Abstract 1455: Next generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cells
Authors: Agorku David , Tomiuk Stefan , Klingner Kerstin , Wild Stefan , Rüberg Silvia , Zatrieb Lisa , Bosio Andreas , Schueler Julia , Hardt Olaf 
Published: August 2015

Abstract
Human tumor xenografts represent the gold standard method for research areas such as drug discovery, cancer stem cell biology, and metastasis prediction. When compared to in vitro cell culture models, human tumor xenografts show a higher validity for most assays (DeRose et al., 2011).
During the growth phase in vivo, xenografted tissue is vascularized and infiltrated by cells of murine origin. The level of infiltration is highly dependent on multiple factors like tumor subtype, growth rate, and region of transplantation. However, even when these factors are kept constant, the amount and composition of infiltrating mouse cells is highly variable. Due to this, molecular downstream analyses such as microarray based expression profiling are challenged by cross hybridization of mouse derived molecules to human probes. In addition, a reduction of sensitivity caused by measuring mouse signals during next-generation sequencing analysis can be expected.
To overcome these limitations, we have developed a fast and easy method allowing for the comprehensive depletion of all cells of mouse origin by using automated tissue dissociation and magnetic cell sorting (MACS). We have performed whole exome sequencing of bulk human tumor xenografts from lung, bladder, and kidney cancer, and compared the results to samples depleted of mouse cells. A significant increase (p &lt; 0.05) in cluster density as well as an average increase in read counts of 33% was observed for the mouse cell depleted samples, indicating improved sample quality. Indeed, we observed a strong reduction of debris and dead cells upon the depletion procedure. We mapped the reads of all samples against human and mouse genomes and determined their putative origin. An average of 12% of reads derived from non-depleted samples was assigned to mouse cells. This amount could be reduced to 0.28% by prior depletion of mouse cells. As 15% of the mouse-derived reads mapped erroneously to human genome (1.9% of total reads) in the non-depleted samples, a strong positive influence of mouse cell depletion (0.04% of total reads) on downstream analyses can be expected. The number of predicted SNPs was 2-fold higher in the non-depleted samples and 56% of SNPs identified in the non-depleted samples were not present anymore after depletion of mouse cells. Furthermore, the increase in coverage from 39-fold (non-depleted) to 56-fold (depleted) resulted in a higher sensitivity of relevant SNP detection after mouse cell depletion. 16% of SNPs identified in the depleted samples were exclusively found in these samples.
Taken together, removal of mouse cells significantly improves the analysis of human tumor xenografts by next generation sequencing. As this effect was observed although a human sequence specific selection has been carried out during exome enrichment, the influence on whole exome and whole transcriptome sequencing are expected to be even more prominent.
Citation Format: David Agorku, Stefan Tomiuk, Kerstin Klingner, Stefan Wild, Silvia Rüberg, Lisa Zatrieb, Andreas Bosio, Julia Schueler, Olaf Hardt. Next generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1455. doi:10.1158/1538-7445.AM2015-1455