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Highly selective capture surfaces on medical wires for fishing tumor cells in whole blood Frank Daniel Scherag, Robert Niestroj-Pahl, Solveigh Krusekopf, Klaus Luecke, Thomas Brandstetter, and Jürgen Rühe Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04219 • Publication Date (Web): 20 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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Analytical Chemistry

Highly selective capture surfaces on medical wires for fishing tumor cells in whole blood. Frank D. Scherag a, Robert Niestroj-Pahl b, Solveigh Krusekopf b, †, Klaus Lücke b, Thomas Brandstetter a and Jürgen Rühe a,* a b

Department of Microsystems Engineering, University of Freiburg, Georges-Köhler-Allee 103, D-79110 Freiburg, Germany GILUPI GmbH, Hermannswerder 20a, 14473 Potsdam

ABSTRACT: The detection of circulating tumor cells (CTCs) in the blood of cancer patients is a challenging task. CTCs are, especially at the early stages of cancer development, extremely rare cells hidden in a vast background of regular blood cells. We describe a new strategy for the isolation of CTCs from whole blood. The key component is a medical wire coated with a multilayer assembly that allows highly specific capture of EpCAM (Epithelial Cell Adhesion Molecule) positive CTCs from blood. The assembly is generated in a layer-by-layer fashion through photochemically induced C,H insertion reactions and consists of a protective layer, which shields the contacting solution from the metal, a protein resistant layer, which prevents non-specific interactions with proteins and a layer containing the EpCAM antibodies. In vitro experiments show that these surfaces can capture tumor cells from whole blood with enrichment factors (specifically vs. non-specifically bound cells) of up to about 3000 compared to the number of leucocytes in the blood. The purity of the isolated cells is greater than 90%. After “fishing” them from the blood, the cells, still bound to the wire, can be genetically analyzed. This demonstrates that this strategy might prove useful for next generation sequencing.

Circulating tumor cells (CTCs) are potent prognostic biomarkers, and their analysis has high potential in personalized cancer treatment.1-5 The ultimate goal is the prevention of metastasis and metastatic recurrence after tumor cells have been released into the blood. Thus information on the extent of the circulation of these cells is of great interest, especially at early stages in tumor development. CTC invasion into the blood can occur during any stage of tumor development, but it might even be provoked by diagnostic or therapeutic procedures (e.g., palpation, biopsy, surgery, drugs).1, 3 Enrichment, detection and counting of CTCs is very important because it allows for statistical predictions concerning the overall survival time and the response of patients to anti-cancer therapies.6 While enrichment and counting is already a big step forward, molecular information and how the cells will respond to therapeutic efforts is still locked away inside them. Consequently, to obtain this information, a prerequisite for any personalized cancer drugs that target selected pathways, analytical downstream processes after cell isolation are needed.7, 8 This is important for several reasons. One of them is in drug development. The differentiation of metastatic cancer cells, namely clone evolution, molecular changes and phenotype generation, causes heterogeneous populations that would adversely affect the course of disease by diminishing the success rate of single target treatments and thus promote disease progression.3, 9-11 Secondly, molecular characterization and genomic profiling of isolated CTCs by e.g. PCR-based methods would also improve the overall sensitivity and specificity of immunostaining detection methods and allow a more reliable determination of the concentration of the cells in the blood sample.1, 7 Finally, and probably most important, highly efficient CTC enrichment must also be easily accessible for molecular investigations at

the single cell level, which focus on differences and subpopulations of tumor cells within the given sample. However, when enrichment and isolation of these cells are studied, and the sensitivity, sample purity, cell viability, and cell recovery are assessed, current technologies are neither ideally suited for the analytical toolbox nor even compatible with molecular downstream technologies, because each of these applications has its own strict sample requirements.12 The selective accumulation of CTCs from the whole blood of a patient (≈ 5 L) is an extraordinarily challenging task because they are of very low abundance. For cases that are relevant in early cancer diagnostics and treatment, the challenge is to find cells present at very low concentrations, even as low as 1-10 cells ml-1.3, 13 This number must be compared to the background of similarly sized nucleated blood cells (≈ 107 cells ml-1) and non-nucleated red blood cells (≈ 109 cells ml-1) and platelets (≈ 108 cells ml-1).13, 14 In recent years many different highly efficient in vitro devices and techniques have been developed that are able to isolate and detect CTCs in whole blood samples.1, 12, 15 These methods can be divided into those that rely on a) affinity enrichment by using biological recognition elements, such as antibodies and aptamers, or b) differences in physical or hydrodynamic properties.12, 16-18. Some devices also combine several physical and biological methods to increase the efficiency of discriminating the target cell from the background.19 Despite the various sophisticated efforts and the advances that have been made to increase sensitivity (efficiency 80100%), selectivity (purity < 70%) and throughput (≤ 10 ml hr1 15, 19, 20 ), several disadvantages inherent to ex vivo and in vitro isolation strategies must be considered such as sample size and loss of information by sample degradation.3, 4 A blood sample

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size of only a few milliliters, usually smaller than 10 ml6, 21 does not necessarily account for all information that would be given by screening the entire blood volume. Thus the potential information in the sample and on the sensor is limited and furthermore will restrict a direct downstream use of isolated cells for methods such as DNA sequencing due to the low sample purity of these from in vitro preparations. An in vivo device however could potentially sample the complete blood volume. An example has been recently presented in form of a CE-certified, (antibody) functionalized and structured medical wire (CellCollector; Gilupi GmbH).4, 22. The device allows capture and identification of EpCAM-/keratin-positive CTCs in vivo. A recent published clinical study demonstrated that capture and isolation of CTCs can be achieved by placing the wire into the arm vein of lung cancer patients for 30 minutes.22 The authors argue that during the 30 minute incubation in the vein, the device is exposed to approximately 1 L of blood, which increases the probability of capturing CTCs.22 Furthermore, in a proof-of-principle trial, the in vivo captured CTCs could be genetically analyzed and compared to the primary tumor tissue.22 Other known approaches use Nylon fibres23 or surface-modified vein indwelling needles24, which have been used in preclinical studies to capture CTCs in vivo. A core requirement for the use of this type of wire-based system is the development of a surface that does not permit nonspecific adsorption of proteins, as this would also lead to non-specific cellular adhesion. However, to allow cell capture, antibodies must be immobilized on the surface that enable the specific binding of the desired cells. In addition, the surface of the device must be non-degradable, non-toxic, nonthrombogenic and must not induce any immune response. Cell and protein repellent coatings are usually realized by hydrophilic polymer brushes, such as polyethylenegylcol (PEG), polyelectrolyte brushes, and surface-attached carbohydrate chains.25, 26 Polyelectrolyte brushes, such as linear polycarboxylates, are susceptible to nonspecific adsorption26 and degradation27, 28. The application of polymeric coatings to surfaces has to be compatible with the in vivo application. Monomers and residual solvent from the polymerization process must be completely removed, which in a thin surfaceattached film may be difficult to verify. Currently a very popular approach is the attachment of polymer monolayers to gold plated surfaces. The use of sulfur/gold interactions in such systems, however, can result in low thermal and oxidative stability,29, 30 which with time may induce significant coating degradation. The predominant covalent antibody coupling method employed is based on carbodiimide activation of carboxyl groups. While this surface reaction proceeds with high yield, it could be difficult to control in concerns of pH and unwanted reactions that cause irreversible surface contaminations because the reaction products cannot be purified.31, 32 Furthermore, some non-reacted groups always remain, which could be a source of trouble at later stages of the experiments, so that usually additional blocking procedures, frequently with charged low molecular weight compounds, are employed. As a result, the molecules that were used for blocking and deactivation may themselves cause nonspecific interactions with biomolecules present in the sample or cause an undesired steric hindrance of receptor molecules.33-35 Additionally, the incorporation of charged groups into the surface chemistry will decrease the repellent feature of the coating.26

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To overcome some of these shortcomings, we describe a new coating strategy for the functionalization of medical stainless steel wires. The goal is to establish a multifunctional coating system that is highly reproducible, thermally and mechanically stable, compatible to (at least) short-term in vivo requirements and also applicable in downstream processes such as nucleic acids analyses. To this end, receptor molecules, i.e. anti-EpCAM antibodies, are immobilized on a thin surface-attached hydrogel network using a photoimmoilization strategy.35, 36 Such hydrogel networks are known to be highly protein repellent and non-thrombogenic.26, 37, 38. The investigations on the developed coating strategy focus on the role of antibody surface loads for the in vitro capture performance of spiked tumor cells (SK-BR-3) in whole blood and phosphate buffer (PBS). The influence of antibody surface concentration, the capture specificity, the cytotoxicity and storage stability of the surfaces on device performance and its applicability for nucleic acid based analysis is also elucidated. Experimental Section Medical wire preparation. Stainless steel profile wires (EPflex Feinwerktechnik GmbH, Germany) having a diameter of 0.5 mm and a length of 160 mm were partially twisted by hand on 40 mm with 2 turns cm-1 with a self-made setup. The flat wire was vertically fixed and strained between a screw clamp and the drill adapter of a laboratory stirrer while applying a normal force of roughly 1 N. For a torsion of 2 turns cm1 , the wire was overwound with 2.5 turns cm-1 and then turned back. The blank stainless steel triple helix wires were a product from gilupi GmbH, Potsdam. Prior to antibody immobilization the wires were pre-coated with a multilayer as previously described.39 In brief, cleaned stainless steel surfaces were incubated for one hour with 1 mM octadecyl-phosphonic acid (ODP; Sigma–Aldrich, Germany) dissolved in ethanol. Layers composed of PS-BP (styrene copolymer containing 5 % photoactive benzophenone units) and subsequently PDMAA-BP (dimethylacrylamide copolymer containing also 5 % photoactive benzophenone units) were each applied by dip coating (30 mm min-1) the wires into solutions of ethyl acetate and ethanol respectively. The concentrations of the benzophenone containing copolymers were chosen as: polystyrene, c = 5 mg ml-1 for triple helices and 10 mg ml-1 for flat wires; polydimethylacrylamide, PDMAA, c = 5 mg ml-1 for all wires. The coatings were sequentially dried in air and cross-linked by UV irradiation (3 J cm-2 at 365 nm on each side) in a Bio-Link™ UV-crosslinker (VWR International GmbH, Germany). For the immobilization of the antibodies on the multilayer coating, we applied a droplet of 8 µl containing the anti-EpCAM antibodies (clone hu-HEA125, InVivo GmbH, Germany) to a nonirradiated PDMAA-BP layer. During deposition the wire was horizontally rotated (40 rpm) around the longitudinal axis and vertically tilted so that the drop rolled over the moving wire surface. Finally, air-dried antibodies were immobilized on the surface of the wire by UV-irradiation, which crosslinks the non-irradiated PDMAA layer and links adjacent antibody molecules simultaneously while forming a network. The functionalized wires were stored until use at 4°C in PBS buffer containing sodium azide (0.01 %). In vitro cell capture experiments. EpCAM positive SKBR-3 breast cancer cells (CLS, Cell Lines Service GmbH, Germany) were grown and prepared as described.4 EDTAblood samples obtained from healthy donors were spiked with


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35,000 SK-BR-3 cells ml-1. Cell capture experiments with twisted flat wires were carried out with a PBS solution spiked with SK-BR-3 (35,000 cells ml-1) and K562-A2 (150,000 cells ml-1), an EpCAM negative leukemia cell line. Functionalized medical wires were incubated in fully filled 2 ml vials (Eppendorf) on a rotational shaker (Stuart SB3 Tube Rotator, Bibby Scientific Limited) at 2 rpm for 30 min. Staining and counting bound cells. After cell capture, the functionalized medical wires were washed in PBS, followed by incubation in PBS containing 2% (w/v) BSA (Carl Roth, Germany) for 30 min at RT. Identification of surface bound cells was done by immunocytochemical staining. Each wire was incubated with a FITC-conjugated mouse monoclonal antibody directed to EpCAM [1:100 in PBS (10 µg/ml); Acris Antibodies GmbH, Germany], a phycoerythrin (PE)conjugated rabbit antibody directed to CD45 [1:25 in PBS (2 µg ml-1); Life Technologies GmbH, Germany] and a PEconjugated antibody directed to CD16 [1:50 in PBS (2 µg ml1 ); ExBio, Czech Republic]. Cells were counterstained with Hoechst 33342 [1 µg ml-1 in PBS, Sigma Aldrich]. Stained cells were evaluated using an Axio Imager, A1m microscope (Zeiss, Germany) equipped with an AxioCam digital camera system and the AxioVision 4.6 Software. EpCAM ELISA with medical wires. For comparison and semi-quantitative evaluation of antibody immobilization on functionalized wires, an adapted ELISA assay was performed. After counting surface bound and stained cells, each wire was incubated with a horseradish peroxidase (HRP)-conjugated anti-human-IgG(Fab2) antibody [1:5000 in PBS (0,1 µg ml-1); Bio-Rad AbD Serotec GmbH, Germany]. The HRP mediated reaction with 3,3′,5,5′-Tetramethylbenzidine (TMB,) was measured with a plate-reader (Omega Spectrostar, BMG Labtech) and the OD values (450 nm/620 nm) were recorded. Cytotoxicity tests. To investigate potential cytotoxic effects of the new coating system, we performed direct cell contact and material elution tests in vitro, based on the requirements for a class IIa medical device, as outlined in the ISO Guideline 10993-5 (www.iso.org) and as previously described.4 For the elution test, three eluates of wires and of reference materials known to be toxic (copper wire, Goodfellow GmbH, Germany) or non-toxic (polytetrafluorethylen wire, PTFE, Goodfellow GmbH) for normal human dermal fibroblasts (NHDF, C12302, PromoCell GmbH, Germany) were generated. Elution was performed with RPMI-medium (FG 1235, Biochrom AG, at 37˚C for 24 h) with a final surface to volume ratio of 3 cm2 ml-1. NHDF cells were exposed to eluates for 48 hours at 37°C. Cell viability was assessed by a colorimetric TTC assay. After removal of the eluates, a yellow tetrazolium compound (EZ4U, Cell Proliferation and Cytotoxicity Assay, Biomedica GmbH & Co. KG, Austria) was added (3 h, 37˚C) and was converted to a brick-red formazan by mitochondrial cellular activity. Change in color was recorded at 450 nm by use of a microtiter plate reader (Omega SpectroStar, BMG Labtech, Germany). For direct contact tests, the wires and reference materials were placed on a layer of adherent NHDF, followed by a 24 h incubation of this set-up at 37˚C. Nucleic acid analysis. Functionalized twisted flat wires containing surface bound SK-BR-3 tumor cells were used for PCR-based analytical methods. A direct dipstick PCR39 was done by cutting the functional twisted area of the wire into 20 mm segments and depositing them into polycarbonate capillaries (GeneOn GmbH, Germany). A liquid DNA-extraction was

done directly on the wire by applying 8 µl of NukEx Plus reagent (gerbion GmbH & Co. KG, Germany). After incubation for 10 min. on the moving (rotated and tilted) wire surface the released nucleic acids could be retrieved by applying an 8 µl droplet of pure water that moves at least once over the whole surface area. The uptake of eluted nucleic acids could also be repeated and fractionated. A master mix for all samples was prepared from the HotStar Taq PCR-Kit (QIAGEN, Germany) with a final concentration of 0.025 U µl-1 Taq DNA polymerase, 160 nM of each dNTP, a standard concentration of PCR-buffer containing 4 mM MgCl2 and 1 x EvaGreen® dye (Biotium Inc., CA, USA) was added for real-time PCRanalysis. The DNA-template of the positive control had a final concentration of 5 ng µl-1 human genomic DNA (Female, G1521, Promega GmbH, Germany). A concentration of 0.08 nM of each primer (TIB Molbiol GmbH, Germany) was added for the amplification of a 92-base pair fragment of the PIK3CA exon 9 on human chromosome 3. The forward sequence was 5´-GCTCAAAGCAATTTCTACACGAGA-3´ and the reverse sequence was 5´TCCATTTTAGCACTTACCTGTGAC-3´.40 Amplifications were performed using a Roche LightCyclerTM1.5. The temperature cycling was set as follows: Primary activation at 95°C for 15 min. followed by 60 cycles of 95°C for 30 sec. and 67°C for 30 sec. The heating rate for the melting curve analysis was set 0.1°C per second. Gels for Electrophoresis were prepared with 1.5% (w/v) Agarose (Merck, Germany) in 1x TAE buffer (Carl Roth, Germany) containing 1x GelRed nucleic acid stain (Biotium Inc., CA, USA). Samples contained a mixture of 5 µl PCR-products and 1 µl loading buffer (PEQLAB, Germany). Running buffer was also 1x TAE buffer and 90 V was applied for 45 min. Results and Discussion Surface coating and antibody immobilization. Binding of the antibodies to the wire surfaces with a controlled surface density was achieved via photoimmobilization of benzophenone containing copolymer networks following a strategy described previously.35, 36, 41 Briefly, first the stainless steel wire substrate was coated with a self-assembled monolayer (SAM) consisting of a phosphonic acid octadecyl ester. Onto this monolayer a styrene copolymer (PS) containing photoactive benzophenone (BP) moieties was deposited and crosslinked by brief UV irradiation. This process essentially inverts the process described by Pahnke et al, who describe a phosphonic acid anchored photoactive SAM for polymer layer generation.41 During the process described here, the BP units become activated upon UV exposure and induce crosslinking of the polymer. Simultaneously some groups in the PS network that are by coincidence located directly at the surface, attack C,H groups contained in the phosphonic acid SAM, so that the two layers become covalently attached to each other. Since the applied polystyrene layers are homogeneous, cover the whole surface, and are not swollen by the aqueous environment, they act as barrier layers and prevent any direct contact of the metal with the surrounding medium and/or leaching of metal ions, which would impair the function of proteins in a contacting solution.39 As topmost layer, a thin cell and protein repellent hydrogel layer (≅ 100 nm film thickness) based on poly(dimethylacrylamide) (PDMAA) was generated by depositing a PDMAA polymer containing again BP-units (PDMAA-BP) on top of the PS layer.26, 37, 38 The



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PDMAA hydrogel does not permit a penetration of the layer by proteins through entropic shielding and size exclusion.26, 37, 38 Prior to irradiation, however, a droplet of the antibody solution was distributed on the wire surface and air dried. During this the wire was rotated along the longitudinal axis and tilted thereby enabling a rather homogeneous distribution of the antibody solution. The photochemical process leads to simultaneous crosslinking and surface-attachment of the hydrogel and attachment of a covalently bound protein monolayer. A schematic representation of the complete coating composition and its mechanism is depicted in figure 1.

Figure 1. Schematic representation for the complete multilayer coating of medical metal wires. Coatings were obtained by sequential deposition of: 1. a self-assembled monolayer of octadecylphosphonic acid on a stainless steel surface; 2. PS-co-5%MABP followed by UV-crosslinking; 3. PDMAA-co-5%-MABP; 4. droplet deposition, distribution and air-drying of an antibody solution followed by UV-crosslinking. The polymer layer thicknesses were determined on gold substrates by SPR.

Receptor load and cell capture in phosphate buffer. The surface concentration of the immobilized receptors may have a large effect on the capture performance concerning selectivity and the total number of captured cells. In a first line of experiments this surface concentration of the antibody was varied. To this end, identical volumes of antibody solution with varying antibody concentration were used for the coating. The approximate surface area of a twisted flat wire (figure S1) that can effectively interact with its surrounding is 47 mm2. Solutions of the anti-EpCAM antibodies, with protein contents ranging between 0.0625 µg and 32 µg were applied so that surface loads between 1.3 and 680 ng mm-2 were physically deposited. The approximate molecular weight of an IgG based antibody is 150 kDa, so the corresponding molecular densities were roughly between 9 x 10-15 and 4.5 x 10-12 mol mm-2 or 5.3

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x 10-3 to 2.8 molecules nm-2 assuming a homogenous distribution. Thus at the highest concentration applied (without irradiation), the deposited number of molecules would greatly exceed that of a monolayer. The surface contraction of the bound antibodies after irradiation and extraction was evaluated by a colorimetric ELISA, in which the OD values represent the relative amount of immobilized antibodies present on the final surface and in the assay. As shown in figure 2a, at low concentrations the OD values correlated directly with the amounts of deposited antibodies after photoimmobilization and washing, but levelled off at antibody concentrations of about 170 ng mm-2 (≈1 pmol mm-2 ≈ 0.6 molecules nm-2). In reference experiments, layer thicknesses of covalently bound antibody attached to hydrogel coated silicon wafers were measured by imaging ellipsometry. Depending on the amount of antibody deposited, the thicknesses of the layers ranged between 2 and 6 nm (figure S3), which compares well with the hydrodynamic radius of an IgG-based antibody, which has been reported to about 5.5 nm.42, 43

Figure 2. Influence of the antibody load prior to photo immobilization on the in vitro capture performance of twisted flat wires in PBS. a) Mean cell counts per wire and OD values (n=5 wires) representative for the amount of functional antibodies. Labelled photographs show fluorescence cell pictures with 3D wire shape overlay. b) Percentage purity of SK-BR-3 on wires relative to mean values of both cell types. c) Enrichment factor of SK-BR-3 to K562-A2 on wires relative to the value in bulk solution.

Any excess protein molecules that are not accessible to the reactive species generated by the activation of the BP-groups cannot be bound and are washed away. The capacity of binding an analyte, such as cells or secondary antibodies to measure OD values by ELISA, depends on the number of immobilized, accessible and functional antibody binding sites which in turn depends on the initial surface load. A schematic representation of surfaces having different loads of physically deposited antibodies prior to irradiation and washing and their


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resultant binding capacity of analytes (cells and proteins) in assays is shown in figure 3. It should be noted that the layer thicknesses of the deposited layers are not completely homogeneous when complex wire geometries are employed. For example in the capillary gaps of the triple helix wire higher film thicknesses are observed compared to the areas in between (figure S2).

Figure 3. Schematic representation (not drawn to scale) showing the influence of different amounts of physically deposited antibodies prior to photoimmobilization on surface attached polymer layers on the assay performance and binding capacity. Shown are four surfaces with increasing load (from top to bottom) of deposited antibodies on non-irradiated PDMAA-BP layers. After UV irradiation (hν) non immobilized antibodies will be washed away.

The coated wires were exposed to phosphate buffer (PBS, 2 ml) that had been spiked with EpCAM-positive SK-BR-3 tumor cells (35,000 ml-1) and EpCAM-negative K562-A2 leukemia cells (150,000 ml-1). After a thorough washing of the wires all intact cells that bound to the capture surface were immunocytochemically stained, counted and categorized into EpCAM positive (carcinoma cells) or negative (leukemia). The high cell loads were chosen due to statistical considerations and are not representative for cancer patients. Furthermore, examinations of increasing the in-vitro capture efficiencies were not considered in the experimental set-up. The number of specifically captured target cells increased relative to the amount of antibodies available on the wire surface, whereas the number of non-specifically attached leukocytes did not change significantly and remained low (figure 2a). Antibody loads in the initial coating solution above 170 ng mm-1 did not further enhance the number of specifically bound cells in agreement with the results on antibody immobilization. Enrichment can be expressed by the percentage of target cells in reference to all captured cells44 or simply by the number of nonspecifically adsorbed nucleated cells per ml of applied blood15. Flat wires loaded with 85 ng mm-2 or more antiEpCAM antibody resulted in an enrichment of specifically captured target cells of more than 1000 with a purity of 99%

(< 10 nonspecific cells ml-1) and SK-BR-3 enrichment factors ranging between 600 and 1000 depending on the experimental conditions (figure 2b and 2c; table S1). This compares favorably with results obtained when microfluidic devices were used for cell isolation by affinity enrichment or negative depletion, which reported purities of about 60-70 %19, 20, 44 and > 300 nonspecific cells ml-1.15, 19 It should be noted that size and shape of the wire have a direct effect on the capture performance under flow conditions due to different surface areas. The blood flow velocity in a vein can be affected by varying the surface area and the space filling by the wire, which in turn determines contact time45, 46 and shear forces18, 47 of passing cells. When the diameter or the cross-sectional area of the wire increases, the flow velocity and shear rates, to which the cells are exposed, are increased. In comparison to a round wire of the same size (each Ø 0.5 mm), the twisted flat wire (figure S1) results in a decrease of the cross-section of 76 % accompanied by a surface area decrease of 25 %. For a triple helix wire structure with the same dimensions (figure S2) the effective surface area is 72 mm2. Thus the cross section is decreased by 34%, while the surface area is increased by 14% when compared to a round wire shape having the same diameter. This is important to bear in mind when absolute values of attached cells are discussed in reference to different wire shapes under flow conditions. Cell capture in whole blood. The results of the in vitro model experiments strongly depend on the details of sample manipulation and the composition of the model solutions. Capture performances can vary due to the extent of antigen expression at the surface of the cultured cells48 or due to variations in matrix constitution of the whole blood, diluted blood and PBS44. To assess the effects of whole blood matrices on the capture performance, we exposed triple helix wires (figure S2) to the blood of healthy donors spiked with EpCAM positive SK-BR-3 tumor cells (35,000 ml-1). The experimental results for antibody surface loads ranging between 0.9 and 444 ng mm-2 are depicted in figure 4a. The selectivity, or more precisely the purity of the isolated cells, did not change significantly when the experiments performed in whole blood were compared to cell isolation in PBS (figure 2a). Purities of 91% for wires with high antibody loads were measured. This result must be evaluated taking into account that the spiked tumor cells contribute only 0.35 % of the total count of nucleated cells, where the background of leucocytes is estimated at ≈ 107 ml-1 in non-diluted blood.3 Erythrocytes, which are even more abundant are not considered in this discussion. Therefore, in samples tested with antibody densities of 56 ng mm-2 and 444 ng mm-2, a selectivity factor of ≈ 3000 was observed (table S2). Wires prepared with antibody concentrations of 7 ng mm2 and 0.9 ng mm-2 showed a lower number of specifically bound cells. Since the number of non-specifically adhering cells was more or less constant, this decreased the tumor cell enrichment factors to ≈ 200 and ≈ 114 respectively (table S2). It should be noted that the number of specifically captured cells from whole blood is smaller by a factor of roughly 5 compared to that when the cells were isolated from PBS solution containing the same target cell concentration. This could be explained by matrix effects,49, 50 which could probably lead to a slightly higher number of nonspecifically adsorbed EpCAM negative background cells and/or a slightly larger cell loss during washing steps. The number of cells bound to the surface in whole blood was not accessible in situ during incu-



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bation due to the opacity of the whole blood samples and could only be determined after removal of the wire from the blood, so that these two cases could be not distinguished. Additionally, it should be noted that the main focus in the experiments described here is placed on the selectivity of the surfaces and no attempt has been made to optimize the flow conditions and accordingly the number of captured cells. Such experiments will be reported in a follow-up communication.

Figure 4. Influence of the antibody load prior to photo immobilization on the in vitro capture performance of triple helix wires in whole blood samples spiked with SK-BR-3 cells. a) Mean cell counts per wire and OD values (n=5 wires) representative for the amount of applied antibodies that increase by a factor of eight. b) Percentage purity of SK-BR-3 on wires relative to mean values of tumor cells and leucocytes. c) Enrichment factor of SK-BR-3 to leucocytes on wires relative to the value in spiked blood.

Cytotoxicity of functionalized wires. For in vivo applications the biocompatibility of the capture device is very important. As a first attempt in this direction, we performed in vitro tests according to the ISO guidelines recommended for class IIa medical devices. To this end, we first examined the shape and proliferation of cultured fibroblast cells (NHDF) in direct contact with the device during incubation for 24 hours. As shown in figure 5a, direct contact of the coated wire surface to cultured cells did not change the shape and proliferation of cultured cells, whereas the presence of copper wires added as controls resulted in strong changes in morphology and growth. Secondly, we extracted the wires carefully with cell culture medium and tested the eluates for effects on the viability of cultured NHDF in solution. The eluates (3 cm2/ml) of three functionalized wires, as well as control eluates of Teflon (PTFE) and copper wires were compared. The mitochondrial activity of cells was measured by a quantifiable substrate metabolism (TTC/TPF Assay) after 48 hours of incubation in eluates and control media. Extracts of PTFE and cell collector wires did not change the cell metabolism rates significantly (figure 5b) and can be assumed to be non-toxic since the related mean cell activity was clearly above 80%. In

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contrast to this, copper extracts, as well as buffer media containing 1% (w/v) sodium azide or 10% (v/v) ethanol resulted in a strong decrease of cell activity of more than 50%, measured by the mitochondrial substrate metabolism [reduction of triphenyl tetrazolium chloride (TTC) to 1,3,5triphenylformazan (TPF)].

Storage stability. To investigate the functional stability of EpCAM coated wires, they were stored for prolonged periods of time at 4°C in a sterile PBS buffer containing 0.01 % sodium azide. In figure 6 the result of the cell counts per wire is shown as a function of storage time. Three wires of three different batches were examined each 3, 14, and 19 weeks after production. As a result, the capture performance, as well as the relative antibody concentrations on the wire surface did not change significantly. Furthermore, a good reproducibility of the coating and antibody immobilization process was observed. Samples produced in different batches showed very small variations in the antibody surface concentrations as well as the capture efficiency and specificity.

Figure. 5. Evaluation and comparison of the cell morphology and cell viability in the presence of cell collectors and reference materials. a) Microscopic images of cultured Fibroblasts (NHDF) in the presence of copper and cell collector wires or without any wire material (Control). Pictures are taken 24 h after incubation. b) Effect of material derived extracts (n = 3) on the viability of Fibroblasts after 48 hr. incubation. Teflon (PTFE), Copper (Cu) and cell collectors (222 ng mm-2 anti-EpCAM) were extracted in culture medium with a surface to volume ratio of 3 cm2 ml-1.

Nucleic acids analysis of wire bound cells. In a further series of experiments it was studied how the isolated cells attached to the surface of the wire-based cell collector could be investigated for gene expression patterns or point mutations. After cell capture, washing, cell fixation, immunocytochemical staining, processing of the microscopic images and counting of the attached cells was performed, genetic analyses were carried out. For quantitative nucleic acid analyses two methods were used: Firstly, the wires were directly used for quantitative real-time PCR. To this end, 20 mm long pieces of the functionalized wires were placed into PCR capillaries filled with reaction mixtures. The mass and size of the wires did not affect the thermal cycling and fluorescent readout of the polycarbonate capillaries and real time experiments were performed.


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Figure 6. Impact of storage in a sterile buffer at 4°C on the in vitro capture performance of functionalized triple helix wires in spiked whole blood samples. Mean cell capture and antibody representative OD values of 3 pooled batches (n = 9; 3 of each batch) as a function of storage time. EpCAM concentration (prior UV-crosslinking) of all coatings was 56 ng mm-2.

Figure 7. Nucleic acids analyses and downstream processing of cells captured by wire-based cell collectors. a) Quantitative realtime nucleic acid analyses (e.g. RT-PCR, q-PCR) were performed in two ways: Wires with a PCR compatible coating and immobilized cells were added directly to PCR capillaries as whole cell sample delivery. The second sample preparation was the cell extraction on the wire. Examples for downstream processes: b) Melting curve analysis (e.g. HRM) were performed directly after PCR. c) Electrophoresis and purification of PCR products. Sequencing of amplified genomic hotspots (carcinogenic mutations) could be performed subsequently.

In the second series of experiments, cell extraction was conducted directly on the wire using volumes of only a few mi-

croliters. In this case a droplet of tissue lysis buffer was added to the functionalized surface and the system was incubated for 10 min while the wire was rotated and tilted in the same way as described above for the antibody deposition. After dilution of the cell extracts by addition of water, the liquid extracts were PCR-ready. Both methods resulted in clear, quantifiable real-time PCR results, without inhibition and were comparable to the positive control (figure 7a). Downstream from PCR, amplification products can be specified by melting curve analysis (figure 7b) and sequencing with or without purification (figure 7c). Both can indicate point mutations and single nucleotide polymorphisms (SNPs). The results of the melting curve analysis as well as the PCR product characterization/purification by gel electrophoresis revealed no differences between the two procedures and the positive control. This indicates that direct dipstick PCR and liquid DNA extraction on the coated and functionalized wire could be employed for genetic analyses of captured and isolated cells. Conclusion For the isolation of rare cells from blood, the cells of interest need to be immobilized on the surfaces through specific interactions, and all other cells must show an extremely weak adhesion. This was achieved by generating a multifunctional coating on the surfaces of the wire used for isolation. A multilayer assembly was generated by photochemically induced C,H-insertion reactions leading to crosslinking and immobilization of the individual layers. The generated assembly is covalently bound to the surface of the wire and consists of a barrier and a protein repellent layer. The barrier layer is effective even under conditions involving thermal stress and high ionic strength, such as those prevalent under PCR conditions. The topmost layer of the multilayer assembly also consists of a photogenerated protein repellent hydrogel, which strongly prevents non-specific cell adhesion.39 To allow specific binding of the desired cells by receptor molecules, a small drop of protein solution was carefully distributed over the wire surface and air dried. During brief UV irradiation the photopolymer layer became crosslinked and simultaneously an antibody monolayer was covalently linked to this surface. This strategy allows for a very simple and effective immobilization of the antibodies with a controlled surface concentration. Additionally, solution application and short light exposure can be performed briefly before use without the requirement of specialized equipment, so the functional devices may be produced upon demand and no long term storage of devices with immobilized antibodies is necessary. After completion of the immobilization process no blocking process is required, and the wires can be directly used after a short wash. The generated surfaces can selectively enrich and separate spiked tumor cells from whole blood samples in vitro. Enrichment factors of up to about 3000 (disregarding nonnucleated cells) and purities of greater than 90% were observed in whole blood when the ratio of the spiked cancer cells to the number of leucocytes present in the sample is considered, which demonstrates the high specificity of the devices. Interestingly, high surface concentrations of the monoclonal antibodies on the protein repellent hydrogel layer did not affect capture specificity. This implies that the general protein and cell repellent characteristics of the hydrogel coating were retained despite an increasing protein density on the surface. This probably reflects a low tendency of the monoclonal anti-



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bodies used to form protein complexes with proteins present in the blood which in turn would induce non-specific cell adhesion. Furthermore, initial cytotoxicity experiments and the long-time stability and reproducibility between different batches of wires are promising results for a short-term, in vivo application of these devices. Cells captured by the wires were not only stained and counted but also genetically analyzed to obtain more differentiated information on the nature of the cells. Here a key advantage of the developed strategy is that the wire coating works like a “cloak of invisibility” for the metal substrate and allows the direct use of captured cells in a PCR reaction. Two different sample preparation methods were successfully tested and reveal a convenient and simple workflow for PCR-based amplifications, analyses and additional downstream processes such as hotspot sequencing.

ASSOCIATED CONTENT Supporting Information 3D Computer modeled views of a twisted flat wire and a triple helix wire; Layer thicknesses in dependence of deposited amounts of antibodies. Tables with mean cell counts and standard deviations, calculated capture ratios and enrichment factors of captured cells for the capture results in PBS and whole blood.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Present Addresses † Metanomics Health GmbH, Tegeler Weg 33, 10589 Berlin.

Author Contributions All authors have given approval to the final version of the manuscript.

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