Abstract 3785: Self-sorting microwells to isolate and expand single circulating tumor cells

Abstract

Abstract Introduction: Circulating tumor cells (CTCs) can be isolated from blood and serve as a source of tumor material. Expansions of CTCs may permit functional treatment-efficacy tests in combination with genetics, epigenetics and proteomics screening. We present a fast workflow to isolate, capture, grow and image cells inside a self-sorting microwell chip using the VyCAP single cell isolation platform. After seeding single cells in a microwell chip, they can grow inside the chip or can be transferred to a tissue culture plate for clonal expansion or downstream analysis. Materials and methods: The Rosette-Sep™ Human CD45 depletion cocktail (Stem Cell Technologies) was used to enrich spiked cells from 1 ml of fresh EDTA blood. Cells were stained with CellTrace™ violet for tracking spiked cells, calcein for live cells, ethidium bromide for dead cells, α-CD45-PERCP as negative marker and α-EpCAM-AF647 as positive marker. After density gradient separation using Ficoll-Paque™, the cell suspension was measured and quantified using flow cytometry for Rosette-Sep™ efficiency. Cell suspensions were seeded in the self-sorting microwell chips using a negative pressure of 40-70 mbar using the VyCAP Filtration system. After seeding single cells in the microwells, the chips were imaged with the VyCAP Puncher system and either placed immediately into culture medium for cell expansion or selected cells were transferred into a 96 well tissue culture plate. Cell expansion in microwell chips and plates was followed up to 14 days. Results: Cultured MDA-MB-231 and MCF-7 cells were single-cell-trapped in the microwells with an efficiency of 82% ± 9% and 74% ± 15% respectively, after direct filtration without enrichment. Immediately after capturing in microwells 93% of the MCF7 cells were viable. Rosette-Sep™ enriched MDA-MB-231 and MCF-7 cells with an efficiency of 73% ± 6% and 74% ± 7% respectively, when measured with flow cytometry. The Rosette-Sep™ procedure followed by capture of the MDA-MB-231 and MCF-7 cells in the microwells resulted in an efficiency of 50% ± 11% and 52% ± 11% respectively. 92% of the cells were found back in a culture plate after punching of single cells from the chip, and of these cells, 80% were alive 4 hours after punching. 82% of these cells were alive and still growing after 14 days. In total 53% of cells spiked in blood, could be found back in the cups of which after punching into individual wells, 77% were alive and growing. Conclusion: We present a single cell capture and isolation method for clonal expansion of viable tumor cells. The VyCAP single cell isolation platform used for these experiments provides an easy separation of the single cells from a CTC enriched cell suspension and allows on-chip expansion as well as immediate separation of the CTCs into culture plates. About 50% of spiked cells could be retrieved inside the microwell chip and of these 77% were alive after punching into individual wells of a culture plate. Citation Format: Joost F. Swennenhuis, Kiki C. Andree, Fikri Abali, Michiel Stevens, Joska Broekmaat, Fiona F. Passanha, Cees J. van Rijn, Leon W. Terstappen. Self-sorting microwells to isolate and expand single circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3785. doi:10.1158/1538-7445.AM2017-3785