Identification of Markers Associated with Highly Aggressive Metastatic Phenotypes Using Quantitative Comparative Proteomics

Materials and Methods

Cell culture. The NM2C5 cell line was a kind gift of Dr. D. Tarin, UCSD Medical center, La Jolla, CA, USA. The M4A4 LM3-2 green fluorescence protein (GFP) (LM3) and M4A4 LM3-4 CL16 GFP (CL16) were obtained from the American Type Culture Collection (Boras, Sweden). All cell lines were derived from the triple-negative breast cancer cell line MDA-MB-435S and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Taastrup, Denmark) supplemented with 10% fetal bovine serum (FBS) (Gibco), and 5% penicillin/streptomycin (P/S) (Gibco). Cultures were propagated in a humidified atmosphere of 5% CO₂ and at 37°C. All analyses were performed on cultures passaged no more than 10 times from frozen stock vials to ensure genetic stability.

Orthotopic transplantation of cancer cells into mice. The NM2C5, LM3 and CL16 cell lines were transfected with luciferase 2 (LUC2)-expressing lentiviral particles (In Vivo Imaging Solutions, Fort Collins, CO, USA) using a multiplicity of infection virus particle concentration of 15 according to the manufacturer’s guidelines, generating stable luciferase-expressing lines. Subconfluent NM2C5, LM3 and CL16 cells in culture were washed in phosphate buffered saline (PBS) and harvested using a cell scraper. Cells were washed in PBS and 1×10⁶ tumor cells for inoculation were resuspended in a 1:1 mixture of extracellular matrix from the Engelbreth-Holm-Swarm sarcoma (Sigma-Aldrich, Brøndby, Denmark) and DMEM. Orthotopic transplantation of tumor cells was performed by injecting the cells into the surgically exposed mammary fat pad of anesthetized eight-week-old female CB-17 SCID mice (n=3 per group) (Taconic, Ry, Denmark). All animal experiments were performed at the Animal Core Facility at University of Southern Denmark and approved by The Experimental Animal Committee, The Danish Ministry of Justice. The animals were housed under specific pathogen-free conditions with ad libitum food and drinking water. The mice were euthanized if they showed any adverse signs or symptoms of disease including weight loss, paralysis or general discomfort.

In vivo imaging. Primary tumors were surgically removed five weeks after inoculation when they reached a size of approximately 1.2 cm in diameter and the metastatic burden in these mice was subsequently evaluated weekly for the following three weeks by measuring bioluminescence from spontaneous lung metastases. Relative quantification of metastasis development was performed weekly using whole-body bioluminescent imaging (IVIS-spectrum; Caliper Life Science, Mainz, Germany). Mice were injected with 150 mg D-luciferin/kg body weight (Caliper Life Science) and then anesthetized with isoflurane gas. Images were acquired starting 10 min after luciferin injection. The bioluminescent images were processed using Caliper Life Science Living Image (version 4.0).

Protein labeling using stable isotope labeling by amino acids in cell culture (SILAC). The proteomes of the NM2C5, LM3 and CL16 cells were labeled using SILAC. Cells were cultured in customized DMEM without L-arginine, L-lysine and L-glutamine (Bioconcept, Allschwil, Switzerland) supplemented with 580 mg/l L-glutamine, 28 mg/l L-arginine and 75 mg/l L-lysine, 10% triple 0.1 μm dialyzed FBS (Hyclone, Logan, UT, USA) and 1% P/S (Invitrogen). NM2C5 culture medium was supplemented with ¹³C₆-L-lysine (Cambridge Isotope Laboratories, Andover, MA, USA) and ¹³C₆-L-arginine (Cambridge Isotope Laboratories), while LM3 and CL16 cells were supplemented with ¹²C₆-L-lysine (Sigma Aldrich) and ¹²-L-arginine (Sigma Aldrich). In order to completely incorporate the stable isotopes, the cells were grown for at least five cell doublings in the supplemented growth medium (14). Cultures were propagated in a humidified atmosphere of 5% CO₂ at 37°C.

Isolation of membrane proteins. The isolation procedure was performed as previously detailed (14). In brief, approximately 2×10⁷ cells from each cell line were harvested by scraping, the cells were counted and labeled, and unlabeled cells were mixed at a 1:1 ratio. Following incubation in a hypotonic buffer (10 mM Tris-base, 1.5 mM MgCl₂, 10 mM NaCl, pH 6.8) the mixed population of cells was homogenized in gradient buffer (0.25 mM sucrose, 10 mM HEPES, 100 mM succinic acid, 1 mM EDTA, 2 mM CaCl₂, 2 mM MaCl₂, pH 7.4). The homogenate was centrifuged (1000 ×g for 10 min at 4°C) and the supernatant was collected. The supernatant was centrifuged at 100,000 ×g for 30 min and the pellet, containing crude membranes, was washed and resuspended in a gradient buffer. The membranes were mixed with Percoll containing 10% PBS. By displacing the gradient from the bottom with 2 M sucrose, the membranes were separated into 10 fractions according to their density. To exclude mitochondrial membranes, we measured the activity of mitochondrial succinate dehydrogenase (SDH) conversion of p-iodonitrotetrazolium violet colorimetrically, as previously described (14). Only early fractions with low SDH activity containing the plasma membranes and endoplasmic reticulum (ER) membranes were included in the following procedure, while fractions with high SDH activity containing the mitochondrial membranes were discarded. Protein concentration was determined in triplicate by using a colorimetric-based assay (Bio-Rad, Hercules, CA, USA) in accordance with the manufacturer’s protocol with a bovine serum albumin preparation used as standard (Pierce, Rockford, IL, USA).

Enzymatic digestion. Proteins (∼50 μg) were digested as previously detailed and the tryptic peptides were desalted and concentrated using a modified version of the previously described StageTips (15, 21, 22).

Liquid chromatography – tandem MS. Peptides were separated using an LC-Packings Ultimate 3000 nanoflow system (LC Packings, Amsterdam, the Netherlands). Peptides were loaded at a flow rate of 3 μl/min onto a customized 1-cm precolumn (75 μm inner diameter) of fused silica with kasil-frits retaining Reprosil C18, 3.5-μm reversed-phase particles (Dr. Maisch, GmbH, Ammerbuch-Entringen, Germany). Nanoflow reversed-phase high-performance liquid chromatography (HPLC) was then performed at a flow rate of 0.2 μl/min through a customized 8-12 cm analytical column (50 μm inner diameter), packed with Reprosil C18-AQ, 3-μm reversed-phase particles (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Peptides were eluted directly into the electrospray ionization (ESI) source of a quadrupole time-of-flight (Q-TOF) Premier tandem mass spectrometer (Waters Micromass, Manchester, UK), using a stepped linear gradient (solvent A: 0.1% CH₃COOH, solvent B: 95% acetonitrile, 0.1% CH₃COOH); 0% B for 10 min, 0-35% B 90 min, and 35-100% for 5 min. Mass- and charge-dependent collision energies were used for peptide fragmentation.

MS/MS database search and quantification. Peak list files were created using Masslynx 4.0 (Waters Micromass, Hedehusene, Denmark) with the following processing parameters: background subtraction: polynomial order 10, 10% below curve; smoothing: Savitzky Golay, 3 channels, 2 smooths; centroiding: min peak with at half hight, 4, centroid top, 80%. The data were searched against all human proteins and common contaminants (n=525207) in the SwissProt database as of the 8th of March 2011, using an in-house Mascot server v2.2 (Matrix Science Ltd., London, UK). Search parameters were: 25 ppm precursor ion mass tolerance, 0.1 Da fragment ion mass tolerance, enzyme-specificity: trypsin (C-terminally to R and K, but not PR and PK), 1 missed cleavage, fixed modifications: cystein carbamidomethyl, variable modifications: oxidation (M), label: ¹³C(6) (K and R). The criteria for accepting MS/MS spectra were Mascot scores above 15 and a false discovery rate below 1.5%. Moreover, only peptides with peptide mascot scores above 25, and only peptides that were the best match for the given mass were used for quantification. The false-discovery rate data were assessed against a SwissProt decoy database, essentially as described previously (23), and calculated as follows: false-discovery rate (%) = decoy hits ×100/(SwissProt hits + decoy hits).

Peak list files were recalibrated using the in-house Perl script MSRecal, and relative quantification of stable isotope-labeled peptides was performed using MSQuant v2.0 (24). MSQuant uses extracted ion current (XIC) for quantification, and all peptides fulfilling the criteria mentioned above for MS/MS spectra were quantified, no outlier points were removed. The quantifications were normalized to their collective median and imported via a comma-separated value format to ProteinCenter v2.8.1 (Proxeon A/S, Odense, Denmark), in order to identify for differentially expressed proteins. All protein identifications from the technical replicates from each biological sample were merged (12 LC-MS/MS analyses were merged into four biological samples), and proteins that were 98%, or more, similar were clustered into groups to reduce redundancy. Proteins that were identified in two or more technical repeats were selected for further evaluation.

Differentially expressed proteins had to meet the following criteria: Identification and quantification in two technical replicates by two or more different peptides in NM2C5 vs. CL16 or NM2C5 vs. LM3, and differential expression of >1.5-fold. The quantitative and qualitative data of the differentially expressed proteins were manually inspected using the Mascot search results and MSQuant.

Supplementary data 1 provide the accession number, number of unique peptides and the quantification for each identified protein. The mean and standard deviation for all quantified proteins and the peptides used for quantification are provided in Supplementary data 2. Gene ontology annotations are provided in Supplementary data 3.

Immunocytochemistry and immunohistochemistry. The cell line-derived tumor tissue from mice was established as described above in Orthotopic transplantation of cancer cells into mice; the generation of tissue and cell microarrays and the staining procedures are described elsewhere (15). The following primary antibodies from Sigma Aldrich were used: CD59 (HPA026494, 1:500), chondroitin sulfate proteoglycan 4 (CSPG4) (HPA002951, 1:250) and Leucine-rich repeat-containing 59 (LRRC59) (HPA030827, 1:500). Immunostaining was performed using the PowerVision™ horse radish peroxidase detection system (ImmunoVision Technologies, Burlingame, CA, USA) on a TechMate™ 500 instrument (Dako, Glostrup, Denmark).