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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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Flowchart of enrichment, enumeration, isolation and molecular analysis of CTC by metal filtration-based device.A. Overview of CTC enrichment device which consists of blood reservoir, filter unit and disposal tank. Filter unit (filter cassette) is composed of a palladium (Pd) metal filter placed between upper and lower cassette pieces. Diluted whole blood is applied to the reservoir and filtrated driven by gravity flow without a pump. B. After filtration, filter cassette is detached from the device and set up in combination with cassette holder on the upright fluorescence microscope for enumeration and isolation. C. Single CTC is isolated with micromanipulation using a glass capillary. Isolated CTC moved into PCR plate and DNA/RNA is extracted and amplified. Mutation and/or gene expression analysis is then performed. D. The filter is detached from cassette and is directly stained with immunocytochemistry (ICC) and FISH method.
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pone-0088821-g001: Flowchart of enrichment, enumeration, isolation and molecular analysis of CTC by metal filtration-based device.A. Overview of CTC enrichment device which consists of blood reservoir, filter unit and disposal tank. Filter unit (filter cassette) is composed of a palladium (Pd) metal filter placed between upper and lower cassette pieces. Diluted whole blood is applied to the reservoir and filtrated driven by gravity flow without a pump. B. After filtration, filter cassette is detached from the device and set up in combination with cassette holder on the upright fluorescence microscope for enumeration and isolation. C. Single CTC is isolated with micromanipulation using a glass capillary. Isolated CTC moved into PCR plate and DNA/RNA is extracted and amplified. Mutation and/or gene expression analysis is then performed. D. The filter is detached from cassette and is directly stained with immunocytochemistry (ICC) and FISH method.

Mentions: Enrichment, isolation and molecular analysis of CTC by filtration-based device were overviewed in figure 1. The CTC enrichment device consists of a blood reservoir, filter unit, efflux control unit and waste outlet. The size of the device is approximately 15 (length) x 13 (width) x 20 (height) cm (Figure 1A). The 3D palladium (Pd) filter is composed of an 8 µm-sized pore in the lower layer and a 30 µm-sized CTC capture hole (pocket) in the upper layer with a 34 µm-pitch (Figure 2E). This Pd filter was placed between the upper and lower filter cassette piece (Figure 2A and 2B). Whole blood obtained from posterior caval vein in mice or cubital veins of patients were 10-fold diluted with PBS and applied to the reservoir directly by gravity flow without a peristaltic pump. After filtration of blood, a filter cassette was fixed on-device with 10% formalin in PBS for 10 min, followed by immersion with 0.5 ml of antibody mixture for 30 min at RT, followed by washing with PBS. These procedures were carried out under a clean condition at least S1000 level. After staining, a filter cassette was detached from the device and plugged into the cassette holder (Chuo SEIKI, Tokyo, Japan) on the stage of the upright fluorescence microscope to fix the cassette and to control the depth of PBS above the filter to the optimal position. The fluorescence-positive cells were then counted and photographed by the pathologist before knowing any clinical data of the patients. Single cell isolation of CTC was carried out with a micromanipulator using a glass capillary (Figure 1B). The isolated CTC or CTC pool was transferred to 0.2 ml of PCR tube or a 96 well PCR plate and genomic DNA or RNA was extracted, amplified, and genetic analysis was performed (Figure 1C). When isolating living CTC, 10% formalin fixation process was omitted.


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)

Flowchart of enrichment, enumeration, isolation and molecular analysis of CTC by metal filtration-based device.A. Overview of CTC enrichment device which consists of blood reservoir, filter unit and disposal tank. Filter unit (filter cassette) is composed of a palladium (Pd) metal filter placed between upper and lower cassette pieces. Diluted whole blood is applied to the reservoir and filtrated driven by gravity flow without a pump. B. After filtration, filter cassette is detached from the device and set up in combination with cassette holder on the upright fluorescence microscope for enumeration and isolation. C. Single CTC is isolated with micromanipulation using a glass capillary. Isolated CTC moved into PCR plate and DNA/RNA is extracted and amplified. Mutation and/or gene expression analysis is then performed. D. The filter is detached from cassette and is directly stained with immunocytochemistry (ICC) and FISH method.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3921253&req=5

pone-0088821-g001: Flowchart of enrichment, enumeration, isolation and molecular analysis of CTC by metal filtration-based device.A. Overview of CTC enrichment device which consists of blood reservoir, filter unit and disposal tank. Filter unit (filter cassette) is composed of a palladium (Pd) metal filter placed between upper and lower cassette pieces. Diluted whole blood is applied to the reservoir and filtrated driven by gravity flow without a pump. B. After filtration, filter cassette is detached from the device and set up in combination with cassette holder on the upright fluorescence microscope for enumeration and isolation. C. Single CTC is isolated with micromanipulation using a glass capillary. Isolated CTC moved into PCR plate and DNA/RNA is extracted and amplified. Mutation and/or gene expression analysis is then performed. D. The filter is detached from cassette and is directly stained with immunocytochemistry (ICC) and FISH method.
Mentions: Enrichment, isolation and molecular analysis of CTC by filtration-based device were overviewed in figure 1. The CTC enrichment device consists of a blood reservoir, filter unit, efflux control unit and waste outlet. The size of the device is approximately 15 (length) x 13 (width) x 20 (height) cm (Figure 1A). The 3D palladium (Pd) filter is composed of an 8 µm-sized pore in the lower layer and a 30 µm-sized CTC capture hole (pocket) in the upper layer with a 34 µm-pitch (Figure 2E). This Pd filter was placed between the upper and lower filter cassette piece (Figure 2A and 2B). Whole blood obtained from posterior caval vein in mice or cubital veins of patients were 10-fold diluted with PBS and applied to the reservoir directly by gravity flow without a peristaltic pump. After filtration of blood, a filter cassette was fixed on-device with 10% formalin in PBS for 10 min, followed by immersion with 0.5 ml of antibody mixture for 30 min at RT, followed by washing with PBS. These procedures were carried out under a clean condition at least S1000 level. After staining, a filter cassette was detached from the device and plugged into the cassette holder (Chuo SEIKI, Tokyo, Japan) on the stage of the upright fluorescence microscope to fix the cassette and to control the depth of PBS above the filter to the optimal position. The fluorescence-positive cells were then counted and photographed by the pathologist before knowing any clinical data of the patients. Single cell isolation of CTC was carried out with a micromanipulator using a glass capillary (Figure 1B). The isolated CTC or CTC pool was transferred to 0.2 ml of PCR tube or a 96 well PCR plate and genomic DNA or RNA was extracted, amplified, and genetic analysis was performed (Figure 1C). When isolating living CTC, 10% formalin fixation process was omitted.

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