Abstract A027: A novel two-step tumor cell isolation system, combining acoustophoresis and negative selection of white blood cells using CD45-conjugated elastomeric particles
Authors: Magnusson Cecilia , Undvall Eva , Laurell Thomas , Lilja Hans 
Published: February 2019

Abstract
Introduction: Circulating tumor cells (CTCs) are a promising tool for disease monitoring and for better-targeted therapies for patients with disseminating tumors. However, isolation of CTCs is challenging, due to their scarcity, heterogeneous size, and lack of universal expression markers.
We employ a novel ultrasonic standing wave-based microfluidic technique (acoustophoresis) to enable unbiased, label-free isolation of circulating tumor cells (CTCs). The cell separation is based on intrinsic cell properties, such as size, morphology, density, and compressibility. During acoustophoresis cells/particles get positioned to nodes or antinodes depending on their acoustic contrast factor. The contrast factor depends on the density and compressibility ratios between the particles and the surrounding medium. Both cancer cells and subpopulations of white blood cells (WBCs) partly possess positive acoustic factors and are therefore positioned to pressure nodes during acoustophoresis. By employing negative contrast CD45-conjugated elastomeric particles (EPs) to capture the contaminating WBCs, a negative acoustic complex can be formed that positions the remaining WBCs to the antinodes. This cell separation technique is gentle towards the target cells and tunable to fit various situations, depending on whether throughput, cancer cell recovery, or purity is of major importance. Both paraformaldehyde (PFA) fixed cells and fresh blood can be processed through acoustophoresis. However, acoustophoretic cancer cell separation from fresh blood generates higher WBC contamination than processing of PFA fixed cells, due to overlapping acoustic properties. Therefore, an additional purification step is needed to remove excess contaminating WBCs. Hence, we have added an additional purging step after acoustophoresis, employing CD45-conjugated negative selection of WBCs using negative acoustic contrast EPs.
Material and Methods: The acoustic separation device is a glass/silicon microchip connected to a pressure-driven system. For optimal separation performance, the system is temperature controlled and a prefocusing step is included before the main separation in the first cell separation step. The setup can process samples up to 10 mL, with a processing speed of 1 mL sample in 13 minutes without compromising the cell separation capacity. To evaluate the system performance, 1 mL model samples were processed, consisting of 10,000 DU145 prostate cancer cells spiked in 0.5 mL red blood cell (RBC) lysed blood from healthy donors.
Results and Discussion: In the primary acoustic separation step, an average of 93% of the DU145 cancer cells were collected at the central outlet, whereas 7% were found in the side fraction. A 20-fold WBC depletion was achieved after the primary acoustophoresis-based tumor cell separation step. The central fraction output was subjected to a subsequent one-hour incubation with CD45-conjugated EPs in room temperature. The incubated samples were processed by acoustophoresis to negatively select EP/WBC complexes from cancer cells into separate outlet fractions, with subsequent flow cytometry analysis. Result showed a 98% cancer cell separation efficiency and a total 250-fold WBC depletion from the starting WBC concentration. Although the separation efficiency in the two steps was high, there was some cell loss in the total system, which needs to be further addressed.
We propose a two-step acoustic-based microfluidic technology, consisting of an upstream primary label-free acoustophoretic separation of cancer cells from blood and a subsequent negative WBC depletion. This may lay the foundation for future analytical and clinical studies aimed to validate novel biomarkers for effective treatment management of cancer patients.
Citation Format: Cecilia Magnusson, Eva Undvall, Thomas Laurell, Hans Lilja. A novel two-step tumor cell isolation system, combining acoustophoresis and negative selection of white blood cells using CD45-conjugated elastomeric particles [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A027.