Abstract 5994: Separating cancer cells from neutrophils by gradient acoustic focusing
Authors: Magnusson Cecilia , Charan Mahdi Rezayati , Lilja Hans , Augustsson Per 
Published: June 2022

Abstract
Introduction: A subset of aggressive prostate cancers (PCa) progress to invade surrounding tissue to form distant metastases and shed of circulating tumor cells (CTCs). However, due to a scarcity of CTC among the millions of nucleated blood cells, there is an unmet need to improve CTC assays. Acoustophoresis uses standing ultrasound waves in a flow-through microfluidic chamber to discriminate cells based on the size, density, and compressibility. The technique is label-free as opposed to assays that rely on the cell surface marker EpCAM to detect CTCs. We will use label-free two-step acoustophoresis method as the initial separation step followed by a density medium purging step, to obtain efficient recovery of CTC of very high purity.
Experimental procedure: Acoustic cell separation of CTCs from blood is challenging in reference to overlap of acoustic properties between small-sized CTCs and densely granular WBC. To obtain high recovery of CTC, we must apply a sufficiently large acoustic focusing amplitude which leads to a contaminating cell population comprising 2% white blood cells, (WBCs) that mainly contain eosinophil cells. To obtain high purity cancer cell fractions there is a critical need for an additional cleaning step. Therefore, we used the density medium iodixanol in a purging step where the optimal iodixanol medium concentration is intended to provide one cell type with positive, and the other with negative acoustic contrast.
Preliminary results: show that the granulocytes can be transferred by the ultrasound field into the high-density medium (iodixanol) and can thereby be removed through a first outlet. The cancer cells do not transfer into the iodixanol medium and will be collected in a pure PCa cell fraction in the low-density medium through a second outlet. The samples were processed at a flow rate of 75 μl/min. The limiting factor regarding flow rate when purging the sample from granulocytes is overheating caused by the ultrasound transducer. The separation efficiency for cancer cells was 99.5% with 2.2% contaminating granulocytes. Theoretically, by combining the two separation steps only 0,044% of the WBC would remain in the cancer cell fraction.
Conclusion: It is unclear which are the functional properties that enable a primary tumor to shed CTCs, and comprehensive characterization is urgently needed to identify key features of these processes. CTC fractions of high purity will enable single cell analysis with validated Fluidigm-based RT-PCR panels, mass spectrometry, and RNA-sequencing. We propose to use a two-step acoustic separation method, with a primary acoustic separation step and a second high-density medium purging step.
Citation Format: Cecilia Magnusson, Mahdi Rezayati Charan, Hans Lilja, Per Augustsson. Separating cancer cells from neutrophils by gradient acoustic focusing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5994.