Microfluidic isolation of cancer-cell-derived microvesicles from hetergeneous extracellular shed vesicle populations.

Extracellular shed vesicles, including exosomes and microvesicles, are disseminated throughout the body and represent an important conduit of cell communication. Cancer-cell-derived microvesicles have potential as a cancer biomarker as they help shape the tumor microenvironment to promote the growth of the primary tumor and prime the metastatic niche. It is likely that, in cancer cell cultures, the two constituent extracellular shed vesicle subpopulations, observed in dynamic light scattering, represent an exosome population and a cancer-cell-specific microvesicle population and that extracellular shed vesicle size provides information about provenance and cargo. We have designed and implemented a novel microfluidic technology that separates microvesicles, as a function of diameter, from heterogeneous populations of cancer-cell-derived extracellular shed vesicles. We measured cargo carried by the microvesicle subpopulation processed through this microfluidic platform. Such analyses could enable future investigations to more accurately and reliably determine provenance, functional activity, and mechanisms of transformation in cancer.

Parametric control of collision rates and capture rates in geometrically enhanced differential immunocapture (GEDI) microfluidic devices for rare cell capture.

The enrichment and isolation of rare cells from complex samples, such as circulating tumor cells (CTCs) from whole blood, is an important engineering problem with widespread clinical applications. One approach uses a microfluidic obstacle array with an antibody surface functionalization to both guide cells into contact with the capture surface and to facilitate adhesion; geometrically enhanced differential immunocapture is a design strategy in which the array is designed to promote target cell­obstacle contact and minimize other interactions (Gleghorn et al. 2010; Kirby et al. 2012). We present a simulation that uses capture experiments in a simple Hele-Shaw geometry (Santana et al. 2012) to inform a target-cell-specific capture model that can predict capture probability in immunocapture microdevices of any arbitrary complex geometry. We show that capture performance is strongly dependent on the array geometry, and that it is possible to select an obstacle array geometry that maximizes capture efficiency (by creating combinations of frequent target cell­obstacle collisions and shear stress low enough to support capture), while simultaneously enhancing purity by minimizing nonspecific adhesion of both smaller contaminant cells (with infrequent cell­obstacle collisions) and larger contaminant cells (by focusing those collisions into regions of high shear stress).

Isolation of breast cancer and gastric cancer circulating tumor cells by use of an anti HER2-based microfluidic device.

Circulating tumor cells (CTCs) have emerged as a reliable source of tumor cells, and their concentration has prognostic implications. CTC capture offers real-time access to cancer tissue without the need of an invasive biopsy, while their phenotypic and molecular interrogation can provide insight into the biological changes of the tumor that occur during treatment. The majority of the CTC capture methods are based on EpCAM expression as a surface marker of tumor-derived cells. However, EpCAM protein expression levels can be significantly down regulated during cancer progression as a consequence of the process of epithelial to mesenchymal transition. In this paper, we describe a novel HER2 (Human Epidermal Receptor 2)-based microfluidic device for the isolation of CTCs from peripheral blood of patients with HER2-expressing solid tumors. We selected HER2 as an alternative to EpCAM as the receptor is biologically and therapeutically relevant in several solid tumors, like breast cancer (BC), where it is overexpressed in 30% of the patients and expressed in 90%, and gastric cancer (GC), in which HER2 presence is identified in more than 60% of the cases. We tested the performance of various anti HER2 antibodies in a panel of nine different BC cell lines with varying HER2 protein expression levels, using immunoblotting, confocal microscopy, live cells imaging and flow cytometry analyses. The antibody associated with the highest capture efficiency and sensitivity for HER2 expressing cells on the microfluidic device was the one that performed best in live cells imaging and flow cytometry assays as opposed to the fixed cell analyses, suggesting that recognition of the native conformation of the HER2 extracellular epitope on living cells was essential for specificity and sensitivity of CTC capture. Next, we tested the performance of the HER2 microfluidic device using blood from metastatic breast and gastric cancer patients. The HER2 microfluidic device exhibited CTC capture in 9/9 blood samples. Thus, the described HER2-based microfluidic device can be considered as a valid clinically relevant method for CTC capture in HER2 expressing solid cancers.

Detection of circulating pancreas epithelial cells in patients with pancreatic cystic lesions.

Hematogenous dissemination is thought to be a late event in cancer progression. We recently showed in a genetic model of pancreatic ductal adenocarcinoma that pancreas cells can be detected in the bloodstream before tumor formation. To confirm these findings in humans, we used microfluidic geometrically enhanced differential immunocapture to detect circulating pancreas epithelial cells in patient blood samples. We captured more than 3 circulating pancreas epithelial cells/mL in 7 of 21 (33%) patients with cystic lesions and no clinical diagnosis of cancer (Sendai criteria negative), 8 of 11 (73%) with pancreatic ductal adenocarcinoma, and in 0 of 19 patients without cysts or cancer (controls). These findings indicate that cancer cells are present in the circulation of patients before tumors are detected, which might be used in risk assessment.

Characterization of a hybrid dielectrophoresis and immunocapture microfluidic system for cancer cell capture.

The capture of circulating tumor cells (CTCs) from cancer patient blood enables early clinical assessment as well as genetic and pharmacological evaluation of cancer and metastasis. Although there have been many microfluidic immunocapture and electrokinetic techniques developed for isolating rare cancer cells, these techniques are often limited by a capture performance tradeoff between high efficiency and high purity. We present the characterization of shear-dependent cancer cell capture in a novel hybrid DEP-immunocapture system consisting of interdigitated electrodes fabricated in a Hele-Shaw flow cell that was functionalized with a monoclonal antibody, J591, which is highly specific to prostate-specific membrane antigen expressing prostate cancer cells. We measured the positive and negative DEP response of a prostate cancer cell line, LNCaP, as a function of applied electric field frequency, and showed that DEP can control capture performance by promoting or preventing cell interactions with immunocapture surfaces, depending on the sign and magnitude of the applied DEP force, as well as on the local shear stress experienced by cells flowing in the device. This work demonstrates that DEP and immunocapture techniques can work synergistically to improve cell capture performance, and it will aid in the design of future hybrid DEP-immunocapture systems for high-efficiency CTC capture with enhanced purity.

Microfluidic transport in microdevices for rare cell capture.

The isolation and capture of rare cells is a problem uniquely suited to microfluidic devices, in which geometries on the cellular length scale can be engineered and a wide range of chemical functionalizations can be implemented. The performance of such devices is primarily affected by the chemical interaction between the cell and the capture surface and the mechanics of cell-surface collision and adhesion. As rare cell-capture technology has been summarized elsewhere (E. D. Pratt et al., Chem. Eng. Sci. 2011, 66, 1508-1522), this article focuses on the fundamental adhesion and transport mechanisms in rare cell-capture microdevices, and explores modern device design strategies in a transport context. The biorheology and engineering parameters of cell adhesion are defined; adhesion models and reaction kinetics briefly reviewed. Transport at the microscale, including diffusion and steric interactions that result in cell motion across streamlines, is discussed. The review concludes by discussing design strategies with a focus on leveraging the underlying transport phenomena to maximize device performance.

Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device.

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45- cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2-400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.

Immunocapture of prostate cancer cells by use of anti-PSMA antibodies in microdevices.

Patients suffering from cancer can shed tumor cells into the bloodstream, leading to one of the most important mechanisms of metastasis. As such, the capture of these cells is of great interest. Circulating tumor cells are typically extracted from circulation through positive selection with the epithelial cell-adhesion molecule (EpCAM), leading to currently unknown biases when cells are undergoing epithelial-to-mesenchymal transition. For prostate cancer, prostate-specific membrane antigen (PSMA) presents a compelling target for immunocapture, as PSMA levels increase in higher-grade cancers and metastatic disease and are specific to the prostate epithelium. This study uses monoclonal antibodies J591 and J415-antibodies that are highly specific for intact extracellular domains of PSMA on live cells-in microfluidic devices for the capture of LNCaPs, a PSMA-expressing immortalized prostate cancer cell line, over a range of concentrations and shear stresses relevant to immunocapture. Our results show that J591 outperforms J415 and a mix of the two for prostate cancer capture, and that capture performance saturates following incubation with antibody concentrations of 10 micrograms per milliliter.