Review: Isolation and Detection of Tumor-Derived Extracellular

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Review: Isolation and Detection of Tumor-derived Extracellular Vesicles Parissa Ziaei, Clifford E. Berkman, and M. Grant Norton ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00267 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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ACS Applied Nano Materials

Review: Isolation and Detection of Tumor-derived Extracellular Vesicles Parissa Ziaeia, Clifford E. Berkmanb, M. Grant Nortona,c,d a

Materials Science and Engineering Program , Washington State University, Pullman, WA 99164, USA b

c

Department of Chemistry, Washington State University, Pullman, WA 99164, USA

School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164, USA d

Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA

Corresponding author: Parissa Ziaei, Materials Science and Engineering Program, Washington State University, Pullman, WA 99164, USA E-mail: [email protected] Abstract Exosomes play a significant role in cancer progression and are potentially useful biomarkers for non-invasive diagnostics and therapeutic treatments. Although exosomes are difficult to study due to their small, inconsistent sizes and challenging purification processes, new micro- and nanotechnologies have been recently developed that seek to overcome these limitations. In this review, we examine and compare isolation and detection techniques for various types of extravesicles (EVs) including exosomes, which have sizes 200nm. Various microfluidic devices that offer better EV purity, higher recovery rates, lower costs, decreased isolation times, and low sample volumes compared to conventional techniques are described with an emphasis on the importance of micro- and nano-based technologies to isolate and detect EVs for the point-of-care acquisition and diagnosis of cancer. Key word Exosome isolation, exosome detection, extravesicles, microvesicles, microfluidic device

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1. Introduction In eukaryote cells, membrane vesicles are secreted by budding from the membrane of the donor compartment and transfer to the acceptor compartment. Vesicles such as microvesicles, ectosomes, microparticles and exovesicles are released from the plasma membrane of platelets [1], tumors [2, 3], neutrophils [4] and dendritic cells [5]. Alternatively, membrane vesicles can form inside the multivesicular endosome (MVE). By fusing of the MVE to the plasma membrane, the released membrane vesicles are called exosomes. Exosomes have sizes < 200 nm diameter and contain a mixture of proteins, mRNA, miRNA, and lipids, with signature characteristics of their parent cells [6]. Exosomes can be excreted both by normal and cancerous cells including epithelial and tumor cells [7]. Excreted exosomes have been isolated from cell-culture media and also from body fluids including blood, urine, saliva, amniotic fluid, and malignant pleural effusion [8-13]. Other factors that affect the quantity and composition of secreted exosomes include cell type, cell cycle, and stage of cancer [14]. For instance, some cell types such as T cells or resting B cells release trace amounts of exosomes while dendritic cells and macrophages release much higher amounts [14-18] Exosomes play a key role in intracellular communication and cancer progression in the tumor microenvironment [19, 20]. As shown in Figure 1, exosomes interact with other cells through different mechanisms as they are released: either by fusion to the membrane (1), adhesion to receptors on the plasma membrane (2), or endocytosis (3) [21, 22]. Ultimately, internalized exosomes could be degraded in the lysosome [14]. After degradation of the exosomes, released antigenic peptides and peptide-MHC complexes can inhibit or promote immune responses [3, 14, 17]. Many studies reveal that tumor-derived exosomes suppress immune responses and induce immune tolerance [23, 24].


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Although recent reviews in this area have been published [25, 26] we believe the current review is merited because it comprehensively examines each available isolation and detection technique in this rapidly evolving field and compares them based on their sensitivity and accuracy. Also, we review recently published double-filtration, acoustic nano-filter, and viscoelastic flow techniques that compensate for the limitation of low purity, recovery, and in some cases, clogging in physical-based isolation techniques. Several techniques have been introduced to characterize the surface markers, protein and RNA cargo associated with EVs.

Figure 1. Illustration of protein and RNA transfer by exosomes and MVs. 2. Techniques to Isolate Exosomes Exosomes with the variety of exosomal protein markers are involved in cancer progression [27, 28]. They are also critical in the development of liver disease and neurodegenerative diseases [29, 30], immune system disorders [31], intercellular signaling, blood coagulation, and waste 3 ACS Paragon Plus Environment


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management such as the removal of the redundant transferrin receptor from the exosome surface [32]. Because of the increasing interest in exosomes and their potential use in therapeutics or as biomarkers for the early diagnosis of disease, researchers are working on developing isolation and purification techniques for exosomes [21]. Isolation techniques are frequently based on physical properties of extracellular vesicles such as their size and density. Other approaches use immunoaffinity techniques where the exosomes specifically bind to certain receptors [33]. 2.1. Isolation based on size One of the most common techniques to separate exosomes from other EVs with different sizes and masses is ultracentrifugation, which typically requires a sequence of centrifugation steps eventually reaching speeds up to 200,000g. This technique is time-consuming (>4 h), the equipment is relatively expensive (> $100 k), provides low exosome recovery typically 100-fold higher concentration of mRNA and >25-fold higher purity compared with ultracentrifugation [44]. Additionally, the concentration of CD9/CD81 positive EVs was detected by using on-disc ELISA for 30 min and the obtained concentration of CD9/CD81 for Exodisc was greater than ultracentrifugation by a factor of thirteen. Lee et al. developed an acoustic nano-filter device, which is illustrated in Figure 2V. This device separates microvesicles based on their size and density [45]. The interdigitated transducer electrodes generate ultrasonic waves that apply differential acoustic forces on the microvesicles. Particles in an acoustic field experience forces proportional to their volume. These forces make the particles migrate. The larger particles, which move faster, are deflected through a larger angle, while the smaller particles remain in the center. The risk of channel clogging was minimized by operating in a continuous-flow manner. The recovery rate obtained by fluorescence intensity measurements was >80% for exosomes and >90% for microvesicles. The same method was used to separate microvesicles from red blood cells. However, the results presented in the supporting documentation do not indicate significant improvement in separation yield with this technique compared to conventional centrifugation and filtration. 7 ACS Paragon Plus Environment


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II

I

III

IV

V

Figure 2I. The structure of a ciliated micropillar structure for trapping exosomes. (Redrawn after Wang et al. [39].) Figure 2II. Illustration of (A) pressure-driven and (B) electrophoresis-driven modes for exosome filtration. (Redrawn after Davies et al. [40].) Figure 2III. Illustration of the operation of a double-filtration microfluidic device. (Redrawn after Liang et al. [42].) Figure 2IV. Schematic of a centrifugal microfluidic platform (Exodisc) with cross-sectional view of the filters. (Redraw after Woo et al. [41].) Figure 2V. Illustration of the operation of an acoustic nano-filter device. Interdigitated transducer electrodes generate a standing surface acoustic wave across the flow direction. (Redrawn after Lee et al. [45].) Recently, a viscoelastic microfluidic system was designed to separate exosomes from larger EVs based on their size without applying any external field [46]. A sample of EVs and a low concentration (0.1%) of polyoxyethylene (PEO) were introduced through separate inlets into the device. PEO causes viscoelasticity of fluids and produces elastic lift forces on vesicles, which 8 ACS Paragon Plus Environment

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create size-dependent lateral motion. The larger vesicles migrate faster than smaller vesicles toward the microchannel centerline, while the smaller exosomes were collected from two side outlets. Although the results showed some exosomes were collected from the centerline along with larger EVs, a high separation purity (>90 %) and exosome recovery (>80%) was obtained. This technique can separate large EVs from exosomes, but cannot purify exosomes when precipitation together with proteins. 2.2. Isolation based on density Density-gradient separation is used to purify exosomes by separating them from large proteins. This technique is performed by loading the sample over a concentrated solution of medium (sucrose or inorganic salts) and applying ultracentrifiguation to extract the exosomes from other particles (proteins) based on their different flotation densities [44]. Although density-gradient separation techniques can improve the purity and recovery rate of exosomes, they require even longer times (21 h) compared with conventional ultracentrifugation and greater technical ability of the user [36, 47]. OptiPrep™ density gradient (ODG) centrifugation with a non-ionic iodixanol-based medium is a more useful method to purify exosomes compared to using sucrose density gradient (SDG), since the latter method cannot separate exosomes from viruses and microvesicles due to their similar size and buoyancy [47]. 2.3. Isolation based on immunoaffinity One conventional technique to isolate exosome is immunoaffinity capture using antibody-coated magnetic beads (IAC-Exos) [48, 49]. A culture medium is incubated with antibody coated magnetic beads for 4 h then the exosome-bound microbeads separated from the media magnetically. The microbeads are washed with PBS and ultracentrifuged for 1 h. The exosomes 9 ACS Paragon Plus Environment


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are eluted from the microbeads or lysed with buffers then analyzed by liquid chromatographytandem mass spectrometry. The data showed a 2-fold increase of exosome markers in IAC-Exos compared to samples obtained by ultracentrifugation and ODG [47]. IAC-Exos like other conventional isolation techniques requires a significant number of preparation and isolation steps. The advantages of microfluidics-based immunoaffinity approaches compared to physical isolation methods are the ability to isolate a specific subpopulation of exosomes by relying on the expression of a specified surface marker as well as improved exosome recovery rates [25, 26]. A further advantage of immunological separation is that it has the ability to be used as a diagnostic device because its primary focus is on capturing tumor-derived exosomes from the total exosome pool. The first microfluidic immunoaffinity method to selectively isolate exosomes from human sera or cell culture was designed by Chen et al. [38] and consisted of a herringbone-structured microfluidic device using microchannels coated with an anti-CD63 antibody: one of the most common protein markers present on the membrane of exosomes [21, 50]. A small volume (100400 μL) of serum was flowed through the device in < 1 h at a rate of 16 μL.min-1. According to the published data, 30 and 4 ng of RNA were recovered from 400 μL of patient and control serum samples, respectively. The authors reported a broad recovery rate ranging from 42 to 94% for four experiments based on RNA extraction, which demonstrates the need for further system optimization. Kanwar et al. [37] selected the same immunoaffinity-based approach as Chen et al. but modified the previous design to perform isolation, detection, and quantification of exosomes directly on-chip. Figure 3I shows the ExoChip device that was assembled by interconnecting several circular chambers through narrow channels in order to increase mixing of the exosomes 10 ACS Paragon Plus Environment

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due to changes in the fluid velocity and thereby extending retention time. Fluid flow simulation studies showed a low velocity in the expanded section to enhance the capture of exosomes. Serum samples from healthy donors or cancer patients were injected in the antibody-coated ExoChip at a flow rate of 50 μL.min-1 for 10 min and incubated for 30 min. The captured exosomes were stained with fluorescent carbocyanine dye (Vybrant™ DiO). The fluorescent intensity was measured by a microplate reader. The results showed an overall 2.34-fold increase of circulating exosomes in the sample from cancer patients compared to healthy controls. The advantages of the ExoChip compared to the earlier work of Chan et al. are more rapid quantification (~20 min) and ease of imaging. Confocal microscope images showed captured EVs with a broad range of sizes (30-300 nm), which indicate both individual exosomes and exosome clusters. An immunomagnetic isolation chip with a cascading microchannel network for multi-stage exosome analysis was designed by He et al. [51]. The device included exosome isolation and enrichment, chemical lysis, protein immune-precipitation, and chemifluorescence-based sandwich immunoassays (Figure 3II). The immunomagnetic method, compared to the surface-based exosome microchips, provides enrichment of captured exosomes and higher capture efficiency due to the larger surface areas involved. The magnetic beads were coated with anti-EpCAM and antiCA125 antibodies for non-small cell lung cancer (NSCLC) and ovarian cancer. Human plasma, pre-mixed with antibody-labeled magnetic beads, was introduced into the chamber through inlet #1. A lysis buffer (10% Triton X-100) was passed through inlet #2 and incubated for 10 min with the captured exosomes. The lysate then flowed into a serpentine channel where antibody-labeled magnetic beads were injected from two side channels to capture the released exosomal proteins. The on-chip assay can be completed in 99.9%) and less damaged vesicles were observed compared with ultracentrifugation. Also, the sensitivity of this technique to detect a biomarker for NSCLC (type 1 insulin growth factor receptor) was 100-fold higher than commercial ELISA kits. Figure 3III illustrates a multiplexed microfluidic device based on the application of an alternating electrohydrodynamic (ac-EHD) field developed to generate a surface shear force, which creates lateral fluid flow within a few nanometers of an electrode surface. This “nanoshearing” phenomenon boosted the number of exosome-antibody (surface bound) collisions, which increased the capture level by a factor of five and tripled the detection sensitivity compared to conventional hydrodynamic flow (pressure-driven) devices [52]. Nanoshearing with a low detection limit (>2700 exosome per μL) was an effective technique to enhance the number of specific captures even at low concentrations. This method also allows for the simultaneous detection of multiple exosome targets by using rapid on-chip naked-eye readouts. For instance, the exosomes were captured via exosome-antibody interaction of anti-HER2 and/or anti-CD9 targets then the horseradish peroxidise (HRP) conjugated anti-HER2 and/or FITC conjugated anti-CD9 were driven in the ac-EHD field. The presence of captured exosomes was rapidly (~ 5 min) detected by the naked eye and the colorimetric solution was used to quantify the amount of captured exosomes by measurement of spectrophotometric intensity. Dudani et al. [53] demonstrated a microfluidic platform using rapid inertial solution exchange (RInSE) to isolate and detect exosomes (Figure 3IV). Antibody-coated polystyrene beads were incubated with exosomes for 30 min before the particles were migrated into the exchange buffer, tris-buffered saline (TBS). The captured beads were collected and lysed for analysis. The amount of RNA extracted from anti-CD63 capture beads was 2.6 times higher than 12 ACS Paragon Plus Environment

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the control bead isolation. The intensity result from flow cytometry histogram analysis indicates an incubation time of 4 h resulted in more captured exosomes and higher average signal intensity as compared with only 30 min incubation. Exosomal microRNAs and messenger RNAs (mRNA) extracted with the anti-CD63 beads increased by a factor of 2.6 above the control beads. Some non-specific adsorption of larger RNA from lysed cells were observed in anti-CD63 and control beads. A microfluidic device based on deterministic lateral displacement (DLD) separates microvesicles from the heterogeneous population of cancer-cell-derived EVs as a function of diameter [54]. DLD separates particles larger or smaller than a critical size through the specific arrangement of posts within a channel [55]. Fluorescent polystyrene microspheres with diameters 51 nm, 190 nm, and 2.01μm were mixed with the sample of EVs (both microvesicles and exosomes) and the solution was pumped to the device with a 250 nm threshold diameter. In Figure 3V microspheres with the diameter of 2.01 μm - above the threshold diameter - were displaced by DLD into the output ε while the remainder of the beads with lower threshold diameter were collected in output δ. Output ε contains the displaced microvesicles and output δ contains the heterogeneous EVs population. This technique demonstrated 39% microvesicle recovery efficiency and 98.5% purity in target output ε. Figure 4I shows the ExoSearch chip designed by Zhao et al. [56] with a continuous flow to isolate blood plasma exosomes in a wide range of preparation volumes (10 μL to 10mL). Briefly, a plasma sample and immunomagnetic ferrite beads are injected from a Y-shaped injector at the same flow rate through the serpentine channel where they uniformly mix to facilitate exosome binding with the beads. The magnetic beads with captured exosomes are retained at a microchamber while a mixture of antibodies labeled with unique fluorescence dyes is injected into 13 ACS Paragon Plus Environment


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the microchamber to stain the exosomes. The capture efficiency was measured by comparing fluorescence intensity of flows at the inlet and outlet chambers. A broad range of capture efficiency from 42% to 97.3% was reported at different flow rates from 50 to 104 nL.min-1. The low flow rate allowed complete mixing of the exosomes and the magnetic bead suspension, which in turn enhances the effective capture. The result demonstrated the significant increase of three exosomal tumor markers (CA-125, 12.4-fold increase, EpCAM, 6.5-fold increase and CD24, 3fold increase) of ovarian cancer compared to a healthy control. Fang et al. [57] created a microfluidic chip for immunocapture and quantification of exosomes. The chip layout is shown in Figure 4II and operates as follows: 1 mL of a plasma sample is mixed with CD63 antibody-coated magnetic nanoparticles (400nm) and introduced into inlet 1. The primary antibody reagents such as HER2 and EpCAM are introduced at 2 μL.min-1 through inlet 2 to specifically detect cancer-derived exosomes. The fluorescently labeled secondary antibody was introduced through inlet 3 to interact with the primary antibodies and the fluorescence was recorded by an inverted fluorescence microscope. The results indicated the higher expression of HER2 and EpCAM for breast cancer patients compared with the healthy control. Another recent isolation and detection approach is the nano-interfaced microfluidic exosome (nano-IMEX) platform based on coating a glass substrate with graphene oxide/polydopamine (GO/PDA) to improve capture efficiency of exosomes because polymerization of highly hydrophilic PDA provides excellent biocompatibility and resistant to biofouling [58]. The nano-IMEX chip illustrated in Figure 4III contains Y-shaped microposts with GO/PDA. In order to immobilize the antibody on the platform, the PDA surface was coated with protein G followed by incubation of anti-CD81. Exosomes extracted from colon cancer cell 14 ACS Paragon Plus Environment

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line COLO-1 were pumped into the nano-IM EX and captured exosomes were incubated by three different biotinylated antibodies (CD63, CD81 and EpCAM) conjugated with the enzyme βgalactosidase. Subsequently, a substrate (di-β-D-galactopyranoside) was introduced to react with the enzyme. The emitted fluorescence was visualized with an inverted epifluorescence microscope and measured with fluorescent ELISA. The nano-interface enables the development of an ultrasensitive exosome ELISA assay with a low detection limit of 50 μL-1 (80 aM), which is one order of magnitude better than that of the most sensitive microfluidic methods. The 3D porous structure of the GO/PDA nano-interface yielded a significantly higher exosome signal and lower non-specific background compared with GO/PEG and PDA-only coating. Nano-IMEX chips were used to detect the expression of exosomal markers in ovarian cancer (OvCa) patients in response to cancer treatment. The expression level for the post-treatment patient was ∼10-fold lower than without treatment.

The microfluidic-based mobile exosome detector (μMED) illustrated in Figure 4IV was designed to quickly (< 1 h) isolate and measure brain-derived exosomes from mild traumatic brain injury (mTBI) [59]. In μMED, the brain-derived exosomes were negatively selected by anti-CD45 and anti-CD61 screening, with positive selection using anti-CD81 followed by labeling for brainderived GluR2. Also, the device was used to monitor different types of mTBI injury by measuring the level of GluR2+ exosomes for contusive brain injury (CCI) and concussive brain injury (blast TBI). The results obtained from mTBI (including both CCI and blast) exosomes represented a 1.8fold increase in the level of GluR2+ compared with a healthy control. Recently, our group has developed a selective capture technology for positive prostate-specific membrane antigen (PSMA+) exosomes. It is known that the prostate tumor enzyme-biomarker, prostate-specific membrane antigen (PSMA), is highly enriched in exosomes excreted by PSMA+ 15 ACS Paragon Plus Environment


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prostate cancer cells such as LNCaP cells [60]. Since PSMA is an attractive diagnostic and therapeutic target, researchers have been looking for developed antibody and small molecules with inhibitory potential against PSMA [61, 62]. TG97 is a known inhibitor of PSMA enzymatic activity, which binds irreversibly in the active site of PSMA and rapidly internalizes in LNCAP and CWR22Rv1 prostate cells [63]. In our research, biotinylated TG97 (biotin-PEG12-TG97) was used to interact with PSMA on tumor-derived exosomes [64]. Figure 4V shows a scanning electron microscope (SEM) image of the nanostructured support. The nanospring mats can be grown on a number of different substrates with >98% open porosity and surface areas of ~260 m2.g-1. The silica surface can readily be functionalized to allow attachment of small molecules, peptides, aptamers, or antibodies [65, 66]. In this specific application, the silica nanosprings were functionalized with GPTMS and biotinylated TG97 to irreversibly interact with PSMA known to be present on exosomes derived from PSMA+ LNCaP cells. The protein concentration of the initial exosome suspensions and the exosome suspensions after incubation with the nanospring supports were determined using the biocinchonic acid (BCA) protein assay. The samples were measured in a spectrophotometer at 562 nm. The difference in exosome protein concentration before and after exposure to the nanosprings substrate was assumed to be proportional to the amount of exosomes retained on the substrate. The BCA assay demonstrates both specific and non-specific exosome capture and highly specific capture efficiency (~50%) for the ligand-activated silica nanostructures [64]. In the future, we plan to construct a microfluidic device by assembling a number of silica nanosprings mats in order to selectively capture tumor-derived exosomes from patient samples. Table 1. Comparison of exosome isolation techniques


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Technique

Recovery

Purity

Sample volume

Time

Ref.

5-8 h

Limit of detection (LOD) N/A

Ultracentrifugation

5-25%

Low

500 mL

OptiPrep™ Density gradient ExoQuick-TCTM

36-65

High

500 μL

21 h

N/A

>100 μL 30 μL

24 h

N/A

[47, 68] [69]

N/A

Low

Ciliated micropillar

60%

N/A

N/A

[39]

100 μL

10 min run and 24 h incubation Passage time of exosomes through the channel 80 %

Acoustic-based

> 80%

>90% separation purity of small exosomes from large EVS N/A

N/A

[46]

50 μL

95%

High

200 μL

1h

42-94%

N/A

2070 exosomes μL-1. The authors compared this technique with the previous isolation and detection techniques (e.g ciliated micropillar, nPLEX, μNMR) and addressed the possibility of obtaining a false result due to the presence of cellular moieties or free proteins expressing the targeted tumor marker along with tumor-derived exosomes. Therefore capturing all 32 ACS Paragon Plus Environment

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exosomes, protein, and cellular moieties using generic exosomal membrane markers and subsequently quantify the tumor-relevant exosomes by tumor-specific detection antibody might help to prevent this issue. Table 2 compares the different sensitivities of each technique.

Table 2. Comparison of different detection techniques.

Technique

LOD (concentration)

Time

Ref

N/A N/A N/A

LOD (size)** >2-5 nm ~1 nm ~1 nm

TEM AFM SEM

>1h >1h >1h

NTA

1×107 mL-1

~50 nm

DLS

108 mL-1

~1 nm

5 min to 1 h per measurement 2 to 5 min per measurement

Review: Isolation and Detection of Tumor-Derived Extracellular

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