Abstract 631: Utilizing Vortex Chip for enumeration and determination of single-cell heterogeneity of circulating tumor cells in prostate cancer
Authors: Pao Edward , Che James , Sollier Elodie , King Andrew , Fan Guoping , Huang Jiaoti , Di Carlo Dino , Rettig Matthew B. , Kulkarni Rajan P. 
Published: August 2015

Abstract
Background: Circulating tumor cells (CTCs) are cells that break away from either the primary tumor or metastatic sites and circulate in the peripheral blood. CTCs are increasingly used as an accessible source of tumor cells (i.e. “liquid biopsy”). We are using a CTC capture technology known as the Vortex Chip, which allows for rapid isolation of highly purified CTCs in less than 1 hour. Moreover, because the Vortex Chip isolates CTCs based on their size, this platform does not require antibody based capture that can potentially bias the isolation of CTC subpopulations to cells that express a specific surface marker, like EpCAM. A new version of the Vortex Chip, Vortex HT, has been used for an increased capture of prostate CTCs.
Methods: The Vortex HT Chip was fabricated using standard lithographic techniques with polydimethylsiloxane (PDMS) polymer. Samples were run using a syringe pump setup to deliver blood to the chip at a constant flow rate, with large cells (including CTCs) trapped in the vortices present within each chamber. Trapped cells were released using PBS rinse buffer and available for immunofluorescence staining (CK, PSA, CD45, DAPI) or other downstream analysis. Individual cells could also be isolated for total RNA amplification and RNA sequencing.
Results: With the new HT chip design, we achieved greater than 10,000-fold enrichment of the cells and an overall purity of greater than 50%. Individual CTCs from patients with advanced metastatic castration resistant prostate cancer (mCRPC) were isolated, immunostained and enumerated: among 13 patients, 100% had more than 1.5 CTCs/mL, while among 9 healthy donors, all had less than 1.5 CTCs/mL; we thus set 1.5 CTC/mL as the cutoff. Proof-of-principle single-cell RNA seq transcriptome analysis was performed on cells isolated from the chip. We isolated three CTCs from two distinct patients and performed RNA sequencing and compared the results to single cell RNA sequencing of three VCaP cell-line cells.
Conclusions: The Vortex Chip HT design has been successfully applied to prostate cancer with an increased throughput. CTCs have been collected from patient samples within less than 1 hour. The individual cells have sufficient RNA for individual single cell RNA seq analysis. Further optimization of these techniques will be necessary to validate these methods and to gain additional insight into heterogeneity of prostate cancer as reflected in CTCs.
Citation Format: Edward Pao, James Che, Elodie Sollier, Andrew King, Guoping Fan, Jiaoti Huang, Dino Di Carlo, Matthew B. Rettig, Rajan P. Kulkarni. Utilizing Vortex Chip for enumeration and determination of single-cell heterogeneity of circulating tumor cells in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 631. doi:10.1158/1538-7445.AM2015-631