CURRENT RESEARCH
Photograph of the CytC sensor platform. Top left: A photo of the sensor layer on PMMA. Top right: A photo of individual three-electrode electrochemical sensors. Bottom: A photo of an assembled CytC sensor platform (Scale bar, 1 cm).
Real-Time Drug Response Monitoring with an Aptamer-Based Sensor Platform
I have developed a multi-well aptasensor platform for label-free, real-time monitoring of Cytochrome C (CytC), a cell death indicator, in microdissected tumor biopsies. Unlike traditional endpoint assays, this platform captures dynamic therapeutic responses, providing a real-time view of how tumors react to different treatments. Aptasensors use nucleic acid aptamers to directly measure ligand binding. Upon binding to their molecular targets, aptamers undergo reversible conformational changes, generating a measurable electrochemical signal.
To enhance signal fidelity and account for baseline drift during prolonged monitoring, we implemented the Kinetic Differential Measurement (KDM) technique. By comparing signals at two distinct frequencies, KDM mitigates background noise, improving the signal-to-noise ratio and enhancing data reliability over extended culture periods. This development provides critical insights into both the timing and effectiveness of various drug treatments.
The platform also incorporates a multiplexed electronic system, allowing simultaneous real-time measurements across multiple wells. Each well contains a set of electrodes, which interface with a printed circuit board, enabling high-throughput and cost-effective CytC monitoring.
Our aptasensor platform holds significant potential for advancing cancer research by enabling a broader understanding of how drugs influence the tumor microenvironment in real-time. This innovation not only optimizes existing treatments but also paves the way for personalized medicine applications, where real-time data on tumor responses can guide more effective therapeutic strategies. Moving forward, we plan to expand the platform's biomarker capabilities, positioning it as an invaluable tool for comprehensive disease modeling and drug screening.
Science Spotlight, Fred Hutch Cancer Center - Cell death on a chip: new tools for a new era of cancer biology
Bioengineering News, University of Washington - A new platform for more effective cancer drug testing using aptamer sensors
Undergraduate student working on the project: Timothy Krilov, Department of Bioengineering, University of Washington
Development of the multiplex system and implementing technology for high-throughput measurement.
Funding: Interdisciplinary Postdoctoral Fellowship in Cancer Research, University of Washington & Fred Hutch Cancer Center
T. N. H. Nguyen, L. Horowitz, *T. Krilov, E. Lockhart, H. L. Kenerson, T. S. Gujral, R. S. Yeung, N. Arroyo-Currás, and A. Folch. “Label-Free, Real-time Monitoring of Cytochrome C Drug Responses in Microdissected Tumor Biopsies with a Multi-Well Aptasensor Platform”, Science Advances. 10: 6 (2024).
Microfluidic Modulation of Tumor Microvasculature
The microvasculature within the tumor microenvironment (TME) plays an essential role in cancer signaling, extending far beyond nutrient delivery. However, controlling the generation and maintenance of microvasculature in ex vivo systems has been challenging, a critical step for establishing cancer models with high clinical biomimicry. To address this, I have developed a microfluidic platform that supports and modulates the microvasculature within microdissected tumor tissues, maintaining vital aspects of the TME. This platform enables controlled perfusion, which enhances vascular integrity and supports the formation and/or maintenance of complex vascular networks within tumor cuboids. Through precise fluid flow control, we observe improved CD31 expression, an endothelial cell marker, compared to static culture conditions. The platform's design includes layered channels and traps that ensure the stability of cuboids while applying targeted flow, making it a versatile tool for investigating vascular dynamics in cancer research.
Our findings demonstrate that continuous flow significantly supports vascular structures in tumor microdissected tumor tissues, offering a controlled environment to study how biophysical and biochemical factors, such as those involving the nitric oxide pathway, affect vascular integrity and function. We have tested the effects of various vascular-targeting agents, including Combretastatin A-4, L-Nω-Nitro arginine methyl ester (L-NNA), and Nitroglycerin and found that controlled flow not only preserves endothelial integrity but also allows for modulating drug responses within the TME. This work provides valuable insights into how vascular health in tumors can be maintained ex vivo, with applications that range from drug screening to personalized cancer therapy, where understanding vascular dynamics is crucial.
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Figure 1. A. Photograph of the microfluidic device: The top panel shows the fully assembled device ready for cuboid culture in the medium; the bottom left depicts microdissected tumor tissues (cuboids); the bottom right focuses on a close-up section of the device displaying cuboids situated in four individual traps. B. A 3D illustration of four cuboids in their traps and the direction of flow inside the device. C. Cross-sectional schematic of the device connected to a motorized syringe pump.
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Figure 2. A. Confocal images of cuboids stained with CD31 being subjected to different flow rates (0.05, 0.1, and 0.15 mL/hr), no-flow control, and orbital shaking for different time points 24 hrs. Scale bar, 100 µm B. Percentage volume of CD31 staining. C. Branch length of all conditions over 24 hrs.
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Figure 3. A. Visualization of NTG treatment displays confocal microscopy images of CD31 immunostained cuboids. These cuboids were treated with NTG at 50 nM and 1 µM concentrations under flow conditions set to 0.1 mL/hr alongside a control group under the same flow without the drug and a static group treated with 50 nM NTG. Scale bar, 100 µm. G. Post-NTG quantitative vascular analysis displays analytical data on the average intensity of CD31 staining, the percentage volume of CD31 staining, branch length, and branch average diameter across all NTG treatment groups compared to controls.
Undergraduate student working on the project: Brandon Nguyen, Department of Bioengineering, University of Washington
Image data analysis using IMARIS: modulation of tumor microvasculature in microdissected cancer tissues.
Funding: Catalytic Collaboration Trainee Award, Brotman Baty Institute, Seattle
T. N. H. Nguyen, L. Horowitz, *B. Nguyen, *T. Krilov, S. Zhu, E. Lockhart, T. S. Gujral, and A. Folch. “Microfluidic Modulation of Microvasculature in Microdissected Tumors”, bioRxiv (/doi.org/10.1101/2024.09.26.615278).