Weian Sheng

Aptamer-Enabled Efficient Isolation of Cancer Cells from Whole Blood Using a Microfluidic Device

Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of ~95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from unprocessed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 min. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.

Multivalent DNA nanospheres for enhanced capture of cancer cells in microfluidic devices.

Isolation of circulating tumor cells (CTCs) from peripheral blood or cancer cells from bone marrow has significant applications in cancer diagnosis, therapy monitoring, and drug development. CTCs are cancer cells shed from primary tumors; they circulate in the bloodstream, leading to metastasis. The extraordinary rarity of CTCs in the bloodstream makes their isolation a significant technological challenge. Herein, we report the development of a platform combining multivalent DNA aptamer nanospheres with microfluidic devices for efficient isolation of cancer cells from blood. Gold nanoparticles (AuNPs) were used as an efficient platform for assembling a number of aptamers for high-efficiency cell capture. Up to 95 aptamers were attached onto each AuNP, resulting in enhanced molecular recognition capability. An increase of 39-fold in binding affinity was confirmed by flow cytometry for AuNP-aptamer conjugates (AuNP-aptamer) when compared with aptamer alone. With a laminar flow flat channel microfluidic device, the capture efficiency of human acute leukemia cells from a cell mixture in buffer increased from 49% using aptamer alone to 92% using AuNP-aptamer. We also employed AuNP-aptamer in a microfluidic device with herringbone mixing microstructures for isolation of leukemia cells in whole blood. The cell capture efficiency was also significantly increased with the AuNP-aptamer over aptamer alone, especially at high flow rates. Our results show that the platform combining DNA nanostructures with microfluidics has a great potential for sensitive isolation of CTCs and is promising for cancer diagnosis and prognosis.

An ensemble of aptamers and antibodies for multivalent capture of cancer cells

We developed an optimized ensemble of aptamers and antibodies that functions as a multivalent adhesive domain for the capture and isolation of cancer cells. When incorporated into a microfluidic device, the ensemble showed not only high capture efficiency, but also superior capture selectivity at a high shear stress (or high flow rate).

Tumor cell capture patterns around aptamer-immobilized microposts in microfluidic devices

Circulating tumor cells (CTCs) have shown potential for cancer diagnosis and prognosis. Affinity-based CTC isolation methods have been proved to be efficient for CTC detection in clinical blood samples. One of the popular choices for affinity-based CTC isolation is to immobilize capture agents onto an array of microposts in microchannels, providing high CTC capture efficiency due to enhanced interactions between tumor cells and capture agents on the microposts. However, how the cells interact with microposts under different flow conditions and what kind of capture pattern results from the interactions have not been fully investigated; a full understanding of these interactions will help to design devices and choose experimental conditions for higher CTC capture effeciency. We report our study on their interaction and cell distribution patterns around microposts under different flow conditions. Human acute lymphoblastic leukemia cells (CCRF-CEM) were used as target cancer cells in this study, while the Sgc8 aptamer that has specific binding with CCRF-CEM cells was employed as a capture agent. We investigated the effects of flow rates and micropost shapes on the cell capture efficiency and capture patterns on microposts. While a higher flow rate decreased cell capture efficiency, we found that the capture pattern around microposts also changed, with much more cells captured in the front half of a micropost than at the back half. We also found the ratio of cells captured on microposts to the cells captured by both microposts and channel walls increased as a function of the flow rate. We compared circular microposts with an elliptical shape and found that the geometry affected the capture distribution around microposts. In addition, we have developed a theoretical model to simulate the interactions between tumor cells and micropost surfaces, and the simulation results are in agreement with our experimental observation.

“Tissue is the Issue”: Circulating Tumor Cells in Pancreatic Cancer

Pancreatic cancer (PCa) is the fourth most common cause of all cancer-related deaths in the USA. Currently, the overall median survival is less than 1 year and 5-year survival rate is about 5 % [1]. The poor outcome for PCa is largely due to the fact that the vast majority of patients (pts) are diagnosed at an advanced disease stage or with occult metastases that soon reveal themselves despite no clinical or radiographic clues. Therefore, early and accurate diagnosis is critical to appropriately risk stratify and intervene to improve the clinical outcomes of pts with PCa. Diagnostic modalities in current use include cross-sectional imaging and the use of the non-specific serum biomarker carbohydrate antigen 19-9 (CA-19-9) and occasionally carcinoembryonic antigen (CEA). Radiographic imaging of PCa can be challenging due to the potential absence of a discrete pancreatic mass or presence of alternate symptom etiologies including pancreatic inflammation. Even in the setting of an appropriate clinical context with supportive imaging and serum biomarkers, assurance of histological or cytological confirmation of PCa is directly related to adequate tissue acquisition [2]. Methodologies for pancreatic tissue acquisition are invasive, of relative low diagnostic yield, and associated with potential complications such as pancreatitis, bleeding, duodenal perforations, and infections [3]. Therefore, novel non-invasive or minimally invasive methods of accurate PCa cell acquisition represent a clinical unmet need. Progression of most solid cancers is associated with intravasation of cancer cells into the pt’s circulatory system with dissemination to metastatic sites. These circulating tumor cells (CTCs), measured by collection and detection of epithelial cells in the peripheral circulation, are known to be detectable in pts with solid tumors from early through advanced disease [4]. In addition to diagnostic sampling, CTCs have many potential clinical applications in the management of patients with solid tumors. These include, but are not limited to, risk stratification/prognostication, monitoring of response to therapy, characterizing the tumor’s molecular alterations, screening for early relapse, and other potential ways to personalize therapy [5–9]. In this brief report, we describe the successful isolation and molecular characterization of CTCs from a pt with PCa in whom traditional acquisition of tissue for diagnosis and management was unsuccessful despite multiple traditional attempts.

Abstract 416: Tumor cell isolation using antibody/aptamer-based multivalent binding

Isolation and enumeration of circulating tumor cells (CTCs) in peripheral blood have been used for cancer diagnosis, prognosis, and theragnosis. The majority of the CTC isolation methods, including the FDA-approved CellSearch®, employ one antibody to capture tumor cells. Using such a monovalent-binding capture agent likely results in lower cell capture efficiency than multivalent-binding methods, which tend to have stronger binding force (or lower dissociation constant). We have investigated in a multivalent-binding-based tumor cell isolation method by immobilizing a mixture of aptamers and antibodies on the surfaces of microfluidic devices. Since aptamers are much smaller in the size (8-15 kDa) than antibodies (150 kDa), aptamers and antibodies could interact with different receptors on cell surfaces, resulting in a configuration that one cell simultaneously interacts with multiple different types of capture agents. The microfluidic devices we used are in the size of a microscope slide and each of them consists of an inlet, eight parallel channels connected via consecutive bifurcation, and an outlet. There are micromixers inside the channels for enhanced interactions. Biotinylated aptamers and biotinylated antibodies were coated onto microchannel surfaces using avidin-biotin chemistry. We tested the devices by isolating human leukemia cells (CCRF-CEM) in devices immobilized with a mixture of sgc8 aptamers and protein tyrosine kinase-7 (PTK7) antibodies. Both sgc8 aptamers and anti-PTK7 exhibited strong binding with CCRF-CEM cells according to flow cytometry. An antibody-to-aptamer ratio of 1:300 showed the highest capture efficiency among the range we studied (from 1:30 to 1:3000). We found that the antibody-aptamer surfaces had higher capture efficiency than antibody-alone or aptamer-alone surfaces; the degree of enhancement increased with the flow rate. Control experiments using non-specific aptamer (TD05) and non-specific antibody (anti-EpCAM) were carried out to verify the benefits of multivalent binding. We also found that the multivalent-binding surfaces have higher specificity than monovalent-binding surface, especially at a high flow rate. A calibration curve was obtained by spiking leukemia cells into human whole blood. In summary, antibody/aptamer-coated multivalent-binding surfaces in microfluidic devices showed higher capture efficiency and specificity than monovalent-binding surfaces, suggesting their potential for the isolation and enumeration of CTCs for clinical applications. Citation Format: Z. Hugh Fan, Jinling Zhang, Weian Sheng. Tumor cell isolation using antibody/aptamer-based multivalent binding. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 416.

Abstract IA21: Enumeration of circulating tumor cells for studying cancer drug sensitivity

More than 90% of cancer deaths result from metastasis, hence understanding of metastasis and detection of cancer before spreading are extremely important. During metastasis, some cancer cells escape from the primary tumor and enter the bloodstream, becoming circulating tumor cells (CTCs). Some of these CTCs acquire the capability to colonize secondary sites to form tumors at other organs. As a result, the number of CTCs could be an indicator of cancer drug sensitivity because an effective therapy would shrink the tumor size and reduce the CTC count. Enumerating CTCs is technically challenging because CTCs are extremely rare in the bloodstream, typically a few in billions of normal blood cells. To address this, we have developed microfluidic devices that are able to isolate tumor cells efficiently from peripheral blood. The fundamental advantage of a microdevice over a vial or tube is a much shorter diffusion distance before interacting with the capture agents. The reduction in the dimension from millimeters to microns theoretically increases the interaction opportunities by orders of magnitude. The devices are in the size of a microscope slide. Each device consists of a number of microfabricated hair-thin channels and tens of thousands micropillars, which enhance the probability of their interactions with target cancer cells. Mcromixers have also been integrated in the devices to increase the capture efficiency. We also incorporated gold nanoparticles to enhance the density of capture agents for more interactions with CTCs. Both aptamers and antibodies have been studied for various cancer cells. We immobilized aptamers onto the surfaces of microchannels and micropillars for isolating leukemia cells. The device geometry and the flow rate have been investigated and optimized by studying their effects on the isolation sensitivity and specificity. The capture efficiency of >95% was obtained at the optimum flow rate. In addition, we have applied the devices for isolating CTCs from peripheral bloods of pancreatic cancer patients while CTCs were verified according to the FDA-approved definition, namely EpCAM+ (epithelial cell adhesion molecule), cytokeratin+, DAPI+ (4,6-diamidino-2- phenylindole), and CD45-. CTCs were found in 94% of clinical samples, and none in the healthy control samples. We also compared the aptamers and antibodies in terms of their capture efficiency, and we studied combining them together for cancer cell isolation. We investigated the correlation between the CTC number and the clinical result of pancreatic adenocarcinoma patients who went through a clinic trial, in which a colorectal cancer drug cocktail, FOLFOX (5-fluorouracil, leucovorin, oxaliplatin), was combined with a leukemia/prostate cancer drug dasatinib as chemotherapy treatment. For those patients showing the reduction in the tumor size measured by computed tomograms (CT scans) during the course of anti-cancer treatment, we also observed the decrease in the number of CTCs in their peripheral blood samples. The double-blinded experiments show the strong correlation between the CTC number and cancer drug sensitivity. Citation Format: Z. Hugh Fan, Weian Sheng, Thomas George, Chen Liu. Enumeration of circulating tumor cells for studying cancer drug sensitivity. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA21.

Rapid Capture of Rare Cancer Cells Using a High-Performance Microfluidic Chip

Rare circulating tumor cells (CTCs) are cancer cells that detached from either a primary tumor or metastatic sites. CTCs circulate into bloodstream and become the origin of metastasis, the spread of cancer to distant organs, which is the primary cause of cancer-induced death. This paper describes our development of a microfluidic chip with unique micropillar geometry for rapid capture of CTCs from whole blood. The microscope-slide-sized microchip contains tens of thousands of isotropically-etched elliptical micropillars, which enhanced the interactions between cells and chip surfaces. The microchip was coated with DNA aptamer, an antibody-like molecule which can specifically bind with their target cells. With optimized channel geometry and flow rate, the microchip yielded a capture efficiency of >95% and a purity of >81% when capturing leukemia cells from a cell mixture. Then, the device was applied to capture colorectal tumor cells from whole blood; as few as 10 tumor cells can be efficiently isolated from 1 mL blood in 28 min. We envision that this high-performance microchip is promising for the detection, enrichment and isolation of rare CTCs, and will open up new opportunities for cancer diagnosis and monitoring cancer treatment.Copyright