Removal of lactate dehydrogenase-elevating virus from human-in-mouse breast tumor xenografts by cell-sorting

Abstract

Lactate dehydrogenase-elevating virus (LDV) can infect transplantable mouse tumors or xenograft tumors in mice through LDV-contaminated mouse biological materials, such as Matrigel, or through mice infected with LDV. LDV infects specifically mouse macrophages and alters immune system and tumor phenotype. The traditional approaches to remove LDV from tumor cells, by transplanting tumors into rats or culturing tumor cells in vitro, are inefficient, labor-intensive and time-consuming. Furthermore, these approaches are not feasible for primary tumor cells that cannot survive tissue culture conditions or that may change phenotype in rats. This study reports that fluorescence-activated cell sorting (FACS) is a simple and efficient approach for purifying living primary human breast tumor cells from LDV(+) mouse stromal cells, which can be completed in a few hours. When purified from Matrigel contaminated LDV(+) tumors, sorted human breast tumor cells, as well as tumors grown from sorted cells, were shown to be LDV-free, as tested by PCR. The results demonstrate that cell sorting is effective, much faster and less likely to alter tumor cell phenotype than traditional methods for removing LDV from xenograft models. This approach may also be used to remove other rodent-specific viruses from models derived from distinct tissues or species with sortable markers, where virus does not replicate in the cells to be purified.

Figure 1. Flow cytometry analysis of human tumor cells upon and post sorting

A: Dot plot of bulk tumor cells profiled by the cell size/volume channels, forward scatter area (FSC-A, X axis) and the side scatter area (SSC-A, Y axis). The gate of P1 population excluded cell debris (<50). B: Top panel is a dot plot of P1 cells by side scatter height (SSC-H, X axis) and width (SSC-W, Y axis) to gate potential single cells (P2 population), while bottom panel profiles P2 cells by forward scatter height (FSC-H, X axis) and width (FSC-W, Y axis) to further gate single cells (P3 population). C: Dot plot of P3 cells in the fluorescent channels of DAPI (a viability marker) and H2K^d-PE-cy5 (a mouse stromal cell marker). The P4 population was gated for viable (DAPI⁻) human tumor cells (H2K-PEcy5⁻) for sorting. D: The purity examination of P4 population by flow cytometer after double sorting.

Figure 2. Flow cytometry analysis of sorted human breast tumor cells

Cells were sorted with an extra marker based on single DAPI⁻H2K^d− cells as P4 in Fig 1. A: Representative flow profile of sorted CD44⁺H2K^d− breast tumor cells (P5), by CD44-APC and H2K^d-PE-Cy5 channels (99.4% pure). B: Representative flow profile of sorted tdTomato-labeled tumor cells (98.8% pure). C: Representative flow profile of sorted tdTomato-labeled tumor cells (98.9% pure).