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Imaging, Diagnosis, Prognosis |
Authors' Affiliations: 1 Department of Cancer Biology; 2 Mass Spectrometry Research Center and the Department of Biochemistry; 3 Department of Pathology; 4 Division of Allergy, Pulmonary, and Critical Care Medicine, Veterans Affairs Medical Center; 5 Division of Hematology/Oncology, Department of Medicine; and 6 Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennesse
Requests for reprints: David Carbone, Division of Hematology/Oncology, Department of Medicine, 685 PRB, Vanderbilt University, Nashville, TN 37232-6307. Phone: 615-936-3524; Fax: 615-936-3322; E-mail: d.carbone{at}vanderbilt.edu.
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Abstract |
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Experimental Design: Lung cancer cell lines were cytocentrifuged onto metal-coated, transparent glass slides and used for optimization of fixation, staining, and RBC lysis protocols. Fine-needle aspirates from human tumors and mouse model tumors were used to provide fresh tissue samples for determining the feasibility of this method.
Results: The MALDI-TOF MS compatible fixation and staining techniques provided high-resolution cellular morphology, which allowed identification and selective spotting of tumor cells. The RBC lysis step efficiently removed contaminating RBC yielding spectra nearly free from hemoglobin peaks. Protein profiles of fine-needle aspirates were found highly reproducible and similar to the profiles of the tissue from which they were obtained. Using this method, we were able to differentiate between xenograft tumors derived from two different human cell lines, A549 and H460.
Conclusion: This procedure results in the production of high-quality, cancer cell–specific protein profiles. This highly reproducible technique could be applied to many other types of mixed clinical samples and has the potential to be very useful in the clinical diagnosis, classification, and, potentially, the individualized treatment of cancer patients.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technology that can be used to generate protein signatures from tissue sections that can contain several hundred protein signals (2). In direct tissue profiling experiments, thin tissue sections are cut from fresh frozen biopsies, thaw-mounted on a flat, metal MALDI target plate, and spotted with matrix (typically sinapinic acid). To target the cancerous tissue in the sample, an adjacent tissue section that has been mounted on a glass slide and H&E stained is often used as a rough guide for matrix deposition. With this method, protein profiles have been obtained from tumor tissue sections and successfully analyzed with a class prediction model developed for handling the large data sets produced by MALDI (3–5).
Many clinical samples are fine-needle aspirates that result in a liquid suspension of cells highly contaminated with blood, normal cells, and noncellular debris that are smeared or spun down on slides for analysis without adjacent sections to analyze. To accomplish light microscopic and mass spectrometric analysis of the cells of interest in such a clinical sample, there are two essential requirements that must be met: first, it is necessary for the sample plate to be optically transparent and conductive (for use in the MALDI apparatus); second, a stain must be found that is simultaneously revealing of cellular architecture and compatible with MALDI-TOF MS.
Recently, indium-tin oxide–coated transparent glass slides have been used to mount tissues for MALDI analysis (6, 7). These slides are coated with a 160- to 240-Å-thick layer of indium-tin oxide, which provides the surface necessary for maintaining stable accelerating potential within the source of the time-of-flight mass spectrometer. These conductive glass slides have been found ideal to mount tissue sections with the benefit that the sections can be viewed by light microscopy before MS analysis. Unfortunately, the most common staining methods used in histology, DiffQuik and H&E, have been found to be incompatible with MALDI MS (8, 9). To improve visualization of features, several staining protocols have been successfully used, which include methylene blue and cresyl violet (7). These provide excellent histology with high resolution of tissue architecture and allow high-quality mass spectra to be obtained.
MALDI-TOF MS protein profiling combines speed, simplicity, reproducibility, and sensitivity, making it a very powerful technology. The technological innovation introduced by the use of indium-tin oxide–coated glass slides and MALDI-compatible stains increases this versatility. We have sought to take advantage of these features in designing a new diagnostic tool for the direct analysis of low-abundance cancer cells contained within fine-needle aspirates. We show here that by combining a very simple existing technology, the cytocentrifuge, which is present in every clinical setting, with the simplicity and power of MALDI-TOF MS, a cutting edge diagnostic for lung cancer can be created that should provide a wealth of information and may ultimately have a significant impact on treatment decisions, and potentially, patient outcomes.
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Materials and Methods |
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Cytocentrifugation, fixation, and staining. Cells were washed twice with 10-mL PBS, detached from the bottom of the dish by scraping, and counted. Cells were diluted with PBS to 200,000 cells/mL and, using a double cytofunnel (Thermo Electron, Waltham, MA), 200 and 400 µL of the cell suspension were cytocentrifuged (described below) onto indium-tin oxide–coated glass slides (Delta Technologies Ltd., Stillwater, MN) in a Cytospin3 (Thermo Electron) at 800 rpm for 5 minutes. Erythrocyte lysis buffer (Qiagen USA, Valencia, CA) was used to remove RBC from 60,000 lung cancer cells in 200 µL of normal saline alone, or with 2, 5, and 10 µL of whole blood. Initially, cells were fixed in acetone (Sigma, St. Louis, MO), methanol (VWR, West Chester, PA), or 95% ethanol for 5 minutes after cytocentrifugation. It was later determined that fixation in 95% ethanol provided high-quality cell morphology and was used for all later studies. The cells were stained with 0.5% cresyl violet or 0.15% methylene blue for 1 minute, followed by two 1-minute washes in 70% ethanol (7). Slides were air-dried before the addition of matrix. Samples not analyzed immediately were stored at –80°C until the day of analysis when matrix was added. The H&E-stained slides were prepared using standard techniques.
Simulated and clinical fine-needle aspirates. Tumors were generated by injection of 1 x 106 MC26 murine colon carcinoma cells into the flanks of BALB/c mice or by injection of 1 x 106 NCI-H460 cells into the left flanks and 1 x 106 A549 cells into the right flanks of nude mice. After 30 to 35 days, resulting tumors were removed and placed into ice-cold saline. A 23-gauge needle attached to a 5-mL syringe was inserted into the tumor. Aspirate was placed into 0.2 mL of ice-cold saline. For human samples, a 25-gauge needle attached to a 5-mL syringe was inserted into surgically resected tumor tissue or into adjacent normal tissue contained within the resection specimen immediately after removal of the tumor. As above, the aspirate was placed into ice-cold saline. If the volume of saline was >0.2 mL, the sample was centrifuged at 400 x g and the pellet resuspended in 0.2 mL of ice-cold saline. True fine-needle aspirates were obtained from the clinic in 1 to 2 mL of saline on wet ice without identifiers. Samples were spun down and resuspended in 0.2 mL of ice-cold saline for further processing as described. All samples were processed at the time they were received.
Red cell lysis. Cells with whole blood or fine-needle aspirates in 200 µL of normal saline were placed on ice. Five volumes of erythrocyte lysis buffer (Qiagen USA) were added and the samples were mixed by inversion and placed on ice for 15 minutes with occasional mixing every 3 to 4 minutes. Cells were centrifuged at 400 x g for 5 minutes at 4°C and the supernatant removed with a pipette. The samples were then resuspended in 200 µL of normal saline for cytocentrifugation.
MALDI-TOF MS. For the cell lines, 300 nL of matrix, sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid; Fluka, Buchs, Switzerland) at 20 mg/mL diluted in a solution of 50% acetonitrile, 0.1% trifluoroacetic acid (Fisher Scientific, Pittsburgh, PA), were deposited in a randomly chosen spot within the circle of cells laid down on the slide by cytocentrifugation. This was followed by a second 300 nL of matrix placed directly on top of the first spot to increase crystal density. For the simulated fine-needle aspirates, clusters of cancerous cells, 100 cells (or fewer) to several hundred cells, identified by a pathologist were hand-spotted while visualizing the cells under a microscope at x10 or x20 magnification. MALDI MS analyses were done in an Applied Biosystems Voyager DE-STR time of flight mass spectrometer (Applied Biosystems, Framingham, MA) equipped with a 337-nm nitrogen laser operated at 20 Hz. Samples were analyzed in the linear mode geometry in delayed extraction conditions under 25-kV accelerating potential. The delayed extraction variables were optimized for maximum resolution at 
m/z 15,000. Data were recorded in the m/z range of 2,000 to 70,000 by averaging signals from four independent acquisitions of 250 laser shots each (1,000 shots total). The MALDI MS acquisition method was first calibrated using a mixture of standard calibrating proteins: insulin (MW 5,777.6), cytochrome c (MW 12,360.1), apomyoglobin (MW 16,951.6), and trypsinogen (MW 23,982). Individual spectrums were then internally calibrated using previously identified mass signals. These are, for mouse samples: cytochrome c oxidase (MW 5,443.4), histone H4 (MW 11,306), histone H2A.2 (MW 14,004), and ß hemoglobin when detected (15, 617); and for human samples: cytochrome c oxidase (MW 5,356.2), histone H4 (MW 11,306), histone H2A.2 (MW 14,005), and 
and ß hemoglobin when detected (MW 15,126 and 15,867, respectively).
Tissue section analysis. Ten-micron-thick tissue sections from fresh-frozen tumor biopsies were cut at –15°C using a cryostat (CM3050 S, Leica Microsystems AG, Welzlar, Germany) and thaw-mounted on the conductive glass slides. Serial sections were also cut for subsequent H&E staining. The sections were dried at room temperature before being successively rinsed in 70% and 95% ethanol for 30 seconds, respectively. MALDI matrix was hand-deposited on the sections by double spotting two 300-nL drops using an automatic pipette. MALDI MS analyses were done as described above.
Statistical analysis. Whole spectrum analysis was done as previously described (10) with the exception that classification was not carried out here. Briefly, all mass spectra were exported as ASCII text files and imported into ProTS Data version 1.1 (Biodesix, Steamboat Springs, CO) for baseline correction, normalization by total ion current, and aligning in batch mode. The processed text files were then imported into a script written in Matlab (Jeremy Norris, PhD, Vanderbilt University) to be aligned according to a single m/z column. A standard weighted mean averaging algorithm was then applied (11). In this way, m/z values were filtered according to the highest weight that best differentiated the normal versus cancer groups. Further filtering was carried out to exclude values with weighted mean averages <1.0 (ref. 10; similar in respect to 2 
from the mean control value) and to exclude mean intensity differences that fell below 2-fold (ref. 2; experimentally derived cutoff value often applied for tissue profiling; data not published). The filtered values were then used for peak detection and further evaluated by plotting the whole spectra as compared with the difference spectra in Origin 7.0. Intravariant error and graphs depicting variance throughout the spectra were prepared and plotted with tools provided by Biodesix. The hierarchical clustering analysis and subsequent icicle plot were carried out with Statistica 6.0 (StatSoft, Tulsa, OK).
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Results |
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50 nL) were deposited on the cells using pulled glass capillaries. Given the quality of the MS data recovered with so few cells and considering that cancer cell clumps contained in needle aspirates are frequently in this range, we decided that pursuing the development of methods for analysis of cancer cells in fine-needle aspirates was feasible.
Development of cell fixation and staining techniques compatible with MALDI-TOF MS of cytospins. For optimal morphologic identification, fixation and staining is required. Therefore, we tested three standard fixation techniques including 95% ethanol, methanol, and acetone for compatibility with MALDI MS. Forty thousand A549 lung cancer cells were cytocentrifuged onto an indium-tin oxide–coated slide, fixed in organic solvent, air-dried, and profiled by MALDI MS (Fig. 1A
). Ethanol and methanol showed improved MS data quality with respect to a nonfixed control with higher ion yield (on average, by a factor of 2 in the m/z range 4,000-60,000) and slightly better resolution (e.g., see signal cluster at m/z 18,000 in Fig. 1A). Acetone fixation gave somewhat lower yield and resolution spectra with respect to the control. It was determined that 95% ethanol fixation was the simplest procedure that produced high-quality spectra and this was used for subsequent experiments.
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Erythrocyte removal is a critical step for obtaining MALDI-TOF MS spectra from clinical needle aspirates. Contamination of clinical samples with blood and other cell types, such as polymorphonuclear cells, macrophages, and dendritic cells, is common and a potential problem for MALDI MS protein profiling. The presence of large amounts of hemoglobin from RBC suppresses any other protein signatures in the sample. To reduce the hemoglobin content in fine-needle aspirate samples, an erythrocyte lysis step was added to the protocol before cytocentrifugation. Mass spectra obtained from NCI-H1299 cells treated with erythrocyte lysis buffer showed that cells exposed to erythrocyte lysis buffer generated the identical spectra as untreated cells (data not shown). To test the efficiency of erythrocyte lysis buffer in removing varying amounts of contaminating blood, 6 x 104 NCI-H1299 cells in 0.2 mL of saline were mixed with 2, 5, or 10 µL of whole blood. The effectiveness of erythrocyte removal is seen when comparing cytocentrifuged, erythrocyte lysis buffer–treated samples to untreated samples by light microscopy (Fig. 2
). In the untreated samples, the areas between stained H1299 cells are filled with RBC (Fig. 2A and B). Treated samples are devoid of RBC but do contain polymorphonuclear cells after lysis (Fig. 2C and D). The H1299 cells with blood were also monitored by MALDI MS before and after the erythrocyte lysis buffer treatment. The efficacy of the erythrocyte lysis step is revealed by examining the resulting spectra from cells that were contaminated with 10 µL of whole blood (Fig. 2E). In untreated samples (blue spectrum), dominant and ß hemoglobin peaks at m/z 15,127 and 15,668, respectively, doubly charged species at m/z 7,564 and 7,835, and hemoglobin dimers at m/z 30,253 (2), 30,793 ( + ß), and 31,335 (2ß) are detected. After the erythrocyte lysis step (green spectrum), in samples that contained 10 µL of blood, hemoglobin signals were attenuated to a level below that of abundant cellular proteins, such as histone H4 (at m/z 11,307 and 11,349), with almost complete elimination of peaks due to the doubly charged hemoglobin species and the hemoglobin dimers. Several intense peaks at m/z of 3,372, 3,443, and 3,487 are present in samples that have been contaminated with blood and have previously been identified as defensins, which are proteins present at high levels in neutrophils (12, 13). The intense defensin peaks are indicative of the high abundance of neutrophils in the samples after removal of large numbers of RBC. To determine the extent to which WBC affect the spectra, 10 µL of whole blood in 0.2 mL of saline were treated and the MALDI MS spectra acquired without attempting to avoid matrix deposition on white cells. Whereas the defensins were again very abundant, the overall intensity, and therefore the complexity, of the protein profile was not significant when compared with that of the non–small-cell lung carcinoma cell line alone (data not shown).
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70%; Fig. 4). Almost all of the fine-needle aspirate samples have predominant histone signals that are present in the tissue, but at a lower intensity. Qualitatively, fresh-frozen tissue samples seem to contain more peaks, presumably as a result of additional proteins derived from normal stromal tissue, infiltrating polymorphonuclear cells, macrophages, dendritic cells, or necrotic tissue. Interestingly, the spectrum of a fine-needle aspirate from a tumor with little necrosis and very few infiltrating neutrophils, unlike the tumor analyzed in Fig. 4, gave very low defensin peaks (m/z 3,372 and 3,443) as would be expected (Supplementary Fig. S1). Therefore, due to the large number of signals shared between the tumor and the fine-needle aspirate and the correlation of defensin peaks with numbers of neutrophils, we are confident that spectra obtained from a fine-needle aspirate will be highly informative and accurately reflect the make-up of the tumor from which it is derived, although spectra derived from fine-needle aspirates and tumor sections are clearly not completely equivalent.
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0.57. The above-mentioned results indicate that we can reproducibly harvest and prepare fine-needle aspirates for MALDI-TOF MS.
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From the first experiment, the resulting average spectra shown for each tumor type clearly illustrate distinct differences between the protein profiles (Fig. 6A ). Within the same figure, a difference plot, shown in blue, highlights those peaks that have been found to be statistically different between the two tumor types. The inset in Fig. 6A illustrates an isolated peak that was found to be one of the major discriminators between the two tumor types. This peak is identified in the difference plot by the black arrow. The average peak height is shown as a solid line with 1 SD above and below the average spectra depicted as dotted lines. It should be noted that there is no overlap between the SE plots, indicating a high level of significance between these two tumors with just a single marker.
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Quality spectra from clinical samples. True fine-needle aspirates were obtained from the clinic without identifiers and prepared as described (Fig. 3A). Cresyl violet–stained slides were examined by a pathologist for the presence of tumor cells. Two of the samples were positive for tumor cells and analyzed further by MALDI-MS. The spectra obtained were rich with peaks and of high quality (Fig. 7 ).
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Discussion |
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Reproducibility is prerequisite for any technology if it is to be used for clinical decision-making. From this perspective, we found that multiple samples derived from the same tumor, different tumors, or tumors analyzed on different days generated nearly identical spectra after processing with our novel approach. In two separate experiments, samples generated from tumors that were derived from two different cell lines, H460 and A549, neatly segregated into two distinct groups following a supervised Hierarchical Clustering Analysis. This qualitative analysis clearly shows reproducibility within any single group and further illustrates how this level of reproducibility allows the clear separation between differing groups of samples such as in our case two distinct tumor types. However, in one of the experiments, 96% (46 of 48) of the samples were classified correctly. Of three cell clusters spotted from the same needle pass into the H460 tumor, two were misclassified. One likely explanation for the misgrouping is that this particular pass of the needle came from an area that contained cells undergoing necrosis, which gave some signals, unlike H460 or A549. Difficulties arising from sampling errors are unfortunately intrinsic to blind needle aspirates, but emphasize the importance of attempting to obtain samples from the periphery of the tumor and avoiding potentially necrotic areas. Alternatively, signal contamination from other cell types could possibly come from our current method of manually spotting clumps using a pulled capillary. Although small spots can be achieved, the placement accuracy may in some cases be relatively imprecise. Automated matrix dispensing robots are currently being developed with placement accuracies in the order of 20 µm or better (16), which we intend to use as we refine the technology. Regardless, the overall results were quite encouraging as all other spectra clustered tightly within their respective groups. More work is currently being carried out to further refine this unique and very promising approach and to translate it into the analysis of real clinical specimens.
Many technologies are currently being investigated for refining our ability to diagnose cancer, define cancer type, and predict prognosis and response to treatment. The most developed of these is gene expression profiling using microarray analysis. Indeed, microarray studies have been done using fine-needle aspirates from several types of solid tumors without purification of tumor from nontumor cell types (14, 15, 17–20). However, problems have been encountered in isolating RNA of sufficient quality for microarray analysis and most clinical samples will be too heterogeneous to provide meaningful results.
Additional technologies include multiplex analysis of known biomarkers by bead-based multiplexing, where antibodies are coupled to bead matrices, and high-density single nucleotide polymorphism arrays (21, 22). As previously mentioned, the heterogeneity of most clinical samples complicates the analysis of either technology and could affect the result. By using the combination of erythrocyte lysis buffer to remove RBC and the indium-tin oxide–coated glass slides with MALDI-compatible stains, we have circumvented the problem of potentially interfering contaminants. Directly spotting clusters of cancer cells with matrix by visualizing them under a microscope, with few exceptions, leaves the large majority of contaminating cells untouched and the analysis free from such complications. The equipment to enable image-guided, robotic spotting and spectrum acquisition is currently being developed and should further enhance our ability to precisely target tumor cells.
A major obstacle to progress in this field is the difficulty in obtaining suitable numbers and types of tumor samples from patients necessary for training the specialized algorithms designed to find differential peaks. As current collection protocols in clinical trials change, with requirements that tumor biopsies be taken before and after treatment, the numbers of samples available for analysis should increase dramatically, particularly if fine-needle aspirates are usable for both defining and testing these patterns. With a large number of diagnoses at most institutions being accomplished by fine-needle aspirates, the ability to use these specimens with the technology we describe here should speed our progress. Standardization of the technique will allow collaborations between medical centers and allow a more rapid accumulation of the necessary data.
The MALDI-TOF MS technology has the potential to fill a significant need in the area of diagnostic medicine. The sensitivity makes it especially suited for small sample sizes. Data can be generated with minimal training due to the ease of the technique. The speed of the sample preparation and data acquisition makes the processing of large numbers of samples possible. Finally, the cost of the method, including materials, technician time, and mass spectrometer time, which can be provided by specialized reference institutions for a nominal fee, would be a fraction of the cost of other methods being developed for cancer diagnosis and response prediction. All of these attributes make the use of the cytospin-MALDI-TOF MS technology for the processing of fine-needle aspirates well suited for potential translation into the clinical laboratory.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
J.M. Amann and P. Chaurand contributed equally to this work.
Received 2/ 7/06; revised 5/23/06; accepted 6/20/06.
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-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor. Science 2002;298:995–1000.This article has been cited by other articles:
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  F. Taguchi, B. Solomon, V. Gregorc, H. Roder, R. Gray, K. Kasahara, M. Nishio, J. Brahmer, A. Spreafico, V. Ludovini, et al. Mass Spectrometry to Classify Non-Small-Cell Lung Cancer Patients for Clinical Outcome After Treatment With Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors: A Multicohort Cross-Institutional Study J Natl Cancer Inst, June 6, 2007; 99(11): 838 - 846. [Abstract] [Full Text] [PDF]
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