Abnormal Lysosomal Trafficking and Enhanced Exosomal Export of Cisplatin in Drug-Resistant Human Ovarian Carcinoma Cells

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Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells

Roohangiz Safaei, Barrett J. Larson, of human ovarian carcinoma cells selected for stable Timothy C. Cheng, Michael A. Gibson, resistance to CDDP is markedly reduced in size, and that Shinji Otani, Wiltrud Naerdemann, these cells abnormally sort some lysosomal proteins and Stephen B. Howell and the putative CDDP transporters into an exosomal pathway that also exports CDDP. [Mol Cancer Ther 2005; Rebecca and John Moore University of California at San Diego 4(10):1595–604] Cancer Center, University of California at San Diego, La Jolla, California Introduction Repeated exposure of tumor cells to cisplatin (CDDP) Abstract results in rapid development of resistance, which eventu- Previous work has shown that cisplatin (CDDP) becomes ally contributes to therapeutic failure (1). Resistance to concentrated in lysosomes, and that acquired resistance CDDP is often accompanied by resistance to metalloids to CDDP is associated with abnormalities of protein and is associated with alterations in DNA repair (2), thiol trafficking and secretion. The lysosomal compartment in content (3), and drug accumulation (4). Recent studies have CDDP-sensitive 2008 human ovarian carcinoma cells was linked the expression of several transporters, particularly compared with that in CDDP-resistant 2008/C13*5.25 the copper export proteins ATP7A and ATP7B, with CDDP subline using deconvoluting imaging and specific dyes and resistance and suggest that they play a direct role in the antibodies. The lysosomal compartment in CDDP-resistant efflux of CDDP (5). cells was reduced to just 40% of that in the parental In tumor cells, CDDP accumulates in a variety of vesic- CDDP-sensitive cells (P < 0.002). This was accompanied ular structures, including lysosomes (6). Studies of the by a reduced expression of the lysosome-associated cellular pharmacology of CDDP in cells overexpressing proteins 1 and 2 (LAMP1 and LAMP2) as determined by ATP7A and ATP7B indicate that CDDP is sequestered into both microscopy and Western blot analysis. The CDDP- vesicles belonging to the secretory pathway as well as resistant cells released more protein as exosomes and lysosomes (7, 8). Microscopic analyses with a fluorescein- Western blot analysis revealed that these exosomes conjugated CDDP have confirmed that CDDP accumulates contained substantially more LAMP1 than those released in vesicles that contain ATP7A or ATP7B (9, 10). Additional by the CDDP-sensitive cells. Following loading of the confocal microscopic analysis of fluorescein-conjugated whole cell with CDDP, the exosomes released from 2008/ CDDP in cells treated with drugs that inhibit traffick- C13*5.25 cells contained 2.6-fold more platinum than ing pathways suggests that the accumulation of CDDP those released from sensitive cells. Enhanced exosomal in the secretory compartment may require lysosomal export was accompanied by higher exosomal levels of function (10). the putative CDDP export transporters MRP2, ATP7A, and Lysosomes are an essential part of the vesicular com- ATP7B. Expression profiling identified significant partment and connect the outside medium with many increases in the expression of several genes whose pro- classes of cellular targets in the cytosol, nucleus, mitochon- ducts function in membrane fusion and vesicle traffick- dria, endoplasmic reticulum, and Golgi (reviewed in ref. ing. This study shows that the lysosomal compartment 11). In particular, lysosomes seem to play an important role in the detoxification of heavy metals. Studies in insect cells have shown lysosomal storage of metal conjugates (12), and some heavy metals can cause destabilization and rupture Received 4/4/05; revised 7/8/05; accepted 8/10/05. of lysosomal membranes resulting in cell death (13). Lyso- Grant support: NIH grant CA95298, Department of Defense grant somal abnormalities are often found in drug-resistant cells; DAMD17-03-1-0158, and Foundation for Research. such alterations include aberrant morphology and secreto- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ry phenotypes (14, 15). CDDP-resistant cells release unusu- advertisement in accordance with 18 U.S.C. Section 1734 solely to ally large amounts of lysosomal enzymes and membrane indicate this fact. proteins into their environment (15, 16). Aberrant function Note: R. Safaei and S.B. Howell are Foundation for Research investigators. and expression of the lysosomal H+-pump has also been Requests for reprints: Roohangiz Safaei, Department of Medicine #0819, noted in such CDDP-resistant cells (17). University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093. Phone: 858-822-1117; Fax: 858-822-1111. Although a systematic survey of lysosomal function in E-mail: [email protected] CDDP-resistant cells is lacking, several lines of evidence Copyright C 2005 American Association for Cancer Research. indicate that the development of CDDP resistance is asso- doi:10.1158/1535-7163.MCT-05-0102 ciated with mis-sorting of lysosomal proteins. Lysosomes

Mol Cancer Ther 2005;4(10). October 2005

and plasma membrane proteins involved in CDDP efflux in Opti-MEM containing 1 Ag of the dye/mL at 37jC for originate from trans-Golgi network and are often routed to 30 minutes. The cells were rinsed quickly thrice with PBS multivesicular bodies that also receive endocytosed plasma and fixed by treatment for 15 minutes with 3.7% formal- membrane proteins and receptors. The positioning of dehyde in PBS, permeabilized with 0.3% Triton X-100 in a protein in either the internal or external membrane of PBS for 15 minutes, washed thrice for 15 minutes each with the multivesicular bodies determines whether it recycles PBS, and then stained with 1 Ag/mL Hoechst 33324 and back to the plasma membrane, is destroyed in lysosomes, 0.4 Ag/mL Alexa Flour 647 phalloidin, both dissolved in or is secreted to the extracellular environment via 50- to PBS, for 45 minutes and then mounted on glass slides or 200-nm vesicles known as exosomes (reviewed in ref. 18). A processed for immunostaining. Immunostaining was done link between abnormal sorting of proteins at multivesicular after a 1-hour incubation of fixed and permeabilized bodies and tumorigenesis has been established in trans- samples with 0.3% bovine serum albumin. Primary anti- genic mice (reviewed by ref. 19), and several studies have bodies were diluted in PBS containing 0.3% bovine serum shown that the protein composition of exosomes from albumin and then overlaid on cells for 1 hour. Cells were tumor cells is different from that of nonmalignant cells then washed thrice for 15 minutes, each with PBS, and (18, 20). Mislocalization of lysosomal and plasma mem- incubated for 1 hour with secondary antibodies in PBS and brane proteins has been noted in CDDP-resistant cells 0.3% bovine serum albumin followed by three washes in (17, 21). In addition, disruption of lysosomes with bafi- PBS for 15 minutes each and mounting on glass slides. lomycin A1 (17, 22) and chlorquine (23) has been reported Mounting medium contained 24 g polyvinyl alcohol, 60 to alter CDDP cytotoxicity. Some of the genes whose g glycerol, 60 mL H2O, and 120 mL of 0.2 mol/L Tris-HCl products are involved in lysosome formation seem to (pH 8.5). influence cellular sensitivity to the cytotoxic effect of Deconvoluting microscopy was done at the University of CDDP; these genes include the PI3 kinase (24), rho GTPases California at San Diego Cancer Center Digital Imaging (25), annexins (26), and XIAP (27). Shared Resource using a DeltaVision deconvoluting micro- We report here on further studies of the lysosomal scope system (Applied Precision, Inc., Issaquah, WA.). compartment in human ovarian carcinoma cells that were Images were captured from 0.2-Am sections by Â100, Â60, selected for stable acquired resistance. This work shows and Â40 lenses; SoftWorx software (Applied Precision) that the lysosomal compartment of such cells is markedly was used for deconvoluting data. Image quantification reduced in size and that these cells release protein as was done with Data Inspector program in SoftWorx or exosomes in larger amounts and export CDDP via this by NearCount software. route at increased levels. The resistant cells also abnormally Exosome Harvest sort some lysosomal proteins, including putative CDDP Exosomes were isolated from 2008 and 2008/C13*5.25 transporters, into the exosomal pathway that also exports cells according to the method described by Escola et al. (29) CDDP. with minor modifications. Cells were cultured in 100-mm dishes until 80% confluent. They were then rinsed with Materials and Methods serum-free RPMI 1640 to remove proteins and vesicular Reagents structures already released from the cells or present in the Cell culture media and sera were purchased from Invi- fetal bovine serum. In parallel, one set of cells received trogen (Carlsbad, CA). Antibodies to lysosome-associated fresh serum-free RPMI 1640 alone, whereas another set proteins 1 and 2 (LAMP1 and LAMP2), h-actin, and tubulin was incubated with RPMI 1640 plus 2 Amol/L CDDP for were obtained from Santa Cruz Biotechnology, Inc. (Santa 1 hour in a 37jC humidified tissue culture incubator. The Cruz, CA), and those for MRP2 were from Alexis Bio- supernatants were then removed and the plates rinsed chemicals (San Diego, CA). The specific dyes Alexa Flour thrice with warm RPMI 1640; and then 12 mL of fresh 647 phalloidin, Hoechst 33342, and Lysotracker Red were serum-free RPMI 1640 was layered over cells in each purchased from Molecular Probes (Eugene, OR). Fluores- plate; and cells were incubated for 1 hour at 37jC. cein-conjugated secondary antibodies against mouse, The supernatant from each plate was then transferred to rabbit, and goat immunoglobulin were purchased from a 15-mL conical tube and subjected to two successive Jackson ImmunoResearch Laboratories, Inc. (West Grove, rounds of centrifugation at 10,000 Â g, each for 15 minutes, PA). Other chemicals were purchased from Sigma Co. to remove loose cells and cellular debris. Each supernatant (St. Louis, MO) and Fisher Scientific Co. (Tustin, CA). was then gently transferred to a new SW41 ultracentrifuge CDDP (PLATINOL-AQ) was a gift of Bristol Laboratories tube and centrifuged at 70,000 Â g for 60 minutes. The (Princeton, NJ). pellet was resuspended in 100 AL of lysis buffer containing Cell Culture and Histochemistry 100 mmol/L Tris (pH 7.0), 150 mmol/L NaCl, 5 mmol/L Ovarian carcinoma 2008 cells and the 2008/C13*5.25 EDTA, and 0.25% NP40, mixed thoroughly and an aliquot subline selected for stable acquired CDDP resistance were of 25 AL was taken for measurement of protein concentra- used for all studies (28). Cells were cultured in RPMI with tion using the Bradford kit from BIO-RAD (Hercules, CA). 10% fetal bovine serum in humidified air with 5% CO2. The remainder of the exosomal suspension in each Histochemistry was done on cells 1 day after seeding them experimental group was pooled for polyacrylamide SDS on coverslips. Labeling with Lysotracker Red was done gel analyses.

Mol Cancer Ther 2005;4(10). October 2005

Uptake of PKH67-Labeled Exosomes using the Enhanced Chemiluminescence kit from Amer- Exosomes from 2008 and 2008/C13*5.25 were labeled sham Life Science (Piscataway, NJ). A ChemiImager 4400 with the lipid dye PKH67 Green (Sigma, St. Louis, MO) instrument (Alpha Inotech, San Leandro, CA) was used according to a method previously described (30). Briefly, for determining the density of protein bands. an aliquot of exosomes containing 100 Ag of protein was Expression Profiling mixed with 0.5 Amol/L PKH67 Green and incubated at RNA was harvested and cDNA prepared, labeled, and 37jC for 10 minutes. The mixture was diluted with ice-cold hybridized to cDNA microarrays obtained from the Stanford PBS and centrifuged at 70,000 Â g for 1 hour. Vesicles Functional Genomics Facility (http://www.microarray.org) were washed twice by being resuspended in fresh PBS that contained 43,200 elements representing f29,593 genes and sedimented again by centrifugation at 70,000 Â g for as estimated by UniGene clusters exactly as recently 1 hour. Experiments to determine whether exosomes reported from this laboratory in detail (31). Significance were taken up by 2008 and 2008/C13*5.25 cells were done analysis of microarrays (http://www-stat.stanford.edu/ftibs/ by incubating PKH67 Green–labeled exosomes (10 Agof SAM/index.html) was used to determine which genes total protein) with 1 Â 105 cells in complete medium for were significantly differentially expressed based on the 5 minutes. Cells were then rinsed with PBS thrice and fixed D score (31). and processed for confocal microscopy. Negative controls were prepared of samples of 2008 and 2008/C13*5.25 Results cells in the absence of staining for PKH67 to correct for Reduced Lysosomal Compartment in CDDP-Resis- autofluorescence. tant Cells Measurements of Platinum Content The CDDP-resistant ovarian carcinoma 2008/C13*5.25 Whole cell platinum (Pt) content following a 1-hour cells were selected for acquired resistance by repeated period of incubation with CDDP and a subsequent 1-hour cycles of exposure to CDDP. These cells were developed period of efflux was assayed as previously described (10). 17 years ago and have maintained a stable level of Exosomal content of Pt was measured by rapidly mixing resistance while in continuous culture in the absence of 215 AL of 70% analytic grade nitric acid with 75 ALof additional CDDP exposure. The vital dye Lysotracker Red resuspended exosomes and then incubating the mixture in DN-99 was chosen for its capacity to remain in acidic a65jC water bath overnight. Samples were then diluted compartments of cells following formaldehyde fixation. with an indium (Acros Organics, Tustin, CA) solution to The 2008 and 2008/C13*5.25 cells were exposed to 1 Ag/mL a final concentration of 5% acid and 1 ppb indium. Indium Lysotracker Red in parallel cultures and processed together content was used to correct for flow variation in the throughout the experiment. Figure 1 shows images of these inductivity-coupled plasma mass spectrometry. A Thermo cells in which the filamentous-actin filaments were also Finnigan inductivity-coupled plasma mass spectrometry stained with Alexa Flour 647 phalloidin. CDDP-resistant (model Element 2) from the Analytical Facility at the 2008/C13*5.25 cells were found to contain markedly fewer Scripps Institute of Oceanography was used for measure- lysosomes than the CDDP-sensitive parental 2008 cells. ment of Pt concentration. Pt values were normalized to Quantification of raw images with NearCount software the protein content of the exosome preparation. showed that resistant cells had 2.11 F 0.03-fold (mean F Western Blotting SE) fewer acidic vesicles than their sensitive counterparts Cells were rinsed twice with PBS, scraped with a rubber (P V 0.002). policeman, suspended in 10 mL PBS, and centrifuged for To document that the differences in the numbers of acidic 10 minutes at 2,500 rpm. Cells were lysed in 0.25% NP40 vesicles detected by Lysotracker Red DN-99 were in fact in 100 mmol/L Tris-HCl (pH 8.0) supplemented with pro- due to differences in the number of lysosomes in the two tease inhibitor tablets (complete from Roche Applied types of cells, parallel preparations of 2008 and 2008/ Sciences, Penzberg, Germany) at 4jC and for 30 minutes. C13*5.25 cells were stained with monoclonal antibodies Post-nuclear fractions were obtained by centrifugation of against LAMP1 and LAMP2. These two proteins are both cell lysates for 10 minutes at 600 Â g. Samples containing well-validated markers of lysosomal vesicles (32). The 50 to 100 Ag of protein were electrophoresed on 4% to 10% images presented in Fig. 2 confirm reduction in the lyso- SDS polyacrylamide gels. Gels were blotted onto nitrocel- somal compartment in the CDDP-resistant cells. The num- lulose filters using a Bio-Rad Mini Transblot apparatus ber of LAMP1-containing vesicles was markedly reduced (Bio-Rad, Hercules, CA) and incubated for 1 hour with in the 2008/C13*5.25 cells. The number of vesicles staining 5% fat free dry milk in TBS at room temperature and then positively for LAMP2 was also reduced in the 2008/ overnight in primary antibody mixed with 5% milk in TBS C13*5.25 cells but to a lesser extent than LAMP1. To at 4jC. Blots were washed thrice for 15 minutes each at confirm the results of the immunohistochemical studies, room temperature with 0.025% Tween 20 in TBS. The LAMP1 and LAMP2 levels were measured by Western blot secondary antibody was diluted in 5% fat-free dry milk in analysis of the 2008 and 2008/C13*5.25 cells using post- TBS and added to blots for 1 hour at room temperature. nuclear lysates. Figure 2 (bottom) shows that although Blots were washed again at room temperature thrice for LAMP1 was expressed in the 2008 cells, it was only very 15 minutes each in 0.025% Tween 20 in TBS. The extent of faintly detectable in the 2008/C13*5.25 cells. It also shows specific staining was quantified by chemiluminescence that LAMP2 levels were reduced by >3.2-fold in the 2008/

Mol Cancer Ther 2005;4(10). October 2005

formation and release (see below), an analysis was under- taken of the exosomes released by 2008 and 2008/C13*5.25 cells. A multistep centrifugation procedure was used to produce high quality exosomes released from living cells for subsequent analysis (30). This technique excludes the possibility that the exosomes originate from serum in the tissue culture medium or from dead cells or cell fragments as these are removed before exosome collection. To show that the exosomes released by the CDDP-sensitive and CDDP-resistant cells were functional, they were labeled with PKH67 Green and then incubated with their cell of origin. Figure 3 shows that exosomes from both types of cells were able to reassociate with their cell of origin, although when exposed to exosomes containing equal levels of protein, the 2008/C13*5.25 cells accumulated less PKH67 Green than the 2008 cells. This is consistent with previous observations that exosomes can reenter the cell of origin (33–35). The amount of exosomal protein released into serum-free medium during a 1-hour period was used as an estimate of the differential ability of the 2008 and 2008/C13*5.25 cells to export exosomes. Figure 4 (top) shows that relative to the total cellular protein in the culture, the CDDP-resistant cells released significantly more exosomal protein over this time period (P = 2.3 Â 10À5, n = 20). Western blot analysis of the exosomes from 2008 and 2008/C13*5.25 cells showed that the CDDP-resistant cells contained a substantial amount of LAMP1, whereas this protein was undetectable in the exosomes released from the CDDP-sensitive cells (Fig. 4, bottom). However, the level of LAMP2 was only 1.1-fold higher in the exosomes from the resistant than the sensitive cells. Actin and tubulin were present at similar levels in the exosomes released by both CDDP-sensitive and CDDP-resistant cells. Thus, lysosomal proteins were found in exosomes, and the level of at least one such protein, LAMP1, was much higher in exosomes released by the DPP-resistant cells. Because exosomal export can potentially serve as an alternative route for cells to discard proteins when lysosomal degradation is impaired, the exosomes released from the 2008 and 2008/C13*5.25 cells were subjected to Western blot analysis to measure the level of three other proteins that are believed to play a role in the detoxification of CDDP via sequestration into intracellular vesicles. Figure 1. Comparison of the lysosome content of 2008 and 2008/ Figure 4 (bottom) shows that MRP2 (ABCB2) and the copper C13*5.25 cells. Lysosomes were labeled with Lysotracker Red (artificially colored green). Filamentous-actin was labeled with Alexa Flour 647 efflux transporters ATP7A and ATP7B were all found phalloidin (red). at much higher levels in the exosomes from the CDDP- resistant cells. Quantitative analysis of the Western blots showed that the exosomes from CDDP-resistant cells C13*5.25 cells. Thus, three different analytic approaches contained, respectively, 3.4-, 2.6-, and 2.6-fold higher levels indicated a marked reduction in number of visible MRP2, ATP7A, and ATP7B relative to the levels of these lysosomes and the content of lysosomal proteins in the proteins in exosomes released by the CDDP-sensitive cells. cells selected for CDDP resistance. The finding that the exosomes from 2008 cells contain Analysis of Exosomes Released from CDDP-Sensitive ATP7B was quite surprising, because this protein is not and CDDP-Resistant Cells detectable in whole cell lysates prepared from this cell line, Because expression profiling of the CDDP-sensitive and suggesting that ATP7B is concentrated in the exosomes. CDDP-resistant cell pairs suggested altered levels of The finding that the exosomes from the CDDP-resistant mRNA for some of the genes involved in exosome cells contain much more ATP7B is consistent with our

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earlier observation that the expression of this protein is collected. The Pt content of the cells at the end of the loading markedly increased in 2008/C13*5.25 cells (36). period and at the end of the subsequent 1 hour period of Pt Content of Exosomes Released from CDDP- exosome release and the Pt content of the released Sensitive and CDDP-Resistant Cells exosomes were measured and normalized to protein Recent studies suggest that one mechanism by which content. Figure 5 shows that the amount of Pt/mg CDDP is exported from the cell is via sequestration into exosomal protein was 2.6 F 0.8-fold higher in the exosomes vesicles of the secretory pathway (9). Because exosomes released from the CDDP-resistant cells (P =3.8Â 10À5). represent one of the outputs of this pathway, it was of Taking into account that the 2008/C13*5.25 cells released interest to determine whether exosomes released by the 1.9-fold more exosomal protein over this time period, the CDDP-resistant cells contain more CDDP than those from total exosomal Pt export was 4.9-fold higher from the the sensitive cells following loading of the cells with CDDP. resistant cells, despite the fact that the resistant cells The 2008 and 2008/C13*5.25 cells were exposed to 2 Amol/L accumulated only 41% as much CDDP during the 1-hour CDDP for 1 hour to load them with CDDP. They were loading incubation as the sensitive cells as shown in Fig. 5, then washed to remove all extracellular-free drug and the bottom (see also ref. 36). However, the 0.0037 F 0.00009 exosomes released from the cells over the next 1 hour were pmol of Pt/mg exosomal protein found in the exosomes

Figure 2. Comparison of the levels of the lysosomal proteins LAMP1 and LAMP2 in 2008 and 2008/C13*5.25 cells. Top, deconvoluted confocal images of cells stained with monoclonal antibodies against LAMP1 and LAMP2 (green). Actin was labeled with Alexa Flour 647 phalloidin (artificially colored red). Nuclei were stained with Hoechst 33342 (blue). Bottom, Western blot analysis of whole cell lysates of 2008 and 2008/C13*5.25 cells probed with antibodies against LAMP1, LAMP2, and actin. Each lane was loaded with 25 Agof protein.

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Figure 3. Functionality of exosomes released by 2008 and 2008/ C13*5.25 cells as measured by their reuptake into the cells from which they were released. Exosomes from 2008 and 2008/C13*5.25 cells were labeled with PKH67 Green for 10 min (green) and incubated with their cell of origin for 5 min. Actin is labeled with Alex Flour 647 phalloidin (red). A, 2008 cells labeled with Alexa Flour 647 only. B, 2008 cells labeled with Alex Flour 647 and incubated with PKH67-labeled exosomes from 2008 cells. C, 2008/ C13*5.25 cells labeled with Alexa Flour 647 only. D, 2008/C13*5.25 cells labeled with Alexa Flour 647 and incubated with PKH67-labeled exosomes from 2008/C13*5.25 cells.

released over the first hour from CDDP-sensitive cells 411 genes were found to be significantly differentially represented only 0.037% of the total of 10.08 F 0.87 pmol of expressed between the 2008 and 2008/C13*5.25 cells. As Pt lost from the cells that produced these exosomes during shown in Table 1, among these were nine genes belonging this period of time. In the case of the CDDP-resistant cells, to families whose products are known to be involved in the 0.01818 F 0.00033 pmol of Pt in the exosomes the formation of lysosomes and multivesicular bodies. All represented only 0.075% of the 24.08 F 1.27 pmol lost nine genes were expressed at a higher level in the CDDP- from the cells. Thus, although CDDP-resistant cells export resistant cells. more Pt via the exosomal pathway than CDDP-sensitive cells, this represents only a very small fraction of all the Pt Discussion loss from the cell during the first hour after the end of drug We have previously reported that CDDP becomes localized exposure. in lysosomes in CDDP-sensitive parental human ovarian Expression Pattern of Genes Whose Products Func- carcinoma 2008 cell line (10). In the current study, we tion in Lysosome Formation examined the lysosomal compartment in the 2008/C13*5.25 RNA was harvested from log phase 2008 and 2008/ subline whose degree of CDDP resistance has been stable C13*5.25 cells grown under identical conditions and used for >17 years of continuous culture in the absence of to determine the expression profile by hybridizing the additional drug selection. We confirmed reports from other labeled cDNA derived from reverse transcription of RNA laboratories that the lysosomal compartment is markedly against each other using a cDNA microarray containing abnormal in cells selected for stable acquired CDDP sequences corresponding to 29,593 genes. Each experiment resistance (17, 37). In addition, we discovered that CDDP was repeated six times using independently isolated RNA enters the exosomal pathway, and that CDDP-resistant cells samples. Genes that were significantly differentially export both more CDDP and more putative CDDP trans- expressed were determined using the ‘‘statistical analysis porters via this pathway. of microarrays’’ software (http://www-stat.stanford.edu/ftibs/ Lysosomes are a key station along the path by which SAM/index.html). With variables set such that the number nutrients, pathogens, and cytotoxic agents traffic within of genes falsely discovered is expected to be V1, a total of cells. In the trafficking of metals, they seem to serve as an

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intermediary compartment that receives metals and metalloids from influx pathways and either stores them or distributes them to efflux systems or other destinations in the cell (38, 39). CDDP is known to be concentrated by the copper efflux transporter ATP7A into vesicles of the microsomal fraction (7), and it is likely that ATP7B also concentrates CDDP into such vesicles (9). Fluorescently labeled CDDP colocalizes with lysosomal markers in 2008 cells (40). By virtue of their high content of catabolic enzymes, lysosomes also participate in apoptotic (41), nec rotic (42), and autophagic (43) cell death pathways, often in collaboration with stress management organelles, such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus (11). There is evidence that metalloids can activate these cell death pathways as a direct result of the damage that they do to the lysosomes in which they become concentrated (15, 44). We found major alterations of the lysosome compartment in CDDP-resistant 2008/C13*5.25 cells. The number of visible lysosomes was reduced when imaged with either Figure 5. Platinum content of cells and exosomes. Top, Pt content Lysotracker Red DN-99 or antibodies to LAMP1 or LAMP2 of exosomes released from CDDP-loaded 2008 (open column) and 2008/ C13*5.25 (closed column) cells during the first hour after loading. and the level of expression of LAMP1 and LAMP2 was Pt concentrations are expressed as pmol/mg of exosomal protein. ***, reduced when examined by Western blot analysis. In P V 0.002. Bottom, Pt content of whole 2008 and 2008/C13*5.25 addition, the trafficking of proteins through this compart- cells at the end of a 1-h incubation with 2 Amol/L CDDP (open column) and at the end of a subsequent 1-h period of efflux (closed column). *, ment was abnormal as reflected by both the larger amount P < 0.005. of exosomal protein released by the resistant cells and the altered distribution of the proteins that were found in the exosomes. We have documented similar changes in two other CDDP-resistant ovarian carcinoma cell lines, A2780/ CP and IGROV-1/CP (45). It is noteworthy that, despite the fact that both LAMP1 and LAMP2 levels were decreased in the CDDP-resistant cells, only LAMP1 was found in higher levels in the exosomes. Quantitatively, it is unlikely that increased export of either protein via the exosomal pathway accounts for the marked decrease in their level in the whole cell. Instead, we interpret the fact that LAMP1 levels were increased in exosomes released by the resistant cells, whereas LAMP2 levels were not as evidence of an abnormality of protein trafficking or exosome formation in the lysosomal compartment. It is important to note that the reduction in the size of the lysosomal compartment found in this study is a stable characteristic of the acquired CDDP-resistant phenotype as the 2008/C13*5.25 cells have been grown in the absence of CDDP for many years. The finding that a number of genes coding for proteins involved with the formation of lysosomes and multivesicular bodies were overexpressed at the mRNA level in the CDDP- resistant cells suggests a secondary compensatory response to some primary abnormality that prevents correct assem- bly of lysosomes or enhances their destruction. It was of particular interest to find that several transporters believed to be involved in the movement of CDDP across vesicle membranes were found in exosomes, and that the level of ATP7A, ATP7B, and MRP2 (ABCB2) was higher in the exosomes released from the CDDP-resistant cells than Figure 4. Analysis of exosomal protein content. Top, total amount of those released from sensitive cells. This may reflect a exosomal protein released per mg of total 2008 and 2008/C13*5.25 major abnormality in intracellular protein trafficking such protein. Bottom, Western blot analysis of MRP2, ATP7A, ATP7B, LAMP1, LAMP2, and actin levels in exosomes released from 2008 and 2008/ as has been described in other types of CDDP-resistant cells C13*5.25 cells. Each lane was loaded with 25 Ag of exosomal protein. (17, 37). We speculate that the altered distribution of the

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Table 1. Genes related to lysosomal formation or vesicle trafficking found to be differentially expressed in 2008 and 2008/C13*5.25 cells

c Gene Expression ratio* D score Function

Annexin A10 (ANXA10) 1.9 3.0 Membrane bending, invagination of MVB Decay-accelerating factor for 1.9 2.13 GPI-anchored tetraspanin, complement (CD55, DAF) exported in exosomes Transmembrane 4 superfamily 1.7 2.6 Member of the family of member 2 (TM4SF2) tetraspanin proteins, which are proposed to function in membrane bending, fusion, invagination of MVB Baculoviral IAP repeat–containing 6 1.9 3.723 Involved in membrane (apollon; BIRC6) bending, fusion, invagination of MVB Integrin h1 (fibronectin receptor, 1.7 2.392 Exported in exosomes h polypeptide, antigen CD29 includes MDF2, MSK12; ITGB1) MHC class I C (HLA-C) 1.8 2.617 Exported in exosomes Sorting nexin 5 (SNX5) 2.0 3.007 Early endosomal recycling; binds clathrin; binds to Fanconi anemia complementation group A (FANCA) protein (FANCA is expressed in response to stress) RAB4A, member RAS oncogene 1.5 1.998 Regulates receptor recycling family (RAB4A) from early endosomes Rab3 GTPase-activating protein, 1.6 1.911 Implicated in exocytic noncatalytic subunit (150 kDa; release of neurotransmitters RAB3-GAP150) and hormones; mutation causes Warburg Micro syndrome

Abbreviation: MVB, multivesicular bodies. *Ratio of expression in 2008/C13*5.25 cells relative to that in 2008 cells. cAll d scores were statistically significant.

types of proteins found in exosomes may report on the membranes of the endosomes that are the precursors of CDDP-resistant phenotype. Because exosomes are found in mature multivesicular bodies. CDDP would then be the systemic circulation, this offers a potential route to early expected to exit the cell when the multivesicular bodies detection of the emergence of drug resistance during fuses with the plasma membrane to release its content of treatment. exosomes. In the CDDP-resistant 2008/C13*5.25 cells, a There is now a substantial body of evidence that CDDP is larger fraction of cellular protein was released as exosomes sequestered into intracellular vesicles, some of which per hour and the exosomes contained a larger amount of belong to the secretory pathway (7, 9, 10). Presumably, Pt. Taken together with the fact that the CDDP-resistant many of these vesicles travel directly to, and fuse with, the cells start out with a smaller amount of Pt in the whole plasma membrane releasing CDDP to the outside of the cell cell following incubation with CDDP, and the fact that in a form no longer encapsulated in a lipid membrane. The exosomes are recycled by these cells, it is apparent that a released Pt may be either free drug, a conjugate, or larger fraction of the Pt in the cell enters the exosomal a complex with cellular proteins to which it has become pathway in CDDP-resistant cells. This is consistent with bound. The results reported in this article indicate that the finding of increased amounts of the putative CDDP or CDDP is also exported via exosomes and thus suggest CDDP-conjugate transporters ATP7A, ATP7B, and MRP2 a function for multivesicular bodies, from which these (ABCB2) in the exosomes released by the resistant cells. vesicles originate, in the routing of intracellular CDDP. At what step along the pathway CDDP loading occurs How CDDP enters the exosomal pathway is not entirely remains unknown, but ATP7B has been identified as being clear. Possibly, cytoplasmic CDDP is entrapped in exo- resident in late endosomes (46) and is thus positioned to somes as they form by invagination of the limiting play a role. Although export via exosomes does not account

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Roohangiz Safaei, Barrett J. Larson, Timothy C. Cheng, et al.

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