A rapid and quantitative method to detect human circulating tumor cells in a preclinical animal model

Abstract

BackgroundAs cancer metastasis is the deadliest aspect of cancer, causing 90% of human deaths, evaluating the molecular mechanisms underlying this process is the major interest to those in the drug development field. Both therapeutic target identification and proof-of-concept experimentation in anti-cancer drug development require appropriate animal models, such as xenograft tumor transplantation in transgenic and knockout mice. In the progression of cancer metastasis, circulating tumor cells (CTCs) are the most critical factor in determining the prognosis of cancer patients. Several studies have demonstrated that measuring CTC-specific markers in a clinical setting (e.g., flow cytometry) can provide a current status of cancer development in patients. However, this useful technique has rarely been applied in the real-time monitoring of CTCs in preclinical animal models.MethodsIn this study, we designed a rapid and reliable detection method by combining a bioluminescent in vivo imaging system (IVIS) and quantitative polymerase chain reaction (QPCR)-based analysis to measure CTCs in animal blood. Using the IVIS Spectrum CT System with 3D–imaging on orthotropic-developed breast-tumor-bearing mice.ResultsIn this manuscript, we established a quick and reliable method for measuring CTCs in a preclinical animal mode. The key to this technique is the use of specific human and mouse GUS primers on DNA/RNA of mouse peripheral blood under an absolute qPCR system. First, the high sensitivity of cancer cell detection on IVIS was presented by measuring the luciferase carried MDA-MB-231 cells from 5 to 5x1011 cell numbers with great correlation (R2 = 0.999). Next, the MDA-MB-231 cell numbers injected by tail vein and their IVIS radiance signals were strongly corrected with qPCR-calculated copy numbers (R2 > 0.99). Furthermore, by applying an orthotropic implantation animal model, we successfully distinguished xenograft tumor-bearing mice and control mice with a significant difference (p < 0.001), whereas IVIS Spectrum-CT 3D–visualization showed that blood of mice with lung metastasis contained more than twice the CTC numbers than ordinary tumor-bearing mice. We demonstrated a positive correlation between lung metastasis status and CTC numbers in peripheral mouse blood.ConclusionCollectively, the techniques developed for this study resulted in the integration of CTC assessments into preclinical models both in vivo and ex vivo, which will facilitate translational targeted therapy in clinical practice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3419-x) contains supplementary material, which is available to authorized users.

Nicotinic Acetylcholine Receptor Subtype Alpha-9 Mediates Triple-Negative Breast Cancers Based on a Spontaneous Pulmonary Metastasis Mouse Model

“…MDA-MB-231 cells were transfected with pcDNA3 plasmids expressing the firefly luciferase gene (the gene sequences were originally from luc4.1 ; Chris Contag, Stanford University, Stanford, CA, United States), as described previously ( Tu et al, 2017 ). Briefly, 5 × 10 6 MDA-MB-231 cells were washed twice with phosphate buffered saline (PBS) and mixed with 10 mg of plasmid.…”

α-Tocopherol succinate enhances pterostilbene anti-tumor activity in human breast cancer cells in vivo and in vitro

“…Furthermore, the combination of PS and α-TOS in vivo might be a better breast cancer therapeutic strategy (empty circle vs. reverse triangle, p = 0.02). To determine whether the PS and α-TOS combination could stop cancer metastasis, we used our recently developed novel technique to evaluate CTCs from the xenograft-bearing mice [ 17 ]. Figure 5C shows that the CTC numbers were significantly inhibited in mice that were fed the high Vit.…”

“…( B ) The photon flux for each mouse tumor was measured and calculated. ( C ) Before euthanasia, the blood from each mouse was harvested to measure circulating tumor cells by Q-PCR [ 17 ]. ( D ) At the end of the experiment, the mice were euthanized, and the expression levels of CDK2, AKT, and ERK in their tumor tissues were determined by western blotting.…”

“…Our recently developed circulating tumor cell (CTC) detection assay was used on a xenograft-bearing animal model to determine whether Vit. E and PS exposure could suppress both tumor growth and metastasis [ 17 ]. These data help elucidate whether the use of both α-TP or its analogs and PS as supplements may be beneficial during cancer therapy for the treatment of human breast cancers.…”

The Application of Non-Invasive Apoptosis Detection Sensor (IADS) on Histone Deacetylation Inhibitors (HDACi) Induced Breast Cancer Cell Death

“…MDA-MB-231 cells were transfected with pcDNA3 plasmids expressing the firefly luciferase gene (the gene sequences were originally from luc4.1; Chris Contag, Stanford University, Stanford, CA, USA), as described previously [12]. Briefly, 5×10 6 MDA-MB-231 cells were washed twice with phosphate buffered saline (PBS) and mixed with 10 μg of plasmid.…”