1096 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 40: 1096-1104, 2017 Classification of cancer cell lines using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and statistical analysis VLAD SERAFIM1,2, AJIT SHAH2, MARIA PUIU1, NICOLETA ANDREESCU1, DORINA CORICOVAC3, ALEXANDER E. NOSYREV4, DEMETRIOS A. SPANDIDOS5, ARISTIDES M. TSATSAKIS6, CRISTINA DEHELEAN3 and IULIA PINZARU3 1Center of Genomic Medicine, ‘Victor Babes’ University of Medicine and Pharmacy, Timisoara 300041, Romania; 2Department of Natural Sciences, Middlesex University, London NW4 4BT, UK; 3Department of Toxicology, Faculty of Pharmacy, ‘Victor Babes’ University of Medicine and Pharmacy, 300041 Timisoara, Romania; 4Central Chemical Laboratory of Toxicology, I.M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia; 5Laboratory of Clinical Virology, Medical School; 6Department of Forensic Sciences and Toxicology, Faculty of Medicine, University of Crete, 71003 Heraklion, Greece Received February 2, 2017; Accepted July 12, 2017 DOI: 10.3892/ijmm.2017.3083 Abstract. Over the past decade, matrix-assisted laser desorption/ way for developing a broad‑based strategy for the identification ionization time‑of‑flight mass spectrometry (MALDI‑TOF MS) and classification of cancer cells. has been established as a valuable platform for microbial identi- fication, and it is also frequently applied in biology and clinical Introduction studies to identify new markers expressed in pathological condi- tions. The aim of the present study was to assess the potential The development and application of matrix-assisted laser of using this approach for the classification of cancer cell lines desorption/ionization time‑of‑flight mass spectrom- as a quantifiable method for the proteomic profiling of cellular etr y (MA LDI‑TOF MS) for the identification of microorganisms organelles. Intact protein extracts isolated from different has been revolutionary. Microbial proteomics has attracted tumor cell lines (human and murine) were analyzed using attention worldwide and this has resulted in the development MALDI‑TOF MS and the obtained mass lists were processed of novel identification methods which are rapid, robust and using principle component analysis (PCA) within Bruker relatively inexpensive (1). Biotyper® software. Furthermore, reference spectra were For the identification of microorganisms, MALDI‑TOF MS created for each cell line and were used for classification. Based analysis typically concentrates on intact proteins with a on the intact protein profiles, we were able to differentiate and molecular weight in the range of 2‑20 kDa. The proteins in classify six cancer cell lines: two murine melanoma (B16‑F0 this range are mainly ribosomal proteins which represent and B164A5), one human melanoma (A375), two human breast approximately 30% of the total proteins from a m icrobial cell (2). carcinoma (MCF7 and MDA‑MB‑231) and one human liver Following mass spectral analysis, the raw spectra are processed carcinoma (HepG2). The cell lines were classified according to using a peak recognition algorithm. The data generated cancer type and the species they originated from, as well as by following MALDI‑TOF MS analysis consist of two large sets their metastatic potential, offering the possibility to differentiate of values: mass‑to‑charge (m/z) and intensity. Each m/z value non-invasive from invasive cells. The obtained results pave the has a corresponding intensity value. The signal extraction, also known as peak picking, is often corrupted by noise. Therefore, various algorithms have been developed for obtaining peaks that correspond to true peptide/proteins signals (3). This is a very important step in data analysis as different peak recognition algorithms may have a considerable effect on the peak list, and Correspondence to: Dr Dorina Coricovac, Department of Toxicology, therefore should be adjusted with care (4). Once extracted, the Faculty of Pharmacy, ‘Victor Babes’ University of Medicine and peak lists are compared to a dedicated database which contains Pharmacy, 2 Eftimie Murgu Sq, 300041 Timisoara, Romania E-mail: [email protected] reference mass spectra of known microbial strains. The first such platform ‘MALDI Biotyper’ was developed by Bruker Key words: cancer cell lines, matrix-assisted laser desorption/ Daltonics. Another platform combines the Shimadzu mass ionization time‑of‑flight mass spectrometry, principle component instrumentation and software ‘Launchpad’ with a centralized analysis, reference spectra library, biotyping, intact protein profiling database ‘SARAMIS’ provided by BioMerieux (Marcy l'Etoile, France) (1,5). SERAFIM et al: CLASSIFICATION OF CANCER CELL LINES USING MALDI‑TOF MS 1097 Although mainly used for microbial identification, intact glucose, 2 mM L‑glutamine and supplemented with 10% FBS protein profiles have also been used successfully for the and antibiotic mixture (100 U/ml penicillin and 100 µg/ml strep- characterization and identification of mammalian cell lines (6‑8). tomycin). The human liver carcinoma (HepG2) and human breast Karger et al (6) identified 66 cell lines from 34 species using carcinoma (MCF7) were cultured in EMEM, supplemented with reference spectra library created by MALDI Biotyper, while 10% FBS and antibiotic mixture. The cells were kept in standard Povey et al (8) used partial least squares discriminant analysis conditions as follows: a humidified atmosphere with 5% CO2 at a model to predict the phenotype of recombinant mammalian cell temperature of 37˚C and were passaged every 2‑3 days. lines. The methods currently used for the identification and Scratch assay. The migratory character of the tumor cells used characterization of cancer cells, namely DNA fingerprinting, in this study was examined by the means of a scratch assay. immunohistochemistry and flow cytometry (9‑12), require In brief, 2x105 cells/well were seeded in 12‑well plates in specific reagents which limit the degree of multiplexing. In specific culture medium and when the confluence was appro- addition, these methods require laborious sample preparation priate (85‑90%) a gap/scratch was drawn in the middle of the which leads to an increased analysis time. The proteomic well with a 10 µl tip (13). The capacity of the cells to migrate approach can achieve a level of multiplexing where several cell and fill the gap was monitored for 24 h by acquiring images lines can be analyzed without changing the method parameters at different time points, namely 0, 3 and 24 h using an Optika and without using specialized materials and reagents. Microscopes Optikam Pro Cool 5 and Optika View (Optika, The aim of this study was to assess the potential of using Ponteranica, Italy). MALDI‑TOF MS for the classification of cancer cell lines. To achieve this, the procedure for the taxonomic classification of Protein extraction for MALDI‑TOF MS analysis. Sample microorganisms was adapted. Six cancer cell lines (murine and preparation for MALDI‑TOF MS analysis was performed as human) were used in this study: B16‑F0 and B164A5 (murine follows: the culture medium was discarded from the flasks; the melanoma cells), A375 (human melanoma), HepG2 (human cells were washed with 10 ml PBS and were subsequently incu- liver carcinoma), MCF7 (human breast carcinoma) and bated with 3 ml 0.025% trypsin/EDTA for 3‑5 min. The reaction MDA‑MB‑231 (human breast carcinoma). The statistical was terminated by the addition of 10 ml cell culture medium. analysis was processed using MALDI Biotyper software. These The cells were stained with trypan blue and the cell number data were used for a better observation of differences regarding was established using the Neubauer cell counting chamber. the species and metastatic potential, differences well‑defined The suspension was subsequently centrifuged at 1,700 x g between the two human breast carcinoma cell lines (MCF7 and for 5 min. The supernatant was discarded and the cell pellet MDA‑MB‑231) and two murine melanoma cell lines (B16‑F0 was mixed with 1 ml of ethanol. Following centrifugation at and B164A5). As an end point, a different cell line was applied 1,700 x g for 5 min, the ethanol was removed and the pellet was for an upgraded picture: HepG2 (human liver carcinoma). reconstituted in 70% formic acid at a ratio of 20 µl/1x106 cells. The mixture was left at room temperature for 2 min, and then Materials and methods an equal volume of acetonitrile was added. The samples were then centrifuged at 10,000 x g for 5 min. An aliquot (1 µl) of the Cell lines and reagents. The cancer cell lines used in the supernatant was spotted in duplicate onto the MTP 384 ground present study, B16‑F0 [murine melanoma; CRL‑6322™, steel MALDI target plate (Bruker Daltonics). The sample was American Type Culture Collection (ATCC), Manassas, VA, allowed to dry at ambient temperature and then overlaid with USA], B16 melanoma 4A5 (murine melanoma; 94042254; 1 µl of HCCA (5 mg/ml in a mixture of acetonitrile, water and Sigma‑Aldrich Chemie GmbH, Munich, Germany), A375 trifluoroacetic acid 50:47.5:2.5% v/v). (human melanoma; CRL‑1619™; ATCC), HepG2 (human liver carcinoma; HB8065™; ATCC), MCF7 (human breast MALDI‑TOF MS analysis. The MALDI‑TOF MS instrument carcinoma; HTB22™; ATCC) and MDA‑MB‑231 (human (Bruker Daltonics) was calibrated in a positive ion linear mode breast carcinoma; HTB26™; ATCC), were acquired from in a mass range of 5,000‑20,000 m/z, using Bruker Protein Sigma‑Aldrich Chemie GmbH and ATCC as frozen items. Calibration Standard I containing insulin, ubiquitin I, cyto- The specific reagents for cell culture [Dulbecco's modi- chrome c and myoglobin. Flex Control® (version 3.4) software fied Eagle's medium (DMEM); Eagle's Minimum Essential was used to acquire the data and set the method parameters. The Medium (EMEM)], fetal bovine serum (FBS), antibiotic following settings were used: laser frequency, 2,000 Hz; smart- mixture of penicillin/streptomycin, phosphate‑buffered beam, ‘4_large’; sample rate and digitiser settings, 1.25 GS/sec; saline (PBS), Trypsin/EDTA and trypan blue were acquired accelerator voltage, 20.07 kW; extraction voltage, 18.87 kW; lens from Sigma‑Aldrich Chemie GmbH and ATCC.