Abstract A24: Characterization of a novel p53; BRCA1-deficient claudin-low mammary tumor cell line

Abstract

Abstract Triple-negative breast cancer (TNBC) is a highly diverse and typically aggressive group of cancers. Consequently, elucidating the biology of the various subtypes is necessary for more effective diagnosis and better targeted treatment of this disease. Recently, a novel claudin-low TNBC subtype has been identified. Patients with this subtype have a very poor prognosis, specifically young African-American females. The major focus of our study is to characterize a novel claudin-low cell line cultured from a “humanized” mouse model that is p53 and BRCA1 deficient (K14TRT cell line). Procedures: This cell line has been maintained for more than 40 months and for over 25 subcultures, and was characterized by microscopy, histopathology, genotyping, cytogenetics, and bioinformatics based techniques. Results: Histological analysis supports that the primary K14TRT tumors were highly penetrant, with some having spindloid morphology while others were undifferentiated adenocarcinoams, with large areas of necrosis. As compared to normal mammary cells, K14TRT cells possessed larger variable shaped nuclei with a higher nuclear-to-cytoplasmic ratio. The tumor cells also demonstrated a highly aneuploidy karyotype. When isolated via single cell suspension and subsequently passaged, the K14TRT cells were able to form colonies in soft agar. Tumorigenicity was additionally supported by induction of solid tumors following serial injections of the K14TRT cells into the flanks of nude mice. The primary K14TRT tumor overwhelmingly possessed a basal-like breast tumor profile, whereas the transplanted and serially passaged K14TRT cells diverged to a claudin-low profile, as demonstrated by microarray analysis. These serially passaged cells were highly proliferative, with low to absent expression of tight junction proteins (claudin 3, 4, & 7 and E-cadherin), and typically negative for estrogen receptors (ER-), progesterone receptors (PR-), and HER2/neu (HER2-). Since this cell line did demonstrate an ability to form new tumors in recipient mice we speculated they could possess tumor stem cell properties. There competence for self-renewal and capacity to reestablish tumor heterogeneity was supported by fluorescence-activating cell sorting (FACS) analysis of the primary BRCA1/p53 deficient mammary tumor cells in which two populations were identified. Cells that possess low levels of epithelial marker CD24 and high levels of the B1 integrin marker CD29 have been shown to regenerate entire mammary glands indicative of mammary STEM cells. Strikingly, FACS analysis of the K14TRT primary cell line identified two cell populations, one which expressed markers that were CD24high/CD29high (differentiated) and the other population CD24low/CD29high (undifferentiated stem cell-like profile). The sorted undifferentiated mammosphere population when analyzed by microarray analysis clustered with transplanted and serially passaged K14TRT cells identified as being claudin-low, whereas the differentiated sorted population clustered with the primary basal-like cells. Conclusions: Our current data has demonstrated that the K14TRT cell line is a good candidate for a “humanized” TNBC claudin-low cell line with stem cell properties, and could play a significant role in devising better diagnostic tools and chemotherapeutic strategies for this aggressive tumor subtype. Citation Format: Stephanie T. Dance-Barnes, Ebone Evans, Shanderys Steward, Xiaping He, Jerry Usary, Charles Perou. Characterization of a novel p53; BRCA1-deficient claudin-low mammary tumor cell line. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A24.