Abstract 2392: Primary cultures established from estrogen receptor negative breast tumors: Method and characterization
Authors: Drews-Elger Katherine , Brinkman Joeli A. , Azzam Diana J. , Schlater Amy , Diehl Kathleen M. , El-Ashry Dorraya 
Published: June 2012

Approximately 35% of breast cancers lack expression of estrogen receptor (ER) protein. ER-negative (ER-) breast cancer carries a worse prognosis than ER positive (ER+) breast cancer, but more importantly, it precludes use of less toxic endocrine therapies. Our objective was to establish primary cultures from dissociation of ER- breast tumors in order to provide an alternative cellular model that can be used as a valuable tool for both in vitro and in vivo studies of ER- breast cancer. A total of eight ER- tumors were successfully dissociated into primary cultures: four from ER-/PR-/H2N- and four from ER-/PR-/H2N+ primary breast tumors with a 100% success rate. The epithelial-enriched cell pellet was placed in culture and for experiments carried for a maximum of 30 passages. These cells will be hereafter referred to as “dissociated tumor” (DT) cultures. Cells were grown in 2D culture and their in vitro morphology, proliferation rates, mammosphere and soft agar colony formation ability, and CD44/CD24 surface marker expression (tumor initiating cell (TIC) content) are determined. In addition we have performed gene expression profiling and established their tumorigenic potential in NOD/SCID female mice. Each culture exhibited its own relatively unique morphology; two of the eight cultures grew mainly as suspensions while the remaining six DT cultures grew with a higher percentage of attached, mesenchymal phenotype cells. Proliferation rates ranged from 38 to 60 hours, and while all of the DT cultures had the ability to form mammospheres, five of the eight DTs (DT13, DT16, DT22, DT25 and DT28) formed colonies in soft agar. Analysis of CD44 and CD24 surface markers expression showed that while all DT cultures were CD44 positive, expression of CD24 varied among DTs. Of the eight DT cultures, we found five of them to have a high (&gt;85%) CD44+/CD24-/lo cell content, one the CD44+/CD24high phenotype and two of eight have populations with increasing levels of CD24 expression ranging from -/lo to medium. This CD44+/CD24-/lo phenotype is stable when examined over several passages. Microarray analysis comparing the DT cultures to cancer cell lines showed that they clustered with each other and with several breast cancer cell lines of known ER- status and EGFR or Her2 overexpression status. In agreement with the soft agar assay, DT16, DT22, DT25 and DT28 had the ability to form tumors when injected into the mammary fat pad of female NOD/SCID mice. In summary, our study describes primary cultures of dissociated ER negative breast cancer cells that provide an alternative, primary cell based model that allows both in vitro as well as in vivo experimental approaches. The use of these types of cellular models may lead to a better understanding of ER- breast cancer biology as well as being a valuable tool for screening potential therapeutic options for ER- breast cancer.

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 2392. doi:10.1158/1538-7445.AM2011-2392