James P. Freyer

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.

Mechanisms of light scattering from biological cells relevant to noninvasive optical-tissue diagnostics

We have studied the optical properties of mammalian cell suspensions to provide a mechanistic basis for interpreting the optical properties of tissues in vivo. Measurements of the wavelength dependence of the reduced scattering coefficient and measurements of the phase function demonstrated that there is a distribution of scatterer sizes. The volumes of the scatterers are equivalent to those of spheres with diameters in the range between ~0.4 and 2.0 mum. Measurements of isolated organelles indicate that mitochondria and other similarly sized organelles are responsible for scattering at large angles, whereas nuclei are responsible for small-angle scattering. Therefore optical diagnostics are expected to be sensitive to organelle morphology but not directly to the size and shape of the cells.

Light scattering from cells: the contribution of the nucleus and the effects of proliferative status.

As part of our ongoing efforts to understand the fundamental nature of light scattering from cells and tissues, we present data on elastic light scattering from isolated mammalian tumor cells and nuclei. The contribution of scattering from internal structures and in particular from the nuclei was compared to scattering from whole cells. Roughly 55% of the elastic light scattering at high-angles (> 40 degrees) comes from intracellular structures. An upper limit of 40% on the fractional contribution of nuclei to scattering from cells in tissue was determined. Using cell suspensions isolated from monolayer cultures at different stages of growth, we have also found that scattering at angles greater than about 110 degrees was correlated with the DNA content of the cells. Based on model calculations and the relative size difference of nuclei from cells in different stages of growth, we argue that this difference in scattering results from changes in the internal structures of the nucleus. This interpretation is consistent with our estimate of 0.2 micron as the mean size of the scattering centers in cells. Additionally, we find that while scattering from the nucleus accounts for a majority of internal scattering, a significant portion must result from scattering off of cytoplasmic structures such as mitochondria.

Polarized angular-dependent spectroscopy of epithelial cells and epithelial cell nuclei to determine the size scale of scattering structures

An understanding of the relationship between tissue structures and light scattering from tissue will help facilitate the development and acceptance of noninvasive optical diagnostics including elastic scattering spectroscopy, diffuse reflectance, and optical coherence tomography. For example, a quantitative model of the structures that scatter light in epithelial cells would allow determination of what structures control the characteristics of in vivo light transport measurements and subsequently could provide a detailed relationship between cellular structures and optical measurements. We have determined the size distribution of refractive index structure variations in epithelial cells as well as in nuclei isolated from epithelial cells from measurements of the angular dependence of polarized light scattering. The quantitative size distributions we obtained for both whole cells and isolated nuclei include particles with effective radii of 2 microm to 10 nm or less and contain orders of magnitude more small particles than large particles. These results demonstrate that not only are biological cells very heterogeneous, but so are the nuclei within them. Light scattering is likely sensitive to structures smaller than those commonly investigated by standard pathology methods.

Diffuse backscattering Mueller matrices of highly scattering media

We report on the development of a method that records spatially dependent intensity patterns of polarized light that is diffusely backscattered from highly scattering media. It is demonstrated that these intensity patterns can be used to differentiate turbid media, such as polystyrene-sphere and biological-cell suspensions. Our technique employs polarized light from a He-Ne laser (l=543nm), which is focused onto the surface of the scattering medium. A surface area of approximately 4x4 cm centered on the light input point is imaged through polarization-analysis optics onto a CCD camera. One can observe a large variety of intensity patterns by varying the polarization state of the incident laser light and changing the analyzer configuration to detect different polarization components of the backscattered light. Introducing the Mueller-matrix concept for diffusely backscattered light, a framework is provided to select a subset of measurements that comprehensively describe the optical properties of backscattering media.

FTIR Spectroscopy Demonstrates Biochemical Differences in Mammalian Cell Cultures at Different Growth Stages

We have observed differences in the infrared spectra of viable fibroblast cells depending on whether the cells were in the exponential (proliferating) or plateau (nonproliferating) phase of growth. The spectral changes were observed even after correcting for cell number and volume, ruling out these trivial explanations. Several of the changes occurred for both transformed and normal cell lines and were greater for the normal cell line. The biochemical basis of the spectral changes was estimated by fitting the cell spectra to a linear superposition of spectra for the major biochemical components of mammalian cells (DNA, RNA, protein, lipids, and glycogen). The ratios of RNA/lipid and protein/lipid increased when the cells were in the exponential phase compared to the plateau phase of growth. The fits of cell spectra to individual biochemical components also demonstrated that DNA is a relatively minor spectroscopic component as would be expected biochemically. Contrary to other reports in the literature, our data demonstrate that determining DNA content or structure using Fourier transform infrared spectroscopy data is difficult due to the relatively small amount of DNA and the overlap of DNA bands with the absorption bands of other biochemical components.

Pulsed-gradient spin-echo diffusion studies in NMR imaging. Effects of the imaging gradients on the determination of diffusion coefficients

Studies of self-diffusion by magnetic resonance imaging using variations of the pulsed-gradient spin-echo experiment are complicated by the presence of the imaging-gradient pulses. This problem is particularly severe in NMR microscopy, where the diffusion gradients are of the same order or even smaller than the imaging gradients. Due to cross terms between the diffusion gradient and parallel imaging gradients, the Stejskal-Tanner relation no longer applies. This commonly used equation could result in significant overestimation of the self-diffusion coefficient when used in such instances. The effect of diffusion on signal attenuation in a number of spin-echo diffusion imaging sequences has been analyzed, and analytical expressions including the cross terms with the imaging gradients have been derived. The equations derived were verified experimentally through measurements of the self-diffusion coefficient of water, using high-resolution microimaging (imaging gradients of 10–15 G/cm, pixel size of 23 μm) at 400 MHz. Including the cross terms in the data analysis yields values within the literature range (2.6 × 10−5 cm2/s) for the self-diffusion coefficient of water. Neglecting the cross terms is demonstrated to result in a tenfold over-estimation of the diffusion coefficient. When imaging gradients which are larger than the incremented diffusion gradient are used, the experiment becomes significantly more sensitive to diffusion effects, due to the cross terms between the gradients. This predicted and observed result improves the accuracy of imaging diffusion experiments and may also be applicable in spectroscopic diffusion measurements.

Radiobiology of ultrasoft X rays. I. Cultured hamster cells (V79)

Ultrasoft X rays (approximately less than keV) provide a useful probe for the study of the physical parameters associated with the induction of biological lesions because the spatial scale of their energy depositions is of nanometer dimensions, comparable to that of critical structures within the cell. We report on cell-killing experiments using cultured hamster cells (V79) exposed to carbon K (0.28 keV), aluminum K (1.5 keV), copper K (8.0 keV), and 250 kVp X rays, under oxic and hypoxic conditions, and as a function of cell-cycle phase. Our principal results are: RBE increases with decreasing X-ray energy; OER decreases with decreasing X-ray energy; and cell-cycle response is similar for all X-ray energies. Our RBE results confirm earlier observations using ultrasoft X rays on mammalian cells. The shapes of fitted curves through the data for each energy are statistically indistinguishable from one another, implying that the enhanced effectiveness is purely dose modifying. The results reported herein generally support the view that single-track effects of radiation are predominantly due to very local energy depositions on the nanometer scale, which are principally responsible for observed radiobiological effects.

Characterizing Mammalian cells and cell phantoms by polarized backscattering fiber-optic measurements.

Fiber-optic, polarized elastic-scattering spectroscopy techniques are implemented and demonstrated as a method for determining both scatterer size and concentration in highly scattering media. Measurements of polystyrene spheres are presented to validate the technique. Measurements of biological cells provide an estimate of the average effective scatterer radius of 0.5-1.0 mum. This average effective scatterer size is significantly smaller than the nucleus. In addition, to facilitate use of polarization techniques on biological cells, polarized angular dependent scattering from cell suspensions was measured. The light scattering from cells has properties similar to those of small spheres.

Tumor growth in vivo and as multicellular spheroids compared by mathematical models

In vivo volume growth of two murine tumor cell lines was compared by mathematical modeling to their volume growth as multicellular spheroids. Fourteen deterministic mathematical models were studied. For one cell line, spheroid growth could be described by a model simpler than needed for description of growthin vivo. A model that explicitly included the stimulatory role for cell-cell interactions in regulation of growth was always superior to a model that did not include such a role. The von Bertalanffy model and the logistic model could not fit the data; this result contradicted some previous literature and was found to depend on the applied least squares fitting method. By the use of a particularly designed mathematical method, qualitative differences were discriminated from quantitative differences in growth dynamics of the same cells cultivated in two different three-dimensional systems.