Abstract 3918: Characterizing growth features, allograft generation and transcriptomes of cultured conditionally reprogrammed cells (CRC) prepared from primary triple negative cancer from Brca1-mutant mice
Authors: Alamri Ahmad M. , Groeneveld Svenja , Kang Keunsoo , Dabydeen Sarah , Wang Weisheng , Hennighausen Lothar , Kallakury Bhaskar , Liu Xuefeng , Furth Priscilla A. 
Published: May 2015

Abstract
Background: Efficient acquisition of primary cancer cells for genetic and pharmacological testing is a means towards individualized cancer treatment. Genetically engineered mouse models of triple negative mammary cancer were used to compare culture conditions conditionally reprogrammed cell culture CRC with a rho kinase inhibitor (Y-27632) and irradiated J2 feeder cells, EpiCult™, DMEM for establishment efficiency, differentiation, fibroblast contamination, and allograft generation. Goals: 1) Evaluate mammary cancer progenitor cells by allograft and secondary culture 2) Compare RNA-seq transcriptomes in cells and allografts with different Brca1 gene dosages (100%, 50%, 0%) Methods: Triple negative invasive adenocarcinomas from 4 mouse genotypes, all carrying a p53-null allele, were analyzed: A) 1 mutant Brca1 allele; B) 2 mutant Brca1 alleles; C) 1 mutant Brca1 allele and 1 human aromatase transgene; D) human aromatase transgene. Cancers were divided and growth assessed in CRC, EpiCult™ and DMEM cultures. Illumina (V2scriptseq) RNA-seq libraries were prepared from cell pellets and allografts. One million cells injected into mammary gland fat pads of nude mice generated allografts divided for histology/DNA analyses, secondary culture and RNAseq. Transcript abundance and differentially expressed genes were determined after assembling transcriptomes with Cufflinks. Results: CRC was the most efficient methodology based on rapidity and percentage cancer specimens successfully cultured and paucity of fibroblasts. Brca1 gene dosage made no difference in culture efficiency. Differentiation markers were expressed at significantly higher levels in CRC whereas markers of epithelial mesenchymal transition were expressed higher in EpiCult™. Allografts were derived from CRC and EpiCult™ but not DMEM. Genetic testing confirmed allografts were derived from original endogenous cancer cells. Some individual allografts derived from the same culture exhibited different growth curves and histology, suggesting tumor cell heterogeneity within cultures. Overall, palpable EpiCult™ allografts appeared significantly earlier than CRC allografts (p&lt;0.05 Chi Square). RNAseq analyses distinguished gene expression patterns dependent upon culture condition as distinct from those determined by underlying genetics. Summary: CRC technology was an efficient means of generating primary cancer cell cultures, maintained epithelial cell differentiation better than other methods, had no fibroblast contamination, and preserved cancer progenitor cells.
Citation Format: Ahmad M. Alamri, Svenja Groeneveld, Keunsoo Kang, Sarah Dabydeen, Weisheng Wang, Lothar Hennighausen, Bhaskar Kallakury, Xuefeng Liu, Priscilla A. Furth. Characterizing growth features, allograft generation and transcriptomes of cultured conditionally reprogrammed cells (CRC) prepared from primary triple negative cancer from Brca1-mutant mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3918. doi:10.1158/1538-7445.AM2014-3918