Background: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. Methods: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility) resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. Results: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogenous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC-clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding higher number of CTCs using acoustophoresis. Conclusion: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC-clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables sensitive label-free enrichment of cells with epithelial phenotype in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

CM, PA and TL are inventors on a patent licensed to AcouSort AB, based on the acoustophoresis separation technique, Title: System and method to separate cells and / or particles. HL is named on a patent on assays to measure intact PSA and a patent for a statistical method to detect prostate cancer commercialized by OPKO Health (4KScore). HL receives royalties from sales of this test, has stock in OPKO Health. HL is on SAB for Fujirebio Diagnostics Inc. and has stock in DiaProst AB. AL, HL, PA and TL own stocks in Acousort AB.

zon 2020 Research and Innovation Programme (Grant Agreement No. 852590). TL was supported in part by the Swedish Research Council (grants no. 2018-03672 and 2019-00795) and Knut and Alice Wallenberg Foundation, Grant No. 2012.0023 HL was supported in part by funding from National Institutes of Health/National Cancer Institute (P30-CA008748) and Swedish Cancer Society (Cancerfonden 20 1354 PjF). AJ was supported by Knut and Alice Wallenberg Foundation KAW 2020.0235, Swedish society of medicine and Prostate cancer foundation.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The regional ethics committee at Lund university hospital gave ethical approval for this work (ref. no. 2013/400). The regional ethics committee in Gothenburg gave ethical approval for this work (ref. no. 936-12). The Swedish ethical review authority gave ethical approval for this work. (ref. no. 2020-05818).

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