title: Development of a new rapid isolation device for circulating tumor cells (CTCs) using 3D palladium filter and its application for genetic analysis.
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.
authors: 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.
authors: Akiko
authors: A
authors: Yusa
authors: Department of Gastrointestinal Surgery, Aichi Cancer Center Central Hospital, Japan ; Division of Oncological Pathology, Aichi Cancer Center Research Institute, Japan.
authors: Makoto
authors: M
authors: Toneri
authors: Department of Micro-Nano Systems Engineering, Graduate School of Engineering, Nagoya University, Japan.
authors: Taisuke
authors: T
authors: Masuda
authors: Department of Gastrointestinal Surgery, Aichi Cancer Center Central Hospital, Japan.
authors: Seiji
authors: S
authors: Ito
authors: Department of Biotechnogloy, Graduate School of Engineering, Nagoya University, Japan.
authors: Shuhei
authors: S
authors: Yamamoto
authors: Department of Biotechnogloy, Graduate School of Engineering, Nagoya University, Japan.
authors: Mina
authors: M
authors: Okochi
authors: Department of Breast Oncology, Aichi Cancer Center Central Hospital, Japan.
authors: Naoto
authors: N
authors: Kondo
authors: Department of Breast Oncology, Aichi Cancer Center Central Hospital, Japan.
authors: Hiroji
authors: H
authors: Iwata
authors: Department of Pathology and Molecular Diagnostics, Aichi Cancer Center Central Hospital, Japan.
authors: Yasushi
authors: Y
authors: Yatabe
authors: Optnics Precision Co., Ltd., Japan.
authors: Yoshiyuki
authors: Y
authors: Ichinosawa
authors: Optnics Precision Co., Ltd., Japan.
authors: Seichin
authors: S
authors: Kinuta
authors: Division of Oncological Pathology, Aichi Cancer Center Research Institute, Japan.
authors: Eisaku
authors: E
authors: Kondo
authors: Department of Biotechnogloy, Graduate School of Engineering, Nagoya University, Japan.
authors: Hiroyuki
authors: H
authors: Honda
authors: Department of Micro-Nano Systems Engineering, Graduate School of Engineering, Nagoya University, Japan.
authors: Fumihito
authors: F
authors: Arai
authors: Division of Oncological Pathology, Aichi Cancer Center Research Institute, Japan.
authors: Hayao
authors: H
authors: Nakanishi
mesh_terms: D000328:Adult; D000818:Animals; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000295:instrumentation*; D048429:Cell Size; D004867:Equipment Design; D011905:Genes, ras; D006801:Humans; D021621:Imaging, Three-Dimensional; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D009360:Neoplastic Cells, Circulating; D010165:Palladium / Q000737:chemistry*; D010865:Pilot Projects; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D012878:Skin Neoplasms / Q000097:blood*
chemical_list: D010165:Palladium; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human