Abstract 95: Depletion of mouse cells from human tumor xenografts significantly reduces bias in molecular analysis and improves culture of target cells

Abstract

Abstract Human tumor xenografts represent the gold standard method for research areas like drug discovery, cancer stem cell biology, and metastasis prediction. They can be derived from primary human tumor material, serially transplanted tumor tissue, or cultured cells. When compared to in vitro cell culture models, human tumor xenografts show a higher validity for most assays (Rubio-Viqueira et al., 2009). During the growth phase in vivo, xenografted tissue is vascularized and infiltrated by cells of murine origin including heterogeneous lymphocyte subpopulations, fibroblasts, and endothelial cells. 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, some molecular downstream analyses are challenged. The contaminating mouse cells lead to cross hybridization of mouse derived molecules to human probes on microarrays and a significant reduction of sensitivity caused by measuring mouse signals during next-generation sequencing or proteome analysis. In addition, the culture of human tumor cells is frequently hampered by murine fibroblasts efficiently plating and overgrowing the target cells. To overcome these limitations, we have developed a fast and easy method allowing for the comprehensive depletion of all cells of murine origin. We have dissociated human tumor xenografts as well as normal murine tissue of multiple origins including skin, brain, kidney and lung followed by screening panels of cell surface markers to define an optimized combination of antibodies capable of binding all cells from all murine origins. Subsequently, we generated conjugates of suitable antibodies with paramagnetic nanoparticles (MicroBeads) and titrated all reagents to optimize the depletion efficiency by using magnetic cell sorting (MACS). Even tumors that contain high numbers of mouse cells (> 60%) can be cleaned up to purities of human tumor cells higher than 96% in less than 25 minutes. This depletion step substantially increased sensitivity during empiric cell surface marker screenings as well as during the isolation of tumor cell subpopulations. Finally, we show that downstream molecular analysis and culture of human tumor cells is significantly standardized upon removal of mouse contamination. Taken together, this novel method prevents inaccurate human tumor xenograft analysis caused by contamination with cells of murine origin. As antibodies specific only for mouse cells are used, the target cells stay “untouched” and the procedure can be used for all kinds of xenografted material without the need for a positive marker expressed on the human cells. Citation Format: David Agorku, Andreas Bosio, Olaf Hardt. Depletion of mouse cells from human tumor xenografts significantly reduces bias in molecular analysis and improves culture of target cells. [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 95. doi:10.1158/1538-7445.AM2014-95