Abstract C23: Functional characterization of viable circulating tumor cells using nanowells.

Abstract

Abstract Current characterizations of circulating tumor cells (CTCs) are limited to enumeration, genotyping, and gene expression. Because these assays each require fixation or lysis, it has not been possible to evaluate several important characteristics of live CTCs at the single-cell level. We have interrogated viable, single CTCs using arrays of subnanoliter wells (nanowells), performing functional measurements of viability, migration, and secretion of soluble factors. We developed a process using nanowells to isolate and characterize viable CTCs from whole blood. Each poly(dimethylsiloxane) (PDMS) array comprises 84,762 cubic wells of 275 pL each. CTCs were enriched from whole blood by negative selection against CD45 and loaded onto the array to settle into the wells by limiting serial dilution. Because CTCs are rare, the loading biased the occupancy of the wells to single CTCs or CTCs in preformed clusters, allowing comparisons among individual CTCs. The defined address of each well allowed spatiotemporal tracking of single CTCs, and subsequent registration of data for functional measures on each CTC. Using this approach, we made three types of measurements on viable CTCs isolated from prostate cancer patients: (1) immediate and short-term viability of CTCs, (2) migration and invasion potential of CTCs, and (3) secretion of soluble factors. We found a rare subset of CTCs to possess malignant traits indicative of metastatic potential in late-stage, progressing prostate cancer patients. These CTCs were resistant to anoikis after being in the circulation as they stained Calcein AM+/Annexin V−, and remained viable for at least a week in culture. They were invasive in their epithelial state when embedded in Matrigel. They could also secrete proteases capable of cleaving peptide substrates. However, we did not observe such metastatic potential in every CTC, pointing to the presence of heterogeneity within CTCs and suggesting that enumeration of CTCs alone may be insufficient to understand metastasis or stratify patients. By interrogating the functional behavior of individual CTCs, we identified a rare subset of CTCs with phenotypes consistent with more efficient metastasis. The nanowell technology holds the potential to further investigate the cellular characteristics of rare tumor cells and design therapies to counter metastasis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C23. Citation Format: Xiaosai Yao, Atish D. Choudhury, Yvonne J. Yamanaka, William C. Hahn, K Dane Wittrup, J Christopher Love. Functional characterization of viable circulating tumor cells using nanowells. [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 C23.