Siyang Zheng

Portable Filter-Based Microdevice for Detection and Characterization of Circulating Tumor Cells

Purpose: Sensitive detection and characterization of circulating tumor cells (CTC) could revolutionize the approach to patients with early-stage and metastatic cancer. The current methodologies have significant limitations, including limited capture efficiency and ability to characterize captured cells. Here, we report the development of a novel parylene membrane filter-based portable microdevice for size-based isolation with high recovery rate and direct on-chip characterization of captured CTC from human peripheral blood. Experimental Design: We evaluated the sensitivity and efficiency of CTC capture in a model system using blood samples from healthy donors spiked with tumor cell lines. Fifty-nine model system samples were tested to determine the recovery rate of the microdevice. Moreover, 10 model system samples and 57 blood samples from cancer patients were subjected to both membrane microfilter device and CellSearch platform enumeration for direct comparison. Results: Using the model system, the microdevice achieved >90% recovery with probability of 95% recovering at least one cell when five are seeded in 7.5 mL of blood. CTCs were identified in 51 of 57 patients using the microdevice, compared with only 26 patients with the CellSearch method. When CTCs were detected by both methods, greater numbers were recovered by the microfilter device in all but five patients. Conclusions: This filter-based microdevice is both a capture and analysis platform, capable of multiplexed imaging and genetic analysis. The microdevice presented here has the potential to enable routine CTC analysis in the clinical setting for the effective management of cancer patients. Clin Cancer Res; 16(20); 5011–8. ©2010 AACR.

3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood

Detection of circulating tumor cells has emerged as a promising minimally invasive diagnostic and prognostic tool for patients with metastatic cancers. We report a novel three dimensional microfilter device that can enrich viable circulating tumor cells from blood. This device consists of two layers of parylene membrane with pores and gap precisely defined with photolithography. The positions of the pores are shifted between the top and bottom membranes. The bottom membrane supports captured cells and minimize the stress concentration on cell membrane and sustain cell viability during filtration. Viable cell capture on device was investigated with scanning electron microscopy, confocal microscopy, and immunofluorescent staining using model systems of cultured tumor cells spiked in blood or saline. The paper presents and validates this new 3D microfiltration concept for circulation tumor cell enrichment application. The device provides a highly valuable tool for assessing and characterizing viable enriched circulating tumor cells in both research and clinical settings.

Circulating tumor cells: Advances in isolation and analysis, and challenges for clinical applications

Circulating tumor cells (CTCs) are rare cancer cells released from tumors into the bloodstream that are thought to have a key role in cancer metastasis. The presence of CTCs has been associated with worse prognosis in several major cancer types, including breast, prostate and colorectal cancer. There is considerable interest in CTC research and technologies for their potential use as cancer biomarkers that may enhance cancer diagnosis and prognosis, facilitate drug development, and improve the treatment of cancer patients. This review provides an update on recent progress in CTC isolation and molecular characterization technologies. Furthermore, the review covers significant advances and limitations in the clinical applications of CTC-based assays for cancer prognosis, response to anti-cancer therapies, and exploratory studies in biomarkers predictive of sensitivity and resistance to cancer therapies.

Disseminated and circulating tumor cells: Role in effective cancer management

Dissemination of tumor cells from primary tumors in the circulatory system is an early event in carcinogenesis. The presence of these single disseminated tumor cells (DTC) in peripheral blood, bone marrow and distant organs is the rationale for adjuvant systemic treatment. Detection of DTC in bone marrow aspirates from breast cancer patients and other solid tumors at the primary diagnosis impacts the prognosis of disease. In peripheral blood these cells are termed as circulating tumor cells (CTC). Due to technical difficulties the clinical significance of CTC detection at early stages is less established. This review focuses on available techniques for detection of DTC and CTC, recent technical advances in development of more sensitive microfluidic methods for capture of DTC and CTC and possibilities for further detection and their potential molecular characterization. Not only the clinical significance of DTC but also the presence of cancer stem cells in dissemination clearly demonstrates the need for development of sensitive technologies allowing for definition of biomarkers and molecular targets on cells in dissemination, thus eventually leading to optimization of systemic therapies.

Circulating tumor cell enrichment based on physical properties.

The metastatic dissemination and spread of malignant circulating tumor cells (CTCs) accounts for more than 90% of cancer-related deaths. CTCs detach from a primary tumor, travel through the circulatory system, and then invade and proliferate in distant organs. The detection of CTCs from blood has been established for prognostic monitoring and is predictive of patient outcome. Analysis of CTCs could enable the means for early detection and screening in cancer, as well as provide diagnostic access to tumor tissues in a minimally invasive way. The fundamental challenge with analyzing CTCs is the fact that they occur at extremely low concentrations in blood, on the order of one out of a billion cells. Various technologies have been proposed to isolate CTCs for enrichment. Here we focus on antigen-independent approaches that are not limited by specific capture antibodies. Intrinsic physical properties of CTCs, including cell size, deformability, and electrical properties, are reviewed, and technologies developed to exploit them for enrichment from blood are summarized. Physical enrichment technologies are of particular interest as they have the potential to increase yield and enable the analysis of rare CTC phenotypes that may not be otherwise obtained.

Flexible Micro Spring Array Device for High-Throughput Enrichment of Viable Circulating Tumor Cells

BACKGROUND The dissemination of circulating tumor cells (CTCs) that cause metastases in distant organs accounts for the majority of cancer-related deaths. CTCs have been established as a cancer biomarker of known prognostic value. The enrichment of viable CTCs for ex vivo analysis could further improve cancer diagnosis and guide treatment selection. We designed a new flexible micro spring array (FMSA) device for the enrichment of viable CTCs independent of antigen expression. METHODS Unlike previous microfiltration devices, flexible structures at the micro scale minimize cell damage to preserve viability, while maximizing throughput to allow rapid enrichment directly from whole blood with no need for sample preprocessing. Device performance with respect to capture efficiency, enrichment against leukocytes, viability, and proliferability was characterized. CTCs and CTC microclusters were enriched from clinical samples obtained from breast, lung, and colorectal cancer patients. RESULTS The FMSA device enriched tumor cells with 90% capture efficiency, higher than 10(4) enrichment, and better than 80% viability from 7.5-mL whole blood samples in <10 min on a 0.5-cm(2) device. The FMSA detected at least 1 CTC in 16 out of 21 clinical samples (approximately 76%) compared to 4 out of 18 (approximately 22%) detected with the commercial CellSearch® system. There was no incidence of clogging in over 100 tested fresh whole blood samples. CONCLUSIONS The FMSA device provides a versatile platform capable of viable enrichment and analysis of CTCs from clinically relevant volumes of whole blood.

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78–83%), high retention of cell viability (71–74%), high tumour cell enrichment against leukocytes (1.7–2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4–0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Streamline-Based Microfluidic Devices for Erythrocytes and Leukocytes Separation

In this paper, we report two devices for the continuous size-based separation of particles, such as blood cells, which is an important step for on-chip blood preparation. Unlike previously demonstrated passive fluidic devices for particle separation, the local geometry of the bifurcated side channels was used as a design parameter. The design of the devices was based on 2-D fluidic simulation of a T-shaped model. This novel approach was proved to be effective in predicting device performance. The critical particle size for separation was clearly defined in the bifurcated region by simulation under the established theoretical framework. We validated the operation principle of the devices by separating 5- and 10-mum polystyrene beads. Human leukocytes were also successfully separated from erythrocytes with 97% efficiency. The separation region of the device had a small footprint for the separation of particles in micrometer range, which makes this device a good candidate to be integrated into a lab-on-a-chip system. The particles were collected in different exit channels after they were separated, which facilitated further sensing and processing. Similar to cross-flow filters, particles were separated perpendicular to the flow direction. The filtering effect was achieved with the collection zones established by the fluidic field. Clogging was minimized by designing the minimal channel width of the devices larger than the largest particle diameter. Solvent exchange could be accomplished for particles.

Circulating Tumor Cells in Melanoma Patients

Circulating tumor cells (CTCs) are of recognized importance for diagnosis and prognosis of cancer patients. With melanoma, most studies do not show any clear relationship between CTC levels and stage of disease. Here, CTCs were enriched (∼400X) from blood of melanoma patients using a simple centrifugation device (OncoQuick), and 4 melanocyte target RNAs (TYR, MLANA, MITF, and MIF) were quantified using QPCR. Approximately one-third of melanoma patients had elevated MIF and MLANA transcripts (p<0.0001 and p<0.001, respectively) compared with healthy controls. In contrast, healthy controls had uniformly higher levels of TYR and MITF than melanoma patients (p<0.0001). There was a marked shift of leukocytes into the CTC-enriched fractions (a 430% increase in RNA recovery, p<0.001), and no relationship between CTC levels and stage of disease was found. CTCs were captured on microfabricated filters and cultured. Captured melanoma CTCs were large cells, and consisted of 2 subpopulations, based on immunoreactivity. One subpopulation (∼50%) stained for both pan-cytokeratin (KRT) markers and the common leukocyte marker CD-45, whereas the second subpopulation stained for only KRT. Since similar cells are described in many cancers, we also examined blood from colorectal and pancreatic cancer patients. We observed analogous results, with most captured CTCs staining for both CD-45/KRT markers (and for the monocyte differentiation marker CD-14). Our results suggest that immature melanocyte-related cells (expressing TYR and MITF RNA) may circulate in healthy controls, although they are not readily detectable without considerable enrichment. Further, as early-stage melanomas develop, immature melanocyte migration into the blood is somehow curtailed, whereas a significant proportion of patients develop elevated CTC levels (based on MIF and MLANA RNAs). The nature of the captured CTCs is consistent with literature describing leukocyte/macrophage-tumor cell fusion hybrids, and their role in metastatic progression.

Facile synthesis of magnetic mesoporous hollow carbon microspheres for rapid capture of low-concentration peptides.

Mesoporous and hollow carbon microspheres embedded with magnetic nanoparticles (denoted as MHM) were prepared via a facile self-sacrificial method for rapid capture of low-abundant peptides from complex biological samples. The morphology, structure, surface property, and magnetism were well-characterized. The hollow magnetic carbon microspheres have a saturation magnetization value of 130.2 emu g–1 at room temperature and a Brunauer–Emmett–Teller specific surface area of 48.8 m2 g–1 with an average pore size of 9.2 nm for the mesoporous carbon shell. The effectiveness of these MHM affinity microspheres for capture of low-concentration peptides was evaluated by standard peptides, complex protein digests, and real biological samples. These multifunctional hollow carbon microspheres can realize rapid capture and convenient separation of low-concentration peptides. They were validated to have better performance than magnetic mesoporous silica and commercial peptide-enrichment products. In addition, they can be easily recycled and present excellent reusability. Therefore, it is expected that this work may provide a promising tool for high-throughput discovery of peptide biomarkers from biological samples for disease diagnosis and other biomedical applications.