[CANCER RESEARCH 63, 1943–1953, April 15, 2003] Transcriptional Gene Expression Profiling of Small Cell Lung Cancer Cells1 Nina Pedersen,2 Shila Mortensen,2 Susanne B. Sørensen, Mikkel W. Pedersen, Klaus Rieneck, Lone F. Bovin, and Hans Skovgaard Poulsen3 Department of Radiation Biology, The Finsen Centre [N. P., S. M., M. W. P., S. B. S., H. S. P.] and Institute for Inflammation Research, IIR 7521 [K. R., L. F. B.], National University Hospital, Copenhagen DK-2100, Denmark ABSTRACT expression profiles, which can distinguish between these subclasses, have been revealed by microarray analyses (1–3). A global gene expression analysis using oligonucleotide microarrays Because of the aggressive behavior of SCLC and the very poor was performed on many human small cell lung cancer (SCLC) cell lines in outcome of present treatments, new therapeutic methods for systemic cell culture and/or as xenografts. The expression was compared with the expression profiles of 18 normal tissues. treatment of SCLC are in high demand. Using global gene expression In a hierarchical cluster analysis the cell lines clustered distinctly from analysis we have searched for genes that are highly and/or specifically normal tissues and grouped into four clusters. One cluster consisted of two expressed in all or most of the tumor cells with the aim to identify related cell lines and was markedly different from the other SCLC cell novel potential targets for the development of new therapeutic agents. lines, whereas the rest of the clusters grouped together. Two subclusters These could be surface molecules for direct targeting in radio-, toxin, contained the classical SCLC types and one subcluster the variant SCLC or gene therapy, or molecules to which development of cancer vac- type, thus identifying many genes with differential expression between the cines could be used. Other potential targets are molecules involved in two variants of SCLC. All of the xenografts clustered closest to the cell maintenance of the malignant phenotype, such as oncogenes and lines from which they originated and had the same expression levels as the antiapoptotic molecules, to which inhibitors can be applied or lost cells grown in culture for the majority of genes. activity restored. The analysis confirmed the high expression of many genes identified previously as highly expressed in SCLC cells including neuroendocrine A characteristic of all of tumors and their metastases, both between markers, oncogenes, and genes involved in cell proliferation and division. patients and within a tumor, is their heterogenicity, making the de- The analysis furthermore identified a number of molecules not identified velopment of therapeutic strategies difficult, as some cells invariably previously as expressed in SCLC. Several of these are expressed in low or can escape the treatment. Sufficient material of SCLC tumors from undetectable amounts in the majority of normal tissues and, therefore, are patients is extremely difficult to obtain both in number of specimens potential targets for new therapeutic approaches. By including the pub- and sufficient amounts for microarray analysis. Therefore, we used an lished array profiles of six ressected SCLC tumors from Bhattacharjee et alternative approach and used the expression profiles of 21 SCLC al. (A. Bhattacharjee et al., Proc. Natl. Acad. Sci. USA, 98: 13790–13795, cells lines obtained from five different laboratories and 8 xenografted 2001.), the analysis revealed that most of the novel potential targets tumors from these cell lines to compare to the expression profiles of expressed by SCLC cell lines and xenografts were also expressed in the 17 normal adult tissues. By this analysis, we identified several genes tumors. This analysis demonstrates the value of using cell lines and xenografts highly and specifically expressed by all or most of the SCLC cell for expression profiling, when a limited quantity of tumor material is lines, xenografts, and 6 ressected tumors with no or little expression available. in normal tissues, which could be candidates for therapeutic targeting. In addition, the analysis clearly divided the SCLC cell lines into two INTRODUCTION distinct subclasses with different expression profiles. SCLC4 is an aggressive disease, which is generally disseminated at MATERIALS AND METHODS the time of diagnosis. Initially the cancer is responsive to chemother- apy, but almost always recurs in a resistant form resulting in a 5-year Cell Culture. The following human SCLC cell lines were used: CPH 54A, survival rate of Ͻ5%. SCLC is generally correctly identified by CPH 54B (4), GLC-2, GLC-3, GLC-14, GLC-16, GLC-19, GLC-26, GLC-28 pathological means, wherefore identification of new markers for clas- (5–7), DMS 53, DMS 79, DMS 92, DMS 114, DMS 153, DMS 273, DMS 406, sification of this tumor type is not pertinent. This is in contrast to the DMS 456 (8), NCI H69, NCI N417 (9), MAR 24H and MAR 86MI (10, 11). situation for discrimination of subclasses of other lung tumor forms, CPH 54 A and B were propagated in MEM (Eagle), all of the GLC, NCI, MAR such as adenocarcinomas, for which therapeutic response and survival cell lines and DMS 79 were propagated in RPMI 1640, and all DMS (except DMS 79) were propagated in Waymouth medium, all supplemented with 10% rates can differ markedly despite similar pathology. Differences in FCS. All of the serum and media were obtained from Invitrogen (Tåstrup, Denmark). Received 8/28/02; accepted 2/14/03. Xenografts. Cells (0.5–1.2 ϫ 107) from the cell lines CPH 54A, GLC-3, The costs of publication of this article were defrayed in part by the payment of page GLC-14, DMS 273, NCI H69, NCI N417, and MAR 24H were inoculated charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. bilaterally in the flanks of 12–13-week-old BALB/c nude mice. The mice were 1 Supported by Odin Medical A/S, the Danish Cancer Society, the Danish Medical sacrificed, and the xenografted tumors were harvested when one of the tumors Research Council, The Danish Rheumatism Association, and the A. P. Møller Foundation had reached a maximal diameter of 1 cm. The cell line CPH 136A was only for the Advancement of Medical Science. propagated in nude mice by inoculation of a 2-mm tumor block. Necrotic tissue 2 These two authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Department of Radiation was removed, and the tumors were either processed immediately or stored in Biology, Finsen Centre, Section 6321, National University Hospital, Blegdamsvej 9, RNAlater (Ambion, Cambridgeshire, United Kingdom) for RNA extraction. DK-Copenhagen 2100, Denmark. Phone: 45-35-45-63-03; Fax: 45-35-45-63-01; E-mail: Isolation of RNA. Total RNA from normal, human tissues were obtained skovgaard @rh.dk. from either Clontech (Brøndby, Denmark; fetal brain, brain, lung, kidney, 4 The abbreviations used are: SCLC, small cell lung cancer; RT-PCR, reverse transcrip- tion-PCR; MAGE, melanoma-associated antigen; DDC, L-dopa decarboxylase; GRP, gastrin- heart, trachea, adrenal gland, prostate, salivary gland, and thyroid) or from releasing peptide; SGNE1, secretory granule, neuroendocrine protein 1; ASCL1, achaete scute Ambion (lung, liver, brain, pancreas, spleen, small intestine, skeletal muscle, homologous protein; NCAM1, neural cell adhesion molecule 1; GRIA2, ionotropic glutamate colon, stomach, and testis). Only one sample was analyzed in duplicate (lung receptor 2; GRM8, metabotropic glutamate receptor 8; NPTXR, neuronal pentraxin receptor; RNA from Clontech and Ambion) and one in triplicate (two different batches ITGAE, integrin subunit ␣ E; PTTG1, pituitary tumor transforming gene (securin); TIMP, tissue inhibitor of metalloproteinase; MMP, metalloproteinase; pRB, retinoblastoma protein; brain RNA from Clontech and one from Ambion). The duplicates and tripli- INSM1, insulinoma-associated antigen 1; ENO2, neuron-specific enolase 2 (NSE). cates showed similar expression profiles, and only the results using the RNA 1943 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2003 American Association for Cancer Research. CHIPS ANALYSIS OF SMALL CELL LUNG CANCER from Ambion are shown here. RNA from cell lines in exponential growth was cell lines were distributed in 4 subclasses (A, B, C, and D). The cluster harvested (after trypsinization for adherent cells), and total RNA from ϳ107 D only contains the CPH 54 cell lines and has an expression pattern cells was isolated using the RNeasy kit (Qiagen) according to the manufac- distinctly different from the remainder of the SCLC lines. The 6 turer’s instructions. RNA from xenografted tumors were homogenized in ressected tumors clustered closely together, and closest to the normal TRIzol (Invitrogen) and purified according to the manufacturer’s instruction. tissues and the CPH 54 lines. It has been shown previously that these The TRIzol isolated RNA was additionally purified using the RNAeasy kit SCLC tumors cluster distinctly apart from other forms of lung cancers (Qiagen, Albertslund, Denmark). Affymetrix Oligonucleotide Array. The preparation of biotin cRNA was and normal lung (1). prepared essentially as described in the Affymetrix Expression Analysis Tech- The cell lines GLC-14, -16, and -19 were established from the same nical Manual. Briefly,10 ␮g of RNA was used as template to generate double- patient during longitudinal follow-up. These cell lines have been stranded cDNA using