Abstract

Background: As 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. Methods: In 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. Results: In 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. Conclusion: Collectively, 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.

Original languageEnglish
Article number440
JournalBMC Cancer
Volume17
Issue number1
DOIs
Publication statusPublished - Jun 23 2017

Fingerprint

Circulating Neoplastic Cells
Animal Models
Neoplasms
Cell Count
Neoplasm Metastasis
Lung
Heterologous Transplantation
DNA Primers
Luciferases
Heterografts
Knockout Mice
Pharmaceutical Preparations
Transgenic Mice
Tail
Veins
Flow Cytometry
RNA
Breast Neoplasms

Keywords

  • Cancer metastasis
  • Circulating tumor cells
  • In vivo bioluminescent imaging system
  • Quantitative PCR

ASJC Scopus subject areas

  • Oncology
  • Genetics
  • Cancer Research

Cite this

A rapid and quantitative method to detect human circulating tumor cells in a preclinical animal model. / Tu, Shih Hsin; Hsieh, Yi Chen; Huang, Li Chi; Lin, Chun Yu; Hsu, Kai Wen; Hsieh, Wen Shyang; Chi, Wei Ming; Lee, Chia Hwa.

In: BMC Cancer, Vol. 17, No. 1, 440, 23.06.2017.

Research output: Contribution to journalArticle

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abstract = "Background: As 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. Methods: In 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. Results: In 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. Conclusion: Collectively, 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.",
keywords = "Cancer metastasis, Circulating tumor cells, In vivo bioluminescent imaging system, Quantitative PCR",
author = "Tu, {Shih Hsin} and Hsieh, {Yi Chen} and Huang, {Li Chi} and Lin, {Chun Yu} and Hsu, {Kai Wen} and Hsieh, {Wen Shyang} and Chi, {Wei Ming} and Lee, {Chia Hwa}",
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AU - Tu, Shih Hsin

AU - Hsieh, Yi Chen

AU - Huang, Li Chi

AU - Lin, Chun Yu

AU - Hsu, Kai Wen

AU - Hsieh, Wen Shyang

AU - Chi, Wei Ming

AU - Lee, Chia Hwa

PY - 2017/6/23

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N2 - Background: As 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. Methods: In 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. Results: In 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. Conclusion: Collectively, 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.

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KW - Circulating tumor cells

KW - In vivo bioluminescent imaging system

KW - Quantitative PCR

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