Abstract LB-B08: Somatic mutation detection from liquid biopsy-derived cellular aggregates formed by magnetic 3D bioprinting

Background - A challenge in the analysis of circulating tumor cells (CTCs) is their scarcity and the inability to expand them for further analysis. To overcome this obstacle, we used magnetic 3D bioprinting to form CTC spheroids that could grow. The principle of magnetic 3D bioprinting is the magnetization of cells with nanoparticles and their subsequent printing into spheroids. For this project, CTCs were aggregated into close contact to facilitate interactions and growth in culture. We then demonstrated the ability to perform next generation sequencing (NGS) of the spheroids to detect somatic mutations from renal and prostate cancers. Methods - Blood samples from prostate and kidney cancer patients were enriched for CTCs (Isoflux, Fluxion Biosciences), from a starting blood volume of 7.5-14 mL. CTCs were isolated immunomagnetically for EpCAM+ EGFR+ cells, then enumerated for CK+ CD45-. The cells were then magnetized by incubation with NanoShuttle (NS, Nano3D Biosciences) and printed into spheroids in 384-well plates. After 4 d of growth, the cells were lysed and DNA was amplified by whole genome amplification (WGA) with the NGA kit (Fluxion Biosciences) and quantified via qPCR. Targeted libraries were sequenced using the PGM (ThermoFisher) sequencing instrument; data was analyzed using a customized variant calling/filtering pipeline based on standard Ion Reporter alignment tools and VarSeqTM for variant filtering and functional interpretation. Results - CTCs were successfully aggregated using magnetic 3D bioprinting and grew over 4 d. For both prostate and renal cancers, we then demonstrated the detection of somatic variants within a majority of the samples. Using the commercial Oncomine® test kits (ThermoFisher), we found a median of 5 COSMIC variants (32 total) per sample using cell cultures. Conclusions - We successfully developed a method to aggregate CTCs using magnetic 3D bioprinting, expanded them, and then identified somatic mutations using NGS. This procedure may form the basis of a liquid biopsy-derived molecular testing platform for monitoring urological tumor progression and planning treatment strategies. Citation Format: Hubert Tseng, Robert J. Amato, Reynolds Brobey, Cristian Ionescu-Zanetti, Jeff Jensen, Jacob Gage, Pujan Desai, Angela Liao, Mehdi Dehghani, Kevin Rosenblatt, Glauco Souza. Somatic mutation detection from liquid biopsy-derived cellular aggregates formed by magnetic 3D bioprinting. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B08.