Hyeun Joong Yoon

Sensitive capture of circulating tumour cells by functionalized graphene oxide nanosheets

The spread of cancer throughout the body is driven by circulating tumour cells (CTCs)1. These cells detach from the primary tumour and move from the blood stream to a new site of subsequent tumour growth. They also carry information about the primary tumour and have the potential to be valuable biomarkers for disease diagnosis and progression, and for the molecular characterization of certain biological properties of the tumour. However, the limited sensitivity and specificity of current methods to measure and study these cells in patient blood samples prevent the realization of their full clinical potential. The use of microfluidic devices is a promising method for isolating CTCs2, 3; however, the devices are reliant on three-dimensional structures, which limit further characterization and expansion of cells on the chip. Here we demonstrate an effective approach to isolate CTCs from blood samples of pancreatic, breast and lung cancer patients, by using functionalised graphene oxide nanosheets on a patterned gold surface. CTCs were captured with high sensitivity at low concentration of target cells (73% ± 32.4 at 3–5 cells/mL blood).

Emerging role of nanomaterials in circulating tumor cell isolation and analysis.

Circulating tumor cells (CTCs) are low frequency cells found in the bloodstream after having been shed from a primary tumor. These cells are research targets because of the information they may potentially provide about both an individual cancer as well as the mechanisms through which cancer spreads in the process of metastasis. Established technologies exist for CTC isolation, but the recent progress and future of this field lie in nanomaterials. In this review, we provide perspective into historical CTC capture as well as current research being conducted, emphasizing the significance of the materials being used to fabricate these devices. The modern investigation into CTCs initially featured techniques that have since been commercialized. A major innovation in the field was the development of a microfluidic capture device, first fabricated in silicon and followed up with glass and thermopolymer devices. We then specifically highlight the technologies incorporating magnetic nanoparticles, carbon nanotubes, nanowires, nanopillars, nanofibers, and nanoroughened surfaces, graphene oxide and their fabrication methods. The nanoscale provides a new set of tools that has the potential to overcome current limitations associated with CTC capture and analysis. We believe the current trajectory of the field is in the direction of nanomaterials, allowing the improvements necessary to further CTC research.

Tunable Thermal-Sensitive Polymer–Graphene Oxide Composite for Efficient Capture and Release of Viable Circulating Tumor Cells

A highly sensitive microfluidic system to capture circulating tumor cells from whole blood of cancer patients is presented. The device incorporates graphene oxide into a thermoresponsive polymer film to serve as the first step of an antibody functionalization chemistry. By decreasing the temperature, captured cells may be released for subsequent analysis.

Cascaded spiral microfluidic device for deterministic and high purity continuous separation of circulating tumor cells.

Inertial microfluidics is an emerging class of technologies developed to separate circulating tumor cells (CTCs). However, defining design parameters and flow conditions for optimal operation remains nondeterministic due to incomplete understanding of the mechanics, which has led to challenges in designing efficient systems. Here, we perform a parametric study of the inertial focusing effects observed in low aspect ratio curvilinear microchannels and utilize the results to demonstrate the isolation of CTCs with high purity. First, we systematically vary parameters including the channel height, width, and radius of curvature over a wide range of flow velocities to analyze its effect on size dependent differential focusing and migration behaviors of binary (10 μm and 20 μm) particles. Second, we use these results to identify optimal flow regimes to achieve maximum separation in various channel configurations and establish design guidelines to readily provide information for developing spiral channels tailored to potentially arbitrary flow conditions that yield a desired equilibrium position for optimal size based CTC separation. Finally, we describe a fully integrated, sheath-less cascaded spiral microfluidic device to continuously isolate CTCs. Human breast cancer epithelial cells were successfully extracted from leukocytes, achieving 86.76% recovery, 97.91% depletion rate, and sustaining high viability upon collection to demonstrate the versatility of the device. Importantly, this device was designed without the cumbersome trail-and-error optimization process that has hindered the development of designing such inertial microfluidic systems.

The marrow niche controls the cancer stem cell phenotype of disseminated prostate cancer.

Dissemination of cancer stem cells (CSCs) serves as the basis of metastasis. Recently, we demonstrated that circulating prostate cancer targets the hematopoietic stem cell (HSCs) ‘niche’ in marrow during dissemination. Once in the niche, disseminated tumor cells (DTCs) may remain dormant for extended periods. As the major function of the HSC niche is to maintain stem cell functions, we hypothesized that the niche regulates CSC activities of DTCs. Here we show that DTCs recovered from marrow were significantly enriched for a CSC phenotype. Critically, the conversion of DTCs to CSCs is regulated by niche-derived GAS6 through the Mer/mTOR; molecules previously shown to regulate dormancy. The data demonstrate that the niche plays a significant role in maintaining tumor-initiating prostate cancer in marrow and suggests a functional relationship between CSCs and dormancy. Understanding how the marrow niche regulates the conversion of DTCs to CSCs is critical for the development of therapeutics specifically targeting skeletal bone metastasis and dormancy.

Microfabrication and in Vivo Performance of a Microdialysis Probe with Embedded Membrane

Microdialysis sampling is an essential tool for in vivo neurochemical monitoring. Conventional dialysis probes are over 220 μm in diameter and have limited flexibility in design because they are made by assembly using preformed membranes. The probe size constrains spatial resolution and governs the amount of tissue damaged caused by probe insertion. To overcome these limitations, we have developed a method to microfabricate probes in Si that are 45 μm thick × 180 μm wide. The probes contain a buried, U-shaped channel that is 30 μm deep × 60 μm wide and terminates in ports for external connection. A 4 mm length of the probe is covered with a 5 μm thick nanoporous membrane. The membrane was microfabricated by deep reactive ion etching through a porous aluminum oxide layer. The microfabricated probe has cross-sectional area that is 79% less than that of the smallest conventional microdialysis probes. The probes yield 2-20% relative recovery at 100 nL/min perfusion rate for a variety of small molecules. The probe was successfully tested in vivo by sampling from the striatum of live rats. Fractions were collected at 20 min intervals (2 μL) before and after an intraperitoneal injection of 5 mg/kg amphetamine. Analysis of fractions by liquid chromatography-mass spectrometry revealed reliable detection of 14 neurochemicals, including dopamine and acetylcholine, at basal conditions. Amphetamine evoked a 43-fold rise in dopamine, a result nearly identical to a conventional dialysis probe in the same animal. The microfabricated probes have potential for sampling with higher spatial resolution and less tissue disruption than conventional probes. It may also be possible to add functionality to the probes by integrating other components, such as electrodes, optics, and additional channels.

Correction: The marrow niche controls the cancer stem cell phenotype of disseminated prostate cancer

Supplementary Materials Supplementary Figure S1: Detection of DTCs in murine marrow with HLA-ABC. (A) Flow cytometric analyses for cytokeratin, EpCAM and HLA-ABC expression on prostate cancer and leukemia cell lines in vitro. (B) Experimental DTC model

Abstract 1685: Identification and characterization of EMT/MET signatures in circulating tumor cells isolated from patients with metastatic breast cancer using graphene oxide nano-chip

Disseminated cancer cells identified in the circulatory system of cancer patients has implied to be the key drivers of tumor metastasis. These cells known as circulating tumor cells (CTCs) are emerging as an alternative to tissue biopsies to predict prognostic values, and to monitor evolving tumor heterogeneity through serial drug treatments. Recent technological advances have enabled the detection of CTCs in patient blood samples. However, the enumeration alone seems to be insufficient to obtain information that could benefit any clinical decisions. The limited sensitivity as well as the specificity of current approaches struggle from realizing the full promise of CTCs. We developed a sensitive microfluidic chip using functionalized graphene oxide (GO) nano-sheets to isolate CTCs with improved sensitivity and purity. Blood samples collected from metastatic breast cancer patients were processed through the chip within 6 hours of sampling and the CTCs were identified by the presence of cytokeratin with the absence of CD45 expression. EMT/MET signatures were characterized in the isolated CTCs to investigate its role by assessing gene expression profiles. Protein markers known to be up regulated during the EMT such as Vimentin and N-cadherin was examined along with HER2. mRNA expression of EMT markers were studied simultaneously using a multiplex TaqMan-based qRT-PCR method and compared to healthy controls. CTCs were successfully detected in >95% of patients examined in this study. Cell-to-cell intrapatient heteroheneity was observed in the isolated CTCs. Markers of EMT/MET phenotypes including Vimentin, EpCAM, HER2, CDH1, CDH2 and cytokeratin in the CTC samples were differentially expressed. These studies demonstrate the feasibility of utilizing the GO nano-chip in CTC isolation and characterization from metastatic breast cancer patients. Given the high sensitivity and the reproducibility to measure protein/gene expression levels of CTC biomarkers, we envision that our GO nano-chip may provide potential tool for real-time monitoring of cancer patients on clinical trials. Citation Format: Tae Hyun Kim, Hyeun Joong Yoon, Sunitha Nagrath. Identification and characterization of EMT/MET signatures in circulating tumor cells isolated from patients with metastatic breast cancer using graphene oxide nano-chip. [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 1685.

Abstract 1585: Application of a graphene oxide based microfluidic device (GO Chip) to prostate cancer circulating tumor cell capture and analysis

One in seven men in the United States will be diagnosed with prostate cancer in his life time, in part because of the prevalence of screening for serum levels of prostate specific antigen (PSA). However, this has led to the overtreatment of the disease, leading to recommendations against frequent PSA testing and establishing the need for a more informative biomarker in prostate cancer. Circulating tumor cells (CTCs) are rare cells present in the blood stream of cancer patients at the low frequency on the order of one CTC in one billion normal blood cells. The graphene oxide chip (GO Chip) is a microfluidic CTC capture device with increased sensitivity and purity due to its use of the nanomaterial graphene oxide to present a capture antibody against the epithelial cellular adhesion molecule (EpCAM). In this study, whole blood was collected from 50 prostate cancer patients, including those with metastatic, localized, castrate resistant, and castrate sensitive disease, and analyzed using the GO Chip. Captured cells were stained for cytokeratin, CD45, and DAPI, with nucleated cytokeratin positive cells being denoted as CTCs. CTCs were detected in 47 out of 50 patients, and an average of 5.94 CTCs/mL was recovered from each sample. Correlation of CTCs with surrogate endpoints could improve the utility of CTCs as a liquid biopsy, potentially advancing their application in translational research. Citation Format: Molly Kozminsky, Hyeun Joong Yoon, Nallasivam Palanisamy, Kathleen Cooney, Maha Hussain, Ajjai Alva, Todd Morgan, Sunitha Nagrath. Application of a graphene oxide based microfluidic device (GO Chip) to prostate cancer circulating tumor cell capture and analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1585. doi:10.1158/1538-7445.AM2015-1585

Abstract 376: Capture and release of circulating tumor cells by temperature-sensitive graphene oxide-polymer composite

Introduction and Objective: Recently, various microfluidics-based circulating tumor cells (CTC) isolation technologies have been developed to isolate, enumerate, and characterize CTCs, but there are few studies reporting CTC release after selective capture. Existing devices face impediments to the easy release of captured cells due to the conjugation of antibodies to permanent structures within the devices. Here we present a tunable microfluidic polymer graphene oxide (GO) Chip based on a temperature-sensitive graphene oxide-polymer composite for selective capture and efficient subsequent release of CTCs. Methods: A blend film of GO sheets and thermo-responsive polymers serves as a platform enabling cell capture and release in our microfluidic devices. The polymers with a tunable lower critical solution temperature (LCST) allow for stimulated release of the captured cells when the device temperature is below the LCST. The GO sheets serve as a carrier for phosophlipid-PEG-amine, which is used for the immobilization of an antibody against the epithelial-cell-adhesion-molecule (EpCAM). To investigate the capture efficiency, MCF-7 cells labeled with cell tracker dye were spiked into 1 mL of whole blood and flowed through the microfluidic device. After thoroughly washing the device with PBS at 25 °C to remove any non-specifically bound cells, we dissolved the thermo-responsive composite with 1 mL of PBS at 5 °C in a cold room and collected released cells from the device outlet. The released cells were cytospun and stained to identify CTCs as cytokeratin positive, DAPI positive, and CD45 negative cells. We also tested peripheral blood samples obtained from patients with metastatic breast cancer in a similar manner. Results: The graphene oxide-polymer based CTC-chip showed a capture efficiency of 95.21% at 95.21% and a release efficiency of 92.45% for MCF-7 breast cancer cells. This new GO Chip was also able to isolate viable CTCs from blood samples of metastatic breast cancer patients for further molecular characterizations. Conclusion: We present a sensitive and tunable microfluidic chip based on a GO-polymer composite to capture, release, identify, and characterize extremely rare CTCs. The CTCs released from the chip are viable, suggesting that this polymer based GO Chip offers great potential for clinical applications due to its high-affinity cell capture and the ability to enable various downstream analyses of captured CTCs. Citation Format: Hyeun Joong Yoon, Apoorv Shanker, Yang Wang, Molly Kozminsky, Shamileh Fouladdel, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Jinsang Kim, Sunitha Nagrath. Capture and release of circulating tumor cells by temperature-sensitive graphene oxide-polymer composite. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 376. doi:10.1158/1538-7445.AM2015-376