PDTM-17. IDENTIFICATION OF HUMAN PEDIATRIC BRAIN TUMORS CELLS IN PATIENT DERIVED ORTHOTOPIC XENOGRAFT MOUSE MODELS BY USING SPECIES-SPECIFIC PTGER2 GENE qPCR

Abstract

AbstractBACKGROUNDPediatric 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 PDOX models as well as several paired permanent cell lines using and culture techniques. However, an inherent risk of xenograft models is the contamination of isolates of human tumors with host cells. Therefor strategies to 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.METHODSpecies specific primers based on Alcoster et al., 2011 were used to detect PTGER2 in xenograft and cultured cells. RT-qPCR was carried out using a StepOnePlus™ RT-PCR System. Reaction mix contained SYBR Green master mix, respective primers and 5ng of total gDNA in a total reaction volume of 10μL. The qPCR conditions were as follows: 50°C-2min, 95°C-10min, 40 cycles of (95°C-15sec, 61°C-1min). Primers were additionally controlled 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.RESULTSVarious models of pediatric brain cancer established in our group were investigated for mouse cell contamination. Xenograft as well as cultured cells were routinely analyzed. Our study confirms PTGER2 as a useful target for accurately estimate mouse cell in PDOX models. This enabled us to improve detection and characterization of HLA-ABC/mouse-cocktail double negative population as seen in FACS.CONCLUSIONIn conclusion we have successfully implemented an reliable qRT-PCR approach to determine the human/mouse cell ration in any mixed PDOX cell population.