Abstract P1-01-13: A new workflow comprising size based CTC enrichment followed by in situ labeling and micromanipulation with CellCelector™ enables label-free enrichment, isolation and characterization of circulating tumor cells in breast cancer
Authors: Neubauer HJ , Lampignano R , Neumann MHD , Behrens B , Franken A , Stoecklein N , Niederacher D , Fehm TN 
Published: March 2017

Abstract
Circulating tumor cells (CTCs) are believed to be metastatic precursors of most solid epithelial tumors. Therefore, characterization of their genomic, transcriptomic, and proteomic features may improve cancer diagnosis and prognosis as well as treatment selection and monitoring. CTCs are mostly detected by systems utilizing expression of the epithelial cell adhesion molecule (EpCAM) on CTCs. However, CTCs which have acquired mesenchymal-like features are overlooked although they are believed to be the most malignant cells. Moreover, due to inter cellular heterogeneity single cell isolation by e.g. micromanipulation is required to achieve cellular resolution.
In this project we established a workflow combining the Parsortix™ system to enrich for EpCAMlow/negative CTCs by size and deformability and the CellCelector™ micromanipulator to singularize them for further molecular characterizations. First, our workflow was validated by cell-line spiking experiments into blood from healthy subjects (MCF7). Captured cells were stained in situ for nuclei, cytokeratin and leukocyte markers, were harvested and then micromanipulated as single cells reaching a capturing rate of 62.66% (± 17.79) and a harvesting rate of 72% (± 29.61). Then this workflow was applied to a cohort of metastatic breast cancer blood samples which were previously depleted for EpCAMhigh cells. In 18 out of 22 samples 541 EpCAMhigh CTCs and in 16 samples 257 EpCAMlow/negative potential CTCs were found. Of these 156 EpCAMlow/negative and comparable numbers of EpCAMhigh cells were successfully isolated and lysed for whole genome amplification (Ampli1™). Successful genome-wide amplification was observed in 6.5% of EpCAMlow/negative cells vs. 30% of EpCAMhigh cells indicating lower quality of genomic DNA in the first subpopulation. Sanger sequencing of PCR-amplified exons 9 and 20 of PIK3CA gene revealed heterogeneous mutation status in EpCAMlow/negative cells from the same patient. High quality WGA products were also processed for array-based comparative genomic hybridization (aCGH). Comparison of resulting profiles to 267 archived aCGH profiles from breast cancer single cells identified similar somatic copy number alterations: i.e. chromosome 1p loss and 1q gain confirming the tumor origin of captured EpCAMlow/negative cells. In addition we could also identify some different aberrations which need to be further investigated.
In summary, our workflow combining size based CTC enrichment followed by in situ labeling and micromanipulation is suitable for single tumor cell analysis.
Citation Format: Neubauer HJ, Lampignano R, Neumann MHD, Behrens B, Franken A, Stoecklein N, Niederacher D, Fehm TN. A new workflow comprising size based CTC enrichment followed by in situ labeling and micromanipulation with CellCelector™ enables label-free enrichment, isolation and characterization of circulating tumor cells in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-13.