Mouse fibroblast cell adhesion studied by neutron reflectometry.

Neutron reflectometry (NR) was used to examine live mouse fibroblast cells adherent on a quartz substrate in a deuterated phosphate-buffered saline environment at room temperature. These measurements represent the first, to our knowledge, successful visualization and quantization of the interface between live cells and a substrate with subnanometer resolution using NR. NR data, attributable to the adhesion of live cells, were observed and compared with data from pure growth medium. Independently of surface cell density, the average distance between the center of the cell membrane region and the quartz substrate was determined to be approximately 180 A. The membrane region ( approximately 80 A thick) contains the membranes of cells that are inhomogeneously distributed or undulating, likely conforming to the nonplanar geometry of the supporting adherence proteins. A second region of cell membranes at a greater distance from the substrate was not detectable by NR due to the resolution limits of the technique employed. Attachment of the live cell samples was confirmed by interaction with both distilled water and trypsin. Distinct changes in the NR data after exposure indicate the removal of cells from the substrate.

High-resolution spectral analysis of individual SERS-active nanoparticles in flow.

Nanoparticle spectroscopic tags based on surface enhanced Raman scattering (SERS) are playing an increasingly important role in bioassay and imaging applications. The ability to rapidly characterize large populations of such tags spectroscopically in a high-throughput flow-based platform will open new areas for their application and provide new tools for advancing their development. We demonstrate here a high-resolution spectral flow cytometer capable of acquiring Raman spectra of individual SERS-tags at flow rates of hundreds of particles per second, while maintaining the spectral resolution required to make full use of the detailed information encoded in the Raman signature for advanced multiplexing needs. The approach allows multiple optical parameters to be acquired simultaneously over thousands of individual nanoparticle tags. Characteristics such as tag size, brightness, and spectral uniformity are correlated on a per-particle basis. The tags evaluated here display highly uniform spectral signatures, but with greater variability in brightness. Subpopulations in the SERS response, not apparent in ensemble measurements, are also shown to exist. Relating tag variability to synthesis parameters makes flow-based spectral characterization a powerful tool for advancing particle development through its ability to provide rapid feedback on strategies aimed at constraining desired tag properties. Evidence for single-tag signal saturation at high excitation power densities is also shown, suggesting a role for high-throughput investigation of fundamental properties of the SERS tags as well.

Digital analysis and sorting of fluorescence lifetime by flow cytometry.

Frequency-domain flow cytometry techniques are combined with modifications to the digital signal-processing capabilities of the open reconfigurable cytometric acquisition system (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency (RF)-modulated detector signals, implementing Fourier analysis programming with ORCAS' digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5-25 ns simulated lifetime), pulse widths ranging from 2 to 15 micros, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142 degrees to 1.6 degrees. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 micros and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells, and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a RF-modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to approximately 98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will allow both better dissemination of this technology and better exploitation of the traditionally underutilized parameter of fluorescence lifetime.

Centrifugal Generation of Droplet-Based 3D Cell Cultures.

Quickly and easily producing uniform populations of microsphere-based 3D cell cultures using droplet-based templating methods has the potential to enable widespread use of such platforms in drug discovery or cancer research. Here, we advance the design of centrifuge-based droplet generation devices, describe the use of this platform for droplet generation with controlled cell occupancy, and demonstrate weeklong culture duration. Using simple-to-construct devices and easily implemented protocols, the initial concentration of encapsulated cells is adjustable up to hundreds of cells per microsphere. This work demonstrates the first instance of using centrifugal droplet-generating devices to produce large numbers of cell-encapsulating microspheres. Applications of this versatile methodology include the rapid formation of templated 3D cell culture populations suitable for suspension culture or large batch bioreactor studies that require uniform populations.

Raman spectroscopic characterization of necrotic cell death.

Raman spectroscopy has been used to estimate the biochemical changes due to necrosis in an in vitro model system comprised of a human malignant melanoma cell line (MEL-28). Combined oxygen and glucose deprivation was used to simulate necrotic cell death in tumors. Raman spectroscopy measurements of nonproliferating live cells and dead cells were made at 24, 48, and 72 hours. Quantitative estimates of the biochemical composition of live and dead cells were made by fitting cell spectra to the basis spectra of protein, lipid, RNA, DNA, and glycogen. A decrease in the relative amount of lipid and RNA, and an increase in the relative protein content, were observed in dead cells. A comparison of the spectra indicated the existence of conformational changes in protein and nucleic acids in dead cells. These results suggest that Raman spectroscopy could be used to detect necrotic cell death in tumors.

Light scattering and microarchitectural differences between tumorigenic and non-tumorigenic cell models of tissue.

Differences in light scattering properties of a tumorigenic and a non-tumorigenic model for tissue were demonstrated using a variety of light scattering techniques, the majority of which are in vivo compatible. In addition to determining that light scattering differences exist, models for the microarchitectural changes responsible for the light scattering differences were developed.

Comparison of vibrational spectroscopy to biochemical and flow cytometry methods for analysis of the basic biochemical composition of mammalian cells.

We have conducted an extensive comparison of cellular biochemical composition obtained from infrared and Raman spectra of intact cells with measurements using standard extraction and chemical analysis (including NMR), and flow cytometric assay on fixed cells. Measurements were conducted on a rat fibroblast carcinogenesis model consisting of normal and tumorigenic cells assayed as exponentially growing and plateau-phase cultures. Estimates of protein, DNA, RNA, lipids, and glycogen amounts were obtained from a previous publication in which vibrational spectra were fit to a set of basis spectra representing protein, DNA, RNA, lipids, and glycogen. The Raman spectral estimates of absolute cellular composition were quite similar to the independent biochemical and flow cytometric assays. The infrared spectra gave similar results for protein, lipid, and glycogen but underestimated the DNA content while overestimating the RNA level. When ratios of biochemical concentrations in exponential and plateau-phase cultures were examined, the Raman spectroscopic results were the same, within errors, as the independent methods, in all cases. Several changes in relative biochemical composition due to tumorigenic and proliferative status previously reported using vibrational spectroscopy were confirmed by the independent methods. These results demonstrate that vibrational spectroscopy can provide reliable estimates of the biochemical composition of mammalian cells.

Microenvironmental regulation of proliferation in multicellular spheroids is mediated through differential expression of cyclin-dependent kinase inhibitors.

Multicellular spheroids composed of transformed cells are known to mimic the growth characteristics of tumors and to develop gradients in proliferation with increasing size. This progressive accumulation of quiescent cells is presumably an active process that occurs in response to the microenvironmental stresses that develop within the three-dimensional structure, and, yet, little is known regarding either the signals that induce the cell cycle arrest or the molecular basis for the halt in proliferation. We have previously reported that regulation of cyclin-dependent kinase (CDK) inhibitors (CKIs) differs in monolayer versus spheroid cell culture. In this study, we have examined the expression of three CKIs in EMT6 mouse mammary carcinoma and MEL28 human melanoma spheroids, as a function both of spheroid size and of location within the spheroid. We report that expression of the CKIs p18(INK4c), p21(waf1/cip1), and p27(Kip1) all increase as the spheroid grows and develops a quiescent cell fraction. However, by examining protein expression in discrete regions of the spheroid, we have found that only p18(INK4c) and p27(Kip1) expression positively correlate with growth arrest, whereas p21(waf1/cip1) is expressed predominantly in proliferating cells. Further analysis indicated that, in the quiescent cells, p18(INK4c) is found in increasing association with CDK6, whereas p27(Kip1) associates predominantly with CDK2. In MEL28 cells, CDK2 activity is completely abrogated in the inner regions of the spheroid, whereas in EMT6 cells, CDK2 activity decreases in accordance with a decrease in expression. We also observed a decrease in all cell cycle regulatory proteins in the innermost spheroid fraction, including CDKs, CKIs, and cyclins. Induction of CKIs from separate families, as well as their association with distinct target CDKs, suggests that there may be multiple checkpoints activated to ensure cell cycle arrest in non-growth-conducive environments. Furthermore, because very similar observations were made in both a human melanoma cell line and a mouse mammary carcinoma cell line, our results indicate that these checkpoints, as well as the signal transduction pathways that activate them, are highly conserved.

Evolution and characterization of a new reversibly photoswitching chromogenic protein, Dathail.

We report the engineering of a new reversibly switching chromogenic protein, Dathail. Dathail was evolved from the extremely thermostable fluorescent proteins thermal green protein (TGP) and eCGP123 using directed evolution and ratiometric sorting. Dathail has two spectrally distinct chromogenic states with low quantum yields, corresponding to absorbance in a ground state with a maximum at 389nm, and a photo-induced metastable state with a maximum at 497nm. In contrast to all previously described photoswitchable proteins, both spectral states of Dathail are non-fluorescent. The photo-induced chromogenic state of Dathail has a lifetime of ~50min at 293K and pH7.5 as measured by UV-Vis spectrophotometry, returning to the ground state through thermal relaxation. X-ray crystallography provided structural insights supporting a change in conformation and coordination in the chromophore pocket as being responsible for Dathail's photoswitching. Neutron crystallography, carried out for the first time on a protein from the green fluorescent protein family, showed a distribution of hydrogen atoms revealing protonation of the chromophore 4-hydroxybenzyl group in the ground state. The neutron structure also supports the hypothesis that the photo-induced proton transfer from the chromophore occurs through water-mediated proton relay into the bulk solvent. Beyond its spectroscopic curiosity, Dathail has several characteristics that are improvements for applications, including low background fluorescence, large spectral separation, rapid switching time, and the ability to switch many times. Therefore, Dathail is likely to be extremely useful in the quickly developing fields of imaging and biosensors, including photochromic Förster resonance energy transfer, high-resolution microscopy, and live tracking within the cell.

Biochemical differences in tumorigenic and nontumorigenic cells measured by Raman and infrared spectroscopy.

Both infrared and Raman spectroscopies have the potential to noninvasively estimate the biochemical composition of mammalian cells, although this cannot be unambiguously determined from analysis approaches such as peak assignment or multivariate classification methods. We have developed a fitting routine that determines biochemical composition using basis spectra for the major types of biochemicals found in mammalian cells (protein, DNA, RNA, lipid and glycogen), which is shown to be robust and reproducible. We measured both infrared and Raman spectra of viable suspensions of pairs of nontumorigenic and tumorigenic rat fibroblast cell lines. To model in vivo conditions, we compared nonproliferating, nontumorigenic cells to proliferating, tumorigenic cells. Reproducible differences in biochemical composition were found for both nontumorigenic/tumorigenic cell models, using both spectroscopic techniques. These included an increased fraction of protein and nucleic acids in the tumorigenic cells, with a corresponding decrease in lipid and glycogen fractions. Measurements of each cell type in both the proliferating and nonproliferating states showed that proliferative status was the major determinant of differences in vibrational spectra, rather than tumorigenicity per se. The smallness of the spectral changes associated with tumorgenicity may be due to the subtle nature of the oncogenic change in this system (a single mutant oncogene).

The use of 3-D cultures for high-throughput screening: the multicellular spheroid model.

Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy.

Methods for measuring the infrared spectra of biological cells.

Infrared (IR) spectroscopy of biological cells is a growing area of research, with many papers focusing on differences between the spectra of cancerous and noncancerous cells. Much of this research has been performed using a monolayer of dehydrated cells. We posit that the use of monolayers can introduce artefacts that lead to an apparent but inaccurate measurement of differences between cancerous and noncancerous cells. Additionally, the use of dried cells complicates the extraction of biochemical information from the IR spectra. We demonstrate that using suspensions of viable cells in aqueous suspension reduces measurement artefacts and facilitates determining the concentration of the major biochemical components via a linear least-squares fit of the component spectra to the spectrum of the cells.

Capture of Fluorescence Decay Times by Flow Cytometry.

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction.

Open, reconfigurable cytometric acquisition system: ORCAS.

A digital signal processing (DSP)-based digital data acquisition system has been developed to support novel flow cytometry efforts. The system flexibility includes how it detects, captures, and processes event data. Custom data capture boards utilizing analog to digital converters (ADCs) and field programmable gate arrays (FPGA) detect events and capture correlated event data. A commercial DSP board processes the captured data and sends the results over the IEEE 1394 bus to the host computer that provides a user interface for acquisition, display, analysis, and storage. The system collects list mode data, correlated pulse shapes, or streaming data from a variety of detector types using Linux, Mac OS X, and Windows host computers. It extracts pulse features not found on commercial systems with excellent sensitivity and linearity over a wide dynamic range. List mode data are saved in FCS 3.0 formatted files while streaming or correlated waveform data are saved in custom format files for postprocessing. Open, reconfigurable cytometric acquisition system is compact, scaleable, flexible, and modular. Programmable feature extraction algorithms have exciting possibilities for both new and existing applications. The recent availability of a commercial data capture board will enable general availability of similar systems.

A multiscale model for avascular tumor growth.

The desire to understand tumor complexity has given rise to mathematical models to describe the tumor microenvironment. We present a new mathematical model for avascular tumor growth and development that spans three distinct scales. At the cellular level, a lattice Monte Carlo model describes cellular dynamics (proliferation, adhesion, and viability). At the subcellular level, a Boolean network regulates the expression of proteins that control the cell cycle. At the extracellular level, reaction-diffusion equations describe the chemical dynamics (nutrient, waste, growth promoter, and inhibitor concentrations). Data from experiments with multicellular spheroids were used to determine the parameters of the simulations. Starting with a single tumor cell, this model produces an avascular tumor that quantitatively mimics experimental measurements in multicellular spheroids. Based on the simulations, we predict: 1), the microenvironmental conditions required for tumor cell survival; and 2), growth promoters and inhibitors have diffusion coefficients in the range between 10(-6) and 10(-7) cm2/h, corresponding to molecules of size 80-90 kDa. Using the same parameters, the model also accurately predicts spheroid growth curves under different external nutrient supply conditions.

Raman spectroscopy detects biochemical changes due to proliferation in mammalian cell cultures.

Raman spectra of cells and nuclei from cultures in the plateau (nonproliferating) and exponential (proliferating) phases of growth were measured and show that Raman spectroscopy can monitor changes due to cell proliferation. A simple fitting routine was developed using a basis set (lipid, protein, DNA, RNA) to estimate the relative amounts of biochemical components in cells and nuclei. Using relative amounts and ratios of biochemical components, reproducible differences can be detected and quantified that are not readily apparent by visual analysis of vibrational bands in the spectra. These differences, due to cell proliferation, can be assigned to specific biochemical changes. They include a decrease in the relative lipid and increases in the relative protein and RNA for both nontumorigenic exponential cells and nuclei, and an increase in the relative RNA for tumorigenic exponential cells. The lipid/RNA ratio decreases for nontumorigenic exponential cells and nuclei and tumorigenic exponential cells. The protein/lipid ratio increases for both tumorigenic and nontumorigenic exponential cells and nuclei. Finally, the lipid/DNA ratio decreases for tumorigenic exponential nuclei. This knowledge will be important for Raman detection of rapidly dividing populations of cancer cells in vivo.

Phosphorous metabolites and steady-state energetics of transformed fibroblasts during three-dimensional growth.

Rat1-T1 and MR1 spheroids represent separate transformed phenotypes originated from the same rat fibroblasts that differ in three-dimensional (3D) growth kinetics, histological structure, and oxygenation status. In the present study, (31)P-NMR spectroscopy of perfused spheroid suspensions was used to investigate cellular energetics relative to 3D growth, development of necrosis, and cell cycle distribution. Both spheroid types were characterized by a remarkably low amount of free (inorganic) phosphate (P(i)) and a low phosphocreatine peak. The ratio of nucleoside triphosphate (NTP) to P(i) ranged between 1.5 and 2.0. Intracellular pH, NTP-to-P(i) ratio, and NTP/cell remained constant throughout spheroid growth, being unaffected by the emergence of oxygen deficiency, cell quiescence, and necrosis. However, a 50% decrease in the ratio of the lipid precursors phosphorylcholine and phosphorylethanolamine (PC/PE) was observed with increasing spheroid size and was correlated with an increased G(1)/G(0) phase cell fraction. In addition, the ratio of the phospholipid degradation products glycerophosphorylcholine and glycerophosphorylethanolamine (GPC/GPE) increased with spheroid diameter in Rat1-T1 aggregates. We conclude that changes in phospholipid metabolism, rather than alterations in energy-rich phosphates, reflect cell quiescence in spheroid cultures, because cells in the inner oxygen-deficient zones seem to adapt their energy metabolism to the environmental conditions before necrotic cell destruction.