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Development of a new rapid isolation device for circulating tumor cells (CTCs) using 3D palladium filter and its application for genetic analysis.

Yusa A, Toneri M, Masuda T, Ito S, Yamamoto S, Okochi M, Kondo N, Iwata H, Yatabe Y, Ichinosawa Y, Kinuta S, Kondo E, Honda H, Arai F, Nakanishi H - PLoS ONE (2014)

Bottom Line: Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively.Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers.These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Aichi Science and Technology Foundation, Knowledge Hub Aichi, Priority Research Projects, Japan ; Department of Micro-Nano Systems Engineering, Graduate School of Engineering, Nagoya University, Japan ; Division of Oncological Pathology, Aichi Cancer Center Research Institute, Japan.

ABSTRACT
Circulating tumor cells (CTCs) in the blood of patients with epithelial malignancies provide a promising and minimally invasive source for early detection of metastasis, monitoring of therapeutic effects and basic research addressing the mechanism of metastasis. In this study, we developed a new filtration-based, sensitive CTC isolation device. This device consists of a 3-dimensional (3D) palladium (Pd) filter with an 8 µm-sized pore in the lower layer and a 30 µm-sized pocket in the upper layer to trap CTCs on a filter micro-fabricated by precise lithography plus electroforming process. This is a simple pump-less device driven by gravity flow and can enrich CTCs from whole blood within 20 min. After on-device staining of CTCs for 30 min, the filter cassette was removed from the device, fixed in a cassette holder and set up on the upright fluorescence microscope. Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively. Cell spike experiments demonstrated that the recovery rate of tumor cells from blood by this Pd filter device was more than 85%. Single living tumor cells were efficiently isolated from these spiked tumor cells by a micromanipulator, and KRAS mutation, HER2 gene amplification and overexpression, for example, were successfully detected from such isolated single tumor cells. Sequential analysis of blood from mice bearing metastasis revealed that CTC increased with progression of metastasis. Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers. These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

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Recovery rate of the spiked cancer cells by 3D Pd filter device.A–D. Representative recovered spiked GFP-tagged colonic cancer cells (arrows) showing GFP+/CD45−/Hoechst33342+ phenotypes after filtration of 5000 COLM5-EGFP cells spiked into 2 ml normal blood. Images of GFP (A), PE-CD45 (B), Hoechst33342 (C) and bright field (D) are shown. Bars = 40 µm E–G. Representative recovered spiked GFP non-tagged gastric cancer cells (arrows) showing EpCAM+/CD45−/Hoechst33342+ phenotypes after filtration of 5000 NCI N-87 cells spiked into 2 ml of blood. Images of Alexa488-EpCAM (F), PE-CD45 (G) and Hoechst33342 (H) are shown. Bars = 40 µm H. Recovery rate of spiked tumor cells according to flow rate of the filter device. I. Recovery rate of spiked tumor cells according to the number of tumor cells spiked into blood.
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pone-0088821-g004: Recovery rate of the spiked cancer cells by 3D Pd filter device.A–D. Representative recovered spiked GFP-tagged colonic cancer cells (arrows) showing GFP+/CD45−/Hoechst33342+ phenotypes after filtration of 5000 COLM5-EGFP cells spiked into 2 ml normal blood. Images of GFP (A), PE-CD45 (B), Hoechst33342 (C) and bright field (D) are shown. Bars = 40 µm E–G. Representative recovered spiked GFP non-tagged gastric cancer cells (arrows) showing EpCAM+/CD45−/Hoechst33342+ phenotypes after filtration of 5000 NCI N-87 cells spiked into 2 ml of blood. Images of Alexa488-EpCAM (F), PE-CD45 (G) and Hoechst33342 (H) are shown. Bars = 40 µm H. Recovery rate of spiked tumor cells according to flow rate of the filter device. I. Recovery rate of spiked tumor cells according to the number of tumor cells spiked into blood.

Mentions: On the basis of the above-described study, we used a 3D φ30-H10-pitch34 Pd (or Pd alloy) filter with maximal pore density (100,000/cm2) as the best filter for rapid, gentle enrichment, and subsequent isolation of living CTC. In a spike experiment, two staining methods were used for detection of spiked epithelial tumor cells in the blood. In the spike experiment using GFP-tagged COLM-5 tumor cells, GFP+/CD45−/Hoechst33342+ cells were recognized as tumor cells (Figures 4A–4D). In the spike experiment using GFP non-tagged tumor cells (NCI N-87), cells with EpCAM+/CD45−/Hoechst33342+ and EpCAM−/CD45+/Hoechst33342+ were recognized as tumor cells and leukocytes, respectively (Figures 4E–4G).


Development of a new rapid isolation device for circulating tumor cells (CTCs) using 3D palladium filter and its application for genetic analysis.

Yusa A, Toneri M, Masuda T, Ito S, Yamamoto S, Okochi M, Kondo N, Iwata H, Yatabe Y, Ichinosawa Y, Kinuta S, Kondo E, Honda H, Arai F, Nakanishi H - PLoS ONE (2014)

Recovery rate of the spiked cancer cells by 3D Pd filter device.A–D. Representative recovered spiked GFP-tagged colonic cancer cells (arrows) showing GFP+/CD45−/Hoechst33342+ phenotypes after filtration of 5000 COLM5-EGFP cells spiked into 2 ml normal blood. Images of GFP (A), PE-CD45 (B), Hoechst33342 (C) and bright field (D) are shown. Bars = 40 µm E–G. Representative recovered spiked GFP non-tagged gastric cancer cells (arrows) showing EpCAM+/CD45−/Hoechst33342+ phenotypes after filtration of 5000 NCI N-87 cells spiked into 2 ml of blood. Images of Alexa488-EpCAM (F), PE-CD45 (G) and Hoechst33342 (H) are shown. Bars = 40 µm H. Recovery rate of spiked tumor cells according to flow rate of the filter device. I. Recovery rate of spiked tumor cells according to the number of tumor cells spiked into blood.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921253&req=5

pone-0088821-g004: Recovery rate of the spiked cancer cells by 3D Pd filter device.A–D. Representative recovered spiked GFP-tagged colonic cancer cells (arrows) showing GFP+/CD45−/Hoechst33342+ phenotypes after filtration of 5000 COLM5-EGFP cells spiked into 2 ml normal blood. Images of GFP (A), PE-CD45 (B), Hoechst33342 (C) and bright field (D) are shown. Bars = 40 µm E–G. Representative recovered spiked GFP non-tagged gastric cancer cells (arrows) showing EpCAM+/CD45−/Hoechst33342+ phenotypes after filtration of 5000 NCI N-87 cells spiked into 2 ml of blood. Images of Alexa488-EpCAM (F), PE-CD45 (G) and Hoechst33342 (H) are shown. Bars = 40 µm H. Recovery rate of spiked tumor cells according to flow rate of the filter device. I. Recovery rate of spiked tumor cells according to the number of tumor cells spiked into blood.
Mentions: On the basis of the above-described study, we used a 3D φ30-H10-pitch34 Pd (or Pd alloy) filter with maximal pore density (100,000/cm2) as the best filter for rapid, gentle enrichment, and subsequent isolation of living CTC. In a spike experiment, two staining methods were used for detection of spiked epithelial tumor cells in the blood. In the spike experiment using GFP-tagged COLM-5 tumor cells, GFP+/CD45−/Hoechst33342+ cells were recognized as tumor cells (Figures 4A–4D). In the spike experiment using GFP non-tagged tumor cells (NCI N-87), cells with EpCAM+/CD45−/Hoechst33342+ and EpCAM−/CD45+/Hoechst33342+ were recognized as tumor cells and leukocytes, respectively (Figures 4E–4G).

Bottom Line: Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively.Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers.These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Aichi Science and Technology Foundation, Knowledge Hub Aichi, Priority Research Projects, Japan ; Department of Micro-Nano Systems Engineering, Graduate School of Engineering, Nagoya University, Japan ; Division of Oncological Pathology, Aichi Cancer Center Research Institute, Japan.

ABSTRACT
Circulating tumor cells (CTCs) in the blood of patients with epithelial malignancies provide a promising and minimally invasive source for early detection of metastasis, monitoring of therapeutic effects and basic research addressing the mechanism of metastasis. In this study, we developed a new filtration-based, sensitive CTC isolation device. This device consists of a 3-dimensional (3D) palladium (Pd) filter with an 8 µm-sized pore in the lower layer and a 30 µm-sized pocket in the upper layer to trap CTCs on a filter micro-fabricated by precise lithography plus electroforming process. This is a simple pump-less device driven by gravity flow and can enrich CTCs from whole blood within 20 min. After on-device staining of CTCs for 30 min, the filter cassette was removed from the device, fixed in a cassette holder and set up on the upright fluorescence microscope. Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively. Cell spike experiments demonstrated that the recovery rate of tumor cells from blood by this Pd filter device was more than 85%. Single living tumor cells were efficiently isolated from these spiked tumor cells by a micromanipulator, and KRAS mutation, HER2 gene amplification and overexpression, for example, were successfully detected from such isolated single tumor cells. Sequential analysis of blood from mice bearing metastasis revealed that CTC increased with progression of metastasis. Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers. These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

Show MeSH
Related in: MedlinePlus