A high-performance microsystem for isolating circulating tumor cells

Abstract

A unique flow field pattern in a bio-functional microchannel is utilized to significantly enhance the performance of a microsystem developed for selectively isolating circulating tumor cells from cell suspensions. For high performance of such systems, disposal of maximum non-target species is just as important as retention of maximum target species; unfortunately, most studies ignore or fail to report this aspect. Therefore, sensitivity and specificity are introduced as quantitative criteria to evaluate the system performance enabling a direct comparison among systems employing different techniques. The newly proposed fluidic scheme combines a slow flow field, for maximum target-cell attachment, followed by a faster flow field, for maximum detachment of non-target cells. Suspensions of homogeneous or binary mixtures of circulating breast tumor cells, with varying relative concentrations, were driven through antibody-functionalized microchannels. Either EpCAM or cadherin-11 transmembrane receptors were targeted to selectively capture target cells from the suspensions. Cadherin-11-expressing MDA-MB-231 cancer cells were used as target cells, while BT-20 cells were used as non-target cells as they do not express cadherin-11. The attachment and detachment of these two cell lines are characterized, and a two-step attachment/detachment flow field pattern is implemented to enhance the system performance in capturing target cells from binary mixtures. While the system sensitivity remains high, above 0.95, the specificity increases from about 0.85 to 0.95 solely due to the second detachment step even for a 1 : 1000 relative concentration of the target cells.

Fig. 1

Attachment rates of cells from homogeneous suspensions as a function of the applied flow rate in microchannels functionalized with: (a) EpCAM; Q_C=1.4 and 2.5μl/min with b=5.7 and 7.0 for MDA-MB-231 and BT-20 cells, respectively, and (b) cadherin-11 antibodies; Q_C=1.0 and 0.6μl/min with b=5.7 and 11 for MDA-MB-231 and BT-20 cells, respectively, while Equation 1 is multiplied by a factor of 0.11 to fit the maximum attachment rate of BT-20 cells.

Fig. 2

Example fluorescent images of 1:1,000 MDA-MB-231:BT-20 cell mixture: (a) in suspension prior to loading, and (b) attached cell population after capture in an anti-cadherin-11 functionalized microchannel under a 0.5μl/min flow rate. MDA-MB-231 cells are green labeled and BT-20 cells are labeled in orange.

Fig. 3

Measured sensitivity and specificity of the microfluidic system in isolating MDA-MB-231 cells from binary mixtures with BT-20 cells, in anti-cadherin-11 functionalized microchannels under a 0.5μl/min flow rate, as a function of the cell concentration ratio.

Fig. 4

Detachment rates of MDA-MB-231 and BT-20 cells as a function of the steady-state flow rate, under 0.2ml/min² flow acceleration, in microchannels functionalized with: (a) EpCAM, and (b) cadherin-11 antibodies; the vertical line indicates detachment rates of MDA-MB-231 and BT-20 to be α_D=0.05 and 0.8, respectively, under a 0.3ml/min flow rate.

Fig. 5

Example fluorescent images of the cell population captured from a 1:1,000 MDA-MB-231:BT-20 cell mixture driven through an anti-cadherin-11 functionalized microchannel at a 0.5μl/min flow rate: (a) before, and (b) after an additional detachment step under a 0.3ml/min flow rate obtained at 0.2ml/min² flow acceleration. MDA-MB-231 cells are green labeled and BT-20 cells are labeled in orange. [Note: the two images are not taken at the same channel location.]

Fig. 6

Measured sensitivity and specificity of the microfluidic system in isolating MDA-MB-231 cells from a 1:1,000 MDA-MB-231: BT-20 cell mixture, in anti-cadherin-11 functionalized microchannels, comparison between before and after an additional detachment step under a 0.3ml/min flow rate obtained at 0.2ml/min² flow acceleration.