Abstract A153: Isolation and characterization of high-purity invasive CTCs from men with metastatic castration-resistant prostate cancer (mCRPC).
Authors: Premasekharan Gayatri , Friedlander Terence W. , Ngo Vy T. , Gilbert Elizabeth , Sathaye Priya , Harris Anna , Ryan Charles J. , Paris Pamela L. 
Published: August 2014

Abstract
Introduction: As metastatic biopsies are often difficult and impractical to obtain; isolation, characterization, and genomic profiling of circulating tumor cells (CTCs) from men with metastatic castration resistant prostate cancer (mCRPC) is critical for understanding tumor progression and mechanisms of resistance to treatment. The Vitatex cell-adhesion matrix (CAM) platform captures invasive CTCs in both a cell surface marker- and size-independent manner based on the ability of CTCs to invade a fluorescently-labeled CAM. This platform allows for isolation and characterization of viable CTC sub-populations from individual patients with mCRPC. In this study, we sought to optimize CTC isolation and purification for downstream genomics and to explore epithelial-mesenchymal and stem-like CTC subtypes, all in the context of treatment resistance to Abiraterone, a novel inhibitor of androgen synthesis.
Methods: Forty ml of blood was collected from 23 patients with mCRPC. The CTCs were isolated using the VitaAssay platform and paired metastatic biopsies were obtained for a subset of men. Fluorescence-activated cell sorting (FACS) was coupled with the VitaAssay platform to increase cell purity. Invasive CTCs having ingested the fluorescent CAM were sorted based on CAM+/CD45-/CD14-/DAPI- staining. To complement the FACS data and for quality control, a subset of cases (N=11) were also enumerated using Immunocytochemistry (ICC), where CTCs were identified as CAM+/CD45-/CD14-/DAPI+. Healthy donor blood was used as a control. FACS-sorted stem-like CTCs were defined as CD44+/CAM+/CD45-/CD14-/DAPI- and mesenchymal CTCs were identified with ICC as vimentin+/CAM+/CD45-/DAPI+. Array comparative genomic hybridization (aCGH) of FACS-sorted CAM-avid amplified CTCs using Agilent microarrays was performed to obtain the CTC and matched biopsy genomic profiles.
Results: Enumeration of CTCs by VitaAssay coupled with FACS yielded an average of 72 CTCs per 7.5 ml blood (range 1 to 338) for the 23 patients. Only 4 of the 23 patients had ≤ 2 CTCs per 7.5 ml blood. No CTCs were detected in the healthy control subject after cell sorting. The CTC purity increased post-FACS to ∼90% from ∼1% pre-FACS. Enumeration along with genomic analysis (copy number aberrations) of CTCs and their subpopulations, comparison with matched biopsies and clinical data are currently being investigated.
Conclusions: Combining the VitaAssay platform with FACS allows for recovery of high purity mCRPC CTC subpopulations suitable for downstream molecular and genomic analysis. Ultimately, studies incorporating these optimized CTC methods will allow for better understanding of mechanisms of treatment resistance, disease progression and potentially identify new drug targets for CRPC.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A153.
Citation Format: Gayatri Premasekharan, Terence W. Friedlander, Vy T. Ngo, Elizabeth Gilbert, Priya Sathaye, Anna Harris, Charles J. Ryan, Pamela L. Paris. Isolation and characterization of high-purity invasive CTCs from men with metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A153.