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
Each qPCR reaction plate requires the presence of the standard curve samples, which contain serial dilutions of mouse-only, human-only, or human+mouse mixed samples of known DNA concentration. Standard curves were developed in Microsoft Excel (Microsoft, Redmond, WA) by graphing the mean Threshold Cycle (CT) on the y-axis versus the log initial genomes on the x-axis. A linear trend line was generated, with the equation of the line used to calculate genome number from Mean CT values. "Percent mouse" was estimated by dividing the number of mouse genomes by the sum of both human plus mouse genomes, multiplied by 100. "Percent human" was calculated similarly. The measured genome number was used to back-calculate the measured mass of each species DNA by applying the following equation to each 
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.