Yoshihito Kikuhara

Microcavity array system for size-based enrichment of circulating tumor cells from the blood of patients with small-cell lung cancer

In this study, we present a method for efficient enrichment of small-sized circulating tumor cells (CTCs) such as those found in the blood of small-cell lung cancer (SCLC) patients using a microcavity array (MCA) system. To enrich CTCs from whole blood, a microfabricated nickel filter with a rectangular MCA (10(4) cavities/filter) was integrated with a miniaturized device, allowing for the isolation of tumor cells based on differences in size and deformability between tumor and blood cells. The shape and porosity of the MCA were optimized to efficiently capture small tumor cells on the microcavities under low flow resistance conditions, while allowing other blood cells to effectively pass through. Under optimized conditions, approximately 80% of SCLC (NCI-H69 and NCI-H82) cells spiked in 1 mL of whole blood were successfully recovered. In clinical samples, CTCs were detectable in 16 of 16 SCLC patients. In addition, the number of leukocytes captured on the rectangular MCA was significantly lower than that on the circular MCA (p < 0.001), suggesting that the use of the rectangular MCA diminishes a considerable number of carryover leukocytes. Therefore, our system has potential as a tool for the detection of CTCs in small cell-type tumors and detailed molecular analyses of CTCs.

Development of the automated circulating tumor cell recovery system with microcavity array

Circulating tumor cells (CTCs) are well recognized as useful biomarker for cancer diagnosis and potential target of drug discovery for metastatic cancer. Efficient and precise recovery of extremely low concentrations of CTCs from blood has been required to increase the detection sensitivity. Here, an automated system equipped with a microcavity array (MCA) was demonstrated for highly efficient and reproducible CTC recovery. The use of MCA allows selective recovery of cancer cells from whole blood on the basis of differences in size between tumor and blood cells. Intra- and inter-assays revealed that the automated system achieved high efficiency and reproducibility equal to the assay manually performed by well-trained operator. Under optimized assay workflow, the automated system allows efficient and precise cell recovery for non-small cell lung cancer cells spiked in whole blood. The automated CTC recovery system will contribute to high-throughput analysis in the further clinical studies on large cohort of cancer patients.

Abstract 2370: Development of microcavity array system for size- and deformability-based isolation of circulating tumor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: EpCAM-based enumeration of circulating tumor cells (CTCs) has prognostic value in solid tumors such as advanced breast, colon and prostate cancers. However, currently poor sensitivity has limited the use of CTCs in other types of cancers including non-small cell lung cancer (NSCLC). We have developed a microcavity array (MCA) system integrated with microfluidic device for recovery and enumeration of CTC regardless of EpCAM expression level, allowing isolation of tumor cells on the basis of differences in the size and deformability between tumor and hematologic cells. Shapes and sizes of the cavity were optimized in order to trap tumor cells while letting blood cells flow through the microcavities during whole blood filtration. Enrichment of CTCs, fixation, permeabilization, staining, and counting process were implemented in a microfluidic assay within one integrated device. In this study, we evaluated the sensitivity of the MCA system in detecting CTCs with a preclinical model using cell lines and conducted a clinical feasibility study in NSCLC patients. Methods: A wide range of cancer cell lines derived from breast (MCF-7, Hs578T), colon (SW620), gastric (AGS, SNU-1) and lung (A549, HCC-827, NCI-H358, NCI-H441, NCI-H1650, NCI-H1975, NCI-H69, NCI-H82) were used for spike-in experiments. Cells were spiked into 1 mL of healthy donor blood and then introduced into the MCA system. Trapped cells were stained with Hoechst 33342, FITC-labeled anti-pan cytokeratin antibody and PE-labeled anti-CD45 antibody for subsequent imaging analysis. CTCs were defined as cells with round to oval morphology, a visible nucleus, positive staining for pan-cytokeratin and negative staining for CD45. For the clinical evaluation, 16 advanced NSCLC patients were enrolled into the study and we conducted a head-to-head comparison study with CellSearch system. Results: We obtained a quite high recovery rate regardless of tumor types ranging from 80 to 99% in the cell line spike-in experiments containing EpCAM-negative cell lines (Hs578T, SNU-1). Most of recovered cells were viable and were able to proliferate even after isolation process, suggesting the potential for further biological and molecular analyses of CTCs. In the clinical part of the study, CTCs were detectable in 12 out of 16 patients (count β1 per 7.5 ml) with our system. More CTCs were detected by MCA system (median 13, range 0-313 cells/7.5ml blood) than by CellSearch system (median 0, range 0-37 cells/7.5ml blood) demonstrating statistical superiority (p=0.0132, Wilcoxon test). It is also noteworthy that among patients who had negative CTC count by CellSearch system, several patients had positive CTC count by MCA system, suggesting better detection of EpCAM-negative CTCs. Conclusion: Our results suggest the potential of our MCA system for detection of CTCs in solid tumors including NSCLC and further clinical development should be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2370. doi:1538-7445.AM2012-2370