Abstract
Abstract Introduction: Efatutazone, a PPAR gamma agonist, may affect tumor growth through the induction of terminal cell differentiation. Thus, we hypothesize that efatutazone could affect progenitor cell number. Here, we evaluate progenitor cell number by measuring mammospheres, which is considered one way to measure potential progenitor cells. Methods: Two month old Brca1WT/fl11/Cre/p53+/- (n= 14) and Brca1fl11/fl11/Cre/p53+/- (n= 10) C57Bl/6 mice were randomly placed on either control or treated with efatutazone through the diet (30-mg/kg concentration, F3028, rodent diet, grain-based, 1/2-in pellets; Bio-Serv, Frenchtown, NJ) with necropsy at four months and isolation of primary mammary epithelial cells from thoracic glands for studies of mammosphere formation using SCIVAX 96-well low adhesion nanoculture plates (Organogenix, Inc., Japan) using EpiCult-B Mouse Media (Stem Cell Technology, Inc., Vancouver, Canada) with 0,1,5, and 10% fetal bovine serum (FBS) added. Thoracic mammary gland tissue was frozen at -20°C followed by isolation of RNA and evaluation of PPAR gamma pathway gene expression by real-time RT-PCR using TaqMan® Array Mouse Lipid Regulated Genes (Thermo Fisher Scientific, Inc., Waltham MA). One inguinal gland was fixed for mammary gland whole mount and the other inguinal gland was formalin-fixed and paraffin-embedded for histology. Results: SCIVAX nanoculture plates showed reproducible increases in mammosphere numbers with increasing FBS concentrations (p <0.05 one-way, Kruskal-Wallis). A significant increase in sphere number was seen in Brca1fl11/fl11/Cre/p53+/- compared to Brca1WT/fl11/Cre/p53+/- mice (p<0.05 one-way, Kruskall Wallis). Efatutazone treatment significantly decreased sphere numbers in Brca1WT/fl11/Cre/p53+/- and in Brca1fl11/fl11/Cre/p53+/- mice (p<0.05 one-way, Kruskall Wallis). Expression of PPAR gamma pathway genes were increased at the RNA level with statistically significant increases in Acadvl, Tnf, Alox5 (p<0.05) and Il1B, Srebf2, Hmgcs1, Hmgcr (p<0.01) genes (BootsRatio, http://rht.iconcologia.net/stats/br/several.html) in mice on efatutazone as compared to control diet. Discussion: SCIVAX nanoculture plates can be used to quantitatively evaluate and compare mammosphere numbers between different genotypes and treatment groups. The higher numbers of mammospheres found with loss of both Brca1 copies as compared to one copy is consistent with previously published literature. The decrease in mammosphere numbers with efatutazone treatment could be secondary to its differentiating impact on mammary epithelial cells. Conclusion: While efatutazone statistically significantly reduced mammosphere numbers, the absolute reduction was less the 25%. Citation Format: Sahar J. Alothman, Shan Chao, Weisheng Wang, Priscilla A. Furth. Efatutazone reduces mammosphere formation in Brca1WT/fl11/Cre/p53+/- and Brca1fl11/fl11/Cre/p53+/- mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2234. doi:10.1158/1538-7445.AM2017-2234
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