Establishment and comparative characterization of novel squamous cell non-small cell lung cancer cell lines and their corresponding tumor tissue

Lung Cancer 75 (2012) 45–57

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Establishment and comparative characterization of novel squamous cell non-small cell lung cancer cell lines and their corresponding tumor tissue Sandra Gottschling a,∗ , Anna Jauch b , Ruprecht Kuner c , Esther Herpel d , Karin Mueller-Decker e , Philipp A. Schnabel d , Elizabeth C. Xu f , Thomas Muley f , Holger Sültmann c , Christian Bender c , Martin Granzow b,g , Thomas Efferth h , Hans Hoffmann i , Hendrik Dienemann i , Felix J.F. Herth j , Michael Meister f a

Dept. of Thoracic Oncology, Thoraxklinik/University of Heidelberg, Amalienstr. 5, 69126 Heidelberg, Germany Institute of Human Genetics, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany c Unit Cancer Genome Research, Division of Molecular Genetics, German Cancer Research Center and National Center for Tumor Diseases, Im Neuenheimer Feld 460, 69120 Heidelberg, Germany d Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220, 69120 Heidelberg, Germany e Core Facility Tumor Models, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany f Translational Research Unit, Thoraxklinik/University of Heidelberg, Amalienstr. 5, 69126 Heidelberg, Germany g Quantiom Bioinformatics GmbH & Co. KG, Ringstr. 61, 76356 Weingarten, Germany h Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55099 Mainz, Germany i Dept. of Thoracic Surgery, Thoraxklinik/University of Heidelberg, Amalienstr. 5, 69126 Heidelberg, Germany j Dept. of Pneumology & Critical Care Medicine, Thoraxklinik/University of Heidelberg, Amalienstr. 5, 69126 Heidelberg, Germany b

a r t i c l e

i n f o

Article history: Received 30 November 2010 Received in revised form 28 March 2011 Accepted 23 May 2011 Keywords: Non-small cell lung cancer Squamous cell carcinoma Cell lines Surface marker profile Genotype Gene expression profile

a b s t r a c t Background: Cell lines play an important role for studying tumor biology and novel therapeutic agents. Particularly in pulmonary squamous cell carcinoma (SCC) the availability of cell lines is limited and knowledge about their representativeness for corresponding tumor tissue is scanty. Materials and methods: We established three novel SCC cell lines from fresh tumor tissue of 28 donors, including 8 SCC. Two cell lines were derived from different localizations of the same donor, i.e. primary tumor and lymph node metastasis. This represents a so far unique combination in lung cancer. The genotypes, gene expression profiles and mutational status of epidermal growth factor receptor (EGF-R) and Kirsten rat sarcoma (k-ras) of the cell lines and their corresponding tumor tissue were analyzed and compared. Moreover, the molecular characteristics were related to functional properties of the cell lines. Those comprised proliferation, motility and chemosensitivity. The cell lines were authenticated by single tandem repeat DNA typing. Tumorigenicity was analyzed in a murine xenograft model. Results: Comparative genomic hybridization and multiplex fluorescence in situ hybridization revealed essential genetic similarities between the cell lines and their corresponding tumor tissue, but indicated also some genetic evolution and clonal selection. EGF-R or k-ras mutations were not detected. Gene expression profiling showed various differences between tumor tissue and cell lines affecting gene clusters associated with immune response, adhesion, proliferation, differentiation and angiogenesis. However, there were also common gene expression patterns reflecting the relationship between cell lines and their corresponding tumor tissue. Moreover, the molecular characteristics of the tumor tissue and the descendent cell line were associated with functional properties of the latter. All cell lines showed a unique, heterozygous human DNA profile and one cell line displayed rapid tumor formation in mice. Conclusions: Here, we demonstrate that cell lines represent a useful in vitro system for studying basic mechanisms in lung cancer, but cover only distinct molecular characteristics of the original tumor. Moreover, we present three novel, comprehensively characterized SCC cell lines. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction ∗ Corresponding author at: Dept. of Thoracic Oncology, Thoraxklinik/University of Heidelberg, Amalienstr. 5, 69126 Heidelberg, Germany. Tel.: +49 6221 396 8069; fax: +49 6221 396 1351. E-mail address: [email protected] (S. Gottschling). 0169-5002/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2011.05.020

Lung cancer is the leading cause of cancer-related death in industrial nations and has a mean survival time of 12–18 months [1,2]. Non-small cell lung cancer (NSCLC) accounts for 85–90% of the lung cancer cases and is subdivided into adenocarcinoma (AC),

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squamous cell carcinoma (SCC) and large cell carcinoma (LCC) [1]. SCC shows minor response to antineoplastic agents and thus requires intensified research [1,2]. Due to minor success rates in generating NSCLC (2.6–36%), and particularly SCC (1.0–14%) cell lines, their availability for this purpose is limited [3–6]. Moreover, there is hardly knowledge about their representativeness for corresponding tumor tissue. Wistuba et al. analyzed and compared the morphology, loss of heterozygosity (LOH), microsatellite alterations, expression of HER2/neu and p53, and k-ras and p53 mutations of 12 NSCLC cell lines and their original tissue and found an almost complete concordance [7]. An analog analysis in breast cancer cell lines revealed similar results [8]. Virtanen et al. who performed gene expression profiling of 41 lung cancer cell lines and 44 non-related tumor specimens showed partial clustering of cell lines and tissue even in a heterologous system: four of 8 SCC and 11 of 13 small cell lung cancer (SCLC) cell lines clustered with fresh tumor tissue. However, none of the 11 analyzed AC cell lines clustered with the corresponding tumors [9]. In contrast to these studies, we performed comparative genome-wide genotyping and gene expression profiling of NSCLC cell lines and corresponding tumor tissue, and moreover, we related the molecular characteristics to functional properties of the cell lines. For this purpose three novel SCC cell lines were analyzed. Those had been established from fresh tumor tissue of 28 donors, including 8 SCC. Here, we demonstrate that cell lines represent a useful in vitro system for studying basic mechanisms in lung cancer, but cover only distinct molecular characteristics of the original tumor. Moreover, we present three novel, comprehensively characterized SCC cell lines. 2. Materials and methods 2.1. Donor characteristics and specimen collection Tumor tissue and lymph node metastases (n = 28; 16 AC, 8 SCC, 4 LCC) were obtained from volunteer donors undergoing lung surgery for newly diagnosed NSCLC. The donors had given informed consent following the guidelines of the 2008 revision of the declaration of Helsinki and the local Ethics Committee of the Medical Faculty Heidelberg. Following surgical resection, samples were divided and (i) snap frozen within 30 minutes (min) after resection and stored at −80 ◦ C until total-RNA isolation, and (ii) for cell isolation immediately placed on ice and prepared as described by Sugaya et al. with minor modifications [6]. Details to the donors, cell preparation and culture are given in supplementary Table 1 and supplementary methods. 2.2. Immunophenotyping Tumor cells were detached with 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA; PAA Laboratories GmbH, Pasching, Austria), washed with culture medium (CM), resuspended in fluorescence-activated cell sorting (FACS) buffer containing Dulbecco’s phosphate buffered solution (DPBS), 0.5 mM EDTA (Sigma–Aldrich Chemie GmbH, Munich, Germany) and 1% fetal calf serum (FCS; PAA) and labeled with the following mouseor rabbit-anti-human-IgG antibodies for 15 min at 4 ◦ C: CD9fluorescein-isothyocyanate (FITC), CD34-FITC, CD44-FITC, CD45FITC, CD54-FITC (DakoCytomation GmbH, Hamburg, Germany), CD56-FITC (Abcam plc, Cambridge, UK), CD117-allophycocyanin (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany), synaptophysin (SYP), neuron-specific enolase (NSE), chromogranin A (CHGA), cytokeratin 5/6 (CK5/6) and CK7 (Abcam). SYP, NSE, CHGA, CK5/6 and CK7 staining was performed after fixation and permeabi-

lization with 4% paraformaldehyde/0.1% saponin (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) in DPBS. Non-conjugated antibodies were visualized by secondary FITC- or PE-conjugated goat-anti-rabbit- or rabbit-anti-mouse IgG antibodies (Abcam). For isotype control the primary antibody was omitted. 100,000 labeled cells were acquired and analyzed at passage 5 and 15 using a FACScan-II flow cytometry system running CellQuest software (BD Biosciences, Heidelberg, Germany). 2.3. Immunohistochemistry Hematoxylin-stained tissue sections were deparaffinized and rehydrated. Antigens were retrieved by boiling in target retrieval solution [10 mM sodium citrate pH6 and pH9, respectively; Merck KGaA, Darmstadt, Germany]. Primary antibodies were applied for 40 min followed by automated staining (TechMateTM 500; Dako) using the Dako REALTM Detection System following the manufacturer’s instructions. The following primary antibodies were used: CD9, CD44V6, CD54, CD56, CD117, SYP, NSE, CHGA, CK5/6, CK7. Details to the clones, dilutions and manufacturers are given in the supplementary methods. Tissue sections were analyzed and photographed on an Olympus IX-71 inverted microscope equipped with a ColorView II Camera (Olympus Deutschland GmbH, Hamburg, Germany). All immunohistochemical analyses were performed by the Tissue Bank of the National Center for Tumor Diseases (NCT), Heidelberg. 2.4. Growth kinetics, colony efficiency (CE) and tumorigenicity 10,000 cells of passage 5 were seeded into 75 cm2 flasks and cultured for 14 days. Each other day the cell number was counted in a Neubauer chamber. The CE was determined in a soft agar system [4]. Bottom layers of 0.5% agarose (SeaKem, Rockland, Inc., Rockville, ME, USA) in CM were allowed to harden in 35 mm Petri dishes. 104 –105 cells/ml of passage 5 of each cell line were suspended in CM containing 0.3% agarose. The bottom layer was overlayed with 1 ml of the suspension and incubated in a humidified atmosphere at 37 ◦ C, 5% CO2 . After 21 days colonies containing ≥50 cells were counted under an inverted microscope. The CE was calculated as proportion of colonies per total number of seeded cells. Tumorigenicity of the cell lines was analyzed between passage 9 and 12 in a murine xenograft model and is stated in detail in the supplementary methods [10]. 2.5. Adhesion, migration and invasion For adhesion, 100,000 cells/well were seeded into 24-well dishes. The cells were briefly spun down (30 s, 500 g), cultured and washed after given times on a horizontal shaker. Adherent cells were fixed, stained with 0.5% crystal violet (Sigma) in 99.9% methanol (Roth) and counted under an inverted microscope. Adhesion was calculated as proportion of adherent cells per total number of seeded cells. Migration through 12 ␮m pore size polycarbonate filters (Neuro Probe, Inc., Gaithersburg, USA) was analyzed in Boyden chambers containing 100 ng/ml stromal cell-derived factor 1 alpha (SDF-1˛; PeproTech GmbH, Hamburg, Germany) in the lower compartment. For invasion, filters were coated with matrigel (BD; 50 ␮g protein/filter). All analyses were performed with cells of passage 5. Details to the migration and invasion experiments are given in the supplementary methods. 3. Chemosensitivity 10,000 cells/well of passage 5 were grown in 96-well plates and exposed to various concentrations of cisplatin (0.5, 1, 2, 2.5, 5, 7.5, 10 ␮M; GRY-Pharma GmbH, Kirchzarten, Germany),

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Fig. 1. (A) Morphologies of cell lines and corresponding tissue. Cell lines and corresponding tissue (upper row) display similar morphologies: cells of donor 2106 are mid-size and polygonal with membrane protrusions, prominent nucleoli and intercellular bridges (arrows). Cells of donor 2427 are small, round, show a high nucleus-to-cytoplasm ratio and grow in clusters. In line with poor differentiation, keratinization is lacking except for single cells in tissue 2427. Microphotographs: upper and lower row: scale bar: 50 ␮m, middle row: scale bar: 25 ␮m. (B and C) Surface marker profiles of cell lines and corresponding tissue. Comparison of flow cytometric (B) and immunohistochemical results (C) show similar surface marker profiles of cell lines and corresponding tissue. The marker panel contains antigens associated with multidrug resistance (CD9), haematoendothelial progenitor cells (CD34), leukocytes (CD45), adhesion (CD44, CD54), neuroendocrine differentiation (CD56, SYP: synaptophysin, NSE: neuron-specific enolase, CHGA: chromogranin A), growth/differentiation (CD117), squamous (cytokeratin 5/6 [CK5/6]) and adenocarcinoma differentiation (CK7). Only positive immunohistochemical results are shown. (B) Histograms: grey lines: isotype or secondary antibody control, black areas: primary antibody; x-axis: fluorescence intensity [log], y-axis: cell count. (C) Microphotographs of hematoxylin stained tissue sections: scale bar: 100 ␮m; microphotographs 1, 2, 4–6, 8, 11–14: scale bar: 50 ␮m.

docetaxel (0.05, 0.1, 0.5, 0.75, 1, 5, 7.5, 15 nM; Aventis Pharma Deutschland GmbH, Frankfurt, Germany), gemcitabine (2, 4, 12.5, 25, 35, 350 nM), pemetrexed (0.025, 0.25, 1.25, 2.5, 12.5, 25 mM; Lilly Deutschland GmbH, Bad Homburg, Germany) and vinorelbine (0.5, 1, 2.5, 5, 10, 100 nM; Pierre Fabre Pharma GmbH, Freiburg, Germany) in CM for 24 h. Subsequently the cells were washed with DPBS, maintained for another 72 h in fresh CM and analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as stated in the supplementary methods. Cisplatin-treated cells were additionally analyzed by flow cytometry after staining with the ApoptestTM Kit (Nexins Research, Kattendijke, The Netherlands).

20 h. Hypotonic treatment was performed using 0.0375 M KCl solution (Roth) for 20 min. M-FISH was done as previously described and is stated in detail in the supplementary methods [12]. STR DNA typing of the cell lines was performed by the Leibnitz Institute DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) and is stated in detail in the supplementary methods [13]. Cell lines and tumor tissue were analyzed for EGF-R mutations as previously published [14]. Kras mutation analysis was performed as stated in detail in the supplementary methods. 3.2. Microarray experiments and quantitative real-time polymerase chain reaction (qRT-PCR)

3.1. Nucleic acid isolation Nucleic acid of the cell lines at passage 5, snap-frozen tumor or normal lung tissue was isolated for comparative genomic hybridization (CGH), single tandem repeat (STR) DNA typing, mutation analysis and gene expression profiling. Details to the tumor cell and tissue preparation, RNA and DNA isolation are given in the supplementary methods. CGH, multiplex fluorescence in situ hybridization (M-FISH), STR DNA typing, EGF-R and k-ras mutation analysis CGH experiments were performed as previously described [11]. Details to the method are given in the supplementary methods. For M-FISH analysis, metaphase spreads of subconfluent tumor cells were prepared by adding colcemide (final concentration: 0.04 ␮g/ml; Invitrogen GmbH, Darmstadt, Germany) to the CM for

Gene expression analysis was performed using the Affymetrix U133 Plus 2.0 microarray (Affymetrix, Santa Clara, CA, USA) covering 47,000 transcripts and variants. The microarray data were normalized as previously described and deposited into the NCBI GEO database (GSE25251) [15]. With respect to the small number of hybridizations, an unsupervised approach was used to select genes and distinguish between experimental conditions. Gene ontology (GO) statistics of the 500 most variably expressed genes across all hybridizations was performed as previously described [15]. For hierarchical clustering, a subset of 265 genes was selected from the most prominent and cancer-relevant GO terms. From these genes fold change was calculated between tissue and cell lines, between different donors and between primary tumor and lymph node metastasis. GO terms were assigned to distinct experimen-

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Fig. 1. (Continued )

tal conditions by calculating the fraction of up- or down-regulated genes. Potentially conserved genes that showed similar expression between tissue and corresponding cell line were extracted from hierarchical clustering. Moreover, differential expression of selected genes potentially associated with the chemosensitivity of the cell lines was analyzed by qRT-PCR as previously described and is stated in detail in the supplementary methods [15]. 3.3. Statistics Functional analyses were made in quintuplicate. Results are given as the mean ± standard error of the mean (SEM). Due to the small sample size and descriptive character of the results no statistical analyses were performed.

4. Results 4.1. NSCLC cell lines and corresponding tissue show similar morphologies and surface marker profiles Three squamous cell NSCLC cell lines were established from two primary tumors (2106T, 2427T) and one lymph node metastasis (2106LN) of 28 donors, comprising 8 SCC. Cell lines and corresponding tissue displayed similar morphologies (Fig. 1A) and surface marker profiles (Fig. 1B and C): cells of donor 2106 were mid-size, polygonal and showed intercellular bridges, whereas cells of donor 2427 were small, round and grew in clusters. Consistent with SCC origin, all cell lines expressed CK5/6. However, only a fraction of 2427T cells was CK5/6 positive. Antigens

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Fig. 1. (Continued )

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Table 1 Comparative genomic hybridization (CGH) of cell lines and corresponding tissue.

The CGH shows essential genetic similarities between cell lines and corresponding tissue. Cell lines 2106T and 2427T exhibit slightly more alterations than their related tumor tissue, whereas cell line 2106LN shows less alterations. Aberrations that are exclusively contained in cell line or tissue are highlighted in grey. Threshold: loss 0.85, gain 1.15.

associated with AC histology (CK7), hemato-endothelial progenitors (CD34) or leukocytes (CD45) were lacking. Neuroendocrine markers (CD56, SYP, NSE, CHGA) were negative in 2106T and 2106LN cells, whereas a small proportion of 2427T cells expressed SYP. Other antigens, involved in drug transport (CD9), adhesion (CD44, CD54) or growth/differentiation (CD117) showed differential expression between the donors and between the primary tumor and lymph node metastasis. Immunophenotyping was performed at various passages and revealed a stable antigen profile. Immunohistochemical analyses of the corresponding tumor tissue showed congruent results except for CD9, that was strongly expressed in 2106T tissue, but not in its cell line (supplementary Table 2). 4.2. NSCLC cell lines and corresponding tissue show essential genetic similarities CGH analysis of cell lines and corresponding tissue revealed complex karyotypes with multiple aberrations (supplementary Fig. 1, Table 1). In total, more gains than losses were present. Those affected all autosomes except for chromosomes 13 and 18, whereas losses were predominantly found in chromosomes 3–5, 9, 13 and 18. Several alterations containing oncogenes such

as k-ras or myelocytomatosis viral oncogene homolog (myc) and tumor suppressor genes such as p14, p16, p53, p63, Ras association domain family member 1 (RASSF1A) and retinoblastoma 1 (Rb1) were shared by all cell lines and tissues. The cell lines 2106T and 2427T showed slightly more alterations than their corresponding tissue in terms of larger or additional regions that were lost or amplified indicating some genetic evolution in vitro. In contrast, 2106LN cells exhibited less alterations than their corresponding tissue providing evidence for clonal selection in vivo and/or in vitro. Additional M-FISH analyses of the cell lines revealed several structural aberrations and 40% common changes of the cell lines 2106T and 2106LN (supplementary Fig. 2). Neither cell lines, nor tumor tissue showed EGF-R or k-ras mutations. All cell lines were authenticated by STR DNA typing and showed a unique, heterozygous human DNA profile (supplementary Fig. 3A and B). 4.3. NSCLC cell lines and corresponding tissue show overlapping transcriptomic profiles Genome-wide expression profiling showed striking transcriptomic patterns for cell lines, tissue and the various donors. GO

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Table 2 Gene ontology (GO) statistics of cell lines and corresponding tissue. Name

GO

Genes

Group count

Total count

p value

Immune response

GO:0006955

68

1189

1.29E-G1

Proliferation

GO:0008283

48

745

2.46E-50

Inflammatory response

GO:0006954

42

291

1.89E-34

Differentiation

GO:0030154

66

1810

1.44E-30

Cell-cell signaling

GO:0007267

34

640

5.40E-27

Adhesion

GO:0007155

42

960

2.44E-25

Apoptosis

GO:0006915

29

855

2.95E-11

Motility

GO:0006928

23

383

1.49E-10

Nervous system development

GO:0007399

24

716

3.85E-09

Angiogenesis

GO:0001525

c1qa; cd14; psmb9; igj; il15; hla-dqa1; il1b; cxcl12; cxcl9; ccl18; c1qb; fcgr2a; c1qc; ctss; ccl4; cxcl1; fcer1g; ccl2; il6; il8; trem1; cxc1l0; fcgr1b; il32; fcgr3b; tnfsf10; tnfsf13b; ifitm3; cxcl3; ptprc; il23a; cd55; gzma; ccl8; igkc; Icp2; cd74; igkv4-1; cxcl2; tgfb2; hla-dpb1; fyb; rgs1; trim22; psmb8; serpinb4; hla-dra; arhgdib; ighm; ighg1; serping1; c3; fcgr2b; ets1; ighg3; il7r; slamf7; ccl3; bst2; hla-dqb1; ulbp2; ccl5; gbp1; ereg; hla-dpa1; hla-drb5; cxcr4; gbp3 cd74; gja1; igf2; il11; il15; gcg; timp1; tgfb2; il1b; fgf9; ptgs2; fgg; s100a11; ighm; pin; s100a6; cxcl1; igfbp7; bnc1; pou3f2; il6; rarres1; ets1; gpnmb; il8; cxcl10; tgm2; cdkn2a; jag1; tacstd2; bst2; nox5; areg; cd47; tnfsf13b; hcls1; ehf; rac2; tgfbi; ereg; ptprc; ifiim1; cav2; vsig4; aif1; ifi16; cxcr4; serpinf1 c1qa; cd14; s100a8; cxcl2; lyz; il1b; itgb2; cxcl12; cd163; ptgs2; nr3c1; cxcl9; ccl18; c1qb; hdac9; c1qc; cybb; serpina1; ccl4; cxcl1; fcer1g; serping1; alox5ap; alox5; pla2g7; c3; ccl2; il6; fn1; il8; ccl3; cxcl10; tgm2; tnfaip6 ccl5; cxcl3; il23a; cd55; aif1; cxcr4; s100a9; ccl8 cd14; lgals1; daz1; timp1; il1b; gzmb; itgb2; fgf9; s100a4; prf1; snca; hdac9; s100a6; spry1; fcer1g; mgp; ednrb; ccl2; tubb2b; ckap2; mmp9; l1cam; il6; mafb; tgm2; ppp1r9a; cdkn2a; nox5; sox2; tnfsf13b; tnfsf10; tnfrsf19; cav2; ptprc; epas1; gzma; ifi16; serpinf1; adam18; cd74; gja1; tmem176b; il2rb; sfrp2; il11; lyz; tgfb2; otx2; znf423; anpep; pappa; gpm6b; pou3f2; ets1; bex1; slamf7; tnc; jag1; sulf1; eomes; krt19; hcls1; srgn; ehf; ereg; cxcr4 c1qa; gja1; il11; il15; tgfb2; il1b; itgb2; fgf9; cxcl12; cxcl9; ccl18; snca; s100a6; snai2; ccl4; spry1; gad1; ccl2; mme; il6; il8; ccl3; cxcl10; fst; bst2; areg; tnfaip6; tnfsf10; ccl5; syt4; ereg; cav2; s100a9; ccl8 col7a1; pcdh7; cd44; alcam; col6a3; pcdh18; itgb2; col11a1; nell2; cxcl12; nid1; cd93; arhgdib; mcam; ccl4; cdh11; mgp; ccl2; flrt3; i1cam; slamf7; fn1; il8; tnc; postn; thbs2; cdh10; tgm2; adamdec1; cd300a; cdkn2a; col12a1; il32; cd47; tnfaip6; ccl5; tgfbi; spon1; vcam1; cdh1; Igals3bp; spp1 ccl2; ckap2; cd14; mmp9; cd74; gja1; il6; il2rb; slamf7; Igals1; cdkn2a; il1b; tgfb2; tgm2; itgb2; gzmb; sulf1; nox5; prf1; tnfsf13b; tnfsf10; srgn; tnfrsf19; ptprc; snca; gzma; ifi16; cxcr4; fcer1g ednrb; gja1; fn1; il8; s100a2; cxcl10; ccl3; mmp12; tgfb2; il1b; jag1; itgb2; otx2; ptgs2; ccl5; cav2; enpp2; arhgdib; s100p; ccl4; cxcr4; s100a9; fcer1g ednrb; tubb2b; gpm6b; pou3f2; l1cam; sepp1; gja1; bdnf; bex1; foxg1; pcdh18; ppp1r9a; tgfb2; jag1; otx2; ptprz1; znf423; cav2; snca; ptn; s100a6; cxcr4; serpinf1; cxcl1 ereg; il8; epas1; anpep; tgfb2; serpinf1; cxcr4; il1b; jag1; fgf9; col4a2

11

129

8.92E-07

GO statistics of the 500 most variably expressed genes across all experiments reveals significantly over-represented genes in distinct ontology terms. Shown are GO terms and included genes, counts and p values.

statistics of the 500 most variably expressed genes revealed several cancer-associated GO terms (supplementary Table 3). In total, 265 of the 500 most variably expressed genes were related to common cancer-relevant GO terms (Table 2). Unsupervised clustering of these genes indicated three different gene clusters (Fig. 2A). The largest one reflected broad silencing of genes associated with immune system, proliferation, differentiation and cell adhesion in the cell lines (Fig. 2A and B). In tissue, the most overrepresented cluster (60/68 genes, 88%) contained genes involved in immune response (Fig. 2B). However, there was also a clear association between cell lines and corresponding tissue. A large fraction of genes related to proliferation (60%), adhesion (50%) and angiogenesis (73%) were over-represented in donor 2106, whereas genes

related to nervous system development (50%) were up-regulated in donor 2427 (Fig. 2B, supplementary Table 4). Only very few differences were found between the primary tumor and lymph node metastasis. Those affected predominantly angiogenesisrelated genes, that were over-represented in the primary tumor. 4.4. NSCLC cell lines show donor-specific functional properties that are related to gene expression profiles Assessment of growth kinetics revealed major proliferation and colony efficiency (CE) of 2427T cells and minor proliferation and CE of 2106LN cells (Fig. 3A and B). Adhesion kinetics showed rapid

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Fig. 2. (A and B) Gene expression profiles of cell lines and corresponding tissue. (A) Unsupervised clustering revealed a subset of 265 genes associated with cancer-relevant gene ontology (GO) terms. Those formed three main gene clusters characterizing the cell lines, donor 2106 and donor 2427. (B): The bar plots demonstrate the proportion of genes in an ontology term, that was more than twofold up- (right bars) or down-regulated (left bars) in the comparisons () “tissue vs. cell line”, () “donor 2106 vs. 2427 and () “2106T vs. 2106LN cells”. For the comparisons “donor 2106 vs. 2427 and “2106T vs. 2106LN cells” the expression data of tissue and related cell line were pooled.

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likewise no differential expression, although various sensitivity of the cell lines to cisplatin was confirmed by two different assays.

5. Discussion

Fig. 3. (A and B) Growth kinetics and colonogenicity of the cell lines. (A) The cell lines show different proliferation capacity (2427T > 2106T > 2106LN). x-Axis: time [days], y-axis: expansion [x-fold]; values: mean ± SEM, n = 3. (B) 2106LN cells show less colonogenicity than 2106T and 2427T cells. X-axis: cell line, y-axis: colony forming units (CFU) per total number of seeded cells [%]; values: mean ± SEM, n = 3.

adhesion of all cell lines. However, full adhesion was at first reached by 2106T cells after 2h, whereas 2106LN and 2427T cells took three times longer (Fig. 5A). Likewise, migration was superior in 2106T cells (11.1 ± 1.1%), whereas migration of 2427T and 2106LN cells was 3.5 and 8.5 fold less (supplementary Figs. 4 and 5B). Invasion was equal in 2106T (3.1 ± 0.5%) and 2427T cells (3.0 ± 1.0%) and negligible in 2106LN cells (Fig. 5C). In line with these results, 78% of the genes contained in the GO cluster “cell adhesion” were more than twofold up-regulated in donor 2106. However, 76% of the genes contained in the cluster “proliferation” were up-regulated in donor 2106, although proliferation activity was superior in 2427T cells (Fig. 2B). Cell line 2427T showed rapid tumor formation in nude mice reaching a tumor diameter of 1.5 cm after 28 days. The take rate was 100% per animal and injection site. The tumors reassembled the original basaloid histomorphology and antigen profile showing partial expression of CK5/6 and SYP (Figs. 4 and 5). The cell lines 2106T and 2106LN displayed no tumorigenicity after 28 days. Analysis of chemosensitivity demonstrated, that 2427T cells were most sensitive to cisplatin and gemcitabine, whereas 2106T cells were most sensitive to docetaxel (Fig. 6A–D). The dose–effect curves of pemetrexed and vinorelbine were similar for all cell lines (Fig. 6E and F). Upon data-mining of the transcriptomic profile we considered potential candidate genes for qRT-PCR validation, whose association with substance-specific chemosensitivity to the first-line cytostatics cisplatin, gemcitabine and pemetrexed has been reported. Notably, cytidine deaminase (CDA) was >600 fold up-regulated in 2106T and 2106LN cells, which were less sensitive to gemcitabine, whereas thymidylate synthetase (TYMS) and phosphorybosylglycinamide formyltransferase (GART) showed no differential expression between the cell lines corresponding to the similar dose–effect curves for pemetrexed (supplementary Table 5, 6 and Fig. 5). However, excision repair cross-complementing rodent deficiency repair, complementation group 1 (ERCC1) showed

We established three novel squamous cell NSCLC cell lines derived from two primary tumors (2106T, 2427T) and one lymph node metastasis (2106LN). One cell line (2427T) was highly tumorigenic in nude mice. The success rates of 7% (2/28 NSCLC) and 25% (2/8 SCC) were about in line with previous results reported for the establishment of NSCLC (6–9%) and SCC (1–14%) [4,5,16,17]. Since a defined culture medium for SCC is lacking and keratinization and growth arrest the major obstacles for long-term culture, the availability of pulmonary SCC cell lines is limited [3]. Particularly the combination of a primary tumor- and lymph node metastasis-derived SCC cell line is unique. Success rates in lymph node metastases are very poor [5]. As no specific procedures or medium supplements were used, successful establishment of these cell lines has to be attributed to yet undetermined factors comprised in the tissue properties or culture conditions. All cell lines expressed CK5/6. For this antigen a sensitivity of 79% and specificity of 89% to detect even poorly differentiated SCC has been reported [18–20]. However, 2427T cells showed only partial expression of CK5/6, and moreover, co-expression of the neuroendocrine marker SYP. Besides, gene expression profiling revealed an over-representation of genes associated with nervous system development indicating a SCC with partial neuroendocrine differentiation. Other antigens involved in drug transport (CD9), adhesion (CD44, CD54) and growth/differentiation (CD117) were likewise differentially expressed between the donors. Also primary tumor and lymph node metastasis varied in their antigen profile: 2106LN cells partly expressed CD9 and CD117, whereas 2106T cell did not. CD117 is found in up to 30% of NSCLC and potentially associated with faster division kinetics, cancer stem-like cell properties and a higher metastatic potential [21–26]. Comparison of the surface marker profiles of the cell lines and their corresponding tissue revealed an excellent concordance. However, these analyses also demonstrated intra- and interindividual differences between primary tumor and lymph node metastasis and between the donors. Genotyping by CGH revealed complex karyotypes of cell lines and tissues with an average number of 27 gains and 10 losses. All cell lines and tissues displayed loss of regions containing the tumor suppressor genes Rb1, cyclin-dependent kinase inhibitor 2A, semaphorin 3B, RASSF1A and adenomatous polyposis coli, that are fequently lost in NSCLC [27–30]. Chromosomal regions covering the oncogenes v-myc, jun oncogene and c-abl oncogene 1, receptor tyrosine kinase were consistently amplified. The CGH profiles of cell lines and corresponding tissue showed high concordance: 2106T and 2427T cells shared 95% and 100% (21/22 and 25/25) of the gains and 90% (9/10) and 100% (7/7) of the losses with their corresponding tissue. In line with these results, Wistuba et al. who analyzed microsatellite alterations and LOH in 13 chromosomal regions of 12 NSCLC and 18 breast cancer cell lines found 75–97% concordance with their corresponding tumor tissue [7,8]. However, the SCC cell lines contained additional alterations that were not detected in the original tissue. Moreover, changes in the cell lines covered larger chromosomal regions than in the tissue indicating genetic evolution in vitro. Surprisingly, 2106LN cells exhibited fewer alterations than their tissue. This might reflect clonal selection in vivo and/or in vitro, potentially based on a larger genetic heterogeneity of the lymph node metastasis. Indeed, a paired CGH analysis of Petersen et al. showed considerable chromosomal instability and genetic heterogeneity between primary

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Fig. 4. Tumorigenicity of the cell lines. 5 × 106 cells of each cell line were resuspended in 200 ␮l DPBS/matrigel (1:1, v/v) and subcutaneously injected into both flanks of 6week-old, female NMRI nu/nu mice (n = 3). The tumors were harvested, when the largest diameter reached 1.5 cm. Shown is a 1.4 cm × 1.2 cm sized tumor derived from cell line 2427T (passage 12) that was harvested 28 days after injection. The tumor reassembled the original basaloid histomorphology and antigen profile showing partial expression of cytokeratin 5/6 (CK5/6) and synaptophysin (SYP). Other neuroendocrine markers (CD56, neuron-specific enolase [NSE], chromogranin A [CHGA]) and the adenocarcinoma antigen cytokeratin 7 (CK7) were negative. The cell lines 2106T and 2106LN showed no tumor formation after 28 days. Microphotographs: HE: hematoxylin-eosin, scale bar: 10 ␮m.

S. Gottschling et al. / Lung Cancer 75 (2012) 45–57

Fig. 5. (A–C) Adhesion and motility of the cell lines. The cell lines show different adhesion (A), stromal cell-derived factor 1 alpha (SDF-1˛)-mediated migration (B) and invasion (C). (A) x-Axis: time [h], y-axis: proportion of adherent cells [%]; values: mean ± SEM, n = 3. (B) and (C) SDF-1˛-mediated () and spontaneous () migration or invasion. x-Axis: cell lines, y-axis: migration [%] or invasion [%]; values: mean ± SEM, n = 3.

tumor and metastatic lymph node tissue of ten pulmonary SCC [31]. A more recent analysis using whole genome high resolution single nucleotide polymorphism arrays in 8 sets of primary and metastatic lung cancer revealed >67% common alterations, but also exclusive alterations of the metastatic tissue in 7/8 cases [32]. In our study, the tissue of the lymph node metastasis shared 100% (1722/22) of the gains and 80% (8/10) of the losses with its primary tumor. Besides clonal selection and genetic evolution technical limitations of the applied method have to be considered. Chromosomal CGH recognizes alterations only if present in half of the analyzed cells and if associated with loss or gain of chromosomal fragments ≥10 megabases [33]. Thus, small clones or balanced alterations and structural changes will not be detected by CGH. In fact, M-FISH analyses of the cell lines showed various structural changes, and moreover, an underestimate of losses, likely due to the near triploid karyotype of the tumor cells that impedes detection of monoallelic losses by CGH [34]. All in all, comparative genotyping indicated broad similarities between tumor tissue and corresponding cell line, but also genetic evolution and clonal selection in vitro. Whether such differences contain driver mutations that define the phenotype of a tumor cell or passenger mutations that are negligible for the cellular function has yet to be determined [35,36]. Gao et al. demonstrated that changes in the copy number of a chromosome quantitatively impose a proportional change in the chromosome transcriptome ratio and

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affect genes that are relevant for phenotype determination [37]. Here, whole genome expression analysis demonstrated significant differences between cell lines and tumor tissue. Several relevant gene clusters associated with proliferation, differentiation, adhesion, immune response and angiogenesis were strongly silenced in the cell lines. These expected changes might reflect the fundamental reprogramming of cell signalling processes upon loss of complex interactions and change of the microenvironment during cell culture. Despite these differences, there was a specific transcriptomic activity of the cell line and tissue of a donor. These similarities might help to prioritize tumor-relevant genes and processes for functional studies in cell lines. Cells of donor 2106 showed, for instance, conserved expression of several genes linked to proliferation (cyclin-dependent kinase inhibitor 2A [CDKN2A], epiregulin [EREG], transforming growth factor ˇ2 [TGFB2], jagged 1 [JAG1]), angiogenesis (endothelial PAS domain protein 1 [EPAS1], fibroblast growth factor 9 [FGF9], serpin peptidase inhibitor, clade F, member 1 [SERPINF1]) and adhesion (L1 cell adhesion molecule [L1CAM], melanoma cell adhesion molecule [MCAM], activated leukocyte cell adhesion molecule [ALCAM], CD44, CD47, fibronectin 1 [FN1]). Robust expression of adhesion-related genes might reflect the increased adhesion capacity of these cells, whereas conserved expression of genes associated with nervous system development (brain expressed, X-linked 1 [BEX1], POU class 3 homeobox 2 [POU3F2], synuclein ˛ [SNCA]) might correspond to the partial neuroendocrine differentiation of donor 2427. Differentiation-related genes were consistently conserved in both donors indicating the stability of substantial histologic characteristics during the transition from tissue to cell culture. Although the variability between primary tumor and lymph node metastasis was minor, functional analyses revealed significant differences in the proliferation, motility and partly chemosensitivity of cell lines 2106T and 2106LN. This challenges global genotyping and transcriptomic profiling for characterization of cell lines and tissue. Neither technique considers the contribution of various clones or specific genes characterizing a tissue, disease or process. Indeed, qRT-PCR analysis of selected genes potentially associated with substance-specific chemosensitivity to gemcitabine and pemetrexed revealed accordance between the gene expression and responsiveness of the cell lines [38,39]. However, ERCC1 involved in the repair of cisplatin-induced DNA damage showed no differential expression despite various sensitivity of the cell lines to this drug [40]. This indicates either an activity of a different mechanism or an effect beyond the mRNA level. Thus, analyses of critical genes should precede studies of entire pathways in cell lines and consider multiple aspects of gene expression including protein synthesis and posttranslational modifications. In summary, our data demonstrated that cell lines are a useful tool for systematic analyses of fundamental pathways in cancer, but cover only distinct molecular characteristics of the original tumor and possess considerable intra- and interindividual variability. Moreover, we present three novel, comprehensively characterized SCC cell lines containing the unique combination of a primary tumor- and a lymph node metastasis-derived cell line.

Grant support S.G., H.S., R.K. and C.B. are recipients of the annual joint grant of the German Cancer Research Center and the Thoraxklinik Heidelberg. This work was partly supported by grants of the Deutsche Krebshilfe, Tumorzentrum Heidelberg/Mannheim (A.J.) and German Federal Ministry of Education and Science (FKZ:01GS0890; R.K. and H.S.).

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Fig. 6. (A–F) Chemosensitivity of the cell lines. (A) Dose–effect curve of cisplatin. 2427T is the most sensitive cell line, 2106T the most resistant. x-Axis: cisplatin concentration [␮M], y-axis: proportion of viable cells [%]; mean ± SEM, n = 3. (B) Flow cytometric analysis of annexin V/propidiumiodide stained cells exposed to 2.5 ␮M cisplatin. The tables within the dot plots show the proportions of viable (quadrant lower left), apoptotic (quadrant lower right), secondary necrotic (quadrant upper right) and primary necrotic cells (quadrant upper left). n = 3. (C–F) Dose effect curves of gemcitabine, docetaxel, pemetrexed and vinorelbine. 2427T is most sensitive to gemcitabine, 2106T most sensitive to docetaxel. The cell lines show equal sensitivities to pemetrexed and vinorelbine. X-axis: cytostatics concentration [mM] or [nM], y-axis: proportion of viable cells [%]; values: mean ± SEM, n = 3.

Conflict of interest None declared. Acknowledgements The authors thank Mmes. Christa Stolp (Translational Research Unit, Thoraxklinik/University of Heidelberg), Bettina Walter (Tissue Bank, NCT), Brigitte Schoell and Heidi Holtgreve-Grez (Institute of Human Genetics) for excellent technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.lungcan.2011.05.020. References [1] American Cancer Society: Cancer Statistics; 2008. http://www.cancer. org/docroot/STT/stt 0.asp. [2] Schiller JH, Harrington D, Belani CP, Langer C, Sandler A, Krook J, et al., Eastern Cooperative Oncology Group. Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 2002;346:92–8.

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