A Microfabricated, Flow-Driven Grinding Mill for Mechanical Cell Lysing.

This paper presents the design, microfabrication, and demonstration of a novel microfluidic grinding mill for the lysis of the dinoflagellate, Alexandrium, a neurotoxin-producing genus of algae that is responsible for red tide and paralytic shellfish poisoning. The mill consists of a high-speed, hydrodynamically driven microrotor coupled to a micro grinding mill that lyses robust algal cells by mechanical abrasion with single-pass efficiencies as high as 97%. These efficiencies are comparable to, or better than, current mechanical and chemical lysing methods without adding complications associated with harsh chemical additives that can interfere with subsequent downstream bioanalysis. Release of cytoplasm from lysed algae was confirmed using polymerase chain reaction (PCR) amplification of Alexandrium DNA using dinoflagellate primers.

Development-on-chip: in vitro neural tube patterning with a microfluidic device.

Embryogenesis is a highly regulated process in which the precise spatial and temporal release of soluble cues directs differentiation of multipotent stem cells into discrete populations of specialized adult cell types. In the spinal cord, neural progenitor cells are directed to differentiate into adult neurons through the action of mediators released from nearby organizing centers, such as the floor plate and paraxial mesoderm. These signals combine to create spatiotemporal diffusional landscapes that precisely regulate the development of the central nervous system (CNS). Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity. In vitro methods are preferred for their enhanced experimental clarity but often lack the technical sophistication required for biological realism. In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development. Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo.

Development of a Novel DNA Mono-alkylator Platform for Antibody-Drug Conjugates.

Although microtubule inhibitors (MTI) remain a therapeutically valuable payload option for antibody-drug conjugates (ADC), some cancers do not respond to MTI-based ADCs. Efforts to fill this therapeutic gap have led to a recent expansion of the ADC payload "toolbox" to include payloads with novel mechanisms of action such as topoisomerase inhibition and DNA cross-linking. We present here the development of a novel DNA mono-alkylator ADC platform that exhibits sustained tumor growth suppression at single doses in MTI-resistant tumors and is well tolerated in the rat upon repeat dosing. A phosphoramidate prodrug of the payload enables low ADC aggregation even at drug-to-antibody ratios of 5:1 while still delivering a bystander-capable payload that is effective in multidrug resistant (MDR)-overexpressing cell lines. The platform was comparable in xenograft studies to the clinical benchmark DNA mono-alkylator ADC platform DGN459 but with a significantly better tolerability profile in rats. Thus, the activity and tolerability profile of this new platform make it a viable option for the development of ADCs.

Discovery and Optimization of a STING Agonist Platform for Application in Antibody Drug Conjugates.

While STING agonists have proven to be effective preclinically as anti-tumor agents, these promising results have yet to be translated in the clinic. A STING agonist antibody-drug conjugate (ADC) could overcome current limitations by improving tumor accessibility, allowing for systemic administration as well as tumor-localized activation of STING for greater anti-tumor activity and better tolerability. In line with this effort, a STING agonist ADC platform was identified through systematic optimization of the payload, linker, and scaffold based on multiple factors including potency and specificity in both in vitro and in vivo evaluations. The platform employs a potent non-cyclic dinucleotide STING agonist, a cleavable ester-based linker, and a hydrophilic PEG8-bisglucamine scaffold. A tumor-targeted ADC built with the resulting STING agonist platform induced robust and durable anti-tumor activity and demonstrated high stability and favorable pharmacokinetics in nonclinical species.

Discovery and Preclinical Characterization of XMT-1660, an Optimized B7-H4-Targeted Antibody-Drug Conjugate for the Treatment of Cancer.

Antibody-drug conjugates (ADC) achieve targeted drug delivery to a tumor and have demonstrated clinical success in many tumor types. The activity and safety profile of an ADC depends on its construction: antibody, payload, linker, and conjugation method, as well as the number of payload drugs per antibody [drug-to-antibody ratio (DAR)]. To allow for ADC optimization for a given target antigen, we developed Dolasynthen (DS), a novel ADC platform based on the payload auristatin hydroxypropylamide, that enables precise DAR-ranging and site-specific conjugation. We used the new platform to optimize an ADC that targets B7-H4 (VTCN1), an immune-suppressive protein that is overexpressed in breast, ovarian, and endometrial cancers. XMT-1660 is a site-specific DS DAR 6 ADC that induced complete tumor regressions in xenograft models of breast and ovarian cancer as well as in a syngeneic breast cancer model that is refractory to PD-1 immune checkpoint inhibition. In a panel of 28 breast cancer PDXs, XMT-1660 demonstrated activity that correlated with B7-H4 expression. XMT-1660 has recently entered clinical development in a phase I study (NCT05377996) in patients with cancer.

Fabrication and characterization of a solid-state nanopore with self-aligned carbon nanoelectrodes for molecular detection.

Stochastic molecular sensors based on resistive pulse nanopore modalities are envisioned as facile DNA sequencers. However, recent advances in nanotechnology fabrication have highlighted promising alternative detection mechanisms with higher sensitivity and potential single-base resolution. In this paper we present the novel self-aligned fabrication of a solid-state nanopore device with integrated transverse graphene-like carbon nanoelectrodes for polyelectrolyte molecular detection. The electrochemical transduction mechanism is characterized and found to result primarily from thermionic emission between the two transverse electrodes. Response of the nanopore to Lambda dsDNA and short (16-mer) ssDNA is demonstrated and distinguished.

A microfluidic approach to rescue ALS motor neuron degeneration using rapamycin.

TAR DNA-binding protein-43 (TDP-43) is known to accumulate in ubiquitinated inclusions of amyotrophic lateral sclerosis affected motor neurons, resulting in motor neuron degeneration, loss of motor functions, and eventually death. Rapamycin, an mTOR inhibitor and a commonly used immunosuppressive drug, has been shown to increase the survivability of Amyotrophic Lateral Sclerosis (ALS) affected motor neurons. Here we present a transgenic, TDP-43-A315T, mouse model expressing an ALS phenotype and demonstrate the presence of ubiquitinated cytoplasmic TDP-43 aggregates with > 80% cell death by 28 days post differentiation in vitro. Embryonic stem cells from this mouse model were used to study the onset, progression, and therapeutic remediation of TDP-43 aggregates using a novel microfluidic rapamycin concentration gradient generator. Results using a microfluidic device show that ALS affected motor neuron survival can be increased by 40.44% in a rapamycin dosage range between 0.4-1.0 µM.

The applications of in situ electron energy loss spectroscopy to the study of electron beam nanofabrication.

An in situ electron energy loss spectroscopy (EELS) technique has been developed to investigate the dynamic processes associated with electron-beam nanofabrication on thin membranes. In this article, practical applications germane to e-beam nanofabrication are illustrated with a case study of the drilling of nanometer-sized pores in silicon nitride membranes. This technique involves successive acquisitions of the plasmon-loss and the core-level ionization-loss spectra in real time, both of which provide the information regarding the hole-drilling kinetics, including two respective rates for total mass loss, individual nitrogen and silicon element depletion, and the change of the atomic bonding environment. In addition, the in situ EELS also provides an alternative method for endpoint detection with a potentially higher time resolution than by imaging. On the basis of the time evolution of in situ EELS spectra, a qualitative working model combining knock-on sputtering, irradiation-induced mass transport, and phase separation can be proposed.

Nanopore with Transverse Nanoelectrodes for Electrical Characterization and Sequencing of DNA.

A DNA sequencing device which integrates transverse conducting electrodes for the measurement of electrode currents during DNA translocation through a nanopore has been nanofabricated and characterized. A focused electron beam (FEB) milling technique, capable of creating features on the order of 1 nm in diameter, was used to create the nanopore. The device was characterized electrically using gold nanoparticles as an artificial analyte with both DC and AC measurement methods. Single nanoparticle/electrode interaction events were recorded. A low-noise, high-speed transimpedance current amplifier for the detection of nano to picoampere currents at microsecond time scales was designed, fabricated and tested for future integration with the nanopore device.

Discovery and Optimization of HKT288, a Cadherin-6-Targeting ADC for the Treatment of Ovarian and Renal Cancers.

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Directing the spatial patterning of motor neuron differentiation in engineered microenvironments.

Embryonic development of the spinal cord proceeds through a carefully orchestrated temporal and spatial sequence of chemical cues to provide precise patterning of adult cell types. Recreating this complex microenvironment in a standard cell culture dish is difficult, if not impossible. In this paper, a microfluidic device is used to recapitulate, in vitro, the graded patterning events which occur during early spinal cord development. The microdevice design is developed using COMSOL modeling, with which the spatiotemporal profiles of multiple, diffusible morphogens are simulated. Four independently addressed source/sinks are employed to generate two overlapping orthogonal gradients within a cell culture chamber, mimicking the dorsoventral and anteroposterior axes of the developing embryo. Mouse embryonic stem cells are directed therein to differentiate into motor neurons in a spatially organized manner, reminiscent of a neural tube.

Biomolecule detection via target mediated nanoparticle aggregation and dielectrophoretic impedance measurement.

A new biosensing system is described that is based on the aggregation of nanoparticles by a target biological molecule and dielectrophoretic impedance measurement of these aggregates. The aggregation process was verified within a microchannel via fluorescence microscopy, demonstrating that this process can be used in a real time sensor application. Positive dielectrophoresis is employed to capture the nanoparticle aggregates at the edge of thin film electrodes, where their presence is detected either by optical imaging via fluorescence microscopy or by measuring the change in electrical impedance between adjacent electrodes. The electrical detection mechanism demonstrates the potential for this method as a micro total analysis system (microTAS).

High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response.

Profiling candidate therapeutics with limited cancer models during preclinical development hinders predictions of clinical efficacy and identifying factors that underlie heterogeneous patient responses for patient-selection strategies. We established ∼1,000 patient-derived tumor xenograft models (PDXs) with a diverse set of driver mutations. With these PDXs, we performed in vivo compound screens using a 1 × 1 × 1 experimental design (PDX clinical trial or PCT) to assess the population responses to 62 treatments across six indications. We demonstrate both the reproducibility and the clinical translatability of this approach by identifying associations between a genotype and drug response, and established mechanisms of resistance. In addition, our results suggest that PCTs may represent a more accurate approach than cell line models for assessing the clinical potential of some therapeutic modalities. We therefore propose that this experimental paradigm could potentially improve preclinical evaluation of treatment modalities and enhance our ability to predict clinical trial responses.

A field-deployable colorimetric bioassay for the rapid and specific detection of ribosomal RNA.

Rapid and specific on-site detection of disease-causing or toxin-producing organisms is essential to public health and safety. Many molecular recognition methods target ribosomal RNA sequences due to their specificity and abundance in the cell. In this work RNA targets were identified and quantified using a colorimetric bioassay. Peptide nucleic acid (PNA) probes were used to capture RNA targets, and a micrococcal nuclease digestion was performed to remove all non-target nucleic acids, including single base mismatches flanked by adenines or uracils. Perfectly-matched PNA-RNA hybrids remained intact and were detected using the symmetrical cyanine dye 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)). Assay applicability to complex samples was demonstrated using mixtures containing RNA sequences from two related, harmful algal bloom-causing Alexandrium species. Target RNA was detected even in mixtures with mismatched sequences in excess of the perfect match. The fieldability of the assay was tested with a portable two-wavelength colorimeter developed to quantify the dye-indicated hybridization signal. The colorimeter sensing performance was shown to be comparable to a laboratory spectrophotometer. This quick, inexpensive and robust system has the potential to replace laborious identification schemes in field environments.

Combination of antibody that inhibits ligand-independent HER3 dimerization and a p110α inhibitor potently blocks PI3K signaling and growth of HER2+ breast cancers.

We examined the effects of LJM716, an HER3 (ERBB3) neutralizing antibody that inhibits ligand-induced and ligand-independent HER3 dimerization, as a single agent and in combination with BYL719, an ATP competitive p110α-specific inhibitor, against HER2-overexpressing breast and gastric cancers. Treatment with LJM716 reduced HER2-HER3 and HER3-p85 dimers, P-HER3 and P-AKT, both in vitro and in vivo. Treatment with LJM716 alone markedly reduced growth of BT474 xenografts. The combination of LJM716/lapatinib/trastuzumab significantly improved survival of mice with BT474 xenografts compared with lapatinib/trastuzumab (P = 0.0012). LJM716 and BYL719 synergistically inhibited growth in a panel of HER2+ and PIK3CA mutant cell lines. The combination also inhibited P-AKT in HER2-overexpressing breast cancer cells and growth of HER2+ NCI-N87 gastric cancer xenografts more potently than LJM716 or BYL719 alone. Trastuzumab-resistant HER2+/PIK3CA mutant MDA453 xenografts regressed completely after 3 weeks of therapy with LJM716 and BYL719, whereas either single agent inhibited growth only partially. Finally, mice with BT474 xenografts treated with trastuzumab/LJM716, trastuzumab/BYL719, LJM716/BYL719, or trastuzumab/LJM716/BYL719 exhibited similar rates of tumor regression after 3 weeks of treatment. Thirty weeks after treatment discontinuation, 14% of mice were treated with trastuzumab/LJM716/BYL719, whereas >80% in all other treatment groups were sacrificed due to a recurrent large tumor burden (P = 0.0066). These data suggest that dual blockade of the HER2 signaling network with an HER3 antibody that inhibits HER2-HER3 dimers in combination with a p110α-specific inhibitor in the absence of a direct HER2 antagonist is an effective treatment approach against HER2-overexpressing cancers.

An electron microscopy investigation of the structure of porous silicon by oxide replication.

The morphology of porous silicon is studied by scanning electron microscopy (SEM) by making an oxide replica of the pore structure. Highly branched n-type porous silicon samples were prepared and a replica was formed by oxidation of the pores followed by selective removal of the silicon substrate to expose the oxide pores. Scanning and transmission electron microscopy images confirmed many previously held assumptions about porous silicon formation, including the fractal structure and crystallographic propagation; they also provided a clearer understanding of the details of pore formation. The replica procedure also provides a platform for a more facile and comprehensive analysis of the porous silicon morphology.

Solid-phase direct write (SPDW) of carbon via scanning force microscopy.

The fabrication of carbon nanostructures by direct writing with a scanning force microscope is described. A conductive atomic force tip is used to collect carbon from a glassy carbon substrate and redeposit it onto a gold thin film under voltage control. The resulting patterns are examined with atomic force microscopy and Auger electron spectrometry. Writing of carbon lines with widths as small as 40 nm is demonstrated.

Frequency dependence of gold nanoparticle superassembly by dielectrophoresis.

Dielectrophoresis is an effective method for capturing nanoparticles and assembling them into nanostructures. The frequency of the dielectrophoretic alternating current (ac) electric field greatly influences the morphology of resultant nanoparticle assemblies. In this study, frequency regimes associated with specific gold nanoparticle assembly morphologies were identified. Gold nanoparticles suspended in water were captured by microelectrodes at different electric field frequencies onto thin silicon nitride membranes. The resultant assemblies were examined by transmission electron microscopy. For this system, the major frequency-dependent influence on morphology appears to arise not from the Clausius-Mossotti factor of the dielectrophoretic force itself, but instead from ac electroosmotic fluid flow and the influence of the electrical double layer at the electrode-solution interface. Frequency regimes of technological interest include those forming one-dimensional nanoparticle chains, microwires, combinations of microwires and nanoparticle chains suitable for nanogap electrode formation, and dense three-dimensional assemblies with very high surface area.

Micromachined, silicon filament light source for spectrophotometric microsystems.

A miniature broadband light source is a critical element in a spectrophotometric microsystem. The design, fabrication, and characterization of a highly stable, miniature broadband light source that comprises filaments of single-crystal silicon are presented. Electrical current versus voltage and radiant emittance spectra under constant voltage bias are measured and related to filament dimensions. A maximum stable operating temperature for these filaments is estimated to be 1200 K. Resistance drift is demonstrated to be less than 0.5% over a 10-h period of continuous operation with visible incandescence. Emittance spectra of a multifilament array, measured at three different electrical biases, are presented and shown to compare well with theoretical blackbody radiation spectra. A continuous, total radiated power of 10.7 mW was achieved with a 1 mm x 1 mm filament array with peak emittance at lambda=2.7 micrometers.