Real-time PCR-based assay to quantify the relative amount of human and mouse tissue present in tumor xenografts

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Tumor response and biomarkers for Top1 inhibitors in topotecan-responsive xenograft models.

<p>Abbreviations: C, control group; d, day; IP, intraperitoneal; IV, intravenous; NA, not applicable; ND, not determined QD×5, treated for 5 sequential days at designated dose; T, treated group.</p>a<p>. No mice were tumor-free by study day 70.</p>b<p>. A death is considered treatment-related if the animal dies within 15 days of the last treatment and either the tumor weight is less than the lethal burden in the control mice or its net body weight loss at death is 20% greater than the mean net weight change in the control animals at death or sacrifice.</p>c<p>. Data from topotecan and NSC 724998 treated A375 xenografts in athymic nude mice were previously published by our group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050494#pone.0050494-Kinders1" target="_blank">[30]</a>.</p

Dose-dependent decrease in Top1 levels in A375 xenografts.

<p>Tumor biopsies were collected from A375 xenografts 2, 4, and 7 hours after a single dose of 0.033 to 1.0 MTD topotecan or water vehicle control (n = 4/cohort). Box plots represent interquartile range, 10^th and 90^th percentile whiskers, and median Top1 levels; diamonds represent group mean. Asterisks (*), treatment cohort mean was statistically different from vehicle mean at same time point, with a significance level of P≤0.05, as determined by Student's one-tailed <i>t</i>-test.</p

Biological variation observed in baseline Top1 levels in xenograft models and patient samples.

<p>Abbreviations: PBMCs, peripheral blood mononuclear cells; SD, standard deviation.</p>a<p>. Xenograft data include Top1 levels measured in untreated and vehicle-treated (water) A375 and HCT 116 xenograft bearing mice and vehicle-treated (water) Colo829 and SK-MEL-28 mice from all experiments.</p>b<p>. Top1 levels measured in tumor biopsies from patients at baseline. Top1 levels expressed as an average of all measurements above the LLQ; each patient had Top1 levels above the LLQ for at least 2 of 3 protein loads.</p>c<p>. Top1 levels measured in PBMC samples from patients at baseline. Top1 levels expressed as an average of all measurements above the LLQ. In one patient, the Top1 levels were below the LLQ, in this case the LLQ was used as the Top1 measurement.</p

Top1 levels in topotecan-responsive and -nonresponsive xenograft models.

<p>Mice treated with water vehicle or single dose 1.0 MTD topotecan for the designated times were assessed for Top1 levels using the validated immunoassay. (A) Mice bearing A375, topotecan-responsive xenografts were collected 2, 4, or 7 hours after treatment (n = 4/cohort). (B) HCT 116 colon cancer xenografts were collected 2, 5, and 24 hours after treatment and compared to baseline (0 h, no vehicle). n = 3–6 topotecan-treated mice/cohort and n = 12 in the 0 h cohort. (C) Colo829 xenografts were collected 4, 7, or 24 hours after treatment (n = 3/cohort). (D) SK-MEL-28 xenografts were collected 2, 5, or 24 hours after treatment (n = 6/cohort). Box plots represent interquartile range, 10^th and 90^th percentile whiskers, and median Top1 levels; diamonds represent group mean. Asterisks (*), treatment cohort mean was statistically different from the vehicle mean at same time point (or baseline in the HCT 116 cohorts), with a significance level of P≤0.05, as determined by Student's one-tailed <i>t</i>-test.</p

Validation of assay performance.

<p>(A) Top1 levels were measured in serial dilutions of three A375 and three HCT 116 xenograft extract samples using the Top1 immunoassay. (B) Total Top1 levels from the diluted extracts in panel A normalized to 1 μg/mL. (C) Comparison of Top1 levels in 24 matched samples and 6 control extracts, measured by two independent laboratories, the Pharmacodynamic Assay Development and Implementation Section (PADIS) laboratory and the National Clinical Target Validation Laboratory (NCTVL). Sample dilution and analysis were performed independently by both laboratories and Top1 levels were compared across sites.</p

Baseline Top1 levels in clinical biopsy samples.

<p>Top1 levels measured in extracts prepared from pre-treatment tumor needle biopsies from 6 patients enrolled in NCI clinical trials. Top1 levels were assayed using three different protein loads for each extract. Top1 levels in biopsy extracts for the 0.5 μg/mL protein load of patients 3 to 6 were below the LLQ of the assay.</p

Top1 levels measured in xenografts treated with topotecan compared to two indenoisoquinoline topoisomerase inhibitors.

<p>(A) Top1 levels in A375 xenografts collected 4 or 7 hours following single dose treatment (MTD indicated) with topotecan or the indenoisoquinoline NSC 724998 (n = 5–6 mice/cohort). (B) HCT 116 xenografts and (C) Colo829 xenografts collected 4 hours after mice were treated with the indicated doses and drugs. (n = 3–6 mice/cohort; NSC 724998 had 2 mice/cohort). Box plots represent interquartile range, 10^th and 90^th percentile whiskers, and median Top1 levels; diamonds represent group mean. Dashed lines in panels B and C indicate the mean Top1 levels in a matched untreated cohort (0 h) for these xenograft models. Asterisks (*), treatment cohort mean was statistically different from vehicle mean at same time point, with a significance level of P≤0.05, as determined by Student's one-tailed <i>t</i>-test.</p