Xiangjun Zheng

Detachment of captured cancer cells under flow acceleration in a bio-functionalized microchannel

Attachment, deformation and detachment of N-cadherin expressing prostate and breast cancer cell lines in a functionalized microchannel under hydrodynamic loading have been studied. N-cadherin antibodies are immobilized on the microchannel surface to capture the target cancer cells, PC3N and MDA-MB-231-N, from a homogeneous cell suspension. Although difficult, a significant fraction of moving cells can be captured under a low flow rate. More than 90% of the target cells are captured after a certain incubation time under no flow condition. The mechanical response of a captured cancer cell to hydrodynamic flow field is investigated and, in particular, the effect of flow acceleration is examined. The observed cell deformation is dramatic under low acceleration, but is negligible under high acceleration. Consequently, the detachment of captured cells depends on both flow rate and flow acceleration. The flow rate required for cell detachment is a random variable that can be described by a log-normal distribution. Two flow acceleration limits have been identified for proper scaling of the flow rate required to detach captured cells. A time constant for the mechanical response of a captured cell, on the order of 1 min, has been identified for scaling the flow acceleration. Based on these acceleration limits and time constant, an exponential-like empirical model is proposed to predict the flow rate required for cell detachment as a function of flow acceleration.

A high-performance microsystem for isolating circulating tumor cells

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.

Cell receptor and surface ligand density effects on dynamic states of adhering circulating tumor cells

Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. The cell motion is analyzed with the aid of a simplified physical model featuring a receptor-coated rigid sphere moving above a solid surface with immobilized ligands. The motion of the sphere is described by the Langevin equation accounting for the hydrodynamic loadings, gravitational force, receptor-ligand bindings, and thermal fluctuations; the receptor-ligand bonds are modeled as linear springs. Depending on the applied shear flow rate, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion. Of particular interest is the fraction of captured circulating tumor cells, defined as the capture ratio, via specific receptor-ligand bonds. The cell capture ratio decreases with increasing shear flow rate with a characteristic rate. Based on both experimental and theoretical results, the characteristic flow rate increases monotonically with increasing either cell-receptor or surface-ligand density within certain ranges. Utilizing it as a scaling parameter, flow-rate dependent capture ratios for various cell-surface combinations collapse onto a single curve described by an exponential formula.

Adhesion dynamics of circulating tumor cells under shear flow in a bio-functionalized microchannel

The adhesion dynamics of circulating tumor cells in a bio-functionalized microchannel under hydrodynamic loading is explored experimentally and analyzed theoretically. EpCAM antibodies are immobilized on the microchannel surface to specifically capture EpCAM-expressing target breast cancer cells MDA-MB-231 from a homogeneous cell suspension in shear flow. In the cross-stream direction, gravity is the dominant physical mechanism resulting in continuous interaction between the EpCAM cell receptors and the immobilized surface anti-EpCAM ligands. Depending on the applied shear rate, three dynamic states have been characterized: firm adhesion, rolling adhesion and free rolling. The steady-state velocity under adhesion- and free-rolling conditions as well as the time-dependent velocity in firm adhesion has been characterized experimentally, based on video recordings of target cell motion in functionalized microchannels. A previously reported theoretical model, utilizing a linear spring to represent the specific receptor–ligand bonds, has been adopted to analyze adhesion dynamics including features such as the cell–surface binding force and separation gap. By fitting theoretical predictions to experimental measurements, a unified exponential decay function is proposed to describe the target cell velocity evolution during capture; the fitting parameters, velocity and time scales, depend on the particular cell–surface system.

Kinematics of Specifically Captured Circulating Tumor Cells in Bio-Functionalized Microchannels

The attachment kinematics of cancer cells under hydrodynamic loading in antibody-functionalized microchannels has been studied. Epithelial-cell-adhesion-molecule antibodies are immobilized on the microchannel surface for specific capture of the target cancer cells from homogeneous cell suspensions. The specific interaction between the cancer cell receptors and the immobilized antibodies under static conditions is demonstrated. The capture efficiency of the target cells from homogeneous suspensions under applied hydrodynamic flow field has been investigated, revealing a characteristic shear stress. Applying a lower stress allows the capture of most target cells, while the capture efficiency drops sharply with an increasing shear stress. The captured cells are spatially distributed along the microchannel; both the velocity and the distance travelled by cells prior to capture are measured. The characteristic time and length scales for cell capture are determined, and a log-normal statistical distribution is proposed to describe the observations. Furthermore, a first-order kinetic model for receptor-ligand bond formation provides a rough estimate of the cell adhesion rate constant. Under a low shear stress, the on-rate is much higher than the off-rate, allowing capture of most loaded cells. The off-rate constant increases exponentially with an increasing shear stress, such that above the characteristic stress level, most loaded cells avoid capture.

Isolation of viable cancer cells in antibody-functionalized microfluidic devices

Microfluidic devices functionalized with EpCAM antibodies were utilized for the capture of target cancer cells representing circulating tumor cells (CTCs). The fraction of cancer cells captured from homogeneous suspensions is mainly a function of flow shear rate, and can be described by an exponential function. A characteristic shear rate emerges as the most dominant parameter affecting the cell attachment ratio. Utilizing this characteristic shear rate as a scaling factor, all attachment ratio results for various combinations of receptor and ligand densities collapsed onto a single curve described by the empirical formula. The characteristic shear rate increases with both cell-receptor and surface-ligand densities, and empirical formulae featuring a product of two independent cumulative distributions described well these relationships. The minimum detection limit in isolation of target cancer cells from binary mixtures was experimentally explored utilizing microchannel arrays that allow high-throughput processing of suspensions about 0.5 ml in volume, which are clinically relevant, within a short time. Under a two-step attachment/detachment flow rate, both high sensitivity (almost 1.0) and high specificity (about 0.985) can be achieved in isolating target cancer cells from binary mixtures even for the lowest target/non-target cell concentration ratio of 1:100 000; this is a realistic ratio between CTCs and white blood cells in blood of cancer patients. Detection of CTCs from blood samples was also demonstrated using whole blood from healthy donors spiked with cancer cells. Finally, the viability of target cancer cells released after capture was confirmed by observing continuous cell growth in culture.

Dynamic states of adhering cancer cells under shear flow in an antibody-functionalized microchannel

Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. A simplified physical model was adopted to analyze the cell motion; it features a rigid sphere, with receptors on its surface, moving above a solid surface with distributed ligands. The cell motion is described by the Langevin equation where the hydrodynamic interactions, gravitational drift force, receptor-ligand binding force, and thermal fluctuations are all taken into account. The receptor-ligand bonds are modeled as Hookean springs. In this study, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion depending on the flow shear rate. The numerical simulations allow exploring effects of numerous parameters such as cell-receptor and surface-ligand density.

Flow Acceleration Effect on Cancer Cell Deformation and Detachment

The effect of flow acceleration, rather than just the flow rate, on the response of an attached cancer cell is for the first time reported. Selective binding of prostate cancer cells to a surface functionalized with anti-N-cadherin antibodies utilizing a microfluidic system under flow conditions has been studied [1]. Here, the behavior of a captured cell under a time-dependent flow field is investigated experimentally and numerically. Under slowly increasing flow rate, the cell deformation is more pronounced resulting in lower drag force on attached cells. Furthermore, the contact area between the cell and the functionalized surface is larger, potentially enhancing the cell adhesion force. Consequently, a higher flow rate is required to detach cells exposed to such a flow field. Numerical simulations have been utilized in effort to quantify the required detachment force. The results confirm that to obtain a similar shear stress, a higher flow rate is needed for attached cells under lower flow acceleration.

On the minimum detection limit of circulating tumor cells in an antibody-functionalized microchannel array

The minimum detection limit in isolation of circulating tumor cells (CTCs) from binary mixtures is experimentally explored utilizing microchannel arrays functionalized with EpCAM antibodies. Hence, the tested binary mixtures contained EpCAM-positive and EpCAM-negative cells serving as target and non-target cells, respectively. The ratio of target to non-target cells in the mixtures ranged from 1:1 to 1:100,000. The dependence of the device performance, particularly its sensitivity and specificity in isolating target cells, on the applied flow shear rate is characterized. The results demonstrate capture of circulating tumor cells from mixtures with high sensitivity and high specificity. The multichannel array devices allow testing of samples within a shorter time, which is critical for high throughput applications.

A high-performance microsystem for isolating viable circulating tumor cells

The attachment and detachment of target cancer cells from homogeneous and binary mixtures in antibody-functionalized microchannels have been studied experimentally. Under the same intermediate flow rate, the attachment rate was found to be higher, and detachment flow rate was lower, for cell lines expressing the target receptor at a higher level. For cells that do not express the target receptor, the attachment rate was much lower but did not diminish, due to non-specific binding, and the detachment rate was much higher. The bio-functional microfluidic system performance in selectively isolating target cells from binary mixtures is quantitatively characterized. While the system sensitivity is typically very high, almost 100%, the specificity is lower than 90%. Applying a unique flow scheme of a slow flow rate, for maximum capture of target cells, followed by a faster flow rate, for maximum removal of non-target cells, the specificity is enhanced to levels above 95%, even for mixtures with target cells present at 1∶1,000 relative concentration ratio.