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Gene Expression Profiles of Epithelial Cells Microscopically Isolated from a Breast-Invasive Ductal Carcinoma and a Nodal Metastasis

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Gene Expression Profiles of Epithelial Cells Microscopically Isolated from a Breast-Invasive Ductal Carcinoma and a Nodal Metastasis Gene expression profiles of epithelial cells microscopically isolated from a breast-invasive ductal carcinoma and a nodal metastasis I. Zucchi*†‡, E. Mento*†, V. A. Kuznetsov†§, M. Scotti*, V. Valsecchi*, B. Simionati¶, E. Vicinanza*, G. Valle¶, S. Pilottiʈ, R. Reinbold**, P. Vezzoni*, A. Albertini*, and R. Dulbecco†† *Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, Via F. lli Cervi 93, 20090 Segrate-Milan, Italy; §Genome Institute of Singapore, Department of Information and Math Sciences, 60 Biopolis Street, Singapore 138672; ¶Centro Ricerche Interdipartimentale Biotecnologie Innovative, University of Padua, 35121 Padua, Italy; ʈIstituto Nazionale Tumori, Via Venezian 31, 20133 Milan, Italy; **Max Planck Institute for Molecular Biomedicine, Cell and Developmental Biology, D48149 Muenster, Germany; and ††Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037 Contributed by R. Dulbecco, November 5, 2004 Expression profiles of breast carcinomas are difficult to interpret This work is a pilot study carried out by following this concept. when they are obtained from tissue in toto, which may contain a We performed SAGE on highly homogeneous populations of large proportion of non-cancer cells. To avoid this problem, we cells microscopically isolated from a primary invasive ductal microscopically isolated cells from a primary invasive ductal carci- carcinoma of the breast and from an axillary node harboring a noma of the breast and from an axillary node harboring a meta- metastatic breast carcinoma. A useful feature of the SAGE static breast carcinoma, to obtain pure populations of carcinoma technology is that databases can be compared directly with each cells (Ϸ500) and used them for serial analysis of gene expression. other. The purity of the cell population is shown in our SAGE The expression profiles generated from both populations of cells libraries by the absence or minimal expression of genes that are were compared with the profile of a disease-free mammary epi- markers of nonepithelial cells such as endothelial and stromal thelium. We showed that the expression profiles obtained are cells, adipose cells, B and T lymphocytes, and macrophages. exclusive of carcinoma cells with no contribution of non-epithelial Materials and Methods cells. From a total of 16,939 unique tags analyzed, we detected 559 MEDICAL SCIENCES statistically significant changes in gene expression; some of these Clinical Information and Cell Microdissection. Samples from both a genes have not been previously associated with breast cancer. We primary invasive ductal carcinoma of the breast and a nodal observed that many of the down-regulated genes are the same in metastasis were obtained at the Istituto Nazionale Tumori both cancers, whereas the up-regulated genes are completely (Milan) after patient consent. For the invasive library construc- different, suggesting that the down-regulation of a set of genes tion, Ϸ500 cancer cells (99% pure) were microscopically isolated may be the basic mechanism of cancer formation, while the from a primary estrogen- and progesterone-receptor-positive up-regulation may characterize and possibly control the state of invasive ductal carcinoma. For the metastatic library, the same evolution of individual cancers. The results obtained may help in amount of cells were also microscopically isolated from a characterizing the neoplastic process of breast cancer. metastatic lymph node derived from an estrogen- and progest- erone-receptor-negative breast-invasive ductal carcinoma. Can- breast cancer ͉ serial analysis of gene expression ͉ cell microdissection ͉ cer cells were microscopically dissected from methylene blue- ␮ carcinoma stained 20- m frozen sections, kept at low temperature during the entire manipulation, by using microneedle aspiration (8); cancer cells are less attached to the connective tissue stroma and reast cancer progresses through a series of stages, starting as are preferentially released by mechanical force. Batypical duct hyperplasia, to ductal carcinoma in situ, inva- sive ductal carcinoma, and finally, metastatic disease. Global cDNA Preparation, Library Construction, and Sequencing. The micro- expression profiling has been extensively used to classify the dissected SAGE libraries were generated by following a protocol disease and to predict its clinical outcome. Most of these studies described (9), with modifications made necessary due to the used array-based platforms, and therefore, were limited to the small number of cells used as starting material. Total RNA was analysis of known but most likely incomplete selection of genes obtained by using the PicoPure RNA isolation kit (Arcturus). A and ESTs. More recently, global changes of gene expression have pre-SAGE linear amplification step was performed with T7 been determined by using serial analysis of gene expression RNA polymerase by using the RiboAmp kit (Arcturus). (SAGE), which does not have this limitation (1, 2). Both microarray and SAGE data suggest that there is considerable Tools for SAGE. Tags were analyzed by using SAGE2000 (www. diversity among breast tumor profiles (1–5). However, these data sagenet.org͞index.htm) and ESAGE software (10). The Na- were obtained primarily from tumor tissue, rather than from a tional Center for Biotechnology Information SAGEmap homogenous population of epithelial cancer cells. The problem (www.ncbi.nlm.nih.gov͞SAGE) and the CGAP SAGEgenie that arises is that, if in the context of a complex tissue, only a (http:͞͞cgap.nci.nih.gov͞SAGE͞AnatomicViewer) databases small proportion of cells corresponds to the cells of interest, were also used. The Gene Expression Level Probability Function many important regulatory genes, often expressed at low levels, was obtained by using the discrete Pareto-like probability func- but essential for determining the pathological cell phenotype, tion (11). The assignment of molecular function of proteins and will be undetected. In fact, purified and unpurified samples were chromosomal location of individual genes was based on the shown to produce different expression profiles (2), because contaminating nontumoral cells may have been present in dif- ferent amounts. For instance, in tumor samples cancer cells may Abbreviations: SAGE, serial analysis of gene expression; INV, invasive; MET, metastatic. be present in proportions ranging from 5% to 50% of the total †I.Z., E.M., and V.A.K. contributed equally to this work. cell mass (6, 7). For these reasons, it is necessary to determine ‡To whom correspondence should be addressed. E-mail: [email protected]. the expression profile on a pure population of carcinoma cells. © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408260101 PNAS ͉ December 28, 2004 ͉ vol. 101 ͉ no. 52 ͉ 18147–18152 Downloaded by guest on September 29, 2021 LocusLink database (www.ncbi.nlm.nih.gov͞projects͞Locus- libraries, when compared with the Br࿝N library. Of these 559 Link͞). tags, 392 (70.1%) correspond to known genes or ESTs, and of the other 167 tags (29.9%), 152 match to multiple genes, and 15 do In Situ Hybridization. In situ hybridization was performed on human not match to any genes. A selection of the 50 most down- breast tissues as described (12). Sense and antisense riboprobes regulated genes is reported in Table 1 and a selection of the 50 were generated by in vitro transcription using T7 or Sp6 polymerase most up-regulated genes is reported in Table 2. Tables 3–5 list primers, from the cloned region of 762 nt, between primers tags down-regulated in the INV and MET libraries, as follows: CXCL6F͞CXCL6R (5Ј-TCATAAAATTGCCCAGTCTTC-3Ј 221 tags matching to known genes, including 40 ribosomal genes and 5Ј-TGTTTTTGGGCTTCTTCATCT-3Ј) of the human are listed in Table 3, 76 multiple-gene-matching tags are re- CXCL6 mRNA sequence NM࿝002993. ported in Table 4, and 3 no-matching tags are reported in Table 5. Tables 6–8 list tags up-regulated in the INV and MET Supporting Information. Tables 3–8 are published as supporting libraries, as follows: 171 tags matching to known genes, including information on the PNAS web site. 30 ribosomal genes, are listed in Table 6, 76 multiple-gene- matching tags are reported in Table 7, and 12 no-matching tags Results are reported in Table 8. Generation of SAGE Libraries from Microdissected Cells. Two SAGE libraries were obtained: one from cancer epithelial cells isolated Genes Down-Regulated in INV and MET Carcinoma Libraries. Our by microscopic dissection from a primary invasive (INV) breast findings are in part similar and in part different in relation to ductal carcinoma (the INV library, 17,306 tags), and the other previously published work (1, 2). In agreement with the earlier from cancer cells microscopically isolated from a lymph node work, we observed that the most dramatic difference in gene harboring a metastatic (MET) breast carcinoma (the MET expression, between cancer and normal cells, involves genes library, 10,363 tags). In both cases, the purified cells were down-regulated in cancer. Of 221 down-regulated genes (Table obtained in very limited amounts (Ϸ500 cells) and, due to the 3), 134 genes are down-regulated in both the INV and MET very limited amount of RNA obtained, amplification was re- libraries, 77 genes are down-regulated only in the INV library, quired. To verify that the RNA amplification preserved the and 10 genes are down-regulated only in the MET library. A original mRNA abundance, an amplification diagnosis test was large fraction
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