Micromanipulation of Live Microdissected Tissues with a Low-Cost Integrated Robotic Platform.

The small amount of human tissue available for testing is a paramount challenge in cancer drug development, cancer disease models, and personalized oncology. Technologies that combine the microscale manipulation of tissues with fluid handling offer the exciting possibility of miniaturizing and automating drug evaluation workflows. This approach minimizes animal testing and enables inexpensive, more efficient testing of samples with high clinical biomimicry using scarce materials. We have developed an inexpensive platform based on an off-the-shelf robot that can manipulate microdissected tissues (µDTs) into user-programmed positions without using intricate microfluidic designs nor any other accessories such as a microscope or a pneumatic controller. The robot integrates complex functions such as vision and fluid actuation by incorporating simple items including a USB camera and a rotary pump. Through the robot's camera, the platform software optically recognizes randomly-seeded µDTs on the surface of a petri dish and positions a mechanical arm above the µDTs. Then, a custom rotary pump actuated by one of the robot's motors generates enough microfluidic lift to hydrodynamically pick and place µDTs with a pipette at a safe distance from the substrate without requiring a proximity sensor. The platform's simple, integrated construction is cost-effective and compact, allowing placement inside a tissue culture hood for sterile workflows. The platform enables users to select µDTs based on their size, place them in user-programmed arrays, such as multi-well plates, and control various robot motion parameters. As a case application, we use the robotic system to conduct semi-automated drug testing of mouse and human µDTs in 384-well plates. Our user-friendly platform promises to democratize microscale tissue research to clinical and biological laboratories worldwide.

Label-Free, Real-Time Monitoring of Cytochrome C Responses to Drugs in Microdissected Tumor Biopsies with a Multi-Well Aptasensor Platform.

Functional assays on intact tumor biopsies can potentially complement and extend genomics-based approaches for precision oncology, drug testing, and organs-on-chips cancer disease models by capturing key determinants of therapeutic response, such as tissue architecture, tumor heterogeneity, and the tumor microenvironment. Currently, most of these assays rely on fluorescent labeling, a semi-quantitative method best suited to be a single-time-point terminal assay or labor-intensive terminal immunostaining analysis. Here, we report integrated aptamer electrochemical sensors for on-chip, real-time monitoring of increases of cytochrome C, a cell death indicator, from intact microdissected tissues with high affinity and specificity. The platform features a multi-well sensor layout and a multiplexed electronic setup. The aptasensors measure increases in cytochrome C in the supernatant of mouse or human microdissected tumors after exposure to various drug treatments. Since the aptamer probe can be easily exchanged to recognize different targets, the platform could be adapted for multiplexed monitoring of various biomarkers, providing critical information on the tumor and its microenvironment. This approach could not only help develop more advanced cancer disease models but also apply to other complex in vitro disease models, such as organs-on-chips and organoids.

miR-181a Promotes Multiple Protumorigenic Functions by Targeting TGFßR3.

Cutaneous squamous cell carcinoma (cSCC) comprises 15‒20% of all skin cancers and has a well-defined progression sequence from precancerous actinic keratosis to invasive cSCC. To identify targets for chemoprevention, we previously reported a cross-species analysis to identify the transcriptional drivers of cSCC development and identified miR-181a as a potential oncomiR. We show that the upregulation of miR-181a promotes multiple protumorigenic properties by targeting an understudied component of TGFß signaling, TGFßR3. miR-181a and TGFßR3 are upregulated and downregulated, respectively, in cSCC. miR-181a overexpression (OE) and TGFßR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT cells and in primary normal human epidermal keratinocytes. In addition, OE of miR-181a or KD of TGFßR3 by short hairpin RNA enhances anchorage-independent survival. miR-181a OE or TGFßR3 KD enhances cellular migration and invasion and upregulation of epithelial‒mesenchymal transition markers. Luciferase reporter assays demonstrate that miR-181a directly targets the 3'-untranslated region of TGFßR3. miR-181a upregulates phosphorylated SMAD3 levels after TGFß2 administration and results in elevated SNAIL and SLUG expression. Finally, we confirm in vivo that miR-181a inhibition compromises tumor growth. Importantly, these phenotypes can be reversed with TGFßR3 OE or KD in the context of miR-181a OE or KD, respectively, further highlighting the physiologic relevance of this regulation in cSCC.

Integrative transcriptomic analysis for linking acute stress responses to squamous cell carcinoma development.

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer and commonly arises in chronically UV-exposed skin or chronic wounds. Since UV exposure and chronic wounds are the two most prominent environmental factors that lead to cuSCC initiation, we undertook this study to test whether more acute molecular responses to UV and wounding overlapped with molecular signatures of cuSCC. We reasoned that transcriptional signatures in common between acutely UV-exposed skin, wounded skin, and cuSCC tumors, might enable us to identify important pathways contributing to cuSCC. We performed transcriptomic analysis on acutely UV-exposed human skin and integrated those findings with datasets from wounded skin and our transcriptomic data on cuSCC using functional pair analysis, GSEA, and pathway analysis. Integrated analyses revealed significant overlap between these three datasets, thus highlighting deep molecular similarities these biological processes, and we identified Oncostatin M (OSM) as a potential common upstream driver. Expression of OSM and its downstream targets correlated with poorer overall survival in head and neck SCC patients. In vitro, OSM promoted invasiveness of keratinocytes and cuSCC cells and suppressed apoptosis of irradiated keratinocytes. Together, these results support the concept of using an integrated, biologically-informed approach to identify potential promoters of tumorigenesis.

Management of the Left Subclavian Artery in TEVAR for Chronic Type B Aortic Dissection.

OBJECTIVES: Left subclavian artery (LSA) revascularization in thoracic endovascular aortic repair (TEVAR) remains controversial. Left subclavian artery coverage without revascularization can cause stroke and death. TEVAR has gained popularity for the treatment of chronic type B aortic dissection (cTBD). Using the Vascular Quality Initiative (VQI) database, we reviewed outcomes of LSA revascularization in TEVAR for cTBD. METHODS: The VQI registry identified 5683 patients treated with TEVAR from July 2010 to July 2016, including 208 repairs for cTBD. We analyzed outcomes per the Society for Vascular Surgery reporting standards. RESULTS: Of the 208 patients, 150 (72.1%) were male with a median age of 65.0 years (interquartile range [IQR], 55.0-72.0). Median aneurysm diameter was 5.7 cm (IQR, 5.0-6.5 cm). Data on the patency of the LSA was available in 131 (63.0%) patients. Twenty-five (19.1%) had occlusion of the LSA without revascularization, while 106 (80.9%) maintained patency or had revascularization. Successful device delivery occurred in all 131 (100%) patients. Maintaining LSA patency did not affect the rate of cerebrovascular accident (P = .16), spinal cord ischemia (P = 1.00), or death (P = 1.00). This was also nonsignificant when analyzing the subgroup of 98 elective cases. There was no difference in the rates of endoleak. Any intervention for the LSA (revascularization or occlusion) led to a longer procedure time (203.6 minutes vs 163.7 minutes, P = .04). CONCLUSIONS: Maintaining LSA patency during TEVAR for cTBD offers no advantage in perioperative morbidity or endoleak. Occlusion of LSA may be performed safely in this cohort and revascularization reserved for those who have anatomy that compromises perfusion to critical organs.

Unveiling prognostics biomarkers of tyrosine metabolism reprogramming in liver cancer by cross-platform gene expression analyses.

Tyrosine is mainly degraded in the liver by a series of enzymatic reactions. Abnormal expression of the tyrosine catabolic enzyme tyrosine aminotransferase (TAT) has been reported in patients with hepatocellular carcinoma (HCC). Despite this, aberration in tyrosine metabolism has not been investigated in cancer development. In this work, we conduct comprehensive cross-platform study to obtain foundation for discoveries of potential therapeutics and preventative biomarkers of HCC. We explore data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Gene Expression Profiling Interactive Analysis (GEPIA), Oncomine and Kaplan Meier plotter (KM plotter) and performed integrated analyses to evaluate the clinical significance and prognostic values of the tyrosine catabolic genes in HCC. We find that five tyrosine catabolic enzymes are downregulated in HCC compared to normal liver at mRNA and protein level. Moreover, low expression of these enzymes correlates with poorer survival in patients with HCC. Notably, we identify pathways and upstream regulators that might involve in tyrosine catabolic reprogramming and further drive HCC development. In total, our results underscore tyrosine metabolism alteration in HCC and lay foundation for incorporating these pathway components in therapeutics and preventative strategies.

Fabrication and ex vivo evaluation of activated carbon-Pt microparticle based glutamate biosensor.

As one of the most abundant neurotransmitters in the brain and the spinal cord, glutamate plays many important roles in the nervous system. Precise information about the level of glutamate in the extracellular space of living brain tissue may provide new insights on fundamental understanding of the role of glutamate in neurological disorders as well as neurophysiological phenomena. Electrochemical sensor has emerged as a promising solution that can satisfy the requirement for highly reliable and continuous monitoring method with good spatiotemporal resolution for characterization of extracellular glutamate concentration. Recently, we published a method to create a simple printable glutamate biosensor using platinum nanoparticles. In this work, we introduce an even simpler and lower cost conductive polymer composite using commercially available activated carbon with platinum microparticles to easily fabricate highly sensitive glutamate biosensor using direct ink writing method. The fabricated biosensors are functionality superior than previously reported with the sensitivity of 5.73 ± 0.078 nA µM-1 mm-2, detection limit of 0.03 µM, response time less than or equal to 1 s, and a linear range from 1 µM up to 925 µM. In this study, we utilize astrocyte cell culture to demonstrate our biosensor's ability to monitor glutamate uptake process. We also demonstrate direct measurement of glutamate release from optogenetic stimulation in mouse primary visual cortex (V1) brain slices.

In Vivo Glutamate Sensing inside the Mouse Brain with Perovskite Nickelate-Nafion Heterostructures.

Glutamate, one of the main neurotransmitters in the brain, plays a critical role in communication between neurons, neuronal development, and various neurological disorders. Extracellular measurement of neurotransmitters such as glutamate in the brain is important for understanding these processes and developing a new generation of brain-machine interfaces. Here, we demonstrate the use of a perovskite nickelate-Nafion heterostructure as a promising glutamate sensor with a low detection limit of 16 nM and a response time of 1.2 s via amperometric sensing. We have designed and successfully tested novel perovskite nickelate-Nafion electrodes for recording of glutamate release ex vivo in electrically stimulated brain slices and in vivo from the primary visual cortex (V1) of awake mice exposed to visual stimuli. These results demonstrate the potential of perovskite nickelates as sensing media for brain-machine interfaces.

Printable Nonenzymatic Glucose Biosensors Using Carbon Nanotube-PtNP Nanocomposites Modified with AuRu for Improved Selectivity.

Nonenzymatic glucose biosensors have the potential for a more reliable in vivo functionality due to the reduced risk of biorecognition element degradation. However, these novel sensing mechanisms often are nanoparticle-based and have nonlinear responses, which makes it difficult to gauge their potential utility against more conventional enzymatic biosensors. Moreover, these nonenzymatic biosensors often suffer from poor selectivity that needs to be better addressed before being used in vivo. To address these problems, here we present an amperometric nonenzymatic glucose biosensor fabricated using one-step electrodeposition of Au and Ru nanoparticles on the surface of a carbon-nanotube-based platinum-nanoparticle hybrid in conductive polymer. Using benchtop evaluations, we demonstrate that the bimetallic catalyst of Au-Ru nanoparticles can enable the nonenzymatic detection of glucose with a superior performance and stability. Furthermore, our biosensor shows good selectivity against other interferents, with a nonlinear dynamic range of 1-19 mM glucose. The Au-Ru catalyst has a conventional linear range of 1-10 mM, with a sensitivity of 0.2347 nA/(µM mm2) ± 0.0198 (n = 3) and a limit of detection of 0.068 mM (signal-to-noise, S/N = 3). The biosensor also exhibits a good repeatability and stability at 37 °C over a 3 week incubation period. Finally, we use a modified Butler-Volmer nonlinear analytical model to evaluate the impact of geometrical and chemical design parameters on our nonenzymatic biosensor's performance, which may be used to help optimize the performance of this class of biosensors.

Simple Fabrication of Flexible Biosensor Arrays Using Direct Writing for Multianalyte Measurement from Human Astrocytes.

Simultaneous measurements of glucose, lactate, and neurotransmitters (e.g., glutamate) in cell culture over hours and days can provide a more dynamic and longitudinal perspective on ways neural cells respond to various drugs and environmental cues. Compared with conventional microfabrication techniques, direct writing of conductive ink is cheaper, faster, and customizable, which allows rapid iteration for different applications. Using a simple direct writing technique, we printed biosensor arrays onto cell culture dishes, flexible laminate, and glass to enable multianalyte monitoring. The ink was a composite of PEDOT:PSS conductive polymer, silicone, activated carbon, and Pt microparticles. We applied 0.5% Nafion to the biosensors for selectivity and functionalized them with oxidase enzymes. We characterized biosensors in phosphate-buffered saline and in cell culture medium supplemented with fetal bovine serum. The biosensor arrays measured glucose, lactate, and glutamate simultaneously and continued to function after incubation in cell culture at 37 °C for up to 2 days. We cultured primary human astrocytes on top of the biosensor arrays and placed arrays into astrocyte cultures. The biosensors simultaneously measured glucose, glutamate, and lactate from astrocyte cultures. Direct writing can be integrated with microfluidic organ-on-a-chip platforms or as part of a smart culture dish system. Because we print extrudable and flexible components, sensing elements can be printed on any 3D or flexible substrate.

Ex vivo electrochemical measurement of glutamate release during spinal cord injury.

Excessive glutamate release following traumatic spinal cord injury (SCI) has been associated with exacerbating the extent of SCI. However, the mechanism behind sustained high levels of extracellular glutamate is unclear. Spinal cord segments mounted in a sucrose double gap recording chamber are an established model for traumatic spinal cord injury. We have developed a method to record, with micro-scale printed glutamate biosensors, glutamate release from ex vivo rat spinal cord segments following injury. This protocol would work equally well for similar glutamate biosensors.

Facile fabrication of flexible glutamate biosensor using direct writing of platinum nanoparticle-based nanocomposite ink.

Glutamate excitotoxicity is a pathology in which excessive glutamate can cause neuronal damage and degeneration. It has also been linked to secondary injury mechanisms in traumatic spinal cord injury. Conventional bioanalytical techniques used to characterize glutamate levels in vivo, such as microdialysis, have low spatiotemporal resolution, which has impeded our understanding of this dynamic event. In this study, we present an amperometric biosensor fabricated using a simple direct ink writing technique for the purpose of in vivo glutamate monitoring. The biosensor is fabricated by immobilizing glutamate oxidase on nanocomposite electrodes made of platinum nanoparticles, multi-walled carbon nanotubes, and a conductive polymer on a flexible substrate. The sensor is designed to measure extracellular dynamics of glutamate and other potential biomarkers during a traumatic spinal cord injury event. Here we demonstrate good sensitivity and selectivity of these rapidly prototyped implantable biosensors that can be inserted into a spinal cord and measure extracellular glutamate concentration. We show that our biosensors exhibit good flexibility, linear range, repeatability, and stability that are suitable for future in vivo evaluation.

APOBEC mutation drives early-onset squamous cell carcinomas in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.

Anti-biofouling implantable catheter using thin-film magnetic microactuators.

Here we report on the development of polyimide-based flexible magnetic actuators for actively combating biofouling that occurs in many chronically implanted devices. The thin-film flexible devices are microfabricated and integrated into a single-pore silicone catheter to demonstrate a proof-of-concept for a self-clearing smart catheter. The static and dynamic mechanical responses of the thin-film magnetic microdevices were quantitatively measured and compared to theoretical values. The mechanical fatigue properties of these polyimide-based microdevices were also characterized up to 300 million cycles. Finally, the biofouling removal capabilities of magnetically powered microdevices were demonstrated using bovine serum albumin and bioconjugated microbeads. Our results indicate that these thin-film microdevices are capable of significantly reducing the amount of biofouling. At the same time, we demonstrated that these microdevices are mechanically robust enough to withstand a large number of actuation cycles during its chronic implantation.

REST represses miR-124 and miR-203 to regulate distinct oncogenic properties of glioblastoma stem cells.

Background: Glioblastoma (GBM) is one of the most common, aggressive, and invasive human brain tumors. There are few reliable mechanism-based therapeutic approaches for GBM patients. The transcriptional repressor RE1 silencing transcriptional factor (REST) regulates the oncogenic properties of a class of GBM stem-like cells (high-REST [HR]-GSCs) in humans. However, it has been unclear whether REST represses specific targets to regulate specific oncogenic functions or represses all targets with overlapping functions in GSCs. Methods: We used genome-wide, biochemical, and mouse intracranial tumorigenic assays to identify and determine functions of microRNA (miR) targets of REST in 2 independent HR-GSC lines. Results: Here we show that REST represses 2 major miR gene targets in HR-GSCs: miR-203, a new target, and miR-124, a known target. Gain of function of miR-124 or miR-203 in HR-GSCs increased survival in tumor-bearing mice. Importantly, the increased survival of tumor-bearing mice caused by knockdown of REST in HR-GSCs was reversed by double knockdown of REST and either miR-203 or miR-124, indicating that these 2 miRs are critical tumor suppressors that are repressed in REST-mediated tumorigenesis. We further show that while miR-124 and the REST-miR-124 pathways regulate self-renewal, apoptosis and invasion, miR-203 and the REST-miR-203 pathways regulate only invasion. We further identify and validate potential mRNA targets of miR-203 and miR-124 in REST-mediated HR-GSC tumor invasion. Conclusions: These findings indicate that REST regulates its miR gene targets with overlapping functions and suggest how REST maintains oncogenic competence in GSCs. These mechanisms could potentially be utilized to block REST-mediated GBM tumorigenesis.

Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates.

Cutaneous squamous cell carcinoma (cuSCC) comprises 15-20% of all skin cancers, accounting for over 700,000 cases in USA annually. Most cuSCC arise in association with a distinct precancerous lesion, the actinic keratosis (AK). To identify potential targets for molecularly targeted chemoprevention, here we perform integrated cross-species genomic analysis of cuSCC development through the preneoplastic AK stage using matched human samples and a solar ultraviolet radiation-driven Hairless mouse model. We identify the major transcriptional drivers of this progression sequence, showing that the key genomic changes in cuSCC development occur in the normal skin to AK transition. Our data validate the use of this ultraviolet radiation-driven mouse cuSCC model for cross-species analysis and demonstrate that cuSCC bears deep molecular similarities to multiple carcinogen-driven SCCs from diverse sites, suggesting that cuSCC may serve as an effective, accessible model for multiple SCC types and that common treatment and prevention strategies may be feasible.