Functional profiling of circulating tumor cells with an integrated vortex capture and single-cell protease activity assay

Abstract

Tumor cells are hypothesized to use proteolytic enzymes to facilitate invasion. Whether circulating tumor cells (CTCs) secrete these enzymes to aid metastasis is unknown. A quantitative and high-throughput approach to assay CTC secretion is needed to address this question. We developed an integrated microfluidic system that concentrates rare cancer cells >100,000-fold from 1 mL of whole blood into ∼50,000 2-nL drops composed of assay reagents within 15 min. The system isolates CTCs by size, exchanges fluid around CTCs to remove contaminants, introduces a matrix metalloprotease (MMP) substrate, and encapsulates CTCs into microdroplets. We found CTCs from prostate cancer patients possessed above baseline levels of MMP activity (1.7- to 200-fold). Activity of CTCs was generally higher than leukocytes from the same patient (average CTC/leukocyte MMP activity ratio, 2.6 ± 1.5). Higher MMP activity of CTCs suggests active proteolytic processes that may facilitate invasion or immune evasion and be relevant phenotypic biomarkers enabling companion diagnostics for anti-MMP therapies.

Keywords: cell secretion; circulating tumor cells; liquid biopsy; microfluidics; protease.

Fig. 1.

Size-based purification and encapsulation of cells (SPEC) followed by fluorescence analysis of enzyme secretion (1). Large cells are trapped in microvortices, while smaller cells and molecules are washed away with a wash buffer (2). An MMP-cleavable peptide substrate solution is introduced through another fluid exchange (3). We dissipate the vortices by lowering the flow rates and release captured cells into the substrate solution. A pinch valve is opened to the droplet generator in synchrony with vortex dissipation (4). The droplets float away from the generation region due to buoyancy differences with the oil (5). The cells can then be incubated and imaged in the large reservoir section of the droplet generator. An imaging cytometer can also be used to image the droplets and contained cells in flow (6 and 7).

Fig. 2.

Vortex-based cell release and step emulsification. (A) The flow rate decays quickly when the pressure is released to dissipate the vortices. Beads tracked in the exit region of the vortex were used to calculate the decay. (B) Droplet generation remains stable and monodisperse at flow rates from 0.001 to 0.002 mL/min per channel but becomes polydisperse beyond these flow rates. (C) There is minimal loss between the cells captured in the vortex device and the cells in the droplets, because of the continuous flow integrated system.

Fig. 3.

MMP assay performance. (A) We test the detection limits of the assay using serial dilutions of known concentrations of collagenase. A linear correlation exists between droplet intensity and number of molecules reacted for greater than 700 molecules per droplet up to 300,000 molecules per droplet, indicating a large dynamic range for detection. These correlations can be used to estimate the time-averaged number of molecules secreted by single cells. (B) The signal becomes saturated for concentrations of greater than 300,000 molecules per droplet, and there is little time dependence of the signal beyond 3 h.

Fig. 4.

(A) MMP secretion levels vary across cell lines. Lung cancer cell lines (A549 and HCC827) and prostate cancer cell lines (VCaP, LnCaP, and PC3) secrete varying levels of MMPs. Only droplets with single viable cells were measured, and intensity was normalized as a ratio of empty drop levels. (B) Lung cancer cells were interrogated for MMP secretion at 3 and 20 h.

Fig. 5.

CTCs were collected into microdroplets from seven prostate cancer patients and four healthy volunteers. (A) Cells that were negative for CD31, CD66c, and CD45 and positive for prostate-specific membrane antigen or had a large nucleus were classified as CTCs. Six out of seven patient samples were observed to have CTCs with above-background levels of MMP secretion. (B) The ratios of CTC to leukocyte MMP activity levels were higher in patients with progressive disease and correlated with higher PSA levels.