Hakho Lee - Academia.edu (original) (raw)

Papers by Hakho Lee

JCI insight, Jan 5, 2018

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. ... more Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.

Journal of Extracellular Vesicles, 2019

ABSTRACTTumour cells release diverse populations of extracellular vesicles (EVs) ranging in size,... more ABSTRACTTumour cells release diverse populations of extracellular vesicles (EVs) ranging in size, molecular cargo, and function. We sought to characterize mRNA and protein content of EV subpopulations released by human glioblastoma (GBM) cells expressing a mutant form of epidermal growth factor receptor (U87EGFRvIII) in vitro and in vivo with respect to size, morphology and the presence of tumour cargo. The two EV subpopulations purified from GBM U87EGFRvIII cancer cells, non‐cancer human umbilical vein endothelial cells (HUVEC; control) and serum of U87EGFRvIII glioma‐bearing mice using differential centrifugation (EVs that sediment at 10,000 × g or 100,000 × g are termed large EVs and small EVs, respectively) were characterized using transmission electron microscopy (TEM), confocal microscopy, nanoparticle tracking analysis (NTA), flow cytometry, immunofluorescence (IF), quantitative‐polymerase chain reaction (qPCR), droplet digital polymerase chain reaction (ddPCR) and micro‐nucl...

Nature Biomedical Engineering, 2018

Users may view, print, copy, and download text and data-mine the content in such documents, for t... more Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

ACS photonics, Jan 21, 2018

Extracellular vesicles (EVs), including exosomes, are nanoscale membrane particles shed from cell... more Extracellular vesicles (EVs), including exosomes, are nanoscale membrane particles shed from cells and contain cellular proteins whose makeup could inform cancer diagnosis and treatment. Most analyses have focused on surface proteins while analysis of intravesicular proteins has been more challenging. Herein, we report an EV screening assay for both intravesicular and transmembrane proteins using a nanoplasmonic sensor. Termed iNPS (intravesicular nanoplasmonic system), this platform used nanohole-based surface plasmon resonance (SPR) for molecular detection. Specifically, we i) established a unified assay protocol to detect intravesicular as well as transmembrane proteins; and ii) engineered plasmonic substrates to enhance detection sensitivity. The resulting iNPS enabled sensitive (0.5 L sample per marker) and high-throughput (a 10 × 10 array) detection for EV proteins. When applied to monitor EVs from drug-treated cancer cells, the iNPS assay revealed drug-dependent unique EV pro...

ACS nano, Jan 20, 2018

Sepsis is an often fatal condition that arises when the immune response to an infection causes wi... more Sepsis is an often fatal condition that arises when the immune response to an infection causes widespread systemic organ injury. A critical unmet need in combating sepsis is the lack of accurate early biomarkers that produce actionable results in busy clinical settings. Here, we report the development of a point-of-care platform for rapid sepsis detection. Termed IBS (integrated biosensor for sepsis), our approach leverages (i) the pathophysiological role of cytokine interleukin-3 (IL-3) in early sepsis and (ii) a hybrid magneto-electrochemical sensor for IL-3 detection. The developed platform produces test results within 1 h from native blood samples and detects IL-3 at a sensitivity of <10 pg/mL; this performance is >5-times faster and >10-times more sensitive than conventional enzyme-linked immunoadsorbent assays, the current gold standard. Using clinical samples, we show that elevated plasma IL-3 levels are associated with high organ failure rate and thus greater risk o...

ACS nano, Jan 4, 2018

Extracellular vesicles (EV) are a family of cell-originating, membrane-enveloped nanoparticles wi... more Extracellular vesicles (EV) are a family of cell-originating, membrane-enveloped nanoparticles with diverse biological function, diagnostic potential, and therapeutic applications. While EV can be abundant in circulation, their small size (∼4 order of magnitude smaller than cells) has necessitated bulk analyses, making many more nuanced biological explorations, cell of origin questions, or heterogeneity investigations impossible. Here we describe a single EV analysis (SEA) technique which is simple, sensitive, multiplexable, and practical. We profiled glioblastoma EV and discovered surprising variations in putative pan-EV as well as tumor cell markers on EV. These analyses shed light on the heterogeneous biomarker profiles of EV. The SEA technology has the potential to address fundamental questions in vesicle biology and clinical applications.

ACS nano, Nov 20, 2017

Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated... more Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are non-invasive alternatives, but are late markers with low specificity. We here report a new urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as surrogate marker for inflammation. We optimized iKEA to detect T-cell derived EVs, and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients, and achieved high detection accuracy (91.1%). Fast, non-invasive, and cost-effective, iKE...

Science translational medicine, May 24, 2017

Pancreatic ductal adenocarcinoma (PDAC) is usually detected late in the disease process. Clinical... more Pancreatic ductal adenocarcinoma (PDAC) is usually detected late in the disease process. Clinical workup through imaging and tissue biopsies is often complex and expensive due to a paucity of reliable biomarkers. We used an advanced multiplexed plasmonic assay to analyze circulating tumor-derived extracellular vesicles (tEVs) in more than 100 clinical populations. Using EV-based protein marker profiling, we identified a signature of five markers (PDAC(EV) signature) for PDAC detection. In our prospective cohort, the accuracy for the PDAC(EV) signature was 84% [95% confidence interval (CI), 69 to 93%] but only 63 to 72% for single-marker screening. One of the best markers, GPC1 alone, had a sensitivity of 82% (CI, 60 to 95%) and a specificity of 52% (CI, 30 to 74%), whereas the PDAC(EV) signature showed a sensitivity of 86% (CI, 65 to 97%) and a specificity of 81% (CI, 58 to 95%). The PDAC(EV) signature of tEVs offered higher sensitivity, specificity, and accuracy than the existing s...

Chemical communications (Cambridge, England), Jan 9, 2017

We herein report a biomimetic technique to modify plastic substrates for bioassays. The method fi... more We herein report a biomimetic technique to modify plastic substrates for bioassays. The method first places a polydopamine adhesion layer to plastic surface, and then grows conformal silica coating. As proof of principle, we coated plastic microbeads to construct a disposable filter for point-of-care nucleic acid extraction.

Nanomedicine: Nanotechnology, Biology and Medicine, 2017

This study evaluates μNMR technology for molecular profiling of tumor fine needle aspirates and p... more This study evaluates μNMR technology for molecular profiling of tumor fine needle aspirates and peripheral blood of melanoma patients. In vitro assessment of melanocyte (MART-1, HMB45) and MAP kinase signaling (pERK, pS6K) molecule expression was performed in human cell lines, while clinical validation was performed in an IRB-approved study of melanoma patients undergoing biopsy and blood sampling. Tumor FNA and blood specimens were compared with BRAF genetic analysis and cross-sectional imaging. μNMR in vitro analysis showed increased expression of melanocyte markers in melanoma cells as well as increased expression of phosphorylated MAP kinase targets in BRAF-mutant melanoma cells. Melanoma patient FNA samples showed increased pERK and pS6K levels in BRAF mutant compared with BRAF WT melanomas, with μNMR blood circulating tumor cell level increased with higher metastatic burden visible on imaging. These results indicate μNMR technology provides minimally invasive pointof-care evaluation of tumor signaling and metastatic burden in melanoma patients.

Scientific Reports, 2016

Lens-free digital in-line holography (LDIH) is a promising technology for portable, wide field-of... more Lens-free digital in-line holography (LDIH) is a promising technology for portable, wide field-of-view imaging. Its resolution, however, is limited by the inherent pixel size of an imaging device. Here we present a new computational approach to achieve sub-pixel resolution for LDIH. The developed method is a sparsity-based reconstruction with the capability to handle the non-linear nature of LDIH. We systematically characterized the algorithm through simulation and LDIH imaging studies. The method achieved the spatial resolution down to one-third of the pixel size, while requiring only single-frame imaging without any hardware modifications. This new approach can be used as a general framework to enhance the resolution in nonlinear holographic systems.

Theranostics, 2016

Low-cost, rapid and accurate detection technologies are key requisites to cope with the growing g... more Low-cost, rapid and accurate detection technologies are key requisites to cope with the growing global cancer challenges. The need is particularly pronounced in resource-limited settings where treatment opportunities are often missed due to the absence of timely diagnoses. We herein describe a Holographic Assessment of Lymphoma Tissue (HALT) system that adopts a smartphone as the basis for molecular cancer diagnostics. The system detects malignant lymphoma cells labeled with marker-specific microbeads that produce unique holographic signatures. Importantly, we optimized HALT to detect lymphomas in fine-needle aspirates from superficial lymph nodes, procedures that align with the minimally invasive biopsy needs of resource-constrained regions. We equipped the platform to directly address the practical needs of employing novel technologies for "real world" use. The HALT assay generated readouts in <1.5 h and demonstrated good agreement with standard cytology and surgical pathology.

ACS combinatorial science, Jun 28, 2016

Molecular targeting using ligands specific to disease markers has shown great promise for early d... more Molecular targeting using ligands specific to disease markers has shown great promise for early detection and directed therapy. Bead-based combinatorial libraries have served as powerful tools for the discovery of novel targeting agents. Screening platforms employing magnetic capture have been used to achieve rapid and efficient identification of high-affinity ligands from one-bead-one-compound (OBOC) libraries. Traditional manual methodologies to isolate magnetized "hit" beads are tedious and lack accuracy, while existing instruments to expedite bead sorting tend to be costly and complex. Here, we describe the design and construction of a simple and inexpensive microfluidic magnetic sorting device using standard photolithography and soft lithography approaches to facilitate high-throughput isolation of magnetized positive hit beads from combinatorial libraries. We have demonstrated that the device is able to sort magnetized beads with superior accuracy compared to convent...

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Lab on a Chip, 2016

A smartphone-based assay is developed to detect soluble protein markers in point-of-care settings.

Advanced healthcare materials, Jan 20, 2015

A novel dual marking hydrogel system is reported for radiological and laparoscopic localization o... more A novel dual marking hydrogel system is reported for radiological and laparoscopic localization of lesions. Bioorthogonally crosslinked hydrogel containing both tantalum and India ink can be rapidly formed inside the body after injecting precursors, and stably located for several days as a long-term biocompatible carrier for markers.

Chemical reviews, Jan 10, 2015

This section reviews various MNP-based bioassay systems. We broadly categorized them as magnetic ... more This section reviews various MNP-based bioassay systems. We broadly categorized them as magnetic sensors and actuators, according to their primary use of MNPs, and discuss representative examples in each category. 2.1. Magnetic detection Signals from MNP-labeled biological objects are often measured by magnetometers. 37 Based on the detection mechanism, magnetometers can be categorized into volumetric or surface-based sensors. 38 The volumetric sensors measure analytical signals coming from the entire detection volume, which makes assays simple and fast. The sensors' resolving power, however, can be restricted, because the acquired signal is an ensemble average of the whole volume. Representative examples of volumetric sensors include NMR devices, magnetic susceptometors, and conventional superconducting-quantum-interference-devices (SQUIDs). Surface-based sensors directly detect individual magnetic objects near the sensing elements. These sensors generally achieve higher sensitivity and finer resolution than volumetric ones, but target samples should be placed in close proximity of the sensor surface. Such an arrangement limits the assay configuration, and typically causes the assays to be more time-Lee et al.

Nature communications, Jan 11, 2015

Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical proble... more Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

Proceedings of the National Academy of Sciences of the United States of America, Jan 17, 2013

Ascites tumor cells (ATCs) represent a potentially valuable source of cells for monitoring treatm... more Ascites tumor cells (ATCs) represent a potentially valuable source of cells for monitoring treatment of ovarian cancer as it would obviate the need for more invasive surgical biopsies. The ability to perform longitudinal testing of ascites in a point-of-care setting could significantly impact clinical trials, drug development, and clinical care. Here, we developed a microfluidic chip platform to enrich ATCs from highly heterogeneous peritoneal fluid and then perform molecular analyses on these cells. We evaluated 85 putative ovarian cancer protein markers and found that nearly two-thirds were either nonspecific for malignant disease or had low abundance. Using four of the most promising markers, we prospectively studied 47 patients (33 ovarian cancer and 14 control). We show that a marker set (ATCdx) can sensitively and specifically map ATC numbers and, through its reliable enrichment, facilitate additional treatment-response measurements related to proliferation, protein translatio...