Background: As cancer metastasis is the deadliest aspect of cancer, causing 90% of human deaths, evaluating the\nmolecular mechanisms underlying this process is the major interest to those in the drug development field. Both\ntherapeutic target identification and proof-of-concept experimentation in anti-cancer drug development require\nappropriate animal models, such as xenograft tumor transplantation in transgenic and knockout mice. In the\nprogression of cancer metastasis, circulating tumor cells (CTCs) are the most critical factor in determining the\nprognosis of cancer patients. Several studies have demonstrated that measuring CTC-specific markers in a clinical setting\n(e.g., flow cytometry) can provide a current status of cancer development in patients. However, this useful technique has\nrarely been applied in the real-time monitoring of CTCs in preclinical animal models.\nMethods: In this study, we designed a rapid and reliable detection method by combining a bioluminescent in vivo\nimaging system (IVIS) and quantitative polymerase chain reaction (QPCR)-based analysis to measure CTCs in\nanimal blood. Using the IVIS Spectrum CT System with 3Dââ?¬â??imaging on orthotropic-developed breast-tumor-bearing mice.\nResults: In this manuscript, we established a quick and reliable method for measuring CTCs in a preclinical animal mode.\nThe key to this technique is the use of specific human and mouse GUS primers on DNA/RNA of mouse peripheral blood\nunder an absolute qPCR system. First, the high sensitivity of cancer cell detection on IVIS was presented by measuring the\nluciferase carried MDA-MB-231 cells from 5 to 5x1011 cell numbers with great correlation (R2 = 0.999). Next, the MDA-MB-\n231 cell numbers injected by tail vein and their IVIS radiance signals were strongly corrected with qPCR-calculated copy\nnumbers (R2 > 0.99). Furthermore, by applying an orthotropic implantation animal model, we successfully distinguished\nxenograft tumor-bearing mice and control mice with a significant difference (p < 0.001), whereas IVIS Spectrum-CT 3Dââ?¬â??\nvisualization showed that blood of mice with lung metastasis contained more than twice the CTC numbers than ordinary\ntumor-bearing mice. We demonstrated a positive correlation between lung metastasis status and CTC numbers\nin peripheral mouse blood.\nConclusion: Collectively, the techniques developed for this study resulted in the integration of CTC assessments into\npreclinical models both in vivo and ex vivo, which will facilitate translational targeted therapy in clinical practice.
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