David K. Wood

Point-of-care diagnostics for noncommunicable diseases using synthetic urinary biomarkers and paper microfluidics

Significance Noncommunicable diseases, including cardiovascular disease and cancer, are growing worldwide but are challenging to diagnose because biomarkers that can accurately detect them in patients are lacking. Here, we designed nanoscale agents that are administered to reveal the presence of diseased tissues by producing a biomarker in the urine that can be detected using paper strips similar to a home pregnancy test. Using mouse models, we show that we can detect diseases as diverse as solid cancer and blood clots using only a single injection of our diagnostic followed by urine analysis on paper. This platform does not require expensive instruments, invasive procedures, or trained medical personnel, and may allow low-cost diagnosis of diseases at the point of care in resource-limited settings. With noncommunicable diseases (NCDs) now constituting the majority of global mortality, there is a growing need for low-cost, noninvasive methods to diagnose and treat this class of diseases, especially in resource-limited settings. Molecular biomarkers combined with low-cost point-of-care assays constitute a potential solution for diagnosing NCDs, but the dearth of naturally occurring, predictive markers limits this approach. Here, we describe the design of exogenous agents that serve as synthetic biomarkers for NCDs by producing urinary signals that can be quantified by a companion paper test. These synthetic biomarkers are composed of nanoparticles conjugated to ligand-encoded reporters via protease-sensitive peptide substrates. Upon delivery, the nanoparticles passively target diseased sites, such as solid tumors or blood clots, where up-regulated proteases cleave the peptide substrates and release reporters that are cleared into urine. The reporters are engineered for detection by sandwich immunoassays, and we demonstrate their quantification directly from unmodified urine; furthermore, capture antibody specificity allows the probes to be multiplexed in vivo and quantified simultaneously by ELISA or paper lateral flow assay (LFA). We tailor synthetic biomarkers specific to colorectal cancer, a representative solid tumor, and thrombosis, a common cardiovascular disorder, and demonstrate urinary detection of these diseases in mouse models by paper diagnostic. Together, the LFA and injectable synthetic biomarkers, which could be tailored for multiple diseases, form a generalized diagnostic platform for NCDs that can be applied in almost any setting without expensive equipment or trained medical personnel.

Nanoparticles that sense thrombin activity as synthetic urinary biomarkers of thrombosis.

Thrombin is a serine protease and regulator of hemostasis that plays a critical role in the formation of obstructive blood clots, or thrombosis, that is a life-threatening condition associated with numerous diseases such as atherosclerosis and stroke. To detect thrombi in living animals, we design and conjugate thrombin-sensitive peptide substrates to the surface of nanoparticles. Following intravenous infusion, these “synthetic biomarkers” survey the host vasculature for coagulation and, in response to substrate cleavage by thrombin, release ligand-encoded reporters into the host urine. To detect the urinary reporters, we develop a companion 96-well immunoassay that utilizes antibodies to bind specifically to the ligands, thus capturing the reporters for quantification. Using a thromboplastin-induced mouse model of pulmonary embolism, we show that urinary biomarker levels differentiate between healthy and thrombotic states and correlate closely with the aggregate burden of clots formed in the lungs. Our results demonstrate that synthetic biomarkers can be engineered to sense vascular diseases remotely from the urine and may allow applications in point-of-care diagnostics.

Single cell trapping and DNA damage analysis using microwell arrays

With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24- and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.

Critical role for lysyl oxidase in mesenchymal stem cell-driven breast cancer malignancy

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to differentiate into multiple mesoderm lineages in the course of normal tissue homeostasis or during injury. We have previously shown that MSCs migrate to sites of tumorigenesis, where they become activated by cancer cells to promote metastasis. However, the molecular and phenotypic attributes of the MSC-induced metastatic state of the cancer cells remained undetermined. Here, we show that bone marrow-derived human MSCs promote de novo production of lysyl oxidase (LOX) from human breast carcinoma cells, which is sufficient to enhance the metastasis of otherwise weakly metastatic cancer cells to the lungs and bones. We also show that LOX is an essential component of the CD44-Twist signaling axis, in which extracellular hyaluronan causes nuclear translocation of CD44 in the cancer cells, thus triggering LOX transcription by associating with its promoter. Processed and enzymatically active LOX, in turn, stimulates Twist transcription, which mediates the MSC-triggered epithelial-to-mesenchymal transition (EMT) of carcinoma cells. Surprisingly, although induction of EMT in breast cancer cells has been tightly associated with the generation of cancer stem cells, we find that LOX, despite being critical for EMT, does not contribute to the ability of MSCs to promote the formation of cancer stem cells in the carcinoma cell populations. Collectively, our studies highlight a critical role for LOX in cancer metastasis and indicate that the signaling pathways controlling stroma-induced EMT are distinct from pathways regulating the development of cancer stem cells.

A Biophysical Indicator of Vaso-occlusive Risk in Sickle Cell Disease

Sickle cell patients are stratified according to risk of vaso-occlusion on the basis of the dynamic biophysical properties of their blood as measured ex vivo in a microfluidic system. Going with the Flow … or Not In healthy people, blood flows freely throughout the body, delivering oxygen to tissues via the molecule hemoglobin. For those with sickle cell disease, red blood cells carry a mutated form of hemoglobin, called hemoglobin S, that changes cells into a rigid sickle, or crescent, shape and causes blood flow to slow markedly. Although sickle cell disease can be detected through genetic and molecular tests, there is no objective biomarker of clinical outcome. Wood et al. therefore devised a microfluidic device that can mimic this vaso-occlusive event, allowing them to detect biophysical changes in blood—a new type of marker that could stratify sickle cell patients on the basis of disease severity. Wood et al. collected blood from 29 patients: 23 of which were classified as having “severe” disease, and 6 of which were “benign.” The blood was flowed through a microfluidic device consisting of a channel coupled to an oxygen reservoir. As sickle cells become deoxygenated, they adopt their stiff, sickle shape. In their device, such deoxygenation would result in a drop in flow velocity, which could be measured as a change in conductance. This biophysical marker helped the authors to successfully identify benign versus severe samples, without using less reliable markers like white blood cell count and hemoglobin S fraction. Wood and colleagues could also predict a patient’s response to therapy, such as a blood transfusion or small-molecule drugs. It may never be possible to predict the exact timing of vaso-occlusion, but the ex vivo biophysical test proposed by Wood et al. can objectively identify patients who are not responding well to treatment or transfusion. Conversely, the test could pinpoint those who may benefit from such therapies, putting more patients on treatment regimens that make their red blood cells just go with the flow. The search for predictive indicators of disease has largely focused on molecular markers. However, biophysical markers, which can integrate multiple pathways, may provide a more global picture of pathophysiology. Sickle cell disease affects millions of people worldwide and has been studied intensely at the molecular, cellular, tissue, and organismal level for a century, but there are still few, if any, markers quantifying the severity of this disease. Because the complications of sickle cell disease are largely due to vaso-occlusive events, we hypothesized that a physical metric characterizing the vaso-occlusive process could serve as an indicator of disease severity. Here, we use a microfluidic device to characterize the dynamics of “jamming,” or vaso-occlusion, in physiologically relevant conditions, by measuring a biophysical parameter that quantifies the rate of change of the resistance to flow after a sudden deoxygenation event. Our studies show that this single biophysical parameter could be used to distinguish patients with poor outcomes from those with good outcomes, unlike existing laboratory tests. This biophysical indicator could therefore be used to guide the timing of clinical interventions, to monitor the progression of the disease, and to measure the efficacy of drugs, transfusion, and novel small molecules in an ex vivo setting.

High-bandwidth radio frequency Coulter counter

We demonstrate a method for high-bandwidth, high-sensitivity particle sensing and cell counting in a microfluidic system. Our approach employs a tuned radiofrequency probe, which forms the radiofrequency analog of a Coulter counter. By measuring the reflected rf power, this approach provides an unprecedented detection rate, with a theoretical bandwidth in excess of 10 MHz. Particle detection was performed in a continuous flow mode in a microfluidic channel, using 15μm diameter polystyrene beads suspended in a sucrose-saline solution. We demonstrate 30 kHz counting rates and show high-resolution bead time-of-flight data, comprising the fastest electronic particle detection on-chip to date.

Microfabricated high-throughput electronic particle detector

We describe the design, fabrication, and use of a radio frequency reflectometer integrated with a microfluidic system, applied to the very high-throughput measurement of micron-scale particles, passing in a microfluidic channel through the sensor region. The device operates as a microfabricated Coulter counter [U.S. Patent No. 2656508 (1953)], similar to a design we have described previously, but here with significantly improved electrode geometry as well as including electronic tuning of the reflectometer; the two improvements yielding an improvement by more than a factor of 10 in the signal to noise and in the diametric discrimination of single particles. We demonstrate the high-throughput discrimination of polystyrene beads with diameters in the 4-10 microm range, achieving diametric resolutions comparable to the intrinsic spread of diameters in the bead distribution, at rates in excess of 15 x 10(6) beads/h.

Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors

A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects.

Standard fluorescent imaging of live cells is highly genotoxic.

Fluorescence microscopy is commonly used for imaging live mammalian cells. Here, we describe studies aimed at revealing the potential genotoxic effects of standard fluorescence microscopy. To assess DNA damage, a high throughput platform for single cell gel electrophoresis is used (e.g., the CometChip). Light emitted by three standard filters was studied: (a) violet light [340–380 nm], used to excite DAPI and other blue fluorophores, (b) blue light [460–500 nm] commonly used to image green fluorescent protein (GFP) and Calcein AM, and (c) green light [528–553 nm], useful for imaging red fluorophores. Results show that exposure of samples to light during imaging is indeed genotoxic even when the selected wavelengths are outside the range known to induce significant damage levels. Shorter excitation wavelengths and longer irradiation times lead to higher levels of DNA damage. We have also measured DNA damage in cells expressing enhanced GFP or stained with Calcein AM, a widely used green fluorophore. Data show that Calcein AM leads to a synergistic increase in the levels of DNA damage and that even cells that are not being directly imaged sustain significant DNA damage from exposure to indirect light. The nature of light‐induced DNA damage during imaging was assessed using the Fpg glycosylase, an enzyme that enables quantification of oxidative DNA damage. Oxidative damage was evident in cells exposed to violet light. Furthermore, the Fpg glycosylase revealed the presence of oxidative DNA damage in blue‐light exposed cells for which DNA damage was not detected using standard analysis conditions. Taken together, the results of these studies call attention to the potential confounding effects of DNA damage induced by standard imaging conditions, and identify wavelength, exposure time, and fluorophore as parameters that can be modulated to reduce light‐induced DNA damage.

Multiscale Cues Drive Collective Cell Migration.

To investigate complex biophysical relationships driving directed cell migration, we developed a biomimetic platform that allows perturbation of microscale geometric constraints with concomitant nanoscale contact guidance architectures. This permits us to elucidate the influence, and parse out the relative contribution, of multiscale features, and define how these physical inputs are jointly processed with oncogenic signaling. We demonstrate that collective cell migration is profoundly enhanced by the addition of contract guidance cues when not otherwise constrained. However, while nanoscale cues promoted migration in all cases, microscale directed migration cues are dominant as the geometric constraint narrows, a behavior that is well explained by stochastic diffusion anisotropy modeling. Further, oncogene activation (i.e. mutant PIK3CA) resulted in profoundly increased migration where extracellular multiscale directed migration cues and intrinsic signaling synergistically conspire to greatly outperform normal cells or any extracellular guidance cues in isolation.