Abstract 532: A transcriptional sketch of breast cancer fibroblasts: The validation of cDNA libraries for expression profile study using a high-throughput sequencing technology
Authors: Bromberg Natalia , Molina Gustavo Campos , Ferreira Elisa Napolitano , Carraro Dirce Maria , Brentani Helena , Brentani Maria Mitzi 
Published: June 2012

Abstract
There is now a wide recognition that the cross-talk between cancer and stromal cells may play a crucial role in cancer progression, although the complex underlying molecular mechanisms that occur within the tumor microenvironment are poorly understood. Fibroblasts represent the major cellular component of cancer-associated stroma. Unfortunately at present, a definitive view is lacking on a comparative expression profile between carcinoma associated fibroblasts (CAFs) and fibroblasts derived from normal breast (NAFs). The scarce body of work can not be compared due to differences in methodologies highlighting the importance to evaluate fibroblasts in tumor context and not in culture. Genomic changes in fibroblasts might be the cause for its ability to influence breast cancer cells, hence the identification of differential gene expression through methodologies with high potential of quantitative evaluation not only will complement previous quantitative investigations but could also provide insights on the identification of new prognostic molecular markers for breast cancer. The purpose of this study was the preparation of 3' fragments cDNA libraries from fibroblast samples, its validation on ABI 3130 xl Plataform and the obtainment of a transcriptional snapshot of CAFs and NAFs using the Genome Sequencer FLX Titanium 454 Plataform. Since biological heterogeneity of breast cancers has serious implications on disease treatment and prognostic, total RNAs were isolated from fibroblasts obtained from tissue frozen samples of human invasive ductal breast carcinoma (luminal A subtype), triple negative, fibroadenoma tissues using laser capture microdissection (LCM). Samples were reverse transcribed and amplified, double cDNAs were digested with DpnII restriction enzyme. After recovery of fragments with Streptavidin beads, they were labeled with 454 adaptors containing a specific 6nt TAG. Libraries were pooled before sequencing validation by Sanger method which was held in attempt to confirm the absence of library preparation artefacts (concatamers or linkers with inespecific TAGs). Considering the total high quality sequences presenting a complete adaptor site, 93% also presented complete TAGs and DpnII sites. Moreover, all the studied samples were represented: 19% of total sequences represented luminal A subtype samples while triple negative samples and normal samples presented 40 and 41% of sequences respectively. This work indicates at molecular level, differences among the 3 distinct groups of fibroblasts. The 454 transcriptome sequencing is on going and we expect that this quantitative approach will permit the accurate identification of differential transcriptional portraits associated with changes on breast cancer fibroblasts. Supported by FAPESP: 05/60333-7, 09/10088-7.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 532. doi:10.1158/1538-7445.AM2011-532