Detection of circulating tumor cells in drainage venous blood from colorectal cancer patients using a new filtration and cytology-based automated platform.

Numerous technologies exist to detect circulating tumor cells (CTCs), although reports on cytological detection of CTCs remain limited. We recently developed a cytology-based CTC detection device using glass slides and light microscopy. In this study, we automated this previously manual device to improve its efficiency and cost effectiveness for clinical applications. We conducted a pilot study using this device to compare CTCs in peripheral blood (PB) and draining venous blood (DVB) from patients with colorectal cancer (CRC). The cytology-based automated CTC detection platform consisted of a disposable filtration device with a three-dimensional (3D) metal filter and multichannel automated CTC enrichment device. This platform allowed rapid and gentle filtration of CTCs and their efficient transfer from the filter to glass slides for subsequent Papanicolaou (Pap) and immunocytochemical (ICC) staining. Cytological diagnosis of CTCs was performed by observing permanent glass slide specimens by light microscopy. The current pilot clinical study enrolled CRC patients (n = 26) with stage I-IV tumors, who underwent surgery. PB was collected before surgery, and DVB was obtained from the mesenteric vein immediately after resection. Based on the CTC morphology obtained from PB and DVB samples, we proposed the following cytological criteria for the diagnosis of CTCs: pan-cytokeratin-positive, atypical cells with malignant morphological features identified by Pap staining. The numbers of CTCs defined by these criteria were significantly higher in DVB than PB from CRC patients (p<0.01), and the number of CTCs in DVB was increased significantly with stage progression (p<0.05). These results suggest that DVB may be another potential source of CTCs other than PB for liquid biopsies including downstream analysis. This automated cytology-based CTC detection device therefore provides a unique and powerful tool to investigate the significance of CTCs in CRC patients in a clinical setting

Development of a New Rapid Isolation Device for Circulating Tumor Cells (CTCs) Using 3D Palladium Filter and Its Application for Genetic Analysis

<div><p>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 <i>KRAS</i> 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.</p></div

Flowchart of enrichment, enumeration, isolation and molecular analysis of CTC by metal filtration-based device.

<p><b>A</b>. 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>B</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. <b>C</b>. 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. <b>D</b>. The filter is detached from cassette and is directly stained with immunocytochemistry (ICC) and FISH method.</p

Detection of CTCs from blood in the spontaneous metastasis model of nude mice after sc injection of GFP-tagged COLM-5 human colon cancer cells.

<p><b>A</b>–<b>I</b>. Sequential observation of lung and liver metastasis and detection of CTC in the blood of nude mice (arrows indicate lung metastasis). <b>A</b>–<b>D</b>. Micrometastasis in the lung 1–2 months post-injection (A, B. bright and dark field image of the lung, C. Fluorescence microscopic view of the lung, D. Liver). Bar = 3 mm. <b>E</b>–<b>I</b>. Macroscopic and microscopic metastasis in the lung 2–3 months post-injection (E, F. bright and dark field image of the Lung, G. Fluorescence microscopic view of the lung, H. Liver, I. Subcutaneous tumor). Bar = 3 mm. <b>J</b>–<b>K</b>. Representative single CTC in the blood from mice 1–2 months post-injection (n = 5). <b>L</b>–<b>M</b>. Representative CTC cluster in the blood from mice 2–3 months post-injection (n = 7). Bar = 30 µm. <b>N</b>. Changes in CTC number in the blood with time.</p

Detection and enumeration of CTC from patients with breast cancers.

<p><b>A</b>–<b>E</b>. Representative CTC cluster showing EpCAM+/CD45−/Hoechst33342+ pattern. <b>F</b>–<b>J</b>. Representative single CTC with EpCAM+/CD45−/Hoechst33342+ pattern. Arrows indicate CTC. <b>K</b>. Quantitative comparison of CTC number in blood from 19 patients with metastatic breast cancer (M1) and blood from 13 patients without metastasis (M0) and 12 healthy volunteers. Note that CTC was detected in 3 out of 13 M0 patients, but not detected at all in healthy volunteers. (A, F: Alexa488-EpCAM, B, G: PE-CD45, C, H: Hoechst33342, D, I: bright field, E, J: Merge image between EpCAM and bright field. *P<0.05 (vs M1), Bars = 30 µm.</p