Research Article Gene Alterations Identified by Expression Profiling in Tumor-Associated Endothelial Cells from Invasive Ovarian Carcinoma Chunhua Lu,1 Tomas Bonome,3 Yang Li,1 Aparna A. Kamat,1 Liz Y. Han,1 Rosemarie Schmandt,1 Robert L. Coleman,1 David M. Gershenson,1 Robert B. Jaffe,4 MichaelJ. Birrer, 3 and AnilK. Sood 1,2 Departments of 1Gynecologic Oncology and 2Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas; 3Cell and Cancer Biology Branch, National Cancer Institute, Bethesda, Maryland; and 4Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California Abstract the promise of such approaches. However, the full spectrum of Therapeutic strategies based on antiangiogenic approaches differences in the tumor vasculature compared with its normal are beginning to show great promise in clinical studies. counterpart is not known. Identification of additional targets on However, full realization of these approaches requires tumor endothelium may allow opportunities for developing new identification of key differences in gene expression between therapeutic approaches to inhibit angiogenesis in a tumor-specific endothelial cells from tumors versus their normal counter- manner. parts. Here, we examined gene expression differences in Higher levels of proangiogenic cytokines and angiogenesis are purified endothelial cells from 10invasive epithelial ovarian associated with an increased risk of metastasis and poor prognosis cancers and 5 normal ovaries using Affymetrix U133 Plus in ovarian cancer (5, 6). To date, a small number of breast, colon, 2.0microarrays. More than 400differentially expressed genes and brain cancers have been analyzed for gene expression changes were identified in tumor-associated endothelial cells. We in the tumor vasculature using serial analysis of gene expres- selected and validated 23 genes that were overexpressed by sion (7–9). These studies showed the ability to define both tumor- 3.6- to 168-fold using real-time reverse transcription-PCR specific endothelial genes and normal endothelial genes. Whereas and/or immunohistochemistry. Among these, the polycomb selected genes in ovarian cancer vasculature have been character- group protein enhancer of Zeste homologue 2 (EZH2), the ized, there is little information about global gene expression Notch ligand Jagged1, and PTK2 were elevated 3- to 4.3-fold alterations in ovarian cancer endothelium. This lack of data in tumor-associated endothelial cells. Silencing these genes prompted us to carry out expression profiling on purified endothelial individually with small interfering RNA blocked endothelial cells from invasive epithelial ovarian cancers and normal ovaries. cell migration and tube formation in vitro. The present study In recent years, whole genome expression profiling of cancer shows that tumor and normal endothelium differ at the using methods such as microarray and serial analysis of gene molecular level, which may have significant implications for expression has advanced our understanding of the molecular the development of antiangiogenic therapies. [Cancer Res pathways involved in cancer onset and progression. However, 2007;67(4):1757–68] global analysis of gene expression in specific cell populations within the tumor microenvironment is challenging and bulk tissue Introduction expression profiling may, in fact, mask gene changes in different cell types. We have recently used laser capture microdissection to Despite improvements in surgery and chemotherapy, mortality isolate epithelial cells from ovarian cancers for microarray analyses rates in women with advanced ovarian carcinoma have remained (10), which elucidated changes in gene expression specific to the largely unchanged (1). Therefore, novel therapeutic strategies are epithelial tumor cells. Profiling expression changes that occur in needed. It is now well known that growth of tumors, both at the the tumor stroma, including the tumor endothelial cells, will likely primary and metastatic sites, requires a blood supply for expan- provide insights into the mechanisms underlying tumor vascular sion beyond 1 to 2 mm (2). Targeting tumor angiogenesis by inhi- growth, reveal additional targets for antiangiogenic therapies, and biting endothelial cells that support tumor growth is particularly potentially offer new biomarkers for diagnosis and surveillance. promising because of their presumed genetic stability. The recent However, the endothelium is enmeshed in a tissue complex success of a humanized monoclonal antibody (bevacizumab) consisting of vessel wall components, stromal cells, and epithelial against vascular endothelial growth factor in prolonging the lives cells. Only a small fraction of the cells within these tissues are of patients with advanced colon and breast carcinoma (3, 4) shows endothelial. Moreover, gene analysis of specific cell types extracted from chemical reagent–fixed frozen tissue may not be accurate as the gene profile may be altered during the fixation process. In the Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). present study, we immunopurified endothelial cells from human C. Lu and T. Bonome contributed equally to this work. M.J. Birrer and A.K. Sood normal ovarian tissues and invasive epithelial cancers and share senior authorship. investigated the gene expression profile using microarrays. Selected Requests for reprints: Anil K. Sood, Department of Gynecologic Oncology, University of Texas M. D. Anderson Cancer Center, Unit 1362, 1155 Herman Pressler, genes were validated to test the reliability of the microarray Houston, TX 77030. Phone: 713-745-5266; Fax: 713-792-7586; E-mail: asood@ analysis. The gene expression profiles derived in the current study mdanderson.org. I2007 American Association for Cancer Research. define unique alterations in vascular gene expression in epithelial doi:10.1158/0008-5472.CAN-06-3700 ovarian carcinoma. www.aacrjournals.org 1757 Cancer Res 2007; 67: (4).February 15, 2007 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Research Materials and Methods as the actual data set at P < 0.001 was reported as the significance level of the global test. Sample preparation. Fresh tissue samples (5 normal ovaries and 10 Pathway analysis. Differentially regulated genes identified in a series of epithelial high-grade, stage III or IV invasive serous ovarian cancers) were 48 late-stage (III and IV), high-grade (3) microdissected papillary serous obtained from patients undergoing primary surgical exploration at the ovarian carcinomas, as compared with 10 normal ovarian surface epithelial M. D. Anderson Cancer Center after approval from the Institutional Review brushings (10), were categorized by cellular component according to the Board. The minced tissue was digested with collagenase A, elastase, and Gene Ontology ontological hierarchy. Epithelial genes associated with the j DNase 1 at 37 C for 90 min to yield a single-cell suspension. A number of cell membrane, extracellular matrix, and extracellular region were used as negative selections followed, including removal of platelets and RBCs by central nodes to identify signaling pathways modulated in tumor-associated Percoll separation; removal of epithelial cells using M450 beads, which are endothelial cell isolates. This was accomplished using PathwayAssist prebound to BerEP4 antibody; and removal of leukocytes using anti-CD14, version 3.0 software (Iobion Informatics LLC, La Jolla, CA). This software anti-CD45, and anti-CD64 beads (Dynal Biotech, Brown Deer, WI). Positive package contains more than 500,000 documented protein interactions selection was done with P1H12 (CD146) immunobeads (P1H12 antibody acquired from MedLine using the natural language processing algorithm was from Chemicon, Temecula, CA), and the beads linked to secondary MEDSCAN. The proprietary database can be used to develop a biological antibody were from Dynal Biotech. Immunostaining was then done using association network to identify putative coregulated signaling pathways von Willebrand factor and 4¶,6-diamidino-2-phenylindole nuclear staining to using expression data. confirm the purification of endothelial cells. Quantitative real-time PCR validation. Quantitative real-time RT-PCR Total RNA amplification for Affymetrix GeneChip hybridization and was done on 100 ng of double-amplified product from the 15 specimens image acquisition. To successfully generate sufficient labeled cRNA for using primer sets specific for 23 select genes and the housekeeping genes microarray analysis from 25 ng of total RNA, two rounds of amplification GAPDH, GUSB, and cyclophilin. An iCycler iQ Real-time PCR Detection were necessary. For the first-round synthesis of double-stranded cDNA, System (Bio-Rad Laboratories, Hercules, CA) was used in conjunction with 25 ng of total RNA were reverse transcribed using the Two-Cycle cDNA the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Inc., Valencia, CA) ¶ Synthesis Kit (Affymetrix, Santa Clara, CA) and oligo-dT24-T7 (5 -GGCC- according to previously described cycling conditions (13). To calculate the AGTGAATTGTAATACGACTCACTATAGGGAGGCGG-3¶) primer according ÀDDC relative expression for each gene, the 2 T method was used, averaging to the manufacturer’s instructions followed by amplification with the MEGA the CT values for the three housekeeping genes for a single reference gene script T7 Kit