Abstract 1351: A novel application of DEPArrayTM NxT System to isolate circulating tumor cell (CTC)-clusters from patients with metastatic breast cancer (MBC)
Authors: Zhang Qiang , Gerratana Lorenzo , Shah Ami N. , Davis Andrew A. , Flaum Lisa , Zhang Youbin , Behdad Amir , Wehbe Firas , Gradishar William , Platanias Leonidas , Cristofanilli Massimo 
Published: August 2019

Abstract
Introduction: CTCs play a critical role in MBC, and a portion of CTCs may form clusters that contain two or more CTCs bound together which were reported to have up to 50-fold of potential of forming distant metastasis in MBC as compared to individual CTCs. However, genomic characterization of CTCs-clusters remain largely because the enrichment of CTC-clusters is technically challenging. Herein, we describe a novel isolation workflow to select CTC-clusters expressing specific biomarkers for patients with MBC.
Methods: Whole blood samples (7.5ml/each) were collected from 5 patients with stage III/IV BCa patients. CTC enumeration was performed with the FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit targeting the Epithelial Cell Adhesion Molecule antigen for capturing CTCs, and immunofluorescent staining reagents including Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocyte), and anti-HER-2/neu-FLU. Or, using CXC kit includes anti-CCR5-PE. After confirming CTCs-clusters, both CTCs and CTC-clusters were enriched from Celltracks cartridge and were loaded into DEPArrayTM NxT cartridge, and then were isolated and sorted by using fluorescent imaging based DEPArrayTM NxT System.
Results: All these samples were identified having CTCs (≥ 5, between 34 to 208 CTCs) with CTC-clusters (between 1 to 16) by CellSearch analysis. The images of CTCs and CTC-clusters were displayed in CellBrowserTM by the DEPArrayTM NxT System. According to multiple channels assigned for PE, DAPI, APC and FITC, the CTCs and CTC-clusters were classified and then sorted based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45-, with additional markers of HER2 or CCR5. The targeted CTCs and CTC-clusters were routed, parked and then recovered under the direction of Recovery ManagerTM. By using this technique, we successfully recovered and harvested 27/34 (DEPArray harvest/CellSearch diagnosis), 54/151, 57/115, 75/196, and 115/208 CTCs from each CellSearch cartridge sample respectively. Meanwhile, we collected 1/1 (100%), 6/16 (37.5%), 1/1 (100%), 3/11 (27.3%), and 4/4 (100%) of CTC-clusters respectively, as each CTC-cluster includes average of 2.85 CTCs. CTC-clusters at diameter as much as 45µm could be routed and recovered smoothly. Furthermore, CTC-Clusters with biomarkers of interest such as HER2+ or CCR5+ expression were separated into different tubes. The white blood cells from the same samples were also selected successfully as the controls.
Conclusions: We first reported a new workflow for CTC-clusters isolation. With further optimization, this feasible and reliable strategy will help streamline isolation of single CTC and CTC-clusters in MBC patients for genomic analysis providing the opportunity to gain new insights on the molecular mechanisms associated with the metastasis process.
Citation Format: Qiang Zhang, Lorenzo Gerratana, Ami N. Shah, Andrew A. Davis, Lisa Flaum, Youbin Zhang, Amir Behdad, Firas Wehbe, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. A novel application of DEPArrayTM NxT System to isolate circulating tumor cell (CTC)-clusters from patients with metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1351.