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Genome-Wide Transcriptional Analysis of Carboplatin Response in Chemosensitive and Chemoresistant Ovarian Cancer Cells

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Genome-Wide Transcriptional Analysis of Carboplatin Response in Chemosensitive and Chemoresistant Ovarian Cancer Cells 1605 Genome-wide transcriptional analysis of carboplatin response in chemosensitive and chemoresistant ovarian cancer cells David Peters,1,2 John Freund,2 and Robert L. Ochs2 variety of disease processes, including cancer. Patterns of global gene expression can reveal the molecular pathways 1 Department of Pharmacology and Therapeutics, University of relevant to the disease process and identify potential new Liverpool, United Kingdom and 2Precision Therapeutics, Inc., Pittsburgh, Pennsylvania therapeutic targets. The use of this technology for the molecular classification of cancer was recently shown with the identification of an expression profile that was Abstract predictive of patient outcome for B-cell lymphoma (1). We have recently described an ex vivo chemoresponse In addition, this study showed that histologically similar assay for determining chemosensitivity in primary cultures tumors can be differentiated based on their gene expression of human tumors. In this study, we have extended these profiles. Ultimately, these unique patterns of gene expres- experiments in an effort to correlate chemoresponse data sion may be used as guidelines to direct different modes of with gene expression patterns at the level of transcription. therapy. Primary cultures of cells derived from ovarian carcinomas Although it is widely recognized that patients with the of individual patients (n = 6) were characterized using same histologic stage and grade of cancer respond to the ChemoFx assay and classified as either carboplatin therapies differently, few clinical tests can predict indi- sensitive (n = 3) or resistant (n = 3). Three representa- vidual patient responses. The next great challenge will be tive cultures of cells from each individual tumor were then to use the power of post-genomic technology, including subjected to Affymetrix gene chip analysis (n = 18) using microarray analyses, to correlate gene expression patterns U95A human gene chip arrays. Data were analyzed using with individual patient responses to clinical therapies. the dCHIP software package. We identified a significant The major objective of the current study was to determine number of genes whose expression patterns were altered whether predictive gene expression patterns can be between carboplatin chemosensitive and chemoresistant identified that correlate with the results of an ex vivo cells, in normal culture conditions and in the presence chemoresponse assay done on primary cultures of cells of carboplatin for either 2 or 72 hours. Among these derived from ovarian carcinomas of individual patients. differentially expressed genes, we found a significant Specifically, we used Affymetrix gene chip microarrays to proportion to be associated with apoptosis, cell-cell analyze gene expression patterns in 18 cell cultures that communication, cell adhesion, DNA repair, and cell pro- were functionally characterized as either chemoresponsive liferation. In general, the molecular phenotype displayed or chemosensitive to carboplatin in vitro. by chemoresistant cells was reflective of an extended life span in culture in the presence of carboplatin and the Materials and Methods genes that define this phenotype are potential biomarkers Patients for the prognostic management of ovarian cancer patients. Tumors were submitted to Precision Therapeutics, Inc. [Mol Cancer Ther 2005;4(10):1605–16] (Pittsburgh, PA) for assessment of chemoresponsiveness to oncologist-selected chemotherapeutic agents. They were Introduction tested using the ChemoFx phenotypic chemoresponse Gene expression profiling at the level of transcription by assay that assesses tumor cell viability in the presence of oligonucleotide microarray analysis is a powerful molecu- a range of drug concentrations (see below; Table 1). lar tool for the discovery of genes that are involved in a Primary Cell Culture and the ChemoFx Assay Precision Therapeutics’ ChemoFx assay involves the isolation, short-term growth, and drug dosage treatment of epithelial cells derived from solid tumors (2, 3). At the Received 11/22/04; revised 7/19/05; accepted 8/10/05. time of surgical ‘‘debulking,’’ pieces of solid tumor are Grant support: Precision Therapeutics, Inc. obtained by the surgeon or pathologist and placed in The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked tissue culture transport medium for overnight shipment to advertisement in accordance with 18 U.S.C. Section 1734 solely to the laboratories of Precision Therapeutics. Upon arrival, indicate this fact. the tumor is minced into small pieces and placed with Requests for reprints: David Peters, Department of Pharmacology and cell culture medium into multiple small flasks for cell Therapeutics, University of Liverpool, The Sherrington Buildings, Ashton Street, Liverpool, L69 3GE United Kingdom. Phone: 44-151-794-5477. outgrowth. Over time, cells move out of the tumor pieces E-mail: [email protected] and form a monolayer on the bottom of the flask. Once Copyright C 2005 American Association for Cancer Research. enough cells have migrated out of the ex vivo explant doi:10.1158/1535-7163.MCT-04-0311 pieces, they are then trypsinized and reseeded into Mol Cancer Ther 2005;4(10). October 2005 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. 1606 Genome-Wide Response to Carboplatin Table 1. Patient profiles and RNeasy (Qiagen, Valencia, CA) protocols. Twenty micrograms of purified RNA were then used as a template Patient Tumor Tumor Cell ChemoFx c for double-stranded cDNA synthesis primed using a T7- no. type site kill (%)* response (dT)24 oligonucleotide. Double-stranded cDNA was then used as a template for biotin-labeled cRNA preparation 1 PSA Liver 66 S using T7 RNA polymerase. Resulting cRNA was fragmented 2 PSA Ovary 37 S 3 PSA Ovary 53 S and hybridized to Affymetrix GeneChip Human Genome 4 PSA Ovary 8 R U95 version 2 (HG-U95A) oligonucleotide microarrays. 5 PSA Ovary 3 R Affymetrix data were analyzed using the dCHIP 6 PSA Omentum 9 R software package (4). The 18 DAT files generated by the Affymetrix Microarray Suite version 5.0 were converted Abbreviations: PSA, papillary serous adenocarcinoma; S, sensitive; R, into DCP files using dCHIP,3 as described previously by resistant. Li and Wong (4). The DCP files were normalized, and raw *% Cell kill at the highest drug dose (1,000 mmol/L). cIn vitro response to carboplatin. gene expression data generated. Normalization was carried out according to the Invariant Set Normalization method as described by Li and Wong (5). The resulting microtiter plates for either the ChemoFx assay (described gene list was then filtered such that only genes whose below) or for immunohistochemistry analysis for the expression levels satisfied the following criteria were identification of malignant epithelial cells, as previously included: 0.5 < SD/mean >10, gene called P (present) in described (2). 20% of samples, and expression level >20 in >50% of For the ChemoFx assay, f350 cells in 10 AL of medium samples. Global comparisons of data derived from are seeded into 60-well microtiter plates and allowed to resistant and sensitive cells was done as follows (where attach and grow for 24 hours. After 24 hours in culture, 10 E = experimental samples and B = baseline samples): AL of six doses of 2Â drug are dissolved in HBSS and Emean/Bmean >2 or Bmean/Emean >2; Emean À Bmean >100 j added to each well for 2 hours at 37 C. For the present or Bmean À Emean >100; and P for testing Emean = Bmean À study, carboplatin was added in concentrations of 20, 50, < 0.03. Thep t statistic was computed as (mean1 100, 200, 500, and 1,000 mmol/L. Plates are then rinsed mean2)/ [SE(mean1)2 + SE(mean2)2] and its P computed rapidly with HBSS four times to remove any drug residue, based on the t distribution, and the degree of freedom and then fresh medium is added for 72 hours. After the is set according to the Welch-modified two-sample t test. 72 hours of recovery period, the medium and any The clustering algorithm used is as follows: the distance nonadherent dead cells are rinsed off with HBSS, and between two genes is defined as 1 À r, where r is the the remaining cells are fixed for 5 minutes in 95% ethanol Pearson correlation coefficient between the standardized containing the DNA intercalating blue fluorescent dye, expression values (make mean = 0 and SD = 1) of the 4V,6-diamidino-2-phenylindole. The number of cells per two genes across the samples used.4 Full data sets are well was then quantitated by counting fluorescent nuclei available [accession no. GSE1926 (Gene Expression Omnibus using an operator-controlled, computer-assisted image series)].5 analysis system (Zeiss, Hertfordshire, United Kingdom) customized for Precision Therapeutics. A complete dose- response curve was generated for each drug evaluated. Results The data are presented graphically as the cytotoxic index. The experiments done in this study were designed to (a) The cytotoxic index (% kill) is defined as 1 À [no. of cells identify genes whose expressions were altered in primary in treated wells / no. of cells in control wells] Â 100. For cultures of carboplatin-sensitive versus carboplatin-resis- each drug dose, the cytotoxic index was calculated using tant ovarian carcinoma cells; (b) to determine the differ- two columns of untreated cells in the plate as the negative ences in genomic response of chemosensitive versus control. Drug responses were scored from 0 to 5, with the resistant cells to 2 or 72 hours of carboplatin exposure score determined by the number of drug doses where the in vitro; and (c) to analyze the temporal genomic response cytotoxic index was z35%. For the purposes of the present to carboplatin in either chemosensitive or resistant cells study, a drug response score of 0 to 1 was considered independently of each other. Affymetrix data sets were resistant and a score of 3 to 5 was considered sensitive.
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