Abstract 819: Transcriptomic profiling of two canine mammary cancer cell lines for translational oncology studies
Authors: Fukumasu Heidge , Cordeiro Yonara G. , Xavier Pedro L. , Rochetti Arina L. , Alexandre Pamela A. , Mori Claudia C. , Freitas Silvio H. , Strefezzi Ricardo F. 
Published: July 2017

Abstract
Dogs develop spontaneous cancers as humans and shares a variety of features including histology, genomic alterations, molecular targets, biological behavior and response to conventional therapies. Although dogs can be considered good models for translation of new cancer treatments, there is a lack of established preclinical in vitro models of canine cancers. Therefore, our aim here was to establish two canine mammary cancer cell lines and characterize their transcriptomic profile to speed the translation of new cancer treatments from dogs to humans. Mammary cancer samples were obtained from female dogs that underwent surgery. Pieces of the tumors were FFPE for histopathology and the other fragments were washed with PBS, minced and dissociated with hyaluronidase and collagenase. Cells were filtered and put in culture with DMEM-F12, 5%FBS and 1% pen-strep. After 5 passages, FBS was increased to 10%. After passage 15, cells were characterized for doubling time, karyotype, immunocytochemistry for cytokeratin, vimentin and α-smooth muscle actin, matrigel-invasion assay and tumor growth in nude mice. At last, RNA-seq was performed on total RNA extracted with TRIZOL for functional enrichment analysis of differentially expressed (DE) genes from cancer cell lines. From 49 tumor samples, we characterized 2 cell lines named M5 and M25, originally from a comedocarcinoma and a mixed carcinoma, respectively. Both cell cultures showed spindle-shape cells and were positive for cytokeratin, vimentin and α-smooth actin, suggesting myoepithelial origin. The doubling time was 26.0h for M5 and 42.8h for M25 cells. Karyotyping analysis showed moderate aneuploidy for both cell cultures. M25 cell line presented significant higher invasion potential than M5 cells and formed tumors in mice but the latter didn’t. Histopathology of these tumors resembled the original tumor in the dog. An average of 16.2 million paired-end 100 bp reads were sequenced per replicate (three for each cell culture) with an average of 81.1% alignment to the reference genome. A total of 11,260 and 11,476 genes with more than 1 count per million were detected in M5 and M25 cell lines. M5 cell line have 404 and 394 up- and downregulated genes in comparison to M25 cell line, respectively. Gene ontology of DE genes showed the more malignant cell (M25) significantly enriched for pathways related to drug metabolism, ECM-receptor interaction, cell cycle, DNA replication, focal adhesion and cell differentiation. In conclusion, M25 cell line is more malignant than the M5 which is supported by their transcriptome. Thus, it is possible to use these cells as a comparative translational model for drug development especially if one wishes to work with molecular targets as described above.
Citation Format: Heidge Fukumasu, Yonara G. Cordeiro, Pedro L. Xavier, Arina L. Rochetti, Pamela A. Alexandre, Claudia C. Mori, Silvio H. Freitas, Ricardo F. Strefezzi. Transcriptomic profiling of two canine mammary cancer cell lines for translational oncology studies [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 819. doi:10.1158/1538-7445.AM2017-819