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At least 19 recordsLinked to original sources
PubMed2002

Anchorage-independent multi-cellular spheroids as an in vitro model of growth signaling in Ewing tumors.

Little is known about the growth-signaling pathways that govern the proliferation of Ewing tumor (ET) cells either in vitro or in vivo. We have studied signal transduction pathways in ET cell lines and compared kinase expression levels and proliferation rates with primary tumors. Cell lines were studied both as conventional adherent monolayers and as anchorage-independent multi-cellular spheroids. Importantly, we observed significant differences between these in vitro models and found that ET spheroids were more closely related to primary tumors with respect to cell morphology, cell-cell junctions, proliferative index and kinase activation. Monolayer ET cells demonstrated serum-dependent phosphorylation of ERK1/2 and AKT and constitutively high serum-independent cyclin D1 protein expression. However, when ET cells were placed in suspension culture, there was immediate serum-independent activation of ERK1/2 and AKT. In addition, cyclin D1 protein expression was completely blocked until stable multi-cellular spheroids had formed, indicating that cell-cell adhesion is necessary for the proliferation of anchorage independent ET cells. This reduction in cyclin D1 expression was post-transcriptional and could be mimicked in monolayer cells by treatment with phosphatidyl inositol-3 kinase (PI3K) inhibitors. Moreover, PI3K inhibition significantly reduced ET cell proliferation and, in primary ET samples, cyclin D1 expression correlated with expression of activated AKT. Thus, the PI3K-AKT pathway appears to be critical for the proliferation of ET cells both in vitro and in vivo and tumor cell growth in vivo may be better represented by the study of anchorage-independent multi-cellular spheroids.

Artificial Gene Fusion↗
PubMed1983

Methodological aspects of microelectrode measurements in cellular spheroids.

Different types of oxygen microelectrodes have been tested in measurements on cellular spheroids. The shape of the oxygen gradients varied strongly depending on size and type of the spheroids. No significant differences in the results were obtained when different types of electrodes were applied. All measurements were made in a perfusion chamber. The shape of the gradients did not vary with time in the perfusion chamber. The reproducibility was found good in repeated measurements using the same spheroid. No mechanical or chemical disturbances were seen during the penetration of the spheroids. Changes in the medium flow rate through the chamber did not drastically change the shape of the oxygen gradients. Almost no convection could be seen at the bottom of the chamber close to the spheroids. The composition of the medium was found to be of importance. Lock's solution containing glucose was found to be satisfactory. The potential signals in the double barrel electrodes allowed an accurate determination of the position when the electrode hit the spheroid surface. The information gained from microelectrode measurements in spheroids might be valuable for the understanding of effects of new tumor treatment modalities in which hypoxic cell sensitizers or high LET radiation are utilized.

Animals↗
PubMed1984

Liquid-overlay culture of cellular spheroids.

The capacity of different types of human cells to form and grow as spheroids was tested by using suspension culture over a bottom surface, which prevented cell attachment (liquid-overlay culture). The best and most convenient bottom surface so far tested was the bottom of a normal culture dish covered with a thin film of agarose. The agarose film was allowed to dry prior to the addition of medium. Several different human cell lines were tested, and it was found that many types of tumor cells formed and grew as spheroids, Large variations in growth rate, cell morphology, thickness of the viable cell layer and PO2 profiles were found among the tested spheroids. This variability parallels the variability seen in solid tumors. The morphology and the growth rate of solid tumors vary a great deal, depending on the type and position of the tumors (Ackerman and Rosai 1975; Steel 1977). Some tumors have a slow growth rate, with a few proliferating cells, while others grow faster and have higher proportions of proliferative cells (Charbit et al. 1971; Malaise et al. 1973; Friedman 1974; Lightdale and Lipkin 1975; Steel 1977). Local variations in both morphology and proliferation are usually found even within single tumors (Denekamp and Kallman 1973; Ackerman and Rosai 1974). Considering the variability in morphology, growth rate, and radiosensitivity between different types of spheroids, it is not difficult to imagine that wide variations may exist in these variables between different types of tumors or even between different regions within single tumors. The use of a group of different spheroids, showing a spectrum of growth rates, radiosensitivities, etc., will, it is hoped, be helpful in elucidating factors of importance in cancer therapy.

Animals↗
PubMed1988

Penetration of substances into tumour tissue. Model studies using saccharides, thymidine and thymidine-5'-triphosphate in cellular spheroids.

In order to achieve a better understanding of factors involved in drug penetration into poorly vascularized tumour tissue, the penetration of some model substances was studied in vitro. Multicellular human tumour spheroids were used as model system. The test substances were [3H]thymidine and [14C]glucose, both of which are capable of passing easily through cell membranes, and [3H]thymidine-5'-triphosphate, [3H]sucrose and [3H]inulin, all of which are unable to pass directly through cell membranes. The penetration of these substances was studied using a dry histological and autoradiographical method preserving the distribution of water-soluble substances. The two thymidine compounds penetrated very efficiently into the spheroids, and their penetration patterns were rather similar. The saccharides differed somewhat in their penetration properties. Glucose had the fastest penetration and inulin the slowest. After 15 min, however, inulin was also found isotropically distributed within the spheroids. Thus, extracellular penetration seemed to be a possible way for a substance to reach the central parts of a spheroid. The differences between the saccharides could be due to some extent to differences in molecular weight and solubility.

Carbohydrate Metabolism↗
PubMed1984

Penetration and binding of vinblastine and 5-fluorouracil in cellular spheroids.

The kinetics of the penetration and binding of the two commonly used antitumour drugs vinblastine and 5-fluorouracil in nonvascularized tumour tissue were studied. Multicellular human tumour spheroids (glioma U-118 MG and thyroid cancer HTh-7) were used as a model system. Radiolabelled drugs were used in all studies. To avoid disturbances in the distribution of unbound drugs a dry histological technique was used in combination with contact autoradiography. In addition, quantitative measurements of the accumulation and binding of the drugs were made. The results showed that vinblastine penetrated the spheroids less efficiently than 5-fluorouracil. Vinblastine required about 2 h to be isotropically distributed within the studied spheroids, while only a few minutes were required for 5-fluorouracil. Vinblastine seemed to be accumulated in the peripheral parts of the spheroids within 15 min. High concentrations of 5-fluorouracil, isotropically distributed in the spheroids, were observed after 2 h of incubation. Significant amounts (about half) of the accumulated drugs resisted gentle washing for 3 X 20 s plus 15 min in fresh medium. The limited penetration of vinblastine correlated well with a previously observed high resistance of spheroids to treatments of short duration with this drug.

Cell Line↗
PubMed1981

Penetration of substances into tumor tissue--a methodological study on cellular spheroids.

The penetration of [3H]thymidine, [3H]D-leucine, [125I]albumin, and the drugs [3H]5-fluorouracil and [3H]vinblastine into human glioma spheroids (in vitro tumor models) was studied by a method based on rapid freezing, freeze drying, vapor fixation, wax embedding, dry sectioning, and contact autoradiography. No significant disturbances in the distribution of water soluble substances were observed. Thymidine and D-leucine penetrated the whole spheroids relatively fast, whereas albumin showed reduced penetration. The concentration of albumin was highest at the periphery of the spheroids, but only smaller amounts were detected in the deeper regions. A significant difference between the penetration patterns of the drugs studied was also observed. Fluorouracil penetrated rather freely, but the penetration of vinblastine was limited.

Albumins↗
PubMed1983

Penetration of substances into tumor tissue: a methodological study with microelectrodes and cellular spheroids.

A new method was tested for studies of penetration of substances into tumorlike tissue. The penetration of the ions K+, Cl-, and Ca2+ through several layers of tumor cells was demonstrated by using double barrelled, ion sensitive microelectrodes with extra thin tip diameters. Spheroids consisting of human glioma, U-118 MG, and human thyroid cancer, HTh-7, cells were used as models of tumor tissue. A microelectrode was inserted into the center of a spheroid. Thereafter, the concentration of the test substance was increased in the surrounding medium. The change in concentration inside the spheroid was recorded and the penetration pattern evaluated. All three types of tested ions penetrated easily through the spheroids. The K+ ions penetrated most efficiently, and the Ca2+ ions showed the slowest penetration. The Ca2+ ions penetrated somewhat more slowly in the U-118 MG spheroids (which had rather small extracellular spaces) than in the HTh-7 spheroids (which had larger extracellular spaces). Ion sensitive electrodes, which are easily available, were used in this study only to demonstrate the principle. We hope that the method described can be used for penetration studies of various substances. For example, all substances that can be detected by enzyme microelectrodes could be studied. The main advantage of the method is that the complete penetration pattern can be studied as a function of time in individual spheroids. Previously described methods require histological procedures for each analyzed penetration time.

Biological Transport↗
PubMed1983

Electro-physiological measurements in cultured cellular spheroids.

Human Glioma U-118 MG and hamster V-79-379A spheroids were used as models of the nodular structure often seen in poorly vascularized regions of solid tumours. K+, Ca2+ activities and membrane potentials were measured in the spheroids, using microelectrodes. Both stable and peak values were obtained. All values were included in the analysis to allow an accurate estimate of the relative number of electro-physiologically active cells at different depths in the spheroids. The relative number of active cells decreased drastically with depth although most cells in the corresponding regions looked morphologically viable. The amplitude values of potentials and K+ activities showed, however, no significant variations with depth. The Ca2+ activity varied largely between individual cells. The relative number of proliferative cells decreased with depth in the spheroids, in parallel to the decrease in the number of electro-physiologically active cells. The results indicate that the fraction of cells, in poorly vascularized regions, having normal metabolism might be fewer than what can be estimated only on a morphological basis. Thus, the amount of cells with the capacity to contribute to growth might be overestimated when inspecting histological sections.

Animals↗
PubMed1978

Proliferation and viability in cellular spheroids of human origin.

The capacity of cells to form growing tumor-like colonies in culture was tested by a new and unlaborious suspension technique. Six of nine tested cell lines formed spheroids, five of which started to grow. The growing spheroids reached a maximal size determined by balanced cell gain in a proliferative, superficial layer and cell death, particularly at larger depth. Sections of spheroids of different origin showed large variations in the thickness of the viable cell layers and in the shape of the proliferative gradients. The data, taken together with earlier published information, indicate that a difference between rodent and human cells generally exists, the former showing thinner viable and proliferative layers.

Animals↗
PubMed2002

Differential expression of CD44(S) and variant isoforms v3, v10 in three-dimensional cultures of mouse melanoma cell lines.

Multi-cellular spheroids (MCS) generated from tumor cells serve as excellent in vitro models for understanding the mechanisms of tumor progression and micro-metastasis. We have compared the expression of molecular markers with reference to their growth as conventional adherent monolayers (2-D) and anchorage independent cultures (3-D) using two mouse melanoma cell lines, B16F10 and Clone M3. The two cell lines differed in their ability to form spheroids with respect to their aggregation potential, with B16F10 forming large clusters compared to Clone M3. A panel of molecular markers comprising cell adhesion molecules, cyclin dependent kinase inhibitors and members of the cadherin-catenin complex were analyzed by flow cytometry in 2-D and 3-D cultures. There was a distinct difference in the patterns of expression of CD44(S) and variant isoforms v3, v10 in spheroids compared to cells grown as monolayers in both cell lines. Also, there was an increase in cells positive for CDK inhibitor p27 in 3-D cultures from the B16F10 cell line. The expression of alpha and gamma catenin was down regulated in spheroids. As these molecules are implicated in the regulation of cell proliferation, alterations in the expression of these molecules in 3-D cultures compared to their 2-D counterparts suggests the importance of spheroids as experimental model for tumorigenesis.

Animals↗
PubMed2005

Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold.

Primary rat hepatocytes self-assemble into multi-cellular spheroids and maintain differentiated functions when cultured on a two-dimensional (2-D) substrate conjugated with galactose ligand. The aim of this study is to investigate how a functional nanofiber scaffold with surface-galactose ligand influences the attachment, spheroid formation and functional maintenance of rat hepatocytes in culture, as compared with the functional 2-D substrate. Highly porous nanofiber scaffolds comprising of fibers with an average diameter of 760 nm were prepared by electrospinning of poly(epsilon-caprolactone-co-ethyl ethylene phosphate) (PCLEEP), a novel biodegradable copolymer. Galactose ligand with a density of 66 nmol/cm(2) was achieved on the nanofiber scaffold via covalent conjugation to a poly(acrylic acid) spacer UV-grafted onto the fiber surface. Hepatocytes cultured on the galactosylated PCLEEP nanofiber scaffold exhibited similar functional profiles in terms of cell attachment, ammonia metabolism, albumin secretion and cytochrome P450 enzymatic activity as those on the functional 2-D substrate, although their morphologies are different. Hepatocytes cultured on galactosylated PCLEEP film formed 50-300 microm spheroids that easily detached from surface upon agitation, whereas hepatocytes cultured on galactosylated nanofiber scaffold formed smaller aggregates of 20-100 microm that engulfed the functional nanofibers, resulting in an integrated spheroid-nanofiber construct.

Adsorption↗
PubMed2005

Role of reactive oxygen species (ROS), metalloproteinase-2 (MMP-2) and interleukin-6 (IL-6) in direct interactions between tumour cell spheroids and endothelial cell monolayer.

Metastasis is a multistep process involving a variety of direct cell-cell, cell-matrix and paracrine interactions. In the present study, we examined some consequences of direct interaction between tumour cells and endothelial cells in vitro. When multicellular spheroids of two human tumour cell lines (HeLa and Hep-2) were transferred onto a human umbilical vein endothelial cell (HUVEC) monolayer, a peri-spheroidal zone of damaged endothelial cells was observed after 24h co-culture. To determine the cause of this damage, the production levels of superoxide anion (O2-), interleukin-6 (IL-6) and metalloproteinase-2 (MMP-2) were measured both in co-culture and in monocultures of the tumour cell spheroids and endothelial cells. Attachment of HeLa and Hep-2 cellular spheroids to the HUVEC monolayer resulted in 1.6-fold and 2.1-fold increases in O2- release, respectively. Also, the MMP-2 level was five times greater in the co-culture than in the tumour spheroid monoculture. The increase of IL-6 in the co-culture model, on the other hand, was only slight. However, a 2h preincubation of endothelial cells with LPS (10 microg/ml) prior to the transfer of spheroids induced a significant increase in the production of this cytokine compared to an appropriate control (an LPS-activated endothelial cell monolayer). These results strongly suggest that both ROS and MMP-2 are involved in endothelial cell injury when tumour cells cross the endothelial barrier. Moreover, IL-6, which participates in the inflammatory response, may also be involved in the extravasation of tumour cells.

Cell Communication↗
PubMed1991

Cellular energetics measured by phosphorous nuclear magnetic resonance spectroscopy are not correlated with chronic nutrient deficiency in multicellular tumor spheroids.

We have measured the 31P nuclear magnetic resonance spectra of EMT6/Ro multicellular tumor spheroids over a wide range of sizes under constant nutrient conditions which matched those used for culturing the spheroids. The amount of nucleotide triphosphate per cell decreased with spheroid growth, roughly in proportion to the decrease in cell volume. There was no correlation between the intracellular pH, the nucleotide triphosphate:Pi ratio, or the phosphocreatine:Pi ratio and either the spheroid cellularity, the mean cell volume, the S-phase fraction, the clonogenic capacity, or the amount of central necrosis. The phosphoryethanolamine:phosphorylcholine ratio also increased with increasing spheroid size. There was a negative correlation between the phosphoryethanolamine:phosphorylcholine ratio and the S-phase cell fraction or the mean cell volume; this ratio was positively correlated with the extent of central necrosis. The membrane degradation components glycerophosphorylcholine and glycerophosphorylethanolamine showed no significant changes with increasing spheroid size. These results imply that spheroid necrotic areas induced by chronic nutrient deficiencies are "invisible" to 31P nuclear magnetic resonance and that the development of cellular quiescence in spheroids is not caused by a decrease in the steady-state level of high-energy phosphates or a reduced intracellular pH. Together, these data support a model in which cells maintain normal steady-state levels of high energy phosphates until they are very close to necrotic cell death. This implies that the deterioration of 31P nuclear magnetic resonance spectra of tumors with increasing size is not caused by chronic nutrient deficiencies resulting from cells outgrowing the capillary supply, but rather is more related to transient nutrient deprivation phenomena.

Animals↗
PubMed2004

Manipulation of in vitro toxicant sensors in an ultrasonic standing wave.

Multi-cellular spheroids are increasingly employed as in vitro sensors of toxicants and a single spheroid can be used as a test object. An ultrasonic standing wave trap (USWT) can hold small particles in a medium-flowing system. This study investigated the conditions for holding HepG2 spheroids in an USWT and its relevance to use in toxicity testing. It can take many hours to reach a detectable end point of cell damage in a standard cellular in vitro toxicant assay and the process might be accelerated through increased sample flow past the spheroid. A USWT was employed here to levitate and hold HepG2 spheroids stationary against a flow of 3 mm s(-1) when the acoustic pressure amplitude is 1.9 MPa. The ultrasonic drive frequency was 1.64 MHz. Acoustic microstreaming in the standing wave chamber generated 1 mm s(-1) flow past a levitated spheroid-scale (80 microm diameter) latex particle in the absence of sample through-flow. The conditions required to form aggregates of cells of a HepG2 cell line in a single half wavelength ultrasonic standing wave mini-chambers are also described here. It is argued that the manipulation capabilities demonstrated may have potential in increasing the efficiency of in vitro toxicant detection by spheroids. Preliminary, visual (unquantified) fluorescence microscopy observations of spheroids levitated in the standing wave in the presence of the toxicant DL-propranolol do suggest accelerated loss of viability compared with controls.

Biosensing Techniques↗
PubMed1985

A reduction in the in situ rates of oxygen and glucose consumption of cells in EMT6/Ro spheroids during growth.

The rates of consumption of oxygen and glucose by EMT6/Ro cells in multicellular spheroids were measured at various times during normal growth. In situ spheroid cellular consumption rates were similar to those of exponentially growing single cells up to a spheroid diameter of 150 micron. Further growth resulted in decreases in the rates of both oxygen and glucose consumption which were correlated with the increase in spheroid diameter and cell number. At a diameter of 1300 micron, both rates of cellular consumption had decreased by a factor of 2.5. The rates of consumption per unit of nonnecrotic spheroid volume decreased in a similar manner. Measurements with single cells demonstrated that the rate of oxygen consumption was coupled with glucose concentration, and vice versa. The rates of consumption for cells dissociated from small spheroids indicated that there was some effect of the spheroid environment. As the spheroids grew, however, association in the spheroid structure accounted for a smaller proportion of the total observed reduction in the rates of nutrient consumption. The presence of central necrosis also appeared to have no effect on the rates of consumption of these nutrients. Spheroid-derived cells showed a decrease in cell volume with growth as the cells accumulated in a quiescent state. Measurements with single cells demonstrated that oxygen and glucose consumption were correlated with cell volume and with the development of nonproliferating cells. We conclude that the observed decrease in oxygen and glucose consumption with growth in spheroids is largely due to the progressive accumulation of cells in a quiescent state characterized by an inherently lower cellular rate of nutrient utilization.

Animals↗