Spatial control of oxygen delivery to three-dimensional cultures alters cancer cell growth and gene expression.

Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three-dimensional (3D) bioreactor system where cancer cells are grown in Matrigel in modified six-well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3-0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional two-dimensional monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.

Enhanced Sample Handling for Analytical Ultracentrifugation with 3D-Printed Centerpieces.

The centerpiece of the sample cell assembly in analytical ultracentrifugation holds the sample solution between windows, sealed against high vacuum, and is shaped such that macromolecular migration in centrifugal fields exceeding 200 000g can proceed undisturbed by walls or convection while concentration profiles are imaged with optical detection systems aligned perpendicular to the plane of rotation. We have recently shown that 3D printing using various materials allows inexpensive and rapid manufacturing of centerpieces. In the present work, we expand this endeavor to examine the accuracy of the measured sedimentation process, as well as short-term durability of the centerpieces. We find that 3D-printed centerpieces can be used many times and can provide data equivalent in quality to commonly used commercial epoxy resin centerpieces. Furthermore, 3D printing enables novel designs adapted to particular experimental objectives because they offer unique opportunities, for example, to create well-defined curved surfaces, narrow channels, and embossed features. We present examples of centerpiece designs exploiting these capabilities for improved AUC experiments. This includes narrow sector centerpieces that substantially reduce the required sample volume while maintaining the standard optical path length; thin centerpieces with integrated window holders to provide very short optical pathlengths that reduce optical aberrations at high macromolecular concentrations; long-column centerpieces that increase the observable distance of macromolecular migration for higher-precision sedimentation coefficients; and three-sector centerpieces that allow doubling the number of samples in a single run while reducing the sample volumes. We find each of these designs allows unimpeded macromolecular sedimentation and can provide high-quality sedimentation data.

SCORHE: a novel and practical approach to video monitoring of laboratory mice housed in vivarium cage racks.

The System for Continuous Observation of Rodents in Home-cage Environment (SCORHE) was developed to demonstrate the viability of compact and scalable designs for quantifying activity levels and behavior patterns for mice housed within a commercial ventilated cage rack. The SCORHE in-rack design provides day- and night-time monitoring with the consistency and convenience of the home-cage environment. The dual-video camera custom hardware design makes efficient use of space, does not require home-cage modification, and is animal-facility user-friendly. Given the system's low cost and suitability for use in existing vivariums without modification to the animal husbandry procedures or housing setup, SCORHE opens up the potential for the wider use of automated video monitoring in animal facilities. SCORHE's potential uses include day-to-day health monitoring, as well as advanced behavioral screening and ethology experiments, ranging from the assessment of the short- and long-term effects of experimental cancer treatments to the evaluation of mouse models. When used for phenotyping and animal model studies, SCORHE aims to eliminate the concerns often associated with many mouse-monitoring methods, such as circadian rhythm disruption, acclimation periods, lack of night-time measurements, and short monitoring periods. Custom software integrates two video streams to extract several mouse activity and behavior measures. Studies comparing the activity levels of ABCB5 knockout and HMGN1 overexpresser mice with their respective C57BL parental strains demonstrate SCORHE's efficacy in characterizing the activity profiles for singly- and doubly-housed mice. Another study was conducted to demonstrate the ability of SCORHE to detect a change in activity resulting from administering a sedative.

Engineering Tumor Stroma Morphogenesis Using Dynamic Cell-Matrix Spheroid Assembly.

The tumor microenvironment consists of resident tumor cells organized within a compositionally diverse, three-dimensional (3D) extracellular matrix (ECM) network that cannot be replicated in vitro using bottom-up synthesis. We report a new self-assembly system to engineer ECM-rich 3D MatriSpheres wherein tumor cells actively organize and concentrate microgram quantities of decellularized ECM dispersions which modulate cell phenotype. 3D colorectal cancer (CRC) MatriSpheres were created using decellularized small intestine submucosa (SIS) as an orthotopic ECM source that had greater proteomic homology to CRC tumor ECM than traditional ECM formulations such as Matrigel. SIS ECM was rapidly concentrated from its environment and assembled into ECM-rich 3D stroma-like regions by mouse and human CRC cell lines within 4-5 days via a mechanism that was rheologically distinct from bulk hydrogel formation. Both ECM organization and transcriptional regulation by 3D ECM cues affected programs of malignancy, lipid metabolism, and immunoregulation that corresponded with an in vivo MC38 tumor cell subpopulation identified via single cell RNA sequencing. This 3D modeling approach stimulates tumor specific tissue morphogenesis that incorporates the complexities of both cancer cell and ECM compartments in a scalable, spontaneous assembly process that may further facilitate precision medicine.

Whole Animal Feeding FLat (WAFFL): a complete and comprehensive validation of a novel, high-throughput fly experimentation system.

Non-mammalian model organisms have been essential for our understanding of the mechanisms that control development, disease, and physiology, but they are underutilized in pharmacological and toxicological phenotypic screening assays due to their low throughput in comparison with cell-based screens. To increase the utility of using Drosophila melanogaster in screening, we designed the Whole Animal Feeding FLat (WAFFL), a novel, flexible, and complete system for feeding, monitoring, and assaying flies in a high-throughput format. Our 3D printed system is compatible with inexpensive and readily available, commercial 96-well plate consumables and equipment. Experimenters can change the diet at will during the experiment and video record for behavior analysis, enabling precise dosing, measurement of feeding, and analysis of behavior in a 96-well plate format.

Three-dimensional motion tracking for high-resolution optical microscopy, in vivo.

When conducting optical imaging experiments, in vivo, the signal to noise ratio and effective spatial and temporal resolution is fundamentally limited by physiological motion of the tissue. A three-dimensional (3D) motion tracking scheme, using a multiphoton excitation microscope with a resonant galvanometer, (512 × 512 pixels at 33 frames s(-1)) is described to overcome physiological motion, in vivo. The use of commercially available graphical processing units permitted the rapid 3D cross-correlation of sequential volumes to detect displacements and adjust tissue position to track motions in near real-time. Motion phantom tests maintained micron resolution with displacement velocities of up to 200 µm min(-1), well within the drift observed in many biological tissues under physiologically relevant conditions. In vivo experiments on mouse skeletal muscle using the capillary vasculature with luminal dye as a displacement reference revealed an effective and robust method of tracking tissue motion to enable (1) signal averaging over time without compromising resolution, and (2) tracking of cellular regions during a physiological perturbation.

Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer.

A one-of-a-kind high speed optical multichannel spectrometer was designed and built at NIH and described in this journal in 1997 [J.W. Cole, R.W. Hendler, P.D. Smith, H.A. Fredrickson, T.J. Pohida, W.S. Friauf. A high speed optical multichannel analyzer. J Biochem Biophys Methods 1997;35:16-174.]. The most unique aspect of this instrument was the ability to follow an entire time course from a single activation using a single sample. The instrument has been used to study rapid kinetic processes in the photon-driven bacteriorhodopsin photocycle and electron transport from cytochrome c to cytochrome aa3 and from cytochrome aa3 to oxygen. The present paper describes a second generation instrument with a number of important enhancements which significantly improve its capabilities for multichannel kinetic studies. An example application is presented in which the kinetics of photon-induced proton flow across the biological membrane is measured simultaneously with the individual steps of the photocycle determined optically. Matching the time constants for the two processes indicates which molecular transformations are associated with major proton movements.

Physiological Recording in the MRI Environment (PRiME): MRI-Compatible Hemodynamic Recording System.

Hemodynamic recording during interventional cardiovascular procedures is essential for procedural guidance, monitoring patient status, and collection of diagnostic information. Recent advances have made interventions guided by magnetic resonance imaging (MRI) possible and attractive in certain clinical scenarios. However, in the MRI environment, electromagnetic interference (EMI) can cause severe distortions and artifacts in acquired hemodynamic waveforms. The primary aim of this paper was to develop and validate a system to minimize EMI on electrocardiogram (ECG) and invasive blood pressure (IBP) signals. A system was developed which incorporated commercial MRI compatible ECG leads and pressure transducers, custom electronics, user interface, and adaptive signal processing. Measurements were made on pediatric patients (N = 6) during MRI-guided catheterization. Real-time interactive scanning, which is known to produce significant EMI due to fast gradient switching and varying imaging plane orientations, was selected for testing. The effectiveness of the adaptive algorithms was determined by measuring the reduction of noise peaks, amplitude of noise peaks, and false QRS triggers. During real-time gradient-intensive imaging sequences, peak noise amplitude was reduced by 80% and false QRS triggers were reduced to a median of 0. There was no detectable interference on the IBP channels. A hemodynamic recording system front-end was successfully developed and deployed, which enabled high-fidelity recording of ECG and IBP during MRI scanning. The schematics and assembly instructions are publicly available to facilitate implementation at other institutions. Researchers and clinicians are provided a critical tool in investigating and implementing MRI guided interventional cardiovascular procedures.

Measuring Risky Driving Behavior Using an mHealth Smartphone App: Development and Evaluation of gForce.

BACKGROUND: Naturalistic driving studies, designed to objectively assess driving behavior and outcomes, are conducted by equipping vehicles with dedicated instrumentation (eg, accelerometers, gyroscopes, Global Positioning System, and cameras) that provide continuous recording of acceleration, location, videos, and still images for eventual retrieval and analyses. However, this research is limited by several factors: the cost of equipment installation; management and storage of the large amounts of data collected; and data reduction, coding, and analyses. Modern smartphone technology includes accelerometers built into phones, and the vast, global proliferation of smartphones could provide a possible low-cost alternative for assessing kinematic risky driving. OBJECTIVE: We evaluated an in-house developed iPhone app (gForce) for detecting elevated g-force events by comparing the iPhone linear acceleration measurements with corresponding acceleration measurements obtained with both a custom Android app and the in-vehicle miniDAS data acquisition system (DAS; Virginia Tech Transportation Institute). METHODS: The iPhone and Android devices were dashboard-mounted in a vehicle equipped with the DAS instrumentation. The experimental protocol consisted of driving maneuvers on a test track, such as cornering, braking, and turning that were performed at different acceleration levels (ie, mild, moderate, or hard). The iPhone gForce app recorded linear acceleration (ie, gravity-corrected). The Android app recorded gravity-corrected and uncorrected acceleration measurements, and the DAS device recorded gravity-uncorrected acceleration measurements. Lateral and longitudinal acceleration measures were compared. RESULTS: The correlation coefficients between the iPhone and DAS acceleration measurements were slightly lower compared to the correlation coefficients between the Android and DAS, possibly due to the gravity correction on the iPhone. Averaging the correlation coefficients for all maneuvers, the longitudinal and lateral acceleration measurements between iPhone and DAS were rlng=0.71 and rlat=0.83, respectively, while the corresponding acceleration measurements between Android and DAS were rlng=0.95 and rlat=0.97. The correlation coefficients between lateral accelerations on all three devices were higher than with the corresponding longitudinal accelerations for most maneuvers. CONCLUSIONS: The gForce iPhone app reliably assessed elevated g-force events compared to the DAS. Collectively, the gForce app and iPhone platform have the potential to serve as feature-rich, inexpensive, scalable, and open-source tool for assessment of kinematic risky driving events, with potential for research and feedback forms of intervention.

A new strategy for fast radiofrequency CW EPR imaging: direct detection with rapid scan and rotating gradients.

Rapid field scan on the order of T/s using high frequency sinusoidal or triangular sweep fields superimposed on the main Zeeman field, was used for direct detection of signals without low-frequency field modulation. Simultaneous application of space-encoding rotating field gradients have been employed to perform fast CW EPR imaging using direct detection that could, in principle, approach the speed of pulsed FT EPR imaging. The method takes advantage of the well-known rapid-scan strategy in CW NMR and EPR that allows arbitrarily fast field sweep and the simultaneous application of spinning gradients that allows fast spatial encoding. This leads to fast functional EPR imaging and, depending on the spin concentration, spectrometer sensitivity and detection band width, can provide improved temporal resolution that is important to interrogate dynamics of spin perfusion, pharmacokinetics, spectral spatial imaging, dynamic oxymetry, etc.

Tumor-associated endothelial cells display GSTP1 and RARbeta2 promoter methylation in human prostate cancer.

BACKGROUND: A functional blood supply is essential for tumor growth and proliferation. However, the mechanism of blood vessel recruitment to the tumor is still poorly understood. Ideally, a thorough molecular assessment of blood vessel cells would be critical in our comprehension of this process. Yet, to date, there is little known about the molecular makeup of the endothelial cells of tumor-associated blood vessels, due in part to the difficulty of isolating a pure population of endothelial cells from the heterogeneous tissue environment. METHODS: Here we describe the use of a recently developed technique, Expression Microdissection, to isolate endothelial cells from the tumor microenvironment. The methylation status of the dissected samples was evaluated for GSTP1 and RARbeta2 promoters via the QMS-PCR method. RESULTS: Comparing GSTP1 and RARbeta2 promoter methylation data, we show that 100% and 88% methylation is detected, respectively, in the tumor areas, both in epithelium and endothelium. Little to no methylation is observed in non-tumor tissue areas. CONCLUSION: We applied an accurate microdissection technique to isolate endothelial cells from tissues, enabling DNA analysis such as promoter methylation status. The observations suggest that epigenetic alterations may play a role in determining the phenotype of tumor-associated vasculature.

Integration of digital signal processing technologies with pulsed electron paramagnetic resonance imaging.

The integration of modern data acquisition and digital signal processing (DSP) technologies with Fourier transform electron paramagnetic resonance (FT-EPR) imaging at radiofrequencies (RF) is described. The FT-EPR system operates at a Larmor frequency (L(f)) of 300MHz to facilitate in vivo studies. This relatively low frequency L(f), in conjunction with our approximately 10MHz signal bandwidth, enables the use of direct free induction decay time-locked subsampling (TLSS). This particular technique provides advantages by eliminating the traditional analog intermediate frequency downconversion stage along with the corresponding noise sources. TLSS also results in manageable sample rates that facilitate the design of DSP-based data acquisition and image processing platforms. More specifically, we utilize a high-speed field programmable gate array (FPGA) and a DSP processor to perform advanced real-time signal and image processing. The migration to a DSP-based configuration offers the benefits of improved EPR system performance, as well as increased adaptability to various EPR system configurations (i.e., software configurable systems instead of hardware reconfigurations). The required modifications to the FT-EPR system design are described, with focus on the addition of DSP technologies including the application-specific hardware, software, and firmware developed for the FPGA and DSP processor. The first results of using real-time DSP technologies in conjunction with direct detection bandpass sampling to implement EPR imaging at RF frequencies are presented.

3D-Printing for Analytical Ultracentrifugation.

Analytical ultracentrifugation (AUC) is a classical technique of physical biochemistry providing information on size, shape, and interactions of macromolecules from the analysis of their migration in centrifugal fields while free in solution. A key mechanical element in AUC is the centerpiece, a component of the sample cell assembly that is mounted between the optical windows to allow imaging and to seal the sample solution column against high vacuum while exposed to gravitational forces in excess of 300,000 g. For sedimentation velocity it needs to be precisely sector-shaped to allow unimpeded radial macromolecular migration. During the history of AUC a great variety of centerpiece designs have been developed for different types of experiments. Here, we report that centerpieces can now be readily fabricated by 3D printing at low cost, from a variety of materials, and with customized designs. The new centerpieces can exhibit sufficient mechanical stability to withstand the gravitational forces at the highest rotor speeds and be sufficiently precise for sedimentation equilibrium and sedimentation velocity experiments. Sedimentation velocity experiments with bovine serum albumin as a reference molecule in 3D printed centerpieces with standard double-sector design result in sedimentation boundaries virtually indistinguishable from those in commercial double-sector epoxy centerpieces, with sedimentation coefficients well within the range of published values. The statistical error of the measurement is slightly above that obtained with commercial epoxy, but still below 1%. Facilitated by modern open-source design and fabrication paradigms, we believe 3D printed centerpieces and AUC accessories can spawn a variety of improvements in AUC experimental design, efficiency and resource allocation.

Rapid diagnosis of lymph node metastasis in breast cancer using a new fluorescent method with γ-glutamyl hydroxymethyl rhodamine green.

Sentinel lymph node biopsy is performed as a standard procedure in breast cancer surgery, and the development of quick and simple methods to detect metastatic lesions is in high demand. Here, we validated a new fluorescent method using γ-glutamyl hydroxymethyl rhodamine green to diagnose metastatic lymph nodes in breast cancer. One hundred and forty-nine lymph nodes from 38 breast cancer patients were evaluated in this study. Comparison of fluorescent and pathological images showed that this fluorescent method was successful for visualizing breast cancer cells in lymph nodes. This method had a sufficiently high sensitivity (97%), specificity (79%) and negative predictive value (99%) to render it useful for an intraoperative diagnosis of cancer. These preliminary findings suggest that this novel method is useful for distinguishing non-cancerous specimens from those in need of careful examination and could help save time and cost for surgeons and pathologists.

Direct detection and time-locked subsampling applied to pulsed electron paramagnetic resonance imaging.

The application of direct time-locked subsampling (TLSS) to Fourier transform electron paramagnetic resonance (FT-EPR) spectroscopy at radio frequencies (rf) is described. With conventional FT-EPR spectroscopy, the high Larmor frequencies (L(f)) often necessitate the use of intermediate frequency (IF) stages to down convert the received free induction decay (FID) signal to a frequency that can be acquired with common data acquisition technology. However, our research focuses on in vivo studies, and consequently utilizes a FT-EPR system with a L(f) of 300 MHz. This relatively low frequency L(f), in conjunction with the advent of bandpass sampling analog-to-digital conversion and signal processing technologies, has enabled us to omit the IF stage in our FT-EPR system. With this in mind, TLSS techniques have been developed to directly sample the 300 MHz FID signal at a sampling rate of 80 MHz providing a signal bandwidth of 20 MHz. The required modifications to the data acquisition and processing system specific to this application are described. Custom software developed to control the EPR system setup, acquire the signals, and post process the data, is outlined. Data was acquired applying both coherent averaging and stochastic excitation sequences. The results of these experiments demonstrate digital down conversion of the 300 MHz FID signal to quadrature baseband. Direct FID TLSS eliminates many noise sources common in EPR systems employing traditional analog receiver techniques, such as the IF mixer stage in single channel systems, and the quadrature baseband mixer stage in dual channel systems.

Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green.

We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

Expression microdissection: operator-independent retrieval of cells for molecular profiling.

Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.

Stochastic excitation and Hadamard correlation spectroscopy with bandwidth extension in RF FT-EPR.

The application of correlation spectroscopy employing stochastic excitation and the Hadamard transform to time-domain Fourier transform electron paramagnetic resonance (FT-EPR) spectroscopy in the radiofrequency (RF) band is described. An existing, time-domain FT-EPR spectrometer system with a Larmor frequency (L(f)) of 300 MHz was used to develop this technique by incorporating a pseudo-random pulse sequence generator to output the maximum length binary sequence (MLBS, 10- and 11-bit). Software developed to control the EPR system setup, acquire the signals, and post process the data, is outlined. The software incorporates the Hadamard transform algorithm to perform the required cross-correlation of the acquired signal and the MLBS after stochastic excitation. To accommodate the EPR signals, bandwidth extension was accomplished by sampling at a rate many times faster than the RF pulse repetition rate, and subsequent digital signal processing of the data. The results of these experiments showed that there was a decrease in the total acquisition time, and an improved free induction decay (FID) signal-to-noise (S/N) ratio compared to the conventional coherent averaging approach. These techniques have the potential to reduce the RF pulse power to the levels used in continuous wave (CW) EPR while retaining the advantage of time-domain EPR methods. These methods have the potential to facilitate the progression to in vivo FT-EPR imaging of larger volumes.

Performance characteristics of a positron projection imager for mouse whole-body imaging.

INTRODUCTION: We describe a prototype positron projection imager (PPI) for visualizing the whole-body biodistribution of positron-emitting compounds in mouse-size animals. The final version of the PPI will be integrated into the MONICA portable dual-gamma camera system to allow the user to interchangeably image either single photon or positron-emitting compounds in a shared software and hardware environment. METHODS: A mouse is placed in the mid-plane between two identical, opposed, pixelated LYSO arrays separated by 21.8-cm and in time coincidence. An image of the distribution of positron decays in the animal is formed on this mid-plane by coincidence events that fall within a small cone angle perpendicular to the two detectors and within a user-specified energy window. We measured the imaging performance of this device with phantoms and in tests performed in mice injected with various compounds labeled with positron-emitting isotopes. RESULTS: Representative performance measurements yielded the following results (energy window 250-650keV, cone angle 3.5°): resolution in the image mid-plane, 1.66-mm (FWHM), resolution ±1.5-cm above and below the image plane, 2.2-mm (FWHM), sensitivity: 0.237-cps/kBq (8.76-cps/µCi) (18)F (0.024% absolute). Energy resolution was 15.9% with a linear-count-rate operating range of 0-14.8MBq (0-400µCi) and a corrected sensitivity variation across the field-of-view of <3%. Whole-body distributions of [(18)F] FDG and [(18)F] fluoride were well visualized in mice of typical size. CONCLUSION: Performance measurements and field studies indicate that the PPI is well suited to whole-body positron projection imaging of mice. When integrated into the MONICA gamma camera system, the PPI may be particularly useful early in the drug development cycle where, like MONICA, basic whole-body biodistribution data can direct future development of the agent under study and where logistical factors (e.g., available imaging space, non-portability, and cost) may be limitations.

Microfabricated polymeric vessel mimetics for 3-D cancer cell culture.

Modeling tumor growth in vitro is essential for cost-effective testing of hypotheses in preclinical cancer research. 3-D cell culture offers an improvement over monolayer culture for studying cellular processes in cancer biology because of the preservation of cell-cell and cell-ECM interactions. Oxygen transport poses a major barrier to mimicking in vivo environments and is not replicated in conventional cell culture systems. We hypothesized that we can better mimic the tumor microenvironment using a bioreactor system for controlling gas exchange in cancer cell cultures with silicone hydrogel synthetic vessels. Soft-lithography techniques were used to fabricate oxygen-permeable silicone hydrogel membranes containing arrays of micropillars. These membranes were inserted into a bioreactor and surrounded by basement membrane extract (BME) within which fluorescent ovarian cancer (OVCAR8) cells were cultured. Cell clusters oxygenated by synthetic vessels showed a ∼100µm drop-off to anoxia, consistent with in vivo studies of tumor nodules fed by the microvasculature. Oxygen transport in the bioreactor system was characterized by experimental testing with a dissolved oxygen probe and finite element modeling of convective flow. Our study demonstrates differing growth patterns associated with controlling gas distributions to better mimic in vivo conditions.