Acoustic Enrichment of Heterogeneous Circulating Tumor Cells and Clusters from Metastatic Prostate Cancer Patients

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 the 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 heterogeneous 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 a 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 the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity

Acoustic cell separation in a microchannel does not alter PSA secretion by prostate cancer cells.

<p>The androgen receptor (AR) expressing cell lines LNCaP and VCaP were used to evaluate the impact of acoustophoresis on PSA secretion. After acoustophoresis run at 0, 10 and 20 V_pp, the secretion of PSA was measured in the absence or presence of the AR ligand R1881 (1 nM for 24 h) in the LNCaP cell line (a) and in the VCaP cell line (b). Cells not processed through the chip were used as control cells. The graphs show the results from three separate experiments and the data are shown as means ± SD. Significance value <i>P</i><0.05, ns denotes non-significant.</p

Unaltered viability of BV2 microglial cell line following acoustophoretic processing (10V_pp and 20V_pp).

<p>BV2 cells were passed through the acoustophoresis chip with the function generator set at 0, 10 and 20 V_pp. After going through the chip, the BV2 cells were seeded again for 24 and 48 h. Cell viability was measured by XTT (a), apoptotic nuclei appearance (b) and decrease of mitochondrial potential -Ψm- (c), showing no difference between experimental groups. Similar, no difference was detected by clonogenic assay (d) used to study survival and proliferation at 7 days following acoustophoretic processing. The graphs show the results from at least three separate experiments and the data are shown as means ± SD. Significance value <i>P</i><0.05, ns denotes non-significant.</p

Mitochondrial respiratory function in human leukocytes and thrombocytes are not altered following acoustophoresis.

<p>Leukocytes and thrombocytes were passed through the acoustophoresis chip run at 0, 10 and 20 V_pp. Maximal respiration during using both complex I- and complex II-linked substrates for thrombocytes (a) and leukotcytes (b) was unaltered after acoustophoresis, supporting that acoustophoresis does not affect metabolic pathways important for respiration. The remaining respiratory activity following inhibition of ATP synthase with oligomycin, so called Leak or state 4 respiration, was also unaffected by acoustophoresis, in thrombocytes (c) and leukocytes (d). This data confirm no effect of acoustophoresis on inner mitochondrial membrane integrity or changed utilization of proton motive force for other purposes than ADP phosphorylation. Unprocessed cells were used as control cells. The graphs show the results from three separate experiments and the data are shown as means ± SD. Significance value <i>P</i><0.05, ns denotes non-significant.</p

Picture and illustration of the set up.

<p>(a) Photo of the acoustophoresis microfluidic system first presented by Augustsson <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064233#pone.0064233-Augustsson3" target="_blank">[27]</a>. (b) Cross sectional view of cell distribution in the microchannel without ultrasound (left) and with ultrasound forming an ultrasound standing wave (right). (c) Illustration of acouscoustophoresis chip. For the present study, only one of the channel segments was used allowing cells to be exposed to ultrasound.</p

Inflammatory response of BV2 cells upon LPS challenge following acoustophoresis is not changed.

<p>After acoustophoretic processing, BV2 cells were seeded and stimulated the next day with LPS for 24 h. We observed no alteration due to acoustophoretic processing in the expression of iNOS (inducible nitric oxide synthase)(a,b), the release of proinflammatory cytokines IL-1β (χ), IL-12 (d), TNF-α (e) or the anti-inflammatory cytokine IL-10 (f). The graphs show the results from at least three separate experiments and the data are shown as means ± SD. Significance value <i>P</i><0.05, ns denotes non-significant.</p