Lung Primary Samples and Cell lines
Matched specimens of normal lung parenchyma and lung adenocarcinoma tissue were obtained from patients who underwent lobectomy at Istituto Nazionale Tumori (Milan, Italy). Normal human bronchial epithelial cells (HBECs) and lung tumor cell lines were available at the tissue culture repository of the Hamon Center for Therapeutic Oncology Research, UTSW Medical Center (Dallas, TX) . Cell lines that have been used are listed in Additional file 1.
Quantitative real-time PCR
Total RNA was extracted with the RNeasy Midi kit (Qiagen, Valencia, CA) and reverse-transcribed with either the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA). MFSD2A mRNA expression was analyzed using TaqMan gene expression assays (Applied Biosystems, Foster City, CA): MFSD2A (Hs00293017_m1), HPRT1 (Hs99999909_m1), and GAPDH (4352934E).
Intron-spanning primers were designed to validate microarray results of NCI-H520 transfected cells for the following genes: DDIT3, DNAJB9, ELMO3, HRK, IRF1, LAMP3, MX1, NUPR1, PARL, PPP1R15A, RYR1, S100P, TCTA, UHMK1, WIPI1, and HPRT1 (housekeeping control). Amplification mixtures contained cDNA template, Power SYBR® Green PCR Master Mix (Applied Biosystems), and gene-specific PCR primers (sequences of oligonucleotide primers are available upon request).
For the expression levels analysis of genes involved in cell adhesion and cell migration pathways, we have used the TaqMan Array Human Extracellular Matrix & Adhesion Molecules 96-well plate (Applied Biosystems), according to the manufacturer's instructions.
Relative expression values were calculated using the comparative Ct method.
Gene expression profile of NCI-H520 cells transfected with recombinant MFSD2A or empty control vector (4 replicas/each) was analyzed using the Human-8 v3 Expression BeadChips (Illumina Inc., San Diego, CA, USA). Intensity values of each hybridization were quality-checked and the data set was normalized using a cubic spline algorithm, with BeadStudio Version 3 software. A P-value < 0.05 was set as a cutoff to filter reliably detected genes.
Gene expression profile of the 47 NSCLC cell lines was analyzed on the Affymetrix GeneChips HG-U133A and HG-U133B (together 44,928 elements; 23,583 unique genes) according to the manufacturer's protocol. Intensity values were quality-checked and the data set was quantile-normalized.
MFSD2A over-expression and silencing
MFSD2A full-length transcript (NM_032793) was amplified from a pool of human lung cDNAs (primers 5'-ggtcatggccaaaggagaa-3' and 5'-gaggatgctagccagctctgtg-3'), cloned in pEF6/V5-His TOPO vector (Invitrogen), and sequenced. Cell lines were transiently transfected using FuGENE HD Transfection Reagent (Roche, Basel, Switzerland). Stable transfectant clones were obtained after selection with 5 (A549) or 2 (NCI-H596, NCI-H520) μg/ml of blasticidin (Invitrogen) for 2 weeks.
siRNAs targeted against MFSD2A (5'-gcttcacaaagtgccaaccat-3', 5'-catggagagtaacctcatcat-3', 5'-gagtgtcactgggcatttcta-3', 5'-cacggcccatacatcaaactt-3', 5'-ccactgtgaatatgccaagga-3') were purchased from Qiagen. HBECs cells, which express endogenous MFSD2A, were transfected with 25 nM siRNA oligonucleotides using Oligofectamine (Invitrogen) and harvested after 96 hours for quantitative real-time PCR or cell cycle analysis.
Western blots and immunohistochemistry
MFSD2A expression in stably transfected cells was detected by Western blotting with anti-V5-HRP antibody (Invitrogen).
Samples of paraffin-embedded sections of MFSD2A- or empty vector-transfected HEK-293T cells, lung ADCA tissue, and surrounding normal lung tissue were used to prepare histological sections that were immunostained using standard methods after antigen retrieval performed in 0.07 M citrate buffer (pH = 6) at 95°C for 10 min. Mouse polyclonal anti-MFSD2A antibody (H00084879-B01P; Abnova, Taipei City, Taiwan) was used at a 1:170 dilution. Immunoreactive signals were detected with ChemMate DAB (Dako, Glostrup, Denmark).
In vitro assays
Colony formation assay was carried out in stably transfected A549, NCI-H520, and NCI-H596 cells (6 replicas). After 2 weeks of selection, colonies were methanol-fixed, stained with 10% Giemsa, and manually counted.
Cell adhesion was measured using CAFCA (Centrifugal Assay for Fluorescence based Cell Adhesion) assay . Briefly, six well strips were coated with different substrates from Becton-Dickinson (Falcon, Milan, Italy). Cells were labeled with the vital fluorochrome calcein AM (Invitrogen) for 15 minutes at 37°C and aliquoted into the bottom CAFCA miniplates, which were centrifuged to synchronize the contact of the cells with the substrate. The miniplates were incubated for 20 minutes at 37°C and mounted together with a similar CAFCA miniplate to create communicating chambers for reverse centrifugation. The relative number of cells bound to the substrate (i.e. remaining in the wells of the bottom miniplates) and cells that failed to bind to the substrate (i.e. remaining in the wells of the top miniplates) was estimated by top/bottom fluorescence detection with GENios Plus microplate fluorometer (TECAN, Italy). Percentage of adherent cells was determined 8 hours after plating.
Cell migration in response to extracellular matrix substrates was assessed by FATIMA (Fluorescence-Assisted Transmigration Invasion and Motility) assay as described . Briefly, membranes of HTS FluoroBlokTM transwell inserts with 8 μm pores (Becton-Dickinson, Falcon) were coated on the underside with various ECM molecules at 4°C and blocked with 1% BSA. Cells were fluorescently tagged with 5 μg/ml DiI lipophilic dye (Invitrogen) and plated in the upper chamber. Migration was monitored at every hour by independent fluorescence detection from the top (corresponding to non-transmigrated cells) and bottom (corresponding to transmigrated cells) side of the membrane with GENios Plus microplate fluorometer (TECAN).
Cell cycle analysis of stably transfected cells was performed on a FACScan (BD Biosciences, San Jose, CA) or FACSCalibur flow cytometer (BD Biosciences). MFSD2A- or empty vector-transfected cells were harvested and fixed in 70% EtOH over night at 4°C. Cells were then incubated in 500 μl of buffered propidium iodide (PI) staining solution containing 0.05% Triton X-100 (Sigma-Aldrich), 0.1 mg/ml RNase A (Millipore), and 50 g/ml PI (Sigma-Aldrich) in PBS for 30 min at 37°C. Cells were briefly spun down and resuspended in PBS. Flow cytometry data were analyzed using FlowJo software (TreeStar, Ashland, OR) with the aid of the Watson modeling algorithm.
In vivo tumor growth assay
Adult female CD-1 nude mice (purchased from Charles River, Calco, Italy) were injected subcutaneously with 4 × 106 A549 control or MFSD2A-transfected cells (2 independent clones). Tumor size was measured weekly by calipers and animals were sacrificed 8 weeks after injection.
Genes differentially expressed in the two classes of either vector- or MFSD2A-transfected cells were identified using random variance t-statistics . Microarray data were analyzed using BRB ArrayTools developed by Dr. Richard Simon and Amy Peng Lam http://linus.nci.nih.gov/BRB-ArrayTools.html (Illumina experiment) or using in-house Visual Basic software MATRIX 1.4 and the Bioconductor R package affy (Affymetrix experiment). Functional annotation in gene ontology (GO) categories was carried out with the DAVID Functional Annotation Tools . Differences in quantitative measures were assessed by analysis of variance. Correlations between expression levels were expressed by the Spearman's correlation coefficient. Cell cycle parameters by MFSD2A categories of expression (high, low) were analyzed using the generalized linear model, family = quasibinomial, procedure.