THE ISOLATION OF
MORPHOLOGICALLY INTACT AND
BIOLOGICALLY ACTIVE
EXTRACELLULAR VESICLES FROM
THE SECRETOME OF
CANCER-ASSOCIATED ADIPOSE TISSUE
Sarah Jeurissen
Student number: 20058009Supervisor 1: Prof. Dr. H. Denys
Supervisor 2: Dr. A. Hendrix
Master’s dissertation Master of Medicine in Specialist Medicine
RESEARCH PAPER
The isolation of morphologically intact and biologically active extracellular
vesicles from the secretome of cancer-associated adipose tissue
Sarah Jeurissena,b,d, Glenn Vergauwena,c,d, Jan Van Deun a,d, Lore Lapeireb,d, Victoria Depoorter a, Ilkka Miinalainene, Raija Sormunene, Rudy Van den Broeckec,d, Geert Braems c,d, Veronique Cocquytb,d, Hannelore Denysb,d, and An Hendrixa,d
aLaboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, Ghent,
Belgium;bDepartment of Medical Oncology and Department of Gynaecology, Ghent University Hospital, Ghent, Belgium;cDepartment of
Gynaecology, Ghent University Hospital, Ghent, Belgium;dCancer Research Institute Ghent (CRIG), Ghent, Belgium;eBiocenter Oulu and
Departments of Pathology, University of Oulu and Oulu University Hospital, Oulu, Finland
ARTICLE HISTORY Received 26 August 2016 Revised 6 December 2016 Accepted 3 January 2017 ABSTRACT
Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to favor invasive growth and metastasis. Extracellular vesicles (EVs) are nanometer-sized vesicles secreted by different cell types that shuttle proteins and nucleic acids to establish cell-cell communication. To study the role of EVs released by cancer-associated adipose tissue in breast cancer progression and metastasis a standardized EV isolation protocol that obtains pure EVs and maintains their functional characteristics is required. We implemented differential ultracentrifugation as a pre-enrichment step followed by OptiPrep density gradient centrifugation (dUC-ODG) to isolate EVs from the conditioned medium of cancer-associated adipose tissue. A combination of immune-electron microscopy, nanoparticle tracking analysis (NTA) and Western blot analysis identified EVs that are enriched inflotillin-1, CD9 and CD63, and sized between 20 and 200 nm with a density of 1.076– 1.125 g/ml. The lack of protein aggregates and cell organelle proteins confirmed the purity of the EV preparations. Next, we evaluated whether dUC-ODG isolated EVs are functionally active. ZR75.1 breast cancer cells treated with cancer-associated adipose tissue-secreted EVs from breast cancer patients showed an increased phosphorylation of CREB. MCF-7 breast cancer cells treated with adipose tissue-derived EVs exhibited a stronger propensity to form cellular aggregates. In conclusion, dUC-ODG purifies EVs from conditioned medium of cancer-associated adipose tissue, and these EVs are morphologically intact and biologically active.
KEYWORDS
aggregation; breast cancer; characterization; exosomes; function; isolation; proliferation
Introduction
Heterotypic cellular interactions are a prerequisite for primary tumor growth and metastasis.1In breast cancer, adipose tissue is the main component of the tumor envi-ronment. Adipose tissue is no longer regarded as simply an inert store of excess energy, but as an endocrine organ that promotes interaction with cancer cells both through cell-cell and cell-matrix contacts as well as through secreted signaling molecules.2 It is an architecture of mature adipocytes, progenitor cells, endothelial cells, fibroblasts, macrophages and immune cells of which the relative composition changes due to obesity, inflamma-tory conditions and cancer. Multiple cellular elements of the adipose tissue and their products stimulate breast cancer cells toward further progression. Research into
the communication between adipose tissue and cancer cells has mostly been focused on matrix contacts such as type VI collagen and soluble pro-inflammatory factors such as interleukin-6 and oncostatin M among others.3,4 In addition, emerging evidence suggests that extracellular vesicles (EVs) also play a role in tumor environment communications. EVs are nanometer-sized entities which contain numerous proteins, lipids and nucleic acids and are secreted by most cell types.5A role of EVs in cell-to-cell communication was evidenced by the func-tional translation of EV mRNAs from a donor cell by tar-get cells.6 Cancer patients have an increased number of EVs in the circulation and this number correlates with disease progression.7 However, it is not yet known whether these EVs are cancer cell- or host-specific.
CONTACT An Hendrix an.hendrix@ugent.be Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, Ghent, Belgium.
Color versions of one or more of thefigures in the article can be found online atwww.tandfonline.com/kcam. Supplemental data for this article can be accessed on thepublisher’s website.
© 2017 Taylor & Francis
Human abdominal adipose tissue explants produce EVs that modulate monocyte differentiation and alter insulin signaling in adipocytes and liver cells.8,9 Mouse visceral adipose tissue EVs mediate activation of macro-phage-induced insulin resistance.10 Cultures of murine pre-adipocytes have also been shown to release EVs and these EVs stimulate fatty acid-oxidation dependent migration of melanoma cells.11Deng et al., Kranendonk et al. and Lazar et al. have applied differential ultracentri-fugation (dUC) to isolate EVs. dUC is a combination of centrifugation steps with increasing centrifugal strength to sequentially pellet cells (1500 g), larger EVs (10,000 g) and smaller EVs (100,000 g). Lazar et al. and Kranen-donk et al. both washed the 100,000 g pellet in a large volume to reduce non-vesicular proteins, and centrifuged one last time at the same high speed. For some applica-tions it may be advisable to include an extra purification step using density gradient centrifugation.12,13This step eliminates more contaminants, such as proteins non-spe-cifically associated with EVs, or large protein aggregates, which are sedimented by centrifugation but do notfloat on a density gradient.14Kranendonk et al. further
puri-fied the dUC pellet by a sucrose density gradient to obtain high purity EVs.
The results of these pioneer studies warrant further investigation into the role of adipose tissue derived EVs in disease and cancer progression in particular. To be able to fully exploit the potential of EVs, standardized methodology for EV isolation and characterization is a crucial requirement. We prepared secretomes of cancer-associated adipose tissues derived from breast cancer patients and combined differential ultracentrifugation with a density gradient basedfloatation of EVs. Purified EVs were characterized for enriched and non EV-enriched proteins, morphology, size distribution, num-ber and functionality.
Material and methods
Conditioned medium of cancer-associated adipose tissue
Cancer associated adipose tissue (CAAT) was obtained from breast cancer patients undergoing mastectomy at Ghent University Hospital in accordance with local ethics committee and written informed consent was obtained from all subjects. The breast adipose tissue was devoid of fibrosis, washed in sterile phosphate-buffered saline (PBS), cut into pieces of approximately 1–2 mm3
and placed in DMEM/F12 culture medium supple-mented with 100 U/ml penicillin, 100mg/ml streptomy-cin, 2.5 mg/ml fungizone and 0.5% Bovine Serum Albumin (BSA) (Sigma-Aldrich), further called‘control
medium’, in 6-well plates at a ratio of 250 mg of adipose tissue per ml control medium. The 6-well plates were placed on a nutating mixer at 20 rpm (for constant hydration and maximal oxygen supply) (VWR Interna-tional, Radnor, PA) in an incubator at 37C and 5% CO2. After 24 h, the conditioned medium (CM) contain-ing factors of cancer-associated adipose tissue (CMCAAT) was harvested and centrifuged for 10 min at 500 g and 4C. The lipidsfloating on top of the CM were removed with a Pasteur pipette and the CM above the small pellet with debris was further centrifuged for 15 min at 1500 g, 30 min at 10,000 g and filtered through a 0.2 mm filter. CMCAATwas stored at¡80C until experimentation.3
Antibodies and reagents
The following primary and secondary antibodies were used for immunostaining: mouse monoclonal anti-Alix (1:1,000) (2171, Cell Signaling, Danvers, MA, USA), rab-bit polyclonal anti-prohirab-bitin (1:500) (NBP1–40505, Novus Biologicals), rabbit polyclonal anti-calreticulin (1:1,000) (2891, Cell Signaling), antiphospho-CREB (1:1000) (9198, Cell Signaling), anti-CREB (Cell Signal-ing), rabbit monoclonal anti-CD9 (1:1000) (D3H4P, Cell Signaling), mouse monoclonal anti-flotillin-1 (1:1000) (610820, BD Biosciences, Franklin Lakes, NJ, USA), mouse monoclonal anti-GM130 (1:500) (610822, Becton Dickinson, Franklin Lakes, NJ, USA), rabbit polyclonal anti-HSP70 (1:1,000) (EXOAB-HSP70A-1, System Bio-sciences, Mountain View, CA, USA). Secondary antibod-ies coupled to horseradish peroxidase were obtained from Amersham Pharmacia Biotech (Diegem, Belgium). OptiPrepTM was purchased from Axis-Shield PoC (Oslo,
Norway).
Ultracentrifugation
Seventy-five ml of CMCAAT was transferred to open top polyallomer centrifuge tubes (Beckman Coulter, Fuller-ton, CA) and centrifuged for 2 h at 100,000 g and 4C in a swinging bucket rotor (Optima XPN- 80, SW 55 Ti rotor, Beckman Coulter). The pellet was resuspended in 15 ml of PBS and centrifuged again for 2 h at 100,000 g. The resulting pellet was resuspended in 1 ml of PBS and further purified by OptiPrepTM density gradient
centrifugation.
OptiPrepTMdensity gradient centrifugation
A discontinuous iodixanol gradient was used as described by Van Deun et al.13 Solutions of 5%, 10%, 20% and 40% iodixanol were made by mixing appropri-ate amounts of a homogenization buffer (0.25 M sucrose,
1 mM EDTA, 10 mM Tris-HCL, [pH 7.4]) and an iodix-anol working solution. This working solution was pre-pared by combining a working solution buffer (0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl, [pH 7.4]) and a stock solution of OptiPrepTM (60% (w/v) aqueous
iodix-anol solution). The gradient was formed by layering 4 ml of 40%, 4 ml of 20%, 4 ml of 10% and 3.5 ml of 5% solu-tions on top of each other in a 16.8 ml open top poly-allomer tube (Beckman Coulter). 1 ml of resuspended EV pellet was overlaid onto the top of the gradient which was then centrifuged for 18 h at 100,000 g and 4C (SW 32.1 Ti rotor, Beckman Coulter). Gradient fractions of 1 mL were collected from the top of the gradient, diluted to 16 ml in PBS and centrifuged for 3 h at 100,000 g and 4C. The resulting pellets were resuspended in 100 ml PBS and stored at¡80C. The purity of the EV prepara-tions was assessed according to the MISEV guidelines.15
Western blot analysis
To measure protein concentration of isolated EVs, 5ml sample was mixed with 5 mL of Laemmli lysis buffer (0.125 M Tris–HCl [pH 6.8], 10% glycerol, 2.3% sodium dodecyl sulfate [SDS]). Protein concentration was deter-mined using the Bio-Rad DC Protein Assay (Bio-Rad, Hercules, USA). For protein analysis, 10mg of EV pro-tein was lysed in reducing sample buffer (1 M Tris–HCl [pH 6.8], 30% glycerol, 6% SDS, 3% 2-mercaptoethanol, 0.005% bromophenol blue) and boiled at 95C during 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad, Hercules, California, USA). After blocking the membranes, blots were incubated overnight with primary antibodies. Incubation with secondary antibodies was performed after extensive washing of the membranes in PBS with 0.5% Tween20. After final extensive washing, chemiluminescence substrate (West-ernBright Sirius, Advansta, Menlo Park, California, USA) was added and imaging was performed using Proxima 2850 Imager (IsoGen Life Sciences, De Meern, The Netherlands). Quantification of protein bands was performed using Image J software.
Nanoparticle tracking analysis (NTA)
Aliquots of isolated particles were used for NTA using NanoSight LM10 microscope (NanoSight Ltd, Amesbury, UK) equipped with 405 nm laser. For each sample, 3 videos of 60 seconds were recorded and analyzed with overall camera level 13 and detec-tion threshold 5 in standard mode. The measure-ments were performed at ambient temperature which monitored and did not exceed 25C. Recorded videos
were analyzed with NTA Software version 3.1. For optimal measurements, samples were diluted with PBS until particle concentration was within the con-centration range of NTA Software (between 3108 and 5108 particles/ml).16
Immune-electron microscopy
Isolated EVs were deposited and incubated on For-mvar carbon-coated, glow-discharged grids as described previously.13 After 20 min, the grids were
incubated in a blocking serum containing 1% BSA in PBS. Antibodies and gold conjugates were diluted in 1% BSA in PBS. The grids were exposed to the pri-mary anti-CD63 antibody (clone H5C6) (557305, Becton Dickinson) for 20 min, followed by secondary antibody to rabbit anti-mouse IgG (Zymed, San Francisco, CA, USA) for 20 min and protein A-gold complex (10 nm size17) (CMC Utrecht, The Nether-lands) for 20 min. The efficiency of blocking was con-trolled by performing the labeling procedure in the absence of the primary antibody. The grids were stained with neutral uranylacetate and embedded in methylcellulose/uranyl acetate and examined in a Tec-nai Spirit transmission electron microscope (FEI, Eindhoven, The Netherlands). Images were captured by Quemesa charge-coupled device camera (Olympus Soft Imaging Solutions GMBH, Munster, Germany).
Functional assays
MCF-7 cells and ZR75.1 cells were obtained from the ATCC (http://www.lgcstandards-atcc.org). Cells were maintained in‘culture medium’ which is DMEM supple-mented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen).
To analyze the impact of CAAT-derived EVs on phosphorylation of CREB, semi-confluent ZR75.1 cells (1£106cells) were treated with 1£108EVs
(correspond-ing to a dose of 100 EVs/cell) in 2,5 ml control medium and 48 h later lysates were prepared and analyzed by Western blot analysis.
To analyze tumor sphere formation, viable, single MCF-7 cells (5£104cells/well) were transferred in a well of an ultra-low adherence 96-well plate (Corning, Avon, France) in 200 ml of culture medium supplemented or not with 5£109EVs (corresponding to a dose of 10000
EVs/cell). The plate was placed into an IncuCyteTM FLR
imaging system (Essen Biosciences, Welwyn Garden City, UK) within a regular cell culture incubator (37C, 95% humidity, 5% CO2). Tumor spheres were allowed to grow during 4 d and spheroid formation was monitored
every 3 h by IncuCyteTM. Spheroid size increase was
measured by IncuCyteTMsoftware.
Results
Analysis of (non)-EV enriched proteins in EVs isolated from CMCAATby dUC-ODG
EVs were prepared from the secretome of ex vivo-culti-vated breast cancer-associated adipose tissue (CMCAAT). The short, 24 h ex vivo cultivation did not affect adipose tissue integrity, metabolic activity and viability as evi-denced by H&E staining, MTT uptake and monitoring lactate dehydrogenase(LDH) release (data not shown).3 CMCAATwas pre-purified by short-term low-speed centri-fugation steps and subsequently purified by a combination of dUC with ODG (dUC-ODG) (Fig. 1). Western blot demonstrated presence of the EV-enriched proteins HSP70,flotillin-1 and tetraspanin CD9 in lysates from the EVs obtained by the dUC-ODG protocol (Fig. 2A). These EV-enriched proteins are detected in different density fractions (1.076–1.125 g/ml). The adipocyte-specific fatty-acid binding protein 4 (FABP-4) is uniquely present in EV-enriched density fractions. All EV preparations are clear from contaminating cell organelles as indicated by the absence of proteins of the Golgi apparatus (GM130), the mitochondria (prohibitin) or the endoplasmic reticu-lum and apoptotic bodies (calreticulin) (Fig. 2B).
Determination of EV size, number and purity
EV preparations of ODG fractions 8–9 with a corre-sponding density of 1.086–1.103 g/ml were loaded onto carbon-coated grids and analyzed by immune-electron microscopy (Fig. 3A). This revealed a typically heteroge-neous EV population consisting of a few CD63-positive and abundant CD63-negative EVs (33 EVs out of 300 EVs counted on EM images were CD63 positive) with a size range between 20 and 200 nm in diameter. The EV preparations are most enriched in EVs sized smaller than 70 nm. Contaminating protein aggregates were not detected. The dUC-ODG isolation protocol retrieved 2.2£109EVs/g cancer-associated adipose tissue as quan-tified by nanoparticle tracking analysis (NTA). NTA revealed that the modus, i.e. the highest number of par-ticles with similar size, of EV preparations obtained by dUC-ODG is 116 nm (Fig. 3B). This indicates that the most abundant EVs in the preparations sized smaller than 70 nm as analyzed by electron microscopy are not measured by NTA. As such the quantification of the number of particles released/g adipose tissue is likely to be underestimated.
Assessment of the functional activity of dUC-ODG isolated EVs
Next, we determined whether CMCAATEVs obtained by dUC-ODG show functional activity. ZR75.1 breast can-cer cells were treated with 1£ 108EVs, as determined by NTA, supplemented in DMEM containing 0.5%BSA. After 48 h of incubation with either control medium or control medium supplemented with CMCAAT EVs, lysates were prepared and analyzed by Western blot. ZR75.1 showed a higher phosphorylation of CREB serine residue 133 after CMCAAT EV treatment suggesting that
EVs contribute to the proliferative effect of adipose tissue on breast cancer cells (Fig. 4A).
The formation of spherical cellular aggregates (spheres) of MCF-7 cells was monitored via the IncuCyte system over a 5-day time course, allowing comparison of control condition and EV treatment (Fig. 4Band Supple-mentary Movies 1 and 2). Freshly dissociated single MCF-7 cells exhibit a strong and fast propensity to aggregate in culture on ultra-low attachment plates, with the formation of one large sphere being evident as early as 9 h after seeding. This large aggregate becomes more compact over time. Under treatment with 5 £ 109EVs, multiple small spheres are apparent 3 hours after dissoci-ation and treatment. These smaller spheres coalesce with each other to form a large, continuously expanding sphere that becomes larger in size compared with control conditions.
Figure 1.Schematic overview of the dUC-ODG protocol to isolate EVs from cancer-associated adipose tissue-derived conditioned medium (CMCAAT). Approximately 21 g of CAAT was ex vivo culti-vated in control medium. CMCAATwas harvested, centrifuged and
used for further isolation by a combination of differential ultracen-trifugation followed by Optiprep density gradient cenultracen-trifugation.
Discussion
To accurately define EV-specific content, and thus understand the functional significance of intercellular EV communication, there is a growing need for stan-dardized and validated isolation methods to obtain pure EVs. We combined dUC as a pre-purification method (13, 14), with ODG, a floatation-based separation method, and analyzed the EV samples for the presence of (non) EV-enriched proteins, morphology, size and number.
Characteristics of the dUC-ODG protocol are listed inTable 1. We found that the protocol was able to iso-late EVs from conditioned medium of cancer-associ-ated adipose tissue from breast cancer patients, as illustrated by Western blot analysis, immune electron microscopy and NTA. A practical advantage of dUC as a pre-enrichment method is the use of large vol-umes and the EV-containing pellet can be resuspended in a volume of choice, which in general depends on the next step. Clustering or aggregation of EVs may be induced during dUC18 but is not apparent from elec-tron microscopy analysis which is performed after dUC followed by ODG. Kranendonk et al. combined the dUC method with a sucrose density gradient. While both sucrose and iodixanol separate EVs based
on density, reports in literature encourage the use of iodixanol-based gradients for improved separation of EVs from viruses and small apoptotic bodies.19 Also, unlike sucrose, iodixanol is capable of forming iso-osmotic solutions at all densities, thus better pre-serving the size of EVs in the gradient.20
Adipose tissue has been shown to secrete several cyto-kines potentially affecting breast cancer cells but the functional effect of adipose tissue-derived EVs is less clear. Our study demonstrates that highly purified EVs induce phosphorylation of CREB at Serine residue 133. CREB is a bZIP transcription factor that activates target genes through cAMP response elements. CREB is able to mediate signals from numerous physiologic stimuli, resulting in regulation of a broad array of cellular responses. CREB promotes cellular survival and is acti-vated by various signaling pathways including Erk, Ca2C and stress signaling. Some of the kinases involved in phosphorylating CREB at Serine residue 133 are p90RSK, MSK, CaMKIV, and MAPKAPK-2. The increased phosphorylation of CREB in ZR75.1 breast cancer cells by treatment with adipose tissue-derived EVs is in accordance with our previous findings where conditioned medium of cancer-associated adipose tissue from breast cancer patients stimulates CREB phosphory-lation in MCF-7 and T47D breast cancer cells and
Figure 2.Protein analysis of (non) EV-enriched proteins. EVs were isolated from the conditioned medium of cancer-associated adipose tissue by the dUC-ODG protocol. Western blot analysis of (A) EV-enriched proteins (flotillin-1, CD9 and HSP70) and adipocyte-specific protein FABP-4 and (B) cell organelle and apoptotic body proteins (GM130, prohibitin and calreticulin).
induces the differential transcription of CREB target genes (unpublished). This underlines the functional activity of adipose tissue-derived EVs and supports the idea of EVs as an additional player in cell-cell communication.
Real-time imaging on ultra-low attachment plates revealed effects of adipocyte tissue derived EVs on the aggregation of cancer cells. Consistently with the func-tional effect of CREB phosphorylation, aggregates from breast cancer cells treated with EVs were larger in size compared with the control conditions. In addition, live-imaging revealed that in the presence of EVs the cancer cells formed multiple small cellular aggregates before merging together in one single cellular aggregate. The effect on aggregation was already prominent in thefirst 24 h of the experiment excluding that the difference in aggregation is just a reflection of the difference in
proliferation considering that the population doubling time of MCF-7 cells is 24h. This indicates that EVs might change the expression of surface proteins to enhance aggregation, but also that EVs might be scaf-folds to capture and stimulate the growth of cancer cells. In cancer, the activation of endothelial cells by EVs also induces the adhesion of platelets and the for-mation of platelet aggregates.21 This is probably due to an increased expression of platelet adhesion molecules in endothelial cells after EV uptake. The effect of EVs on protein aggregation has been studied in neurologic diseases. EVs catalyze the aggregation of a-synuclein in Parkinson disease22 and presumably act as a seed for amyloid plaque nucleation in Alzheimer disease.23
Simi-larly EVs might induce the formation of protein aggre-gates as scaffolds for cancer cells to adhere. Also, the detachment of adherent cells induces a rapid and
Figure 3.Morphological characterization and quantification of EV preparations by electron microscopy (EM) and nanoparticle tracking analysis (NTA). EVs were isolated from the conditioned medium of cancer-associated adipose tissue by the dUC-ODG protocol. (A) Left: Wide-field EM picture of EVs of fractions 8–9 fsrom the density gradient corresponding to the density of 1.086–1.103 g/ml. Scale bar: 200 nm. Right: Zoom in on CD63-positive and negative EVs. (B) The calculated size distribution of EVs analyzed by NTA depicted as a mean (black line) with standard error (red shaded area). Total particle number, mean particle size and modus are shown.
substantial secretion of EVs, which then concentrate on the cell surfaces and mediate adhesion to various extra-cellular matrix proteins.24Cancer-associated adipose tis-sue derived EVs may also concentrate on the cancer cell surface to enhance adhesion. Fluorescent labeling of EVs to follow their destination (cell surface or cytosol) might improve our understanding of the role of EVs in aggregation of breast cancer cells.
A limitation of this study is the lack of an irrelevant EV control such as EVs from different origin to further strengthen the functional observations of CAAT EVs on proliferation and aggregation. Indeed, the default state of the breast cancer cell cultures is exposure to EVs from fetal bovine serum (FBS) for many passages. Preparation of conditioned medium after depleting FBS results in diminished growth of the cultured cells.25 Adding another source of EVs, especially in the excess as used in the aggregation experiment (a dose of 10000 EVs/cell), might displace or outcompete the FBS EVs almost entirely and influence aggregation of the MCF-7
cells. A second limitation is the use of different doses in the phosphorylation experiments versus the aggregation experiments. Future work should compare dose response effects of purified EVs on functional and bio-chemical cellular activities. In addition, to further understand the functional role of EVs in cell culture experiments it is important to report the experimental parameters of the functional assays.26 Current work shows the ex vivo secretion of EVs by adipose tissue but it doesn’t allow the identification of the cell types responsible for EV production. FABP-4, an adipocyte specific protein, is present in fractions containing EV-enriched proteins suggesting the presence of adipocyte-specific EVs. Although adipocyte EVs are identified, it is speculative to suggest that adipocyte-derived EVs activate a pro-survival pathway in breast cancer cells based on current experimentation. Other cell types such as inflammatory cells, endothelial cells and fibroblasts are part of the stroma vascular fraction in adipose tissue and may contribute to the presence of EVs in adipose tissue-conditioned medium. The relative contribution of each of these cell types to the CAAT-derived EV cock-tail is a subject of further investigation. Cell type-spe-cific EV proteins combined with flow cytometry may enable the identification and relative contribution of adipose tissue-derived EVs. Alternatively, insight in the protein, RNA and lipid content of EVs from cancer-associated adipose tissue can potentially inform us about the different cell types that home the adipose
Figure 4.Stimulation of CREB transcription factor phosphorylation and sphere formation by EVs. EVs were isolated from the conditioned medium of cancer-associated adipose tissue by the dUC-ODG protocol. (A) Western blot analysis of phospho-CREB and total CREB from lysates of ZR75.1 cells under control conditions or treated by 1£108EVs. (B) The number of aggregates formed by MCF7 single cells
seeded in ultra-low attachment plates under control conditions or treated by 5£109
EVs for 24h. Each experiment was performed in quadruplicate.
Table 1.Characteristics of the dUC-ODG protocol implemented to isolate EVs from CMCAAT.
dUC-ODG
Sample volume limitation No
Aggregation of EVs No
Protein interference No
Yield 2.2109EVs/g adipose tissue
tissue and as such broaden our understand of this com-plex tumor environment.
In conclusion, we validated the combined use of dUC and ODG centrifugation to isolate EVs from a complex ex vivo-prepared biofluid. CAAT secretes EVs positive for FABP-4 that stimulate CREB activation and sphere formation of breast cancer cells.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. Acknowledgments
The authors thank the staff of the Electron Microscopy Labora-tory (Biocenter Oulu, University of Oulu, Finland) and Sofie De Geyter, Glenn Wagemans and Davy Waterschoot for excel-lent technical assistance.
Funding
This study was supported by Fund for Scientific Spearheads of the Ghent University Hospital, Concerted Research Actions from Ghent University, the National Cancer Plan (KPC_29_012), Kom op tegen Kanker, a doctoral grant (JVD) from Fund for Scientific Research-Flanders, a postdoctoral grant and Krediet aan Navorsers (AH) from Fund for Scientific Research-Flanders.
ORCID
Jan Van Deun http://orcid.org/0000-0003-1707-6266 Victoria Depoorter http://orcid.org/0000-0003-2409-2280 Geert Braems http://orcid.org/0000-0002-4720-8998
References
[1] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144(5):646-74; PMID:21376230; http://dx.doi.org/10.1016/j.cell.2011.02.013
[2] Lapeire L, Denys H, Cocquyt V, De Wever O. When fat becomes an ally of the enemy: adipose tissue as collabora-tor in human breast cancer. Horm Mol Biol Clin Investig 2015; 23(1):21-38; PMID:26154196; http://dx.doi.org/ 10.1515/hmbci-2015-0018
[3] Lapeire L, Hendrix A, Lambein K, Van Bockstal M, Braems G, Van Den Broecke R, Limame R, Mestdagh P, Vandesompele J, Vanhove C, et al. Cancer-associated adipose tissue promotes breast cancer progression by paracrine oncostatin M and Jak/STAT3 signaling. Cancer Res 2014; 74(23):6806-19; PMID:25252914; http://dx.doi. org/10.1158/0008-5472.CAN-14-0160
[4] Dirat B, Bochet L, Dabek M, Daviaud D, Dauvillier S, Majed B, Wang YY, Meulle A, Salles B, Le Gonidec S, et al. Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion. Can-cer Res 2011; 71(7):2455-65; PMID:21459803; http://dx. doi.org/10.1158/0008-5472.CAN-10-3323
[5] Hendrix A, Hume AN. Exosome signaling in mammary gland development and cancer. Int J Dev Biol 2011; 55 (7–9):879-87; PMID:22161843; http://dx.doi.org/ 10.1387/ijdb.113391ah
[6] Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lot-vall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007; 9(6):654-9; PMID:17486113; http://dx.doi.org/10.1038/ncb1596 [7] Peinado H, Aleckovic M, Lavotshkin S, Matei I,
Costa-Silva B, Moreno-Bueno G, Hergueta-Redondo M, Williams C, Garcıa-Santos G, Ghajar C, et al. Mel-anoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med 2012; 18(6):883-91; PMID:22635005; http://dx.doi.org/10.1038/nm.2753
[8] Kranendonk ME, Visseren FL, van Balkom BW, Nolte-’t Hoen EN, van Herwaarden JA, de Jager W, Schipper HS, Brenkman AB, Verhaar MC, Wauben MH, et al. Human adipocyte extracellular vesicles in reciprocal signaling between adipocytes and macrophages. Obesity (Silver Spring, Md) 2014; 22(5):1296-308; PMID:24339422; http://dx.doi.org/10.1002/oby.20679
[9] Kranendonk ME, Visseren FL, van Herwaarden JA, Nolte-’t Hoen EN, de Jager W, Wauben MH, Kalkhoven E. Effect of extracellular vesicles of human adipose tissue on insulin signaling in liver and muscle cells. Obesity (Silver Spring, Md) 2014; 22(10):2216-23; PMID:25045057; http://dx.doi.org/10.1002/oby.20847 [10] Deng ZB, Poliakov A, Hardy RW, Clements R, Liu C, Liu
Y, Wang J, Xiang X, Zhang S, Zhuang X, et al. Adipose tissue exosome-like vesicles mediate activation of macro-phage-induced insulin resistance. Diabetes 2009; 58 (11):2498-505; PMID:19675137; http://dx.doi.org/ 10.2337/db09-0216
[11] Lazar I, Clement E, Dauvillier S, Milhas D, Ducoux-Petit M, LeGonidec S, Moro C, Soldan V, Dalle S, Balor S, et al. Adipocyte Exosomes Promote Melanoma Aggres-siveness through Fatty Acid Oxidation: A Novel Mecha-nism Linking Obesity and Cancer. Cancer Res 2016; 76 (14):4051-7; PMID:27216185
[12] Zonneveld MI, Brisson AR, van Herwijnen MJ, Tan S, van de Lest CH, Redegeld FA, Garssen J, Wauben MH, Nolte-’t Hoen EN. Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures. J Extracell Vesicles 2014; 3; PMID:25206958; http://dx.doi.org/10.3402/jev. v3.24215
[13] Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Ver-maelen K, Vandesompele J, Bracke M, De Wever O, Hen-drix A. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles 2014; 3; http://dx.doi.org/10.3402/jev. v3.24858
[14] Thery C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture super-natants and biologicalfluids. Curr Protoc Cell Biol 2006; Chapter 3:Unit 3:22.
[15] Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, et al. Minimal experimental require-ments for definition of extracellular vesicles and their
functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 2014; 3:26913; PMID:25536934; http://dx.doi.org/ 10.3402/jev.v3.26913
[16] Gardiner C, Ferreira YJ, Dragovic RA, Redman CW, Sar-gent IL. Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. J Extracell Vesicles 2013; 2; http://dx.doi.org/10.3402/jev.v2i0.19671
[17] Slot JW, Geuze HJ. A new method of preparing gold probes for multiple-labeling cytochemistry. Euro J Cell Biol 1985; 38(1):87-93; PMID:4029177
[18] Linares R, Tan S, Gounou C, Arraud N, Brisson AR. High-speed centrifugation induces aggregation of extracellular vesicles. J Extracell Vesicles 2015; 4:29509; PMID:26700615; http://dx.doi.org/10.3402/ jev.v4.29509
[19] Cantin R, Diou J, Belanger D, Tremblay AM, Gilbert C. Discrimination between exosomes and HIV-1: purifica-tion of both vesicles from cell-free supernatants. J immu-nol Methods 2008; 338(1–2):21-30; PMID:18675270; http://dx.doi.org/10.1016/j.jim.2008.07.007
[20] Dettenhofer M, Yu XF. Highly purified human immuno-deficiency virus type 1 reveals a virtual absence of Vif in virions. J Virol 1999; 73(2):1460-7; PMID:9882352 [21] Terrisse AD, Puech N, Allart S, Gourdy P, Xuereb JM,
Payrastre B, Sie P. Internalization of microparticles by endothelial cells promotes platelet/endothelial cell inter-action underflow. J Thromb Haemos 2010; 8(12):2810-9;
PMID:21029362; http://dx.doi.org/10.1111/j.1538-7836.2010.04088.x
[22] Grey M, Dunning CJ, Gaspar R, Grey C, Brundin P, Sparr E, Linse S. Acceleration of alpha-synuclein aggregation by exo-somes. J Biol Chem 2015; 290(5):2969-82; PMID:25425650; http://dx.doi.org/10.1074/jbc.M114.585703
[23] Dinkins MB, Enasko J, Hernandez C, Wang G, Kong J, Helwa I, Liu Y, Terry AV Jr, Bieberich E. Neutral Sphingomyelinase-2 Deficiency Ameliorates Alz-heimer’s Disease Pathology and Improves Cognition in the 5XFAD Mouse. J Neurosci 2016; 36(33):8653-67; PMID:27535912; http://dx.doi.org/10.1523/ JNEUROSCI.1429-16.2016
[24] Koumangoye RB, Sakwe AM, Goodwin JS, Patel T, Ochieng J. Detachment of breast tumor cells induces rapid secretion of exosomes which subsequently mediate cellular adhesion and spreading. PloS One 2011; 6(9): e24234; PMID:21915303; http://dx.doi.org/10.1371/ journal.pone.0024234
[25] Eitan E, Zhang S, Witwer KW, Mattson MP. Extracellular vesicle-depleted fetal bovine and human sera have reduced capacity to support cell growth. J Extracell Vesicles 2015; 4:26373; PMID:25819213; http://dx.doi. org/10.3402/jev.v4.26373
[26] Dhondt B, Rousseau Q, De Wever O, Hendrix A. Function of extracellular vesicle-associated miRNAs in metastasis. Cell Tissue Res 2016; 365(3):621-41; PMID:27289232; http://dx.doi.org/10.1007/s00441-016-2430-x
KEYWORDS:
Cell Adh Migr. 2017 Mar 4;11(2):196-204. doi: 10.1080/19336918.2017.1279784. Epub 2017 Feb 1.
The isolation of morphologically intact and biologically active
extracellular vesicles from the secretome of cancer-associated
adipose tissue.
Jeurissen S , Vergauwen G , Van Deun J , Lapeire L , Depoorter V , Miinalainen I , Sormunen R ,
Van den Broecke R , Braems G , Cocquyt V , Denys H , Hendrix A .
Abstract
Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to
favor invasive growth and metastasis. Extracellular vesicles (EVs) are nanometer-sized vesicles
secreted by different cell types that shuttle proteins and nucleic acids to establish cell-cell
communication. To study the role of EVs released by cancer-associated adipose tissue in breast
cancer progression and metastasis a standardized EV isolation protocol that obtains pure EVs
and maintains their functional characteristics is required. We implemented differential
ultracentrifugation as a pre-enrichment step followed by OptiPrep density gradient centrifugation
(dUC-ODG) to isolate EVs from the conditioned medium of cancer-associated adipose tissue. A
combination of immune-electron microscopy, nanoparticle tracking analysis (NTA) and Western
blot analysis identified EVs that are enriched in flotillin-1, CD9 and CD63, and sized between 20
and 200 nm with a density of 1.076-1.125 g/ml. The lack of protein aggregates and cell organelle
proteins confirmed the purity of the EV preparations. Next, we evaluated whether dUC-ODG
isolated EVs are functionally active. ZR75.1 breast cancer cells treated with cancer-associated
adipose tissue-secreted EVs from breast cancer patients showed an increased phosphorylation
of CREB. MCF-7 breast cancer cells treated with adipose tissue-derived EVs exhibited a
stronger propensity to form cellular aggregates. In conclusion, dUC-ODG purifies EVs from
conditioned medium of cancer-associated adipose tissue, and these EVs are morphologically
intact and biologically active.
aggregation; breast cancer; characterization; exosomes; function; isolation; proliferation
PMID: 28146372 PMCID: PMC5351718 DOI: 10.1080/19336918.2017.1279784
[Indexed for MEDLINE]
Free PMC Article
Format
:
Abstract
1,2,3 1,4,3 1,3 2,3 1 5 5
4,3 4,3 2,3 2,3 1,3
Author information
Images from this publication.
See all images (4) Free text
NEDERLANDSE SAMENVATTING
De Isolatie van morfologisch intacte en biologisch actieve
extracellulaire vesikels van het secretoom van kanker-geassocieerd
vetweefsel
Inleiding
In borstkanker is vetweefsel het belangrijkste onderdeel van de tumor micro-omgeving. Het is (oa) een endocrien orgaan dat interageert met de kankercellen door cel-cel en cel-matrix contacten, alsook via secretoire molecules. Intussen is er steeds meer evidentie dat extracellulaire vesikels (EV) een belangrijke rol hebben in de communicatie in de tumor-omgeving. EV’s zijn nanometer-grote entiteiten die verschillende proteïnen, lipiden en nucleïnezuren bevatten en door de meeste cellen worden gesecreteerd. De rol van EV’s in cel-cel communicatie werd reeds bewezen door functionele translatie van mRNA van een donor cell, door de target cel. Kankerpatiënten hebben een verhoogd aantal EV’s in hun circulatie, en dit aantal correleert met ziekteprogressie. Het is echter nog niet geweeten of EV’s kanker-specifiek, dan wel gastheer-specifiek zijn. Buikvet-EV’s moduleren monocyt differentiatie en veranderen de insuline signaalcascade van adipocyten en levercellen. In culturen van pre-adipocyten van een muis, sitmuleerden EV’s de vetzuur-oxidatie afhankelijke migratie van melanoomcellen.
Voor isolatie van EV’s werd, onder andere, differentiële ultracentrifugatie (dUC) gebruikt. Hierbij worden sequentieel cellen, grotere, en kleinere EV’s gepelleteerd in de verschillende centrifugestappen. Voor sommige toepassingen is een extra zuiveringsstap, door middel van densiteits gradiënt centrifuge aangewezen. Deze pioneer studies stimuleren om de rol van vetweefsel-afgeleide EV’s te onderzoeken. Echter hiervoor is een gestandardiseerde methode voor EV isolatie en karakteristatie vereist. We isoleerden dan ook EV’s uit kanker-geassocieerd vetweefsel van borstkankerpatiënten, door middel van differentiële ultracentrifugatie, gecombineerd met een densiteitsgradiënt. Deze werden geanalyseerd op inhoud, morfologie, grootte, aantal en functionaliteit.
Materiaal en methoden
Borstkanker geassocieerde vetweefsel (CAAT) werd, na informed consent, verkregen van borstkankerpatiënten in het UZ Gent. Na vrij disseceren, spoelen en versnijden, werd het CAAT, op roterende platen, in medium gecultiveerd.
Na 24u werd het geconditioneerde medium (CMCAAT) gecollecteerd, en na enkele centrifugestappen (10 min 500g, 15 min 1500g, en 30 min 10.000g) en filtratie (door 0,2micron), gestockeerd op -80°.
Tijdens het Ultracentrifugatie-proces, wordt het CMCAAT, in open tubes geültracentrifgueerd (2u, 100.000g), de pellet wordt behouden, heropgelost in een zoutoplossing (PBS), en opnieuw afgedraaid (2u, 100.000g). De resulterende pellet, wordt dan heropgelost in 1ml PBS, en verder gezuiverd door middel van de OptiprepTM density gradient (ODG) centrifugatie. De gradiënt wordt gemaakt door 4 verschillende concentraties van de gradiëntoplossing op elkaar te leggen, en hierbovenop het staal. De gradiënt (met de heropgeloste pellet bovenop) worden dan nog eens 18u gecentrifugeerd op 100.000g. De verschillende gradiënt fracties, worden per 1ml afgehaald, opnieuw in PBS heropgelost en wederom afgedraaid gedurende 3u op 100.000g. Resulterende pellets (=EV’s) werden geresuspendeerd in 100µL PBS en bewaard op -80°. De zuiverheid van de EV’s werd beoordeeld volgens de MISEV guidelines.
De bekomen EV’s werden dan geanalyseerd voor proteïnen, aantal, grootte en morfologie.
Proteïneconcentratie werd gemeten aan de hand van de Bio-Rad DC Protein Assay. De analyse van de eiwitten zelf gebeurde door middel van Western Blot.
Nanoparticle tracking analysis (NTA) werd gebruikt voor de analyse van het aantal en grootte van de partikels. Voor de morfologie, werd gebruik gemaakt van immuun-elektronen microscopie.
Voor de functionele analyse van de EV’s, werden ZR75.1 cellen behandeld met EV’s (equivalent van een 100 EV’s per cel), gedurende 48u en hiervan werden dan lysaten gemaakt voor analyse met Western Blot. Daarnaast werden MCF-7 cellen geïncubeerd in medium, mét of zonder EV’s (equivalent van 10.000 EV’s/cel), gedurende 5 dagen, om de sfeer formatie na te gaan. De formatie werd elke 3u gemonitord via het IncuCyteTM systeem.
Resultaten
EV’s werden bereid uit het secretoom van ex-vivo borstkanker geassocieerd vetweefsel (CMCAAT). De 24u ex-vivo incubatie had géén invloed op de vetweefsel integriteit. Dit CMCAAT werd opgezuiverd, door kortdurende lage centrifugatiestappen, gevolgd door differentiële ultracentrifuge, met optiprep density gradient (dUC-ODG). De bekomen EV’s vertoonden op Western blot de EV-verrijkte proteïnes HSP-70, flottelin-1, en tetraspanine CD9. De EV verrijkte proteïnes werden voornamelijk teruggevonden in 3-opeenvolgende densiteitsfracties (1,076-1,125g/ml), het adipocyt-fatty acid binding protein 4 (FABP-4) enkel in EV-verrijkte fracties. De fracties zijn vrij van cel-organel contaminatie (afwezigheid van proteïnes uit golgi apparaat, mitochondriën en ER).
De densitietsfractie van 1,086-1,103g/ml werd onder immuno-elektronenmicroscopie geanalyseerd, waarbij een heterogene populatie aan het licht kwam. Slechts een klein deel van de EV’s zijn CD63 positief. De grootte varieert tussen 20 en 200nm. De stalen waren meest verrijkt met EV’s kleiner dan 70nm.
Via NTA vinden we 2,2 x 109 EV’s/g CAAT, waarbij de meeste partikels een grootte hebben van 116nm. Hieruit kunnen we concluderen dat een groot aantal EV’s (die kleiner dan 70nm) niet wordt opgepikt door NTA en er aldus waarschijnlijk een onderschatting is van het aantal EV’s per gram CAAT.
Daarna onderzochten we of de bekomen EV’s uit CMCAAT functionele activieit vertoonden. Hiervoor werden ZR75.1 cellen behandeld met EV’s uit CMCAAT, en na 48u zien we dat er een toename is van fosforylatie van CREB ten opzichte van de controle. Hieruit kunnen we besluiten dat EV’s bijdragen aan het proliferatief effect dat vetweefsel heeft op borstkankercellen.
De sfeer-formatie van MCF-7 cellen al/niet behandeld met EV’s uit CMCAAT via het IncuCyte systeem. Normaal hebben gedissocieerde, single MCF7-cellen, neiging om in cultuur aggregaten te vormen, die uiteindelijk een grote sfeer vormen vanaf 9u na zaaien. Deze grote sfeer wordt meer compact met de tijd. Onder behandeling van EV’s ontstaan multipele kleine sferen na 3u, deze kleinere sferen smelten samen tot grotere sferen, die continu blijven expanderen, en groter worden ten opzichte van de controle condities. Discussie
Om een accuraat begrip te hebben van de EV-inhoud en het functioneel belang in de intercellulair EV communicatie, is er nood aan gestandardiseerde en gevalideerde isolatie methoden om zuivere EV’s te bekomen. We combineerden dUC en ODG, en analyseerden de EV stalen op eiwitten, morfologie, grootte en aantal. Het gebruikte protocol was in staat om EV’s te isoleren uit CMCAAT. Een voordeel van dUC is de mogelijkheid om grote volumes te kunnen gebruiken. We gebruikten een iodixanel gradiënt voor ODG, eerder dan een sucrose gradiënt, omdat eerstegenoemde een betere separatie van EV’s van andere kleine partikels zoals virusen en small apoptotic bodies toelaat. Bovendien kunnen met iodixanol iso-osmotische oplossingen op alle densiteiten gevormd worden, die een betere preservatie van de grootte van de EV’s toelaat.
Vetweefsel produceert veel cytokines die borstkankercellen beïnvloeden, maar de rol van vetweefsel-afgeleide EV’s is minder duidelijk. Uit onze studie blijkt dat opgezuiverde EV’s, fosforylatie van CREB induceren. Dit is een transcirptiefactor die target genen activeert. CREB promoot onder andere cel overleving. Het onderlijnt de functionele acitiviteit van vetweefsel-afgeleide EV’s en ondersteunt het idee dat EV’s een bijkomende speler zijn in cel-cel communicatie.
Daarnaast zien we dat onder invloed van de EV’s, borstkankercellen meer en grotere aggregaten vormen. Dit zou kunnen impliceren dat EV’s de expressie van oppervlakte proteïnen veranderen, om zo aggregatie te bevorderen, doch ook dat ze als aanlegsteiger diennen, om borstkankercellen te capteren en te stimuleren tot groei.
Een beperking van de studie, is de afwezigheid van controle EV’s (niet afkomestig van CMCAAT). In de gebruikte controle-arm zit er telkens foetaal bovien seum, wat ook EV’s bevat... Een tweede beperking is het gebruik van verschillende dosissen EV’s in het fosforylatie versus het aggregatie-experiment. Toekomstige experimenten zouden dosis respons effecten, op functionele en biologische cellulaire activiteiten, moeten testen. Huidige studie toont de ex-vivo secretie van EV’s door vetweefsel, maar niet welk celtype verantwoordelijk is voor de EV productie. FABP4 kleurt aan in de fracties met EV-verrijkte proteïne, zodat adipocyten vermoedelijk de bron van de EV’s uit het vetweefsel zijn, doch de contributie van andere cellen (inflammatoire cellen, endotheelcellen, fibroblasten,...) aan de EV-productie is niet duidelijk. Hier zou flowcytometrie, en analyse van het RNA, lipiden en eiwittenprofiel van EV’s mogelijk een antwoord geven op de vragen van welke cellen ze afkomstig zijn.
In conclusie, we valideerden het gecombineerd gebruik van dUC en ODG voor de iolsatie van EV’s uit een complex ex vivo geprepareerde biovloeistof. Het CAAT secreteert EV’s die positief zijn voor FABP-4, die CREB activatie stimuleren, alsook borstkankercel sfeerformatie.
