R. Fabian Pease

Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

Background To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. Methodology/Principal Findings We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. Conclusions/Significance For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of ‘liquid biopsies’ to better model drug discovery.

Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device

The enumeration of rare circulating epithelial cells (CEpCs) in the peripheral blood of metastatic cancer patients has shown promise for improved cancer prognosis. Moving beyond enumeration, molecular analysis of CEpCs may provide candidate surrogate endpoints to diagnose, treat, and monitor malignancy directly from the blood samples. Thorough molecular analysis of CEpCs requires the development of new sample preparation methods that yield easily accessible and purified CEpCs for downstream biochemical assays. Here, we describe a new immunomagnetic cell separator, the MagSweeper, which gently enriches target cells and eliminates cells that are not bound to magnetic particles. The isolated cells are easily accessible and can be extracted individually based on their physical characteristics to deplete any cells nonspecifically bound to beads. We have shown that our device can process 9 mL of blood per hour and captures >50% of CEpCs as measured in spiking experiments. We have shown that the separation process does not perturb the gene expression of rare cells. To determine the efficiency of our platform in isolating CEpCs from patients, we have isolated CEpCs from all 47 tubes of 9-mL blood samples collected from 17 women with metastatic breast cancer. In contrast, we could not find any circulating epithelial cells in samples from 5 healthy donors. The isolated CEpCs are all stored individually for further molecular analysis.

Heat Transfer in Microchannels—2012 Status and Research Needs

Heat transfer and fluid flow in microchannels have been topics of intense research in the past decade. A critical review of the current state of research is presented with a focus on the future research needs. After providing a brief introduction, the paper addresses six topics related to transport phenomena in microchannels: single-phase gas flow, enhancement in single-phase liquid flow and flow boiling, flow boiling instability, condensation, electronics cooling, and microscale heat exchangers. After reviewing the current status, future research directions are suggested. Concerning gas phase convective heat transfer in microchannels, the antagonist role played by the slip velocity and the temperature jump that appear at the wall are now clearly understood and quantified. It has also been demonstrated that the shear work due to the slipping fluid increases the effect of viscous heating on heat transfer. On the other hand, very few experiments support the theoretical models and a significant effort should be made in this direction, especially for measurement of temperature fields within the gas in microchannels, implementing promising recent techniques such as molecular tagging thermometry (MTT). The single-phase liquid flow in microchannels has been established to behave similar to the macroscale flows. The current need is in the area of further enhancing the performance. Progress on implementation of flow boiling in microchannels is facing challenges due to its lower heat transfer coefficients and critical heat flux (CHF) limits. An immediate need for breakthrough research related to these two areas is identified. Discussion about passive and active methods to suppress flow boiling instabilities is presented. Future research focus on instability research is suggested on developing active closed loop feedback control methods, extending current models to better predict and enable superior control of flow instabilities. Innovative high-speed visualization and measurement techniques have led to microchannel condensation now being studied as a unique process with its own governing influences. Further work is required to develop widely applicable flow regime maps that can address many fluid types and geometries. With this, condensation heat transfer models can progress from primarily annular flow based models with some adjustments using dimensionless parameters to those that can directly account for transport in intermittent and other flows, and the varying influences of tube shape, surface tension and fluid property differences over much larger ranges than currently possible. Electronics cooling continues to be the main driver for improving thermal transport processes in microchannels, while efforts are warranted to develop high performance heat exchangers with microscale passages. Specific areas related to enhancement, novel configurations, nanostructures and practical implementation are expected to be the research focus in the coming years.

Thermal conductivity measurements of thin-film resist

In electron-beam and photolithography, local heating can change the resist sensitivity and lead to variations in significant critical dimension. Existing models suffer from the lack of experimental data for the thermal properties of the polymer resist films. We present the measurements of both out-of-plane and in-plane thermal conductivity of thin resist films following different exposure conditions. An optical thermoreflectance technique was used to characterize out-of-plane thermal conductivity; the out-of-plane thermal conductivity of exposed SPR™-700 resist increases as a function of exposure dose. We also designed and fabricated a free-standing micro-electrode structure for measuring the in-plane thermal conductivity and results for poly(methylmethacrylate) films were obtained, indicating that, unlike polyimide films, there is no appreciable anisotropic behavior.

Electric-field-directed growth of carbon nanotubes in two dimensions

The interplay between mechanical and electronic properties in carbon nanotubes leads to interesting characteristics in devices such as a nanotube cross structure. The fabrication of nanotube devices has often been based on random growth or deposition of nanotubes and subsequently searching for those in desired locations with proper orientations. Obviously we want to be able to make such devices controllably. We present data on extending the technique of one-dimensional alignment of nanotubes using electric field to two dimensions in order to make more complicated structures such as nanotube crosses. It appears that nanotubes assemble in the regions of the most intense electric field. Also, they tend to follow the local field lines, even in nonuniform fields. Furthermore, they tend to grow away from negative toward positive polarities.

Transient temperature measurements of resist heating using nanothermocouples

Resist heating is one of the major factors that causes feature size variation and pattern displacement in photomask fabrication. A number of models have been published to predict the rise in temperature during resist heating, but no transient temperature experimental results are available to verify those models. We have fabricated thin film gold/nickel thermocouples with junction areas as small as 100 nm2 on silicon and 500 nm2 on quartz. Microsecond scale transient resist heating measurements were obtained using these thermocouples. Irradiation by a 15 keV, 150 nA electron beam of 1.7 μm radius for 100 μs yielded temperature rises at the resist bottom surface of approximately 62 K on quartz substrates and of 18 K on silicon substrates. Simulation results using a multilayer Green’s function model are in reasonable agreement with these experimental data for smaller temperature rises but tend to overestimate by about 10% for larger rises in temperature. In our experiments, a 100 ms exposure is equivalent to...

Dielectric laser acceleration of sub-100 keV electrons with silicon dual-pillar grating structures

We present the demonstration of high gradient (370 MeV/m) laser acceleration and deflection of sub-relativistic electrons with silicon dual pillar grating structures using both evanescent inverse Smith-Purcell modes and coupled cosh-like modes.

Distributed axis electron beam technology for maskless lithography and defect inspection

The limitations to the throughput of electron beam systems employing a single axis are now well known. Accordingly there is increased activity in the exploration of multiaxis systems. The approach described here features both common focusing and common deflection, thus simplifying the problem of pattern stitching. Another feature, thought by some to be a disadvantage, is that the imaging is at unity magnification. We have designed and built a test stand that presently features one axis (or beamlet) in a large homogeneous magnetic field so that the extension to multiple axes can be seen to be straightforward. With this arrangement we have demonstrated that the resolution can be at least in the 30–50 nm range, that operation at low voltage (down to 260 V) is possible, that we can operate using multiple axes, and that secondary electrons generated by one beamlet can be confined so that it is possible to use this arrangement for high-speed inspection.

High-density waveguide superlattices with low crosstalk

Silicon photonics holds great promise for low-cost large-scale photonic integration. In its future development, integration density will play an ever-increasing role in a way similar to that witnessed in integrated circuits. Waveguides are perhaps the most ubiquitous component in silicon photonics. As such, the density of waveguide elements is expected to have a crucial influence on the integration density of a silicon photonic chip. A solution to high-density waveguide integration with minimal impact on other performance metrics such as crosstalk remains a vital issue in many applications. Here, we propose a waveguide superlattice and demonstrate advanced superlattice design concepts such as interlacing-recombination that enable high-density waveguide integration at a half-wavelength pitch with low crosstalk. Such waveguide superlattices can potentially lead to significant reduction in on-chip estate for waveguide elements and salient enhancement of performance for important applications, opening up possibilities for half-wavelength-pitch optical-phased arrays and ultra-dense space-division multiplexing.

Optical switching based on high-speed phased array optical beam steering

We present a high-speed optical switching scheme based on phased array optical beam steering, and analyze the trade-off between the switch power efficiency, signal-to-noise-ratio, number of output channels, and switching speed. For the proof of concept, a two-channel optical switch has been fabricated, using a high-speed GaAs∕AlGaAs multiple quantum well phase modulator. We demonstrate a beam deflection angle of 100mrad at the fastest ever reported speed of 18GHz, consuming 1.8mW. A signal-to-noise ratio of 8dB is measured at each output channel. The relatively low signal-to-noise ratio can be further improved by increasing the number of phased arrays.