Abstract
There is an unmet clinical need to extract living circulating tumor cells (CTCs) for functional studies and in vitro expansion to enable drug testing and predict responses to therapy in metastatic cancer. Here, we present a novel two-step acoustophoresis (A2) method for isolation of unfixed, viable cancer cells from red blood cell (RBC) lysed whole blood. The A2 method uses an initial acoustofluidic preseparation step to separate cells based on their acoustic mobility. This acoustofluidic step enriches viable cancer cells in a central outlet, but a significant number of white blood cells (WBCs) remain in the central outlet fraction due to overlapping acoustophysical properties of these viable cells. A subsequent purging step was employed to remove contaminating WBCs through negative selection acoustophoresis with anti-CD45-functionalized negative acoustic contrast particles. We processed 1 mL samples of 1:1 diluted RBC lysed whole blood mixed with 10 000 DU145 cells through the A2 method. Additional experiments were performed using 1000 DU145 cells spiked into 1.5 × 106 WBCs in 1 mL of buffer to further elucidate the dynamic range of the method. Using samples with 10 000 DU145 cells, we obtained 459 ± 188-fold depletion of WBC and 42% recovery of viable cancer cells. Based on spiked samples with 1000 DU145 cells, our cancer cell recovery was 28% with 247 ± 156-fold WBC depletion corresponding to a depletion efficacy of ≥99.5%. The novel A2 method provides extensive elimination of WBCs combined with the gentle recovery of viable cancer cells suitable for downstream functional analyses and in vitro culture.
Highlights
Circulating tumor cells (CTCs) in metastatic cancer patients may be exceedingly rare, a noninvasive liquid biopsy can be informative of tumor profile, and circulating tumor cells (CTCs) enumeration has been clinically validated as a prognostic biomarker predictive of overall survival in advanced cancer stages.[1−4] detectable or higher counts of CTC in blood is significantly associated with poor outcomes.[1−3,5] Molecular characterization of enriched CTC populations can provide information on therapeutic targets and drug resistance mechanisms.[5,6]
In the primary acoustic separation step, we found that the separation of unfixed, viable white blood cells (WBCs) from DU145 cancer cells was less efficient compared with that of PFA fixed cells (Figure 2A)
2000 unfixed DU145 cells were spiked into unfixed red blood cell (RBC) lysed blood, in which 94.0% of the DU145 cells focused to the central outlet, with an average WBC
Summary
Circulating tumor cells (CTCs) in metastatic cancer patients may be exceedingly rare, a noninvasive liquid biopsy can be informative of tumor profile, and CTC enumeration has been clinically validated as a prognostic biomarker predictive of overall survival in advanced cancer stages.[1−4] detectable or higher counts of CTC in blood is significantly associated with poor outcomes.[1−3,5] Molecular characterization of enriched CTC populations can provide information on therapeutic targets and drug resistance mechanisms.[5,6] Recently, androgen receptor splice variant 7 (AR-V7) CTCtesting was clinically validated to facilitate decision-making of androgen receptor (AR) signal inhibitor therapy for men with metastatic castration-resistant prostate cancer (mCRPC), which has poor prognosis.[7]. Unfixed blood samples displayed a wider size distribution of the different WBC subpopulations compared with fixed blood samples, as PFA treatment provided more uniform sizes, as discussed above (Figure 2B) and reported by Urbansky et al.[45] Any variability in the proportion of lymphocytes, monocytes, and granulocytes will affect the size distribution and acoustic mobility of the WBC population in a blood sample Both cultured cancer cells and CTCs in clinical samples vary in cell size.[46−48] we would anticipate that experiments using smaller-sized cancer cells spiked into WBCs with a wider size distribution would lead to lower cancer cell recovery with higher WBC contamination. The presence of EPs had no effect on the viability of re-cultured cancer cells as they were washed away through the passages
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