Abstract A197: Size-selective isolation of viable and pure CTCs for molecular analysis using vortex technology.

Circulating tumor cells (CTCs) are cancer cells shed from a primary tumor, which enter the bloodstream and have the potential to metastasize. The detection and isolation of CTCs in bodily fluids can aid in cancer prognosis, characterizing genetic mutations for targeted therapies, and studying the biological mechanisms of metastasis. CTCs are very rare; there may only be tens of CTCs among millions of white blood cells and billions of red blood cells in one milliliter of blood. Current approaches for CTC isolation each have unique advantages and disadvantages: these techniques are often limited by speed, variable gene expression for immunocapture, long sample preparation steps, cost, and ability to deliver viable cells at high purity. Here, we use of arrays of microscale laminar fluid vortices to quickly isolate CTCs from large volumes of blood at high purity and without labels. Under high flow rates, large cells (> 15 μm) become stably trapped in laminar microvortices that form in simple rectangular reservoirs, while smaller red and white blood cells pass through. The large cancer cells are maintained in the microvortices, allowing for solution exchange, followed by release on lowering flow rate. The device is able to purify, enrich, and release a small volume (∼300 μL) of concentrated, viable cancer cells from blood at high throughput, concentration, and purity. The vortex chip successfully separated a variety of cancer cell lines from blood (∼20% capture efficiency), including those from melanoma, ovarian, breast, lung, and prostate cancer cell lines, with ∼10,000 fold enrichment. Captured cells remained viable and could be cultured off-chip. Finally, CTCs were successfully extracted and enumerated from the blood of patients with breast (N=4, 25-51 CTCs per 7.5 mL) and lung (N=8, 23-317 CTCs per 7.5 mL) cancers, and their sizes were measured. Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). Preliminary comparisons with FDA-approved CellSearch system highlighted improved results for CTC enumeration from breast and lung samples. Cells isolated from the vortex chip can also be utilized for downstream molecular analysis. We successfully utilized a qPCR-based approach to detect specific KRAS point mutations from lung cancer cells spiked into pleural fluids and then isolated using the vortex chip and demonstrated improved detection sensitivity. The high purity of our approach should allow for improved molecular sequencing results, and additional molecular analyses of CTCs from patient blood samples are underway, utilizing next generation transcriptome sequencing technologies. This vortex approach offers significant advantages over existing technologies, especially in terms of processing time, low cost, simplicity, cell integrity and purity. The ability to obtain viable CTCs provides flexible opportunities for the clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A197. Citation Format: Elodie Sollier, James Che, Derek E. Go, Westbrook M. Weaver, Nicolas Kummer, Matthew Rettig, Jonathan Goldman, Nicholas Nickols, Dino Di Carlo, Rajan P. Kulkarni. Size-selective isolation of viable and pure CTCs for molecular analysis using vortex technology. [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 A197.