Real-time PCR-based assay to quantify the relative amount of human and mouse tissue present in tumor xenografts
© Alcoser et al; licensee BioMed Central Ltd. 2011
Received: 28 September 2011
Accepted: 16 December 2011
Published: 16 December 2011
Xenograft samples used to test anti-cancer drug efficacies and toxicities in vivo contain an unknown mix of mouse and human cells. Evaluation of drug activity can be confounded by samples containing large amounts of contaminating mouse tissue. We have developed a real-time quantitative polymerase chain reaction (qPCR) assay using TaqMan technology to quantify the amount of mouse tissue that is incorporated into human xenograft samples.
The forward and reverse primers bind to the same DNA sequence in the human and the mouse genome. Using a set of specially designed fluorescent probes provides species specificity. The linearity and sensitivity of the assay is evaluated using serial dilutions of single species and heterogeneous DNA mixtures. We examined many xenograft samples at various in vivo passages, finding a wide variety of human:mouse DNA ratios. This variation may be influenced by tumor type, number of serial passages in vivo, and even which part of the tumor was collected and used in the assay.
This novel assay provides an accurate quantitative assessment of human and mouse content in xenograft tumors. This assay can be performed on aberrantly behaving human xenografts, samples used in bioinformatics studies, and periodically for tumor tissue frequently grown by serial passage in vivo.
Human xenografts implanted and grown in immunodeficient mice are commonly used to expand tumor cell populations for cancer stem cell investigations  and to test anti-cancer drug efficacies or toxicities in vivo . It is often assumed any anti-tumor drug activity is due to targeting pathways in the human cells present in the xenograft, and any associated gene expression data is derived from a mostly human cell population. However, as the implanted xenograft grows in the mouse its human stromal cells are replaced by mouse stromal cells , influencing its microenvironment and resulting in a tumor xenograft that is a heterogeneous mixture of human and mouse derived cell populations. Each cell type may possess different growth rates and react differently to an administered drug. Further, there are many reliable reports in the literature of human xenografts serially passed in vivo transforming adjacent mouse cells into fibrosarcoma-like malignancies [4–8]. Drugs tested on such samples would generate false and misleading data. How can researchers easily verify that a tumor xenograft sample contains only a relatively small number of contaminating mouse cells?
Over the years, several groups have attempted to answer this question using in situ hybridization [9, 10] or immunohistochemical procedures . However, they are slow, labor-intensive endeavors, and are limited by subjective, difficult-to-reproduce measurements. Recently, PCR-based strategies have been developed which take advantage of the ability of species-specific oligomer primers to quickly amplify species-specific genomic DNA sequences. Ono et al  were able to differentiate fourteen different species by targeting their relatively abundant and highly conserved mitochondrial DNA sequences. Cooper et al  took this a step further by performing fourteen species-specific PCR reactions simultaneously in a single PCR tube ("multiplexing"). Each species-specific amplicon was a unique length, different enough from the others to be successfully resolved on a 4% agarose gel. While these methods are certainly not quantitative, they are fast, easy, and accurate. One way to obtain a more quantitative result is to take advantage of DNA sequencing technology to measure gene length variation by PCR-amplification of several markers across different chromosomes then compare the species-specific differences in relative amplicon lengths . This improved method still requires manually measuring peak heights generated by capillary electrophoresis from an ABI 3100 Genetic Analyzer, a robust but still expensive and uncommon piece of laboratory equipment. This method also relies on the use of multiple genomic loci, many of which are located on or near chromosomal regions known to be deleted or amplified in some human cancers.
Real-Time Quantitative PCR (qPCR) instruments have been used to quantify genomic DNA from multiple species in mixed DNA samples: multiple human targets ; rhesus and long-tailed macaques ; feline, bovine, equine, and cervid ; human, cat, and dog . These machines have become commonplace and a 96-well plate can be processed in 90 minutes or less. Therefore, we set out to develop a multiplex qPCR assay that addresses some of the obstacles that limited previous methodologies. Herein we describe the assay methodology, various quality control analyses, and a survey of human xenograft samples to illustrate the kind of real-world results that can be expected from the assay.
Results and Discussion
Initial sequence targeting and validation
Evaluating mixed DNA samples
Species quantification results from a survey of selected xenografts.
in vivo Passage
Initial DNA (ng)
Average % Human ± SD
Sum DNA (ng)
% of Initial DNA
100.0 ± 0.00
12.8 ± 1.84
98.9 ± 1.50
43.2 ± 0.06
81.8 ± 1.42
99.9 ± 0.08
48.4 ± 7.26
55.1 ± 6.38
39.6 ± 7.99
53.1 ± 6.56
33.9 ± 7.76
83.5 ± 5.89
83.6 ± 4.90
99.9 ± 0.09
83.5 ± 3.23
100.0 ± 0.02
100.0 ± 0.00
88.8 ± 0.31
99.3 ± 0.82
90.1 ± 4.26
34.9 ± 6.18
81.0 ± 2.00
This qPCR method allows for quick and accurate estimations of mouse contamination in human xenografts. This is important when accessing anti-tumor drug efficacy in human tumor xenografts and in evaluating any other mixed DNA sample where significant proportions of mouse DNA could influence the interpretation of the results.
Cell lines and xenografts
Human cancer cell lines were obtained from ATCC/LGC (Wesel, Germany) or the DCTD Repository (Frederick, MD) and were cultured in RPMI Medium 1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal calf serum and 2 mM L-glutamine. All cell lines were maintained in a humidified incubator at 37°C in the presence of 5% CO2. Human xenograft samples were implanted subcutaneous in athymic nude mice (Balb/c nu/nu, 4-6 weeks old), which were purchased from Charles River Laboratories (Frederick, MD) and maintained under sterile and controlled conditions of temperature (22-24C), light (12-h light/12-h dark), and humidity (45-65%), with food and water ad libitum. Xenografts used for this study were routinely harvested when they reached 500 mg in size, before a necrotic core develops, and after 1, 4, or 10 serial in vivo passages. Each of the fresh tumors harvested for DNA extraction were divided using surgical scissors into multiple fragments of similar size (~150 mg), each containing regions from both the tumor's core and periphery.
DNA extraction and purification
Tissues and cell lines were processed using a modified version of the REDExtract-N-Amp Tissue PCR kit protocol (Sigma-Aldrich, St. Louis, MO). Briefly, samples (~150 mg wet weight) were incubated overnight in a mixture of 100 μL extraction buffer, 10 μL tissue preparation solution, and 5 μL of 10 mg/ml proteinase K (Life Technologies) at 50°C in a slowly rotating rotisserie oven. Samples were neutralized the next morning by adding 110 μL neutralization buffer and 5 μL 20 mg/mL Purelink RNase-A (Life Technologies), followed by incubations at 37°C and 96°C for 15 minutes each. To quantify DNA for real-time qPCR assays, genomic DNA was purified with the standard phenol-chloroform method and resuspended in 10 mM Tris-Cl buffer, pH 8.0, or diluted in distilled, sterile water. DNA concentrations were measured on a NanoDrop-1000 (Thermo Fisher Scientific, Inc., Waltham, MA).
Qualitative, end-point PCR
Unique human and mouse-specific primer pairs, designed using Primer3 software http://frodo.wi.mit.edu/primer3/, rely on species-specific differences (underlined in Figure 1b) in the forward primers to amplify 189 bp fragments of the prostaglandin E receptor 2 (PTGER2) gene. PCR primers were purchased from Applied Biosystems (ABI) by Life Technologies. PCR was performed using neutralized but unpurified tissue/cell lysate on an ABI-2720 Thermocycler (Life Technologies). PCR conditions: 95°C-5 min, 30 cycles of (94°C-45 sec, 60°C-30 sec, 72°C-90 sec), 72°C-10 min. DNA bands were resolved on a 2% agarose gel + ethidium bromide (0.5 μg/ml).
Real-Time Quantitative PCR (qPCR)
The qPCR primers and probes were designed using Primer3 software  (http://frodo.wi.mit.edu/primer3/) and purchased from ABI (Life Technologies). Target sequences represent regions located in the human and mouse prostaglandin E receptor 2 (PTGER2) genes (see Figure 1A). Real-time qPCR was carried out on an ABI-7500 Real Time PCR System (Applied Biosystems) using custom-labelled species-specific probes (ABI) according to the manufacturer's protocol with 50 ng of total genomic DNA (unless otherwise specified) in 20 μL reaction volumes. The qPCR conditions were as follows: 50°C-2 min, 95°C-10 min, 40 cycles of (95°C-15 sec, 60°C-1 min). The human+mouse forward primer and the common reverse primer listed in Figure 1B were added to each qPCR reaction tube to obtain the same final concentrations (200 nM). Both probes (Figure 1C) were also added to each reaction tube for a final concentration of 200 nM. Samples were usually run in triplicate on the same reaction plate. Samples were assayed on at least three different 96-well reaction plates, often by two different operators, before statistical analysis was performed. All ΔRn thresholds were calculated by default from the 7500 ABI software, v 2.0.5.
Data analysis and statistics
where M = mass of the haploid genome (in grams), Ng = number of base pairs (bp) in haploid genome, and g = grams. The mouse genome is estimated to be 2.651 billion bp (as of NCBI genome Build 36.1), while the human genome is estimated to be 3.038 billion bp (as of NCBI genome Build 36.3). Thus, one haploid mouse genome is approximately 2.9 pg, whereas one human haploid genome is approximately 3.33 pg.
The authors thank Andrea De Biase of Life Technologies for discussions involving TaqMan probes, quenchers, and general qPCR optimization. This project was funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. NCI-Frederick is accredited by AAALACi and follows the Public Health Service Policy on the Care and Use of Laboratory Animals. All animals used in this research project were cared for and used humanely according to the following policies: The U.S. Public Health Service Policy on Human Care and Use of Animals (1996); the Guide for the Care and Use Of Laboratory Animals (NIH publication No. 86-23, 1985); and the U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (1985).
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