reticulum-Golgi) in freshly derived hematopoietic stem cells known as side population; in this study, it was shown that a brief treatment (1.5 hours) of freshly derived mouse bone marrow cells with LY294002, an inhibitor of the Akt effector protein phosphatidylinositol-3-kinase (PI3K), resulted in the rapid translocation of ABCG2 from the plasma membrane to the cytoplasmic compartment. The authors therefore suggested that the PI3K-Akt signaling axis is an important regulator of ABCG2 expression and subcellular localization of the bone marrow–derived side population stem cell phenotype. Another example involves a recent study from our laboratory showing that short-term deprivation of folic acid from the growth medium of ABCG2-overexepressing MCF-7/MR cells resulted in the cytoplasmic confinement of this ABCG2 multidrug-resistant efflux transporter (7). Hence, it is possible that such agents and treatment strategies, which block protein sorting of ABCG2 from the cytoplasmic compartment to the plasma membrane and vesicular membrane, may be used to reverse anticancer drug resistance mediated by these novel ABCG2-rich extracellular vesicles.


   Acknowledgments
 
Grant support: Israel Cancer Association and Star Foundation (Y.G. Assaraf).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank J.M. Fritz for his skillful assistance with the electron microscopy studies, Dr. A.H. Schinkel for the generous gift of Ko143, and Dr. S.E. Bates for the cell lines MCF-7/MR and MCF-7/MR1000 and for providing us with fumitremorgin C and flavopiridol.

Received 6/ 9/05. Revised 8/28/05. Accepted 9/15/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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