Abstract 4874: Application of quantitative analysis of mouse cell contamination and purification of human tumor cells in patient derived orthotopic xenograft mouse models of pediatric brain tumors

Abstract

Abstract Background: Pediatric brain tumors continue to be among the most difficult to cure and are the leading cause of cancer-related deaths in children. Research investigating pediatric brain cancer is still impaired by the lack of cancer models. We established a large panel of orthotopic xenograft models as well as several paired permanent cell lines using in vivo and in vitro culture techniques. However an inherent risk of xenograft models is the contamination of isolates of human tumors with host (mouse) cells. Therefor strategies to correctly and timely identify the human/mouse ratio are essential. In this study we investigated Prostaglandin Receptor EP2 (PTGER2) as a useful target to identifying mouse cell contamination in PDOX models. Method: Species specific primers based on Alcoster et al., 2011 were used to detect PTGER2 in genomic DNA extracts of xenograft and cultured cells. Real-time qPCR was carried out using a StepOnePlus™ Real-Time PCR System. Reaction mix contained SYBR Green select master mix, respective primers and 5 ng of total genomic DNA in a total reaction volume of 10 μL. The qPCR conditions were as follows: 50°C-2 min, 95°C-10 min, 40 cycles of (95°C-15 sec, 61°C-1 min). Primer specificity was controlled in addition by analysis of amplified products on a 1.2% agarose gel. FACS was used to isolate human cells according to anti-Human HLA-ABC Ab and mouse-specific Ab cocktail surface staining and subjected to qPCR. Results: Various models of pediatric brain cancer (PXA, MB, GBM, EPN, ATRT, DIPG, PNET) established in our group were investigated for mouse cell contamination. Cells from xenograft isolates as well as from cultured xenografts were routinely analyzed. Our study confirms PTGER2 as a useful target for accurately estimate mouse cell contamination in brain cancer xenograft models. This further enabled us to improve detection and characterization of HLA-ABC/mouse-cocktail double negative population as seen in FACS. Conclusion: In conclusion we have successfully implemented an easy qRT-PCR approach to determine the human/mouse cell ration in any mixed cell population. Particular for PDOX models it is paramount to have reliable information on mouse cell contamination. Citation Format: Frank K. Braun, Mari Kogiso, Lin Qi, Huiyuan Zhang, Yuchen Du, Holly B. Lindsay, Sibo Zhao, Sarah G. Injac, Xiao-Nan Li. Application of quantitative analysis of mouse cell contamination and purification of human tumor cells in patient derived orthotopic xenograft mouse models of pediatric brain tumors [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 4874. doi:10.1158/1538-7445.AM2017-4874