Microfluidic, Label-Free Enrichment of Prostate Cancer Cells in Blood Based on Acoustophoresis

Circulating tumor cells (CTC) are shed in peripheral blood at advanced metastatic stages of solid cancers. Surface-marker-based detection of CTC predicts recurrence and survival in colorectal, breast, and prostate cancer. However, scarcity and variation in size, morphology, expression profile, and antigen exposure impairs reliable detection and characterization of CTC. We have developed a noncontact, label-free microfluidic acoustophoresis method to separate prostate cancer cells from white blood cells (WBC) through forces generated by ultrasonic resonances in microfluidic channels. Implementation of cell prealignment in a temperature-stabilized (±0.5 °C) acoustophoresis microchannel dramatically enhanced the discriminatory capacity and enabled the separation of 5 μm microspheres from 7 μm microspheres with 99% purity. Next, we determined the feasibility of employing label-free microfluidic acoustophoresis to discriminate and divert tumor cells from WBCs using erythrocyte-lysed blood from healthy volunteers spiked with tumor cells from three prostate cancer cell-lines (DU145, PC3, LNCaP). For cells fixed with paraformaldehyde, cancer cell recovery ranged from 93.6% to 97.9% with purity ranging from 97.4% to 98.4%. There was no detectable loss of cell viability or cell proliferation subsequent to the exposure of viable tumor cells to acoustophoresis. For nonfixed, viable cells, tumor cell recovery ranged from 72.5% to 93.9% with purity ranging from 79.6% to 99.7%. These data contribute proof-in-principle that label-free microfluidic acoustophoresis can be used to enrich both viable and fixed cancer cells from WBCs with very high recovery and purity

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

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

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

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

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