Stromal Cells Derived from Non-Small Cell Lung Cancer and ...



Cell isolation and culture

Non-small cell lung cancer (NSCLC) and corresponding normal lung tissue (NLT) specimens were minced into 1 mm³ pieces and digested for 2h at 37°C in Dulbecco’s modified Eagle medium (DMEM; PAA Laboratories GmbH, Pasching, Austria) containing collagenase (500 U/ml), hyaluronidase (30 U/ml) and DNAse (10 U/ml; all: Sigma-Aldrich Chemie GmbH, Munich, Germany). The cell suspension was washed in phosphate buffered solution (PBS; PAA) and seeded in 75 cm² flasks. The culture medium (CM) contained DMEM, 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (all: PAA). The cultures were maintained in a humidified atmosphere at 37°C, 5% CO2 and the CM was replaced two times a week. The outgrowth of the digested tissue pieces was harvested using 0.25% trypsin/1 mM ethylene diamine tetraacetic acid (EDTA; PAA) and processed as given in the paragraph “identification and selection of MSC”.

Fluorescence-activated cell sorting (FACS) and immunophenotyping

For immunophenotyping, the cells were harvested using 0.25% trypsin/1 mM EDTA, washed in CM and labeled with the following mouse-anti-human IgG1 antibodies for 15 min at 4°C in fluorescence-activated cell sorting (FACS) buffer containing PBS, 1% FCS and 0.5 mM EDTA: CD14-phycoerythrin (PE), CD19-fluorescein-isothyocyanate (FITC), CD29-FITC, CD31-FITC, CD34-FITC, CD45-FITC, CD73-PE, HLA-DR-FITC (all DakoCytomation GmbH, Hamburg,Germany), CD90-FITC/-PE, CD105-FITC, (Serotec GmbH, Düsseldorf, Germany), CD133-PE (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). Mouse IgG1/IgG2 isotype antibodies were used as control. The fibroblast activation protein alpha (FAP) mouse-anti-human IgG1 antibody (EMD Chemicals, Inc., Gibbstown, NJ, USA) was visualized by a secondary PE-conjugated rabbit-anti-mouse IgG antibody (Abcam plc, Cambridge, UK). For control the primary antibody was omitted. Cytoplasmic staining for α-SMA-FITC, pan-cytokeratin-FITC and vimentin-PE (Abcam) was performed after fixation and permeabilization with 4% paraformaldehyde/0.1% saponin (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) in PBS. Dead cells were excluded by propidium iodide staining. 100,000 labeled cells were acquired and analyzed/sorted using a FACScan-II flow cytometry system running CellQuest software (BD Biosciences, Heidelberg, Germany).

Mesenchymal differentiation and induction of α-smooth muscle actin (α-SMA) expression

For adipogenic differentiation, confluent cells were stimulated with CM containing 10-6 M dexamethasone, 100 mg/ml 3-isobutyl-1-methylxanthine, 50 mM indomethacin and 10 mg/ml insulin. For osteogenic differentiation, the CM was supplemented with 10-7 M dexamethasone, 10mM (-glycerophosphate disodium and 50 mg/ml ascorbic acid (all supplements: Sigma-Aldrich Chemie, Munich, Germany). Medium exchanges were performed twice a week. After three weeks, the cells were fixed with 10% formalin (Roth) for 10 min. Lipid droplets were stained with 0.3% oil red-O (Sigma) in 60% isopropanol for 10 min. Calcium deposits were visualized by a 2% aqueous solution of alizarin red S (Sigma), pH 4.2, applied for 3 min.

For induction of α-SMA expression, NSCLC-, NLT-MSC and the uterine leimyosarcoma cell lines SK-LMS-1 which was used as positive control were seeded in eight-well chamber slides (Fisher Scientific GmbH, Schwerte, Germany). After 24h the CM was replaced by tumor cell-conditioned medium derived from three-day cultured A549 cells or fresh CM containing 5 ng/ml transforming growth factor-β (PeproTech GmbH, Hamburg, Germany). The tumor cell-conditioned medium was re-supplemented with 10% FCS (PAA) before use. After five days the cells were analyzed as stated in the paragraph “immunohistochemistry”.

Nulceic acid isolation and polymerase chain reaction (PCR)

DNA and total-RNA were extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). Total-RNA was assessed by an Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit (Agilent Technologies Deutschland GmbH, Böblingen, Germany) and considered for further analysis if the RNA integrity number was ≥ 8.0. Reverse transcription was performed with the Roche Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany) in a Primus 96 advanced Gradient Thermocycler (PEQLAB Biotechnologie, Erlangen, Germany) with anchored-(dT)18 and random hexamer primers according to the following protocol: denaturation of template-primer mixture: 10 min at 25°C, annealing and extension: 60 min at 50°C, inactivation: 5 min at 85°C. The semi-quantitative PCR for peroxisome proliferator-activated receptor γ2 (PPARγ2), osteopontin 1 (OPN1) and the internal control gene glyceraldehyde-3-dehydrogenase (GAPDH) was performed using 0.2 µM of each primer listed in suppl. Tab. 2 and 0.5 U HotStarTaq Plus DNA Polymerase at a MgCl2 concentration of 1.5 mM (all: Qiagen) according to the following protocol (touchdown PCR): activation: 5 min at 95°C, 10 cycles: denaturation: 20 sec at 94°C, annealing: 30 sec at 51°C (PPARγ2) or 55°C (OPN1), extension: 1 min at 72°C, followed by 30 cycles: denaturation: 20 sec at 94°C, annealing: 30 sec at 47°C (PPARγ2) or 51°C (OPN1), extension: 1 min at 72°C, final extension: 10 min at 72°C. 5 µl of each PCR product was analyzed on a 2% agarose gel. Relative expression of PPARγ2 and OPN1 against GAPDH was determined by densitometrical quantification of the bands using Image J 1.44 software (). Expression of the selected candidate genes butyrylcholinesterase (BCHE), clusterin (CLU) and quiescin Q6 sulfhydryl oxidase 1 (QSOX1) was validated by quantitative real-time PCR (qRT-PCR) using Taqman assays in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Weiterstadt, Germany). Ten independent pairs of MSC (4 adenocarcinomas, 3 squamous cell carcinomas, 3 large cell carcinomas) were analyzed in technical triplicates. Gene-specific primers and probes (UPL System, Roche, Mannheim, Germany) were used in combination with QPCR Master Mix Reagent (Abgene, Epsom, UK). Relative quantification was done by using RNA polymerase II (POLR2A) and TATA box binding protein (TBP) as internal control genes. Details on primers and UPL probes are given in suppl. Tab. 3. All primers were synthesized by TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany.

Karyotyping, comparative genomic hybridization (CGH) and multiplex fluorescence in situ hybridization (M-FISH)

Metaphase spreads were obtained according to standard protocols with minor modifications: Subconfluent MSC were arrested in metaphase by adding colcemide (final concentration: 0.08 µg/ml; Sigma) to the CM for 17 hours. Hypotonic treatment was performed using 0.0375 M KCl solution (Roth) for 20 min. The cells were fixed with methanol/acetic acid (3:1, vol:vol) by gently pipetting. Fixation was performed four times prior spreading the cells on glass slides. G-banding was done using standard protocols. For CGH analysis, biotinylated tumor and sex-matched digoxigenin-labeled reference DNA were hybridized to normal metaphase spreads in the presence of Cot-1 DNA and salmon sperm DNA (both Sigma). After hybridization, the biotin-labeled and digoxigenin-labeled probes were detected using FITC and Cy3, respectively. Chromosomes were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma) for identification. Image acquisition, processing and evaluation were performed using a Leica DM RXA epifluorescence microscope (Leica Microsystems Holding GmbH, Bensheim, Germany) equipped with a Sensys CCD camera (Kodak KAF 1400 chip; Photometrics, Tucson, AZ, USA) and controlled by the Leica Q-FISH software (Leica Microsystems Imaging Solutions, Cambridge, UK). Three color images: red for reference DNA, green for tumor DNA and blue for DAPI counterstaining were acquired from at least 20 metaphase spreads per sample. Images were processed using the Leica Q-CGH software. Threshold values for detection of genomic imbalances were set to 0.80 for losses and 1.20 for gains. For multiplex FISH analysis, seven pools of flow-sorted whole chromosome painting probes were amplified by degenerative oligonucleotide primed PCR and directly labeled with DEAC- (diethylaminocoumarin), FITC-, Cy3-, TexasRed-, and Cy5-conjugated nucleotides (all Dyomics GmbH, Jena, Germany) or biotin- and digoxigenin-dUTP (Roche Deutschland Holding GmbH, Mannheim, Germany). After amplification and labeling of the whole chromosome painting probes, the metaphase preparations of the MSC were digested with pepsin (0.5 mg/ml; Sigma) in 0.2 N HCl (Roth) for 10 min at 37°C, washed in PBS, post-fixed in 1% formaldehyde (Roth), dehydrated with a degraded ethanol series and air dried. The slides were denatured in 70% formamide/1 x saline sodium citrate (SSC)/15% dextran sulfate (Roth) for 2 min at 72°C. The hybridization mixture consisting of 50% formamide, 2 x SSC, Cot-1 DNA and the labeled DNA probes was denatured for 7 min at 75°C, pre-annealed for 20 min at 37°C and hybridized to the denatured metaphase preparations of the MSC. After 48h incubation at 37°C the slides were three times washed for 5 min in 2 x SSC at ambient temperature, and subsequently two times for 5 min in 0.2% SSC/0.2% Tween-20 (Roth) at 56°C. For indirectly labeled probes a two-step immunofluorescence detection was performed using biotinylated goat-anti-avidin followed by streptavidin Laser Pro IR790-, and rabbit-anti-digoxin followed by goat-anti-rabbit Cy5.5, respectively (Molecular Probes, Inc. Eugene, OR, USA). The slides were washed in 4 x saline sodium citrate/0.2% Tween-20, counterstained with DAPI and covered with antifade solution (Abbott Laboratories, Maidenhead, UK). Images of at least 20 metaphase spreads were taken separately for each fluorochrome using highly specific filter sets (Chroma Technology, Brattleboro, VT) and processed using Leica MCK software.

Suppl. Figure 1: Chemosensitivity of mesenchymal stem cells (MSC). The dot plots of annexin V/propidium iodide stained cells demonstrate better survival of non-small cell lung cancer- (NSCLC) derived MSC (quadrant lower left). The proportion of apoptotic (quandrant lower right) and necrotic cells (quadrant upper left and right) is lesser than in normal lung tissue- (NLT) derived MSC. As shown in the corresponding microphotographs, NSCLC-MSC layers are more intact than NLT-MSC after exposure to cisplatin. Dot plots and microphotographs of the lower row represent untreated controls. Dot plots: x-axis: fluorescence intensity annexin V, y-axis: fluorescence intensity propidiumiodide; microphotographs: scale bar: 250 µM.

Supplementary references

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SAMD3 (sterile alpha motif domain containing): Kubo H, Shimizu M, Taya V, Kawamoto T, Michida M, Kaneko E, Igarashi A, Nishimura M, Segoshi K, Shimazu Y, Tsuji K, Aoba T, Kato Y . Identification of mesenchymal stem cell (MSC)-transcription factors by microarray and knockdown analyses, and signature molecule-marked MSC in bone marrow by immunohistochemistry. Genes to Cells 2009; 14: 407-424.

EEF1A (eukaryotic translation elongation factor 1 alpha 1): Covas DT, Panepucci RA, Fontes AM, Silva WA Jr, Orellana MD, Freitas MC, Neder L, Santos AR, Peres LC, Jamur MC, Zago MA. Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts. Exp Hematol 2008; 36: 642-654.

EDN1 (endothelin 1): Shi-wen X, Kennedy L, Renzoni EA, Bou-Gharios G, du Bois RM, Black CM, Denton CP, Abraham DJ, Leask A. Endothelin is a downstream mediator of profibrotic responses to transforming growth factor beta in human lung fibroblasts. Arthritis Rheum 2007; 56: 4189-4194.

STXBP2 (syntaxin binding protein 2): Brimhall BB, Sikorski KA, Torday J, Shahsafaei A, Haley KJ, Sunday ME. Syntaxin 1A is transiently expressed in fetal lung mesenchymal cells: potenial developmental roles. Am J Physiol Lung Cell Mol Physiol 1999; 277: 401-411.

PORCN [porcupine homolog (Drosophila)]: Karow M, Popp T, Egea V, Ries C, Jochum M, Neth P. Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression. J Cell Mol Med 2009; 13: 2506-2520.

MTP18 (mitochondrial protein 18 kDa): Tondera D, Santel A, Schwarzer R, Dames S, Giese K, Klippel A, Kaufmann J. Knockdown of MTP18, a novel phosphatidylinositol 3-kinase-dependent protein, affects mitochondrial morphology and induces apoptosis. J Biol Chem 2004; 279: 31544-31555.

ZNF364 (zinc finger protein 364): Zhang H-G, Wang J, Yang X, Hsu HC, Mountz JD. Regulation of apoptosis proteins in cancer cells by ubiquitin. Oncogene 2004; 23: 2009-2015.

PLOD2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2), LOXL2 (lysyl oxidase-like 2): Knippenberg M, Helder MN, Doulabi BZ, Bank RA, Wuisman PI, Klein-Nulend J. Differential effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on gene expression of collagen-modifying enzymes in human adipose tissue-derived mesenchymal stem cells. Tissue Eng Part A 2009; 15: 2213-2225.

ANGPT1 (angiopoietin 1): Metheny-Barlow LJ, Li LY The enigmatic role of angiopoietin-1 in tumor angiogenesis. Cell Res 2003; 13: 309-317.

ANGPT1: Holopainen T, Huang H, Chen C, Kim KE, Zhang L, Zhou F, Han W, Li C, Yu J, Wu J, Koh GY, Alitalo K, He Y. Angiopoietin-1 overexpression modulates vascular endothelium to facilitate tumor cell dissemination and metastasis establishment. Cancer Res 2009; 69: 4656-4664.

ANGPT1: Liu XB, Jiang J, Gui C, Hu XY, Xiang MX, Wang JA. Angiopoietin-1 protects mesenchymal stem cells against serum deprivation and hypoxia-induced apoptosis through the PI3K/Akt pathway. Acta Pharmacol Sin 2008; 29: 815-822.

QSOX1 (quiescin Q6 sulfhydryl oxidase 1): Coppock DL, Kopman C, Scandalis S, Gilleran S. Preferential gene expression in quiescent human lung fibroblasts. Cell Growth Differ 1993; 4: 483-493.

TFPI2 (tissue factor pathway inhibitor 2): Chand HS, Du X, Ma D, Inzunza HD, Kamei S, Foster D, Brodie S, Kisiel W. The effect of human tissue factor pathway inhibitor-2 on the growth and metastasis of fibrosarcoma tumors in athymic mice. Blood 2004; 103: 1069-1077.

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BCHE (butyrylcholinesterase): Song P, Sekhon HS, Fu XW, Maier M, Jia Y, Duan J, Proskosil BJ, Gravett C, Lindstrom J, Mark GP, Saha S, Spindel ER. Activated cholinergic signaling provides a target in squamous cell lung carcinoma. Cancer Res 2008; 68: 4693-4700.

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