Tumor Cell Detection by Mass Spectrometry Using Signal Ion

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Letter

Tumor Cell Detection by Mass Spectrometry using Signal Ion Emission Reactive Release Amplification (SIERRA) Zane Richard Baird, Valentina Pirro, Stephen Ayrton, Adam Hollerbach, Cathleen E Hanau, Karen Marfurt, Mary Foltz, R. Graham Cooks, and Michael Pugia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02043 • Publication Date (Web): 28 Jun 2016 Downloaded from http://pubs.acs.org on June 30, 2016

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Analytical Chemistry

Tumor Cell Detection by Mass Spectrometry using Signal Ion Emission Reactive Release Amplification (SIERRA) Zane Baird†,§, Valentina Pirro§, Stephen Ayrton§, Adam Hollerbach§, Cathleen Hanau†, Karen Marfurt†, Mary Foltz †, R. Graham Cooks§,* and Michael Pugia†,* † §

Siemens Healthcare Diagnostics, 3400 Middlebury Street, Elkhart, IN, USA. Chemistry Department, Purdue University, 560 Oval Drive, West Lafayette, IN, USA.

ABSTRACT: A method is presented for the detection of circulating tumor cells (CTC) using mass spectrometry (MS), through reporter-ion amplification. Particles functionalized with short-chain peptides are bound to cells through antibody-antigen interactions. Selective release and MS detection of peptides is shown to detect as few as 690 cells isolated from a 10-mL blood sample. Here we present proof-of-concept results that pave the way for further investigations.

INTRODUCTION Circulating tumor cells (CTCs) is a collective term used to describe cancer cells of solid tumors or metastases found in transit in the peripheral blood of cancer patients.1,2 Characterization of CTCs is receiving ever-increasing scientific interest because they can provide new insights into the cancer metastatic process,3 novel and more personalized prognostic information and therapeutic targets for cancer treatment.1,4,5 They also have the potential to assist in unravelling drug pharmacodynamics, sensitivity or inherent/acquired drug resistance.6 They could hasten cancer detection, as dissemination of tumor cells from tissue is usually undetected with conventional histopathology and imaging technologies.3,5,7-10 Detection and characterization of CTCs still presents analytical challenges. In fact, CTCs are extremely rare and need to be isolated and detected from a complex biological matrix containing 109 hematological cells per milliliter.5,11 Several methodologies have been proposed.2,4,6 Some simply count the isolated CTCs, as their number is believed to correlate with primary tumor vascularity and invasiveness;2,11 while others allow for CTC enumeration and subsequent genetic sequencing1,12 and/or staining and interrogation of cells via immunochemistry or fluorescence microscopy.6 Techniques differ in analysis time, blood sample volume analyzed, degree of automation, efficiency in CTC isolation, degree of cell viability, and processing procedures,4 even though an enrichment step is typically used to increase analytical sensitivity and detection limits.5 To date, CellSearchTM is the only platform that has received FDA approval for CTC enumeration in whole blood.4,6 It employs antibodies against epithelial cell adhesion molecule (EpCAM) - typically overexpressed in CTCs – coated onto magnetic nanoparticles, combined with cell fixation and staining, to achieve selective and sensitive immunorecognition for visual cell identification and counting.13 Since EpCAM is not universally expressed in CTCs,1,3 other approaches have been proposed, which rely on morphological, physical, or dielectrophoretic properties of CTCs rather than on differential expression of surface markers.4,13 Common

approaches are based on microfiltration1-3 or adhesiveness on surfaces,4,7,13 density gradient centrifugation or viscosity,3 and conventional flow cytometry.6 A vast majority of the assays for CTC enumeration utilize magnetic beads conjugated with antibodies to label CTCs and assure selective and efficient capture away from non-magnetic hematological cells by simply applying a magnetic field.3,7,11 Surface engineering and functionalization with capture (e.g. antibodies, streptavidinbiotin, neutravidin-biotin) or labeling agents, variable sizes, lack of porosity, stability, and ease of preparation are examples of the versatility of magnetic particles, which is why they are extensively researched for diagnostic applications.14,15 Regardless of the manner in which CTC isolation/purification is performed, analysis of the resulting isolate most commonly relies on fluorescence microscopy. This is problematic for a number of reasons. In order to realize specific identification of cells, a cocktail of antibodies and fluorophores is usually employed. However, spectral overlap between different fluorophores limits multiplexing to 6-10 parameters. Additionally, microscopy is time-consuming; each fluorophore necessitates its own filter-set, and automated data acquisition relies on a microscopically flat surface. More recently, mass spectrometry (MS) has been investigated as a simpler and faster approach for CTC enumeration and characterization, owing to its potential for single-cell analysis.16 For example, Chiu et al. proposed to detect gold cluster ions by pulsed laser desorption ionization (LDI) MS where signal intensity is inversely proportional to the number of cells selectively-bound to the surface.5 Huang et al. detected DNA and RNA methylation in CTCs by liquid-chromatography coupled with tandem MS, after lysing the isolated cells and digesting the nucleic acids.12 Wu et al. detected endogenous metabolites from isolated cells by LDI-MS using magnetic capture particles as a matrix to enhance desorption and ionization.15 Here we propose a particle-based immuno-affinity strategy for CTC detection by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS. Enumeration of CTCs is done by immuno-recognition with antibodies conjugated to particle clusters carrying peptide mass labels that are

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selectively released after CTC isolation and quantified by MALDI-TOF-MS. The signal ion emission reactive release amplification (SIERRA) method is proposed for the first time as a strategy to cluster nanoparticles carrying peptide MS labels to magnetic particles conjugated to antibodies, thereby amplifying the number of MS labels carried per antibody in order to reach lower detection limits in the count of CTCs (Figure 1). Inspired by Bendall et al. who developed the mass cytometry technique which utilizes elemental isotope labels,17,18 we propose the use of customizable short-chain peptides as MS labels. For the proposed MS analysis methodology, a cocktail of antibodies conjugated with SIERRA particles carrying different peptide sequences is envisioned as an efficient strategy for multiplexing. The choice of using shortchain mass labels as the tag system for CTC detection and enumeration is motivated by the fact that they can be easily synthesized to provide a portfolio of molecules that can be selectively coupled with epitope-specific antibodies. Peptide analogs can also be synthesized as internal standards, avoiding expensive isotope labeling procedures.

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velop the entire protocol for CTC immuno-recognition by MS analysis in whole blood. MATERIALS AND METHODS Instrumentation

Mass spectra were recorded using a Bruker Daltonic Autoflex Speed™ MALDI-TOF with a polished steel MTP 384 target plate (Bruker Daltonics, Billerica, MA). CTCs were isolated from blood by filtration through Whatman™ Nucleopore™ membranes (8.0 µm pore size, 25 mm diameter) on a Hamilton STARlet™ robotic system (Hamilton Company, Reno, NV). Prior to their use in the filtration system, membranes were welded to Siemens plastic slides with an integrated microstructure.19 Fluorescent microscopy images were taken on a Leica DM5000 (Leica Microsystems GmbH, Wetzlar, Germany) fitted with a DFC365 FX black/white camera with NIR mode. A Lumen 200 fluorescent illumination system (Prior Scientific Inc., Rockland, MA) was used with the A4, L5, N3, and Y5 filter sets for 4’,6-diamidino-2-phenylindole (DAPI), fluorescein, Dylight 550 (Dyl550), and Dylight 650 (Dyl650) fluorophores, respectively. Chemicals

Figure 1. Illustration of SIERRA amplification strategy for the labelling and MS detection of CTCs. Cells are selectively labelled with magnetic particles (violet spheres) bound to nanoparticles (orange spheres), previously conjugated to mass labels (green spheres) which are detected via MS following a chemical cleavage of the disulfide bond between the mass label and nanoparticle.

Here we report proof-of-concept results showing the synthesis of SIERRA particles, the conjugation and release chemistry of a peptide from the SIERRA particles, and its quantitation by MALDI-TOF-MS. The goal of the work in this Letter is to explore the amplification potential and not to present a fully developed methodology nor validate the method for clinical use. For these first experiments, CTCs already conjugated with antibodies carrying SIERRA particles were spiked in whole blood and isolated via filtration, as previously reported,19 and all observations were validated via fluorescence microscopy. Research is ongoing to improve the analytical performance, multiplex and automate the analysis, as well as de-

Peptides consisting of 9 amino acids (IC9-1 and GC9-1), an N-terminus biotin conjugate of IC9-1 (IC9-2), and an Nterminus fluorescein conjugate of IC9-1 (F-IC9) were purchased from Celtek Bioscience (Franklin, TN). The sequence and molecular weight of these peptides are shown in Table 1. Mesoporous propylamine-functionalized silica nanoparticles (NP) with a nominal diameter of 200 nm and 4 nm pores were purchased from Sigma-Aldrich (St. Louis, MO). Sera-mag streptavidin coated magnetic beads (0.756 µm diameter, 10 mg/mL, 3500-4500 pmol/mg binding capacity), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), DAPI, Dyl550 Nhydroxysuccinimide (NHS) ester, Dyl650 NHS ester, and Pierce™ Premium Grade Sulfo-NHS-LC-Biotin were purchased from Thermo Scientific (Fremont, CA). Acetonitrile (ACN) with 0.1% trifluoro acetic acid (TFA), water with 0.1% TFA, α-Cyano-4-hydroxycinnamic acid (HCCA), phosphate buffer saline (PBS), Tween® 20, tris(2carboxyethyl)phosphine hydrochloride (TCEP·HCl), and 1,4Diazabicyclo[2.2.2]octane (DABCO) were acquired from Sigma-Aldrich (St. Louis, MO). Dimethyl sulfoxide (DMSO) was acquired from Fisher Scientific (Hampton, NH). Casein was purchased from Candor Bioscience (Wangen, Germany). Unless otherwise stated, all materials were used as received without further purification. Antibody preparation

Mouse monoclonal antibodies targeting human cytokeratins 8 and 18 (CK8/18) and leukocyte common antigen (CD45) were purchased from ATCC® (Manasas, VA). A CK8/18-biotin-Dyl550 conjugate was prepared by amine reactive crosslinking with N-hydroxysuccimide esters from sulfoNHS-LC-Biotin and Dyl550. CD45-Dyl650 coupling was accomplished in a similar manner, but did not include biotin functionalization. All antibody conjugates were prepared


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Table 1. Short-chain peptides. Mass-label acronym

Amino acids sequence

Molecular weight (g/mol)

IC9-1

Ile-Gly-Met-Thr-Ser-Arg-Tyr-Phe-Cys

1077.3

F-IC9

Fluorescein-Ile-Gly-Met-Thr-Ser-Arg-Tyr-Phe-Cys

1435.6

IC9-2

Biotin-Ile-Gly-Met-Thr-Ser-Arg-Tyr-Phe-Cys

1303.6

GC9-1

Gly-Gly-Met-Thr-Ser-Arg-Tyr-Phe-Cys

1021.2

following standard coupling procedures at Siemens Healthcare Diagnostics (Elkhart, IN). SIERRA particle preparation

Propylamine-functionalized silica nanoparticles (SiNPs) were first treated with SPDP in DMSO to react with freeamines present on the surface of the SiNPs in order to form a site for peptide attachment. IC9-1 and IC9-2 were then linked to the SPDP-treated SiNPs by thiol-disulfide exchange with the sulfhydryl group present on the C-terminal cysteine of IC9-1 and IC9-2. The peptide-labeled SiNPs were then washed several times and finally suspended in PBS. The final washing solution was analyzed via MALDI-TOF-MS to confirm the absence of free peptides in the washing solution. An aliquot of streptavidin coated magnetic beads (ca. 2.7x109 beads) was added to the SiNP suspension and the mixture was placed on a rotary mixer (75 RPM) for 30 minutes at room temperature. Attachment of SiNPs to the magnetic beads to form SIERRA particles was achieved through a streptavidin-biotin interaction as a result of the biotinylated IC9-2 present on the SiNPs. The microcentrifuge tube containing the reaction mixture was placed in a Dynal® MPC-S magnet (Thermo Scientific, Fremont, CA) thus collecting the magnetic particles on the side of the tube. The PBS solution was then removed and the particles washed once more with PBS and finally re-suspended in 1 mL PBS for a final concentration of 2.7x109 magnetic beads/mL A working suspension of about 2.7x107 beads/mL was prepared by diluting 10 µL of the stock to 1 mL in PBS. A separate batch of SIERRA particles was prepared in the same manner, substituting the IC9-1 with a fluorescein-labeled IC9 (F-IC9). These particles (F-SIERRA) were used to validate the attachment of SIERRA particles to cancer cells as determined via fluorescence microscopy. See the Supporting Information (SI) for details on the preparation of SIERRA particles and calculation of final concentration. Cell staining and labeling A solution of approximately 105 SKBR3 cells (ATCC® HB-30™, Manassas, VA) was first stained with the previously prepared CK8/18-biotin-Dyl550 conjugate and DAPI (see SI for more details). A 2-µL aliquot of the stained cells suspended in PBS (1.2 mL) was spotted onto a glass slide and examined via fluorescent microscopy to determine a final cell count of 1.2x104 cells/mL. One hundred µL of the 2.7x107 beads/mL SIERRA particle suspension was mixed with 300 µL of the previously stained cell suspension (ca. 3700 cells) and the mixture was incubated at room temperature for 2 hours on a roller mixer (75 RPM). In a separate tube, the same procedure was carried out with the F-SIERRA particles. Following the 2 hours of incubation, each of these suspensions was centrifuged, washed four times and finally re-suspended in PBS (1.2 mL) for a final concentration of approximately 3700 cells/mL.

Blood filtration and cell isolation Approximately 6 - 9 mL of whole blood was collected from healthy donors into tubes containing potassium ethylenediamine tetraacetic acid (K3EDTA) and 450 µL Transfix® (Vacutest Kima, Padova, Italy). Samples were stored at room temperature for up to 5 days. Twenty-four hours before filtration, the blood samples were spiked with the previously labeled SKBR3 cells in order to have blood tubes containing approximately 1000 cancer cells. Prior to filtration, blood samples were diluted to 20 mL with PBS. The dilute blood samples were filtered in 1 mL increments through Whatman™ Nucleopore™ membranes on a Hamilton STARlet™ robotic system, applying a negative pressure of 10 mbar. After filtration, the cells isolated on the membrane were incubated with 4% formaldehyde in PBS, and then washed with PBS. The isolated cells were then made permeable with 0.2% Triton-X in PBS, and incubated with CD45-Dyl650 (10 µg/mL in casein), and finally with DAPI (1 µg/mL in PBS) according to previously published procedures.19 After filtration, DABCO cover medium was applied to membranes used to isolate cells labeled with F-SIERRA, along with glass coverslips. No cover medium or coverslip was applied to cells labeled with SIERRA particles absent the fluorescein tag. Each membrane was inspected by fluorescence microscopy to determine a final cell-count. Cancer cells were characterized as nucleated (DAPI-positive), CK-positive, and CD45-negative.19 White blood cells were defined as nucleated, CK-negative, and CD45-positive. All membranes were then stored at 2 - 6 °C covered with aluminum foil until MALDI-TOF-MS analysis. MALDI-TOF-MS analysis Membranes which had filtered blood containing SIERRA labeled cells (absent fluorescein label) were removed from the plastic slide support and carefully pushed to the bottom of a 600-µL polypropylene tube. Selective release of the IC9-1 mass label was accomplished through cleavage of the disulfide bond connecting IC9-1 to SiNPs by the addition of 100 µL of 35-µM TCEP·HCl in ACN - water 1:1 (v/v) and vortexing the mixture for 3 minutes. Next, 80 µL of ACN with 0.1% TFA and 20 µL of 2 mg/L GC9-1 (used as internal standard) in ACN with 0.1% TFA was added to the release solution (i.e. final concentration of 200 µg/L GC9-1). All samples were run by first spotting 1.5 µL of the sample solution onto the target plate followed by 1.5 µL of 6.5 mg/mL HCCA (used as a matrix for MALDI) in ACN - water 1:1 (v/v) with 0.1% TFA and letting the spots dry in air at room temperature. MALDI-TOFMS analysis was performed using reflector mode, positive ionization, 250 laser shots per sample, and acquiring full-scan mass spectra over the m/z 700-3500 range. Calibration standards were prepared which contained 0, 25, 50, 100, 150, and 300 µg/L IC9-1 with 200 µg/L GC9-1 in ACN - water 1:1



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(v/v) with 0.1% TFA. Fifty µg/mL is estimated as the limit of detection.

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the fluorescent images it would appear that this may be an underestimate of the number of particles that are bound by a single cell.

RESULTS AND CONCLUSIONS Validation by fluorescence A visual confirmation of SIERRA particle attachment to SKBR3 cells was made by examining the membrane used to filter SKBR3 cells that had been incubated with F-SIERRA particles functionalized with the F-IC9 under a fluorescent microscope (Figure 2). The spots of green fluorescence are indicative of F-SIERRA particles while the blue fluorescence is the result of DAPI staining and is used to visualize the nucleus of the cell in the overlaid image. From this image it is apparent that a large number of SIERRA particles (>100) may be captured by a single cell and that SIERRA particles will retain SiNPs functionalization by a peptide under conditions used to isolate cells from whole blood. Residue unbound particles were not detected on the membrane. Figure 3. MALDI-TOF-MS calibration results for IC9-1 plotted as the ratio of maximum intensity of IC9-1:GC9-1 vs. IC9-1 concentration (black circles) along with the line indicating a 1st-order polynomial fit. The measured response of IC9-1 after release from 690 SIERRA labeled SKBR3 cells is also shown (red triangle) with the resulting calculated concentration indicated by the vertical dotted line.

Figure 2. An illustration of a fluorescently labeled SIERRA particle bound to a cell and a fluorescent image overlay of a single SKBR3 cell that has been stained with DAPI (blue) and is decorated by multiple fluorescein-labelled SIERRA particles (green).

MS analysis The ratio of peak intensities for IC9-1 (m/z 1077) to GC91 (m/z 1021) was recorded for each calibration standard, plotted against the known IC9-1 concentration and a least-squares 1st order polynomial fit was calculated from this data (Figure 3). From the fit of the calibration data and the MS response for IC9-1 from the membrane containing SIERRA-tagged cells, it was determined that the concentration of IC9-1 in the releasing solution was about 150 µg/mL, which corresponds to approximately 1.66x1013 mass labels released from this membrane. Prior to MS analysis this membrane was inspected via fluorescence microscopy to determine a count of ~690 cells isolated after filtration of whole blood spiked with 1000 cells (i.e. 76% recovery which is consistent with previous observations19). The result is a final amplification factor of 2.4x1010 mass labels per cell. This amplification is in agreement with the theoretical amplification of 2.3x1010. A detailed discussion of how this was calculated is given in the SI. This theoretical value assumes only 100 SIERRA particles per cell, and from

Considering a limit of detection for IC9-1 of 50 µg/mL, the cellular detection limit would be approximately 230 cells in 10 mL of blood; however, this represents only a theoretical extrapolation and no experimental results have yet verified such a number. These proof-of-concept results show the relatively simple preparation and use of SIERRA particles as vehicles to label CTCs with numerous small molecules amenable to analysis by MS. Further experiments are necessary to determine the amplification provided by each level of SIERRA particles (i.e. number of peptides per SiNP and SiNPs per magnetic bead) and its reproducibility. Additionally, the cell size and the level of cytokeratin expression by SKBR3 cells are likely to vary from cell-to-cell, which greatly impacts the final amplification.20 All of these factors will contribute to the final ability to accurately enumerate isolated CTCs by MS analysis with the SIERRA method. Conceptually, we envision that many of the isolation strategies proposed in other research studies - based on the use of a magnet to concentrate and recover magnetic beads binding CTCs7-10,12 - could be used for the SIERRA clusters as well, with the added benefit of quantitation and high sensitivity provided by MS. In addition, the high amplification factor can significantly improve detection limits. Such an immunoaffinity amplification system could be useful for applications other than CTC detection as well. For example, immunoaffinity reactions have proved feasible for identification of bacteria in biofluids,21 assays which traditionally require bacterial culturing as an amplification strategy to bring the analytes into a detectable range. By eliminating this step, time of diagnosis may be greatly reduced for time-sensitive medical decisions such as for the treatment of sepsis.22 The SIERRA strategy also provides a unique platform for multiplexed immunoassay development. This is envisioned by preparing different variations of SIERRA particles, each functionalized with an antibody targeting a specific analyte. Re-


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spectively, these SIERRA batches would in turn be associated with SiNPs functionalized with labels that give ions possessing a unique m/z for identification in a mass spectrum. In contrast to fluorescent labeling, which requires a different filter set for each label, the SIERRA approach would allow for simultaneous detection of all targets using single-stage MS detection; the number of which is limited by the mass range and resolution of the MS. However, limitations on the multiplexing strategy are mainly dictated by the magnetic particle size that restricts the loading factor around a cell. Reducing the particle size would exponentially increase the packing number and theoretically allow higher-fold multiplexing; all aspects that still need to be addressed to fully develop the analytical protocol.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional details on the SIERRA particle amplification method (PDF)

AUTHOR INFORMATION Corresponding Authors * Email: [email protected]; [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT Financial support for this work was provided through a grant from Siemens Healthcare Diagnostics (Elkhart, IN USA).

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Tumor Cell Detection by Mass Spectrometry Using Signal Ion

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