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PubMed1976

Internal organization of the circadian timing system in multicellular animals.

Three models of the organization of the circadian timing system in multicellular animals are presented. Each can account for the observed internal synchronization of the various circadian rhythms within the organism and each is also compatible with the known responses of circadian systems to manipulations of environmental time cues. One is a single oscillatory system (Model I) while the other two are multioscillator systems arranged in a hierarchical (Model II) or nonhierarchical (Model III) manner. Experiments that test the predictions of the different models are reviewed. These indicate that the circadian timing system in mammals is organized as a multioscillator system with oscillating concentrations of chemical mediators (nervous or endocrine) internally synchronizing the various potentially-independent oscillators by an entrainment mechanism. However, as yet there is insufficient evidence to indicate whether the oscillators are arranged with a predominantly hierarchical (Model II) or nonhierarchical (Model III) organization.

Adrenal Glands↗
PubMed1993

Macrophages and multicellular tumor spheroids in co-culture: a three-dimensional model to study tumor-host interactions. Evidence for macrophage-mediated tumor cell proliferation and migration.

In a new co-culture model involving multicellular tumor spheroids and different phenotypes of human macrophages, we studied the effects of the latter on migration and proliferation of the human colon carcinoma cell line, HRT-18. The macrophage phenotypes are detectable with monoclonal antibodies and are inducible in culture. 12-O-tetradecanoyl-phorbol-13-acetate-activated macrophages are associated with the phenotype 27E10, which is an acute inflammatory macrophage. The glucocorticoid-induced macrophage phenotype RM3/1 is associated with the down-regulation of inflammation. The phenotype resembling the mature resident macrophage termed 25F9 arises spontaneously in prolonged culture. It could be shown that inflammatory macrophages are localized at invasive areas of the tumor-host interface of colorectal carcinoma, whereas resident and anti-inflammatory macrophages were found in the central tumor region or at well-bordered areas of the tumor-host interface. The results obtained with this co-culture model show that 27E10-associated macrophages stimulate tumor cell migration and inhibit tumor cell proliferation. RM3/1 had only a slight inhibiting effect on proliferation and a slight promoting effect on migration. The 25F9-positive macrophage-stimulated tumor cell proliferation and inhibited migration completely. This investigation indicates that this in vitro system is useful for studying different macrophage effects on tumor cells and that indeed proliferation and migration of tumor cells could be influenced in an opposite manner by different types of macrophages.

Carcinoma↗
PubMedDate not supplied

Growth of prostatic cancer cells, DU 145, as multicellular spheroids and effects of estramustine.

The human prostatic carcinoma cell line DU 145 was grown as multicellular spheroids in vitro. The volume doubling time during the early exponential growth phase was about 5 days. The saturation volume, in the plateau phase of the growth curve, was in the order of 1.4 mm3. The spheroids developed a central degenerative region surrounded by a 0.1-0.3 mm layer of viable cells. The DU 145 spheroid system is planned to be used as a model in studies on chemotherapy and targeted radiotherapy of micrometastases of prostatic cancer. Some effects of the drug estramustine, EM, a conjugate of estradiol and nornitrogen mustard, were analysed in this introductory study. Tritium-labelled estramustine, 3H-EM, bound both in the viable cell layers and in the degenerative region of the spheroid already after 1 hour of incubation which indicated good penetration. The viable cells bound only low levels of 3H-EM while the degenerative region bound 3H-EM to a higher extent. The amount of bound 3H-EM increased after incubation for 24 hours. The binding was nonspecific since it could not be inhibited by pretreatment with an excess of non-radioactive EM. Furthermore, 3H-EM bound to a similar extent in glioma and colon carcinoma spheroids used for comparison. Incubation of DU 145 spheroids for 24 hours with EM (20 mg/ml) induced a growth delay of 6-7 days and a transient increase in the volume of the extracellular spaces for a few days following the treatment. The results showed that the binding of EM to prostate DU 145 cells growing as spheroids was not specific and that the toxic action was limited. An interesting result was that EM works as an extracellular space expander. This might be exploited in combination treatments with other agents.

Autoradiography↗
PubMed1993

Enhancement of heme oxygenase expression and activity in A431 squamous carcinoma multicellular tumor spheroids.

We have investigated the effects of the growth of A431 human squamous carcinoma cells as three-dimensional aggregates (multicellular tumor spheroids) on the expression and enzyme activity of heme oxygenase (HO). We demonstrate that A431 squamous carcinoma cells grown as day 4 spheroids selectively increase the expression of heme oxygenase 1 (HO-1), caused, directly or indirectly, by three-dimensional cell-cell contact effects. Steady-state levels of both mRNA and protein are significantly enhanced in spheroids compared with day 4 monolayers (approximately 13-fold). Because of the similarity of apparent half-lives between monolayers (2.7 h) and spheroids (2.1 h), it appears that the increases are caused at least partly by altered transcriptional rates. Total HO enzyme activity, measured by carbon monoxide production, is also up-regulated (2.6-fold) in spheroids, compared to that in monolayers. This increase indicates that the up-regulation in HO-1 protein expression corresponds to an increase in functional enzyme levels. We propose that HO may play a more complex role in cellular metabolism than would be evident from studies using two-dimensional monolayer cultures.

Blotting, Northern↗
PubMedDate not supplied

Uptake and release of radiolabelled monoclonal antibody Po66 by multicellular aggregates obtained from a lung squamous carcinoma cell line.

Po66, a monoclonal antibody (MAb) directed against lung squamous cell carcinoma, has been shown, when injected intravenously, to be retained for a long time in tumors. This property encouraged trials to use Po66 as an agent of metabolic radiotherapy. The suitability of multicellular spheroids to reproduce the in vivo conditions of irradiation of tumors by Po66 was investigated in the present work. Spheroids were formed from the lung carcinoma cell line SK-MES-1. They resembled morphologically small carcinoma nodules with desmosome-like intercellular junctions at the periphery and a central necrotic core. The cells expressing the antigen recognized by Po66 had a heterogeneous distribution and were predominant in the outer layers of the cell aggregates. Spheroids were exposed to radiolabelled Po66. The MAb diffused slowly and reached a maximal incorporation after 4-12 hours incubation. A control unrelated antibody did not penetrate appreciably. Autohistoradiographic experiments suggested that the antibody accumulated in most cells expressing the antigen. The rate of MAb release from the spheroids was very low (T 1/2 = 163 h). Taken together, the data indicate that spheroids might be a relevant model to investigate the parameters controlling Po66-mediated immunoradiotherapy.

Animals↗
PubMed1996

Therapeutic effect of m-[131I]- and m-[125I]iodobenzylguanidine on neuroblastoma multicellular tumor spheroids of different sizes.

m-[l25I]iodobenzylguanidine (m-[125I]MIBG) has been suggested as an alternative to m-[131I]MIBG for the treatment of metastatic neuroblastoma to achieve a higher radiation dose in micrometastases. To compare these two radiopharmaceuticals, a mathematical model was developed in the present study that allows for the calculation of radiation dose rates within small spherical tumors for different distributions of 131I and 125I. Furthermore, the relationship between tumor size and the therapeutic effects of m-[131I]- and m-[125I]MIBG was studied in vitro using multicellular tumor spheroids of the neuroblastoma cell line SK-N-SH. According to the calculations, higher mean dose rates can be achieved by m-[125I]MIBG than by m-[131I]MIBG up to a tumor diameter of 100 microm when both substances are homogeneously distributed within the tumor. In larger tumors, however, mean dose rates achieved by 131I are up to 8-fold higher. Evaluation of various activity distributions demonstrated that even in tumors of less than 100 microm in diameter, marked heterogeneities of the dose rate can occur when m-[125I]MIBG is not distributed homogeneously. By treatment with m-[131I]MIBG, the growth of tumor spheroids ranging from 100 to 250 microm in diameter was inhibited more effectively in the larger than in the smaller spheroids. The growth inhibition of spheroids treated with m-[125I]MIBG was independent of the spheroid size. In consistency with the calculations, the therapeutic effect of m-[125I]- and m-[131I]MIBG was equal in spheroids with diameters of about 100 microm. In larger spheroids, m-[131I]MIBG induced a more pronounced delay in spheroid growth than m-[125I]MIBG. According to these calculations and in vitro data, m-[125I]MIBG as a single agent does not seem to be a promising alternative to m-[131I]MIBG for treatment of metastatic neuroblastoma. However, the combined use of m-[131I]- and m-[125I]MIBG may be more effective than treatment with m-[131I]MIBG alone.

3-Iodobenzylguanidine↗
PubMedDate not supplied

The reversal of multidrug resistance in multicellular tumor spheroids by SDZ PSC 833.

Multidrug resistance (MDR) is a major impediment to the effective treatment of cancer. We have used multicellular tumor spheroids (MTS) as a model to investigate whether MDR can be reversed in a three dimensional structure. MTS are tightly associated aggregates of tumor cells that exhibit many of the properties of solid tumors. A human MDR breast carcinoma cell line was selected by exposure to taxol under monolayer conditions. The sensitive (parental) and drug-resistant phenotypes were retained when the cells were grown as MTS. Thus, the three dimensional conditions in this novel model system did not affect the MDR phenotype. SDZ PSC 833 is an efficient MDR reversing agent as determined under monolayer conditions and is currently being evaluated in clinical trials. Resistance to taxol and doxorubicin of the MDR cells grown as MTS was almost completely reversed by SDZ PSC 833. Our results suggest that SDZ PSC 833 has the potential to reverse the MDR phenotype in solid tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Invasion of human cartilage by cultured multicellular spheroids of rheumatoid synovial cells--a novel in vitro model system for rheumatoid arthritis.

OBJECTIVE: A new 3-dimensional cell culture system was established to examine the invasion of rheumatoid synovial cells into cartilage. METHODS: Synovial cells from 20 patients with rheumatoid arthritis (RA) and 15 patients with osteoarthritis (OA) were co-cultured as multicellular spheroids with cartilage fragments. RESULTS: After 4 weeks the rheumatoid spheroids eroded the cartilage. The destruction of cartilage was supported by a high expression level of cathepsin D and matrix metalloproteinases. In contrast, the OA tissue showed attachment to the cartilage only. CONCLUSION: Our results indicate that spheroid/cartilage co-cultures seem to be a suitable in vitro model for the study of destructive cellular mechanisms that occur in RA.

Arthritis, Rheumatoid↗
PubMedDate not supplied

Comparison of extracellular matrix in human osteosarcomas and melanomas growing as xenografts, multicellular spheroids, and monolayer cultures.

BACKGROUND: The composition of extracellular matrix in human xenografts and spheroids were compared with the monolayer cultures from which they originated. Collagen I, fibronectin, acetylglucosamine, and acetylgalactosamine were quantitated in two osteosarcomas and one melanoma. METHODS: Using fluorescence microscopy, extracellular matrix constituents in the cellular and extracellular compartment were measured, whereas flow cytometry measured the extracellular matrix constituents bound to the cell surface as well as the total cellular amount including intracellular and surface bound constituents. RESULTS: The fluorescence microscopy measurements, demonstrated that the xenografts contained more or equal quantities of the extracellular matrix constituents compared with the spheroids. Flow cytometric measurements of total cellular amounts, showed that cells from xenografts usually contained more or equal amounts as the spheroid cells, which contained less or equal amounts as the monolayer cells. The surface expression of the extracellular matrix constituents increased or there were no significant differences, comparing cells grown as monolayers, spheroids, and xenografts. CONCLUSIONS: The data shows that multicellular spheroids being an in vitro system of intermediate complexity between monolayer cultures and tumours, contain an extracellular matrix corresponding to some degree to this intermediate position.

Acetylgalactosamine↗
PubMed2000

Extracellular matrix (ECM) components in a very primitive multicellular animal, the dicyemid mesozoan Kantharella antarctica.

One of the most vital synapomorphic properties of metazoans is the presence of an extracellular matrix (ECM), i.e., a complex of proteoglycans, adhesive glycoproteins, and collagens. The genetically controlled ECM mediates between the respective receptors morphogenesis and cell differentiation and is central to gastrulation, i.e., the process which generates the embryonic germ layers, upon which all metazoan body structures are based. However, the primitive metazoans include a phylum, viz. the Dicyemida, which lacks any kind of typical metazoan ECM structures including a basement membrane, and hence does not develop through gastrulation. Since the ECM components fibronectin, laminin, and type IV collagen, all of which are essential constituents of each basement membrane, have been proved to be evolutionary ancient molecules from the lowest metazoans up to vertebrates, antibodies against the respective vertebrate ECM components were employed by electron microscopy to look for these molecules also in the dicyemid mesozoan Kantharella antarctica. As a result, all three protein families showed an immunolabel which was localized intracellularly and intimately associated with the cell membranes as well as with the submembranously arranged delicate filamentous network. The immunolabel was most intense in the fibronectin-like protein, followed by the type IV collagen-like protein and weakest in the laminin-like protein. From an evolutionary point of view, this kind of distribution of ECM components, primarily found in intracellular regions, seems to reflect a very primitive situation of the structures of the respective ECM molecules having not yet reached their definitive position outside the cell, thus generating the complete biological function of typical ECM. Moreover, these results confirm the long-standing presumption that the dicyemid mesozoan body structure might be the missing link between the Protozoa and Metazoa.

Animals↗
PubMed1992

Endogenous ionic currents and DC electric fields in multicellular animal tissues.

Through the use of the non-invasive vibrating probe technique for detecting extracellular ionic currents developed in 1974 [Jaffe and Nuccitelli: J Cell Biol 63:614-628, 1974], embryonic currents have been detected in a wide range of animal systems (recently reviewed in [Nuccitelli, Noninvasive Techniques in Cell Biology. New York: Wiley-Liss, 1990, pp 273-310]. In four of these studies, the corresponding electric field has been measured within the animal tissue. Such measurements of internal electric fields are quite challenging because they involve the insertion of microelectrodes into the developing tissue along specific regions of current flow. This paper reviews the evidence for endogenous transembryonic currents and dc electric fields in animal systems and provides the range of values for such physiological fields. These data should provide a guide to the range of imposed electric field strengths that could influence normal biological functions in living organisms.

Animals↗
PubMed1983

Formation and growth of multicellular spheroids of human origin.

Different types of human cells which normally grow as monolayers or suspension cultures were tested for their capacity to form and grow as spheroids. Sixteen out of the 27 tested tumour cell lines formed spheroids. Nearly all of these spheroids also grew. With only two exceptions the doubling times were longer when the tumour cells grew as spheroids than when they grew in conventional mass culture. Eleven out of 13 tested human non-tumour cells formed small spheroids but of these only the spheroids of lymphoid origin could grow. These lymphoid cells grew faster when aggregated to spheroids than when in single-cell suspension culture. None of the other non-tumour cells, which normally grew as monolayers, could grow as spheroids. The normally monolayer-cultured tumour cells formed symmetrical spheroids with smooth surfaces while the normally suspension-cultured cells formed irregular spheroids with rough surfaces. All large spheroids had a necrotic centre surrounded by a shell of viable cells. The thickness of the viable cell layer varied depending on cell type. The shape and organization of cells within the spheroids also varied largely. The results show that many types of human cells can be cultured as spheroids and that a wide spectrum of morphological appearances and growth rates can be obtained.

Cell Aggregation↗
PubMed1985

The use of multicellular spheroids in establishing human sarcoma cell lines in vitro.

By a new procedure stable monolayer cultures were derived from spheroids in 8 out of 17 different human sarcomas (16 soft-tissue and I osteogenic sarcoma). Eleven of the sarcomas were obtained from patients undergoing surgery, and 6 from BALB/c nude mice carrying s.c. growing xenografts. The new procedure involves aggregation of single-cell suspensions into spheroids and cultivation of these in agar-coated flasks until the growth rate levels off, at which time the spheroids are transferred to uncoated flasks. Cells proliferating from the rim of adhering spheroids are trypsinized and aggregated to form new spheroids. By 3 to 5 such alternations, monolayer cultures were obtained that have now been subcultured for about 6 months. The cell lines all gave rise to colonies in a clonogenic soft-agar system, and upon s.c. injection into athymic nude mice 3 lines tested formed growing tumors. The histology of spheroids formed from late monolayer passages closely resembled that of the original tumors. That the new procedure is superior to other methods of establishing sarcoma cell lines is indicated by the fact that a stable monolayer culture could be obtained directly from the tumors in only 1/8 cases where the above procedure was successful, and in only 2 instances from soft-agar colonies derived from the tumors.

Adolescent↗
PubMed1995

Calibration of misonidazole labeling by simultaneous measurement of oxygen tension and labeling density in multicellular spheroids.

To correlate misonidazole concentrations and oxygen pressures (Po2) at identical locations within EMT6/Ro multi-cell spheroids (mean diameters +/- SD: 867 +/- 20 microns), Po2 measurements were performed with oxygen-sensitive microelectrodes during incubation of these spheroids with tritiated misonidazole (10 mg/I; 445 microCi/mg). In each individual spheroid, Po2 profiles were correlated with the corresponding spatial distribution of misonidazole as quantified by conventional autoradiography and grain counting. To compare the oxygenation status of spheroids in the measuring chamber with that of spheroids in spinner culture, misonidazole labeling was performed in both environments following the same protocol. All experiments were conducted in 20% oxygen and BME or in 5% oxygen and DMEM to obtain spheroids with different degrees of oxygenation. Labeled misonidazole was fairly evenly distributed in the outer, better oxygenated regions of EMT6 spheroids. In contrast, there was an accumulation of the labeled substance near central necrosis where low oxygen tensions were measured. Grain densities were similar at corresponding oxygen pressures under both environmental conditions. Except for some scatter, grain density as a function of oxygen pressure showed little variation in the Po2 range of 20-60 mm Hg, but exhibited a steep increase below 10 mm Hg. The findings imply that a substantial rise in local misonidazole labeling indicates a metabolically active tissue region at low Po2 that is not necessarily identical with the radiobiologically hypoxic cell fraction. A comparison of the labeling densities of spheroids in spinner flasks and in the Po2 measuring chamber indicates that oxygenation of spheroids is better in rotation culture than during microelectrode measurements.

Animals↗
PubMed1985

Increased thermoresistance developed during growth of small multicellular spheroids.

Mammalian cells growing as multicell spheroids, an in vitro model of tumor microregions, have been shown previously to be more resistant than single cells from monolayer cultures to killing by ionizing radiation, hyperthermia, ultrasound, and chemotherapeutic drugs. Although the mechanisms by which cells in spheroids acquire these increased resistances are unknown, available evidence has indicated that intercellular contact mediates the process for ionizing radiation. This investigation was undertaken to evaluate the role of intercellular contact produced during growth of small spheroids on the sensitivity of EMT6/Ro mouse mammary tumor cells to moderate hyperthermia. Increased thermoresistance developed in small spheroids (approximately 70 micron diameter, 25 cells/spheroid), as measured by colony formation, after exposures to different temperatures in the range of 37 to 45 degrees C for periods less than or equal to 2 hr and at 42.5 degrees C for less than or equal to 8 hr. Experiments were performed to determine the relative contributions to this increased thermoresistance of 1) the extent of intercellular contact in spheroids of different cellular multiplicities, 2) differences in membrane damage influenced by trypsin heat treatment sequence, and 3) physiological changes associated with growth of cells as spheroids in suspension compared to monolayer culture. Treatment with trypsin prior to heating sensitized cells to killing by hyperthermia but did not account for the differential thermoresistance between cells from spheroids and monolayers. Spheroid multiplicity in the range of 1.16 to 76.2 cells/spheroid had no significant effect on cell survival after hyperthermia. However, cells grown in spinner suspension culture were more thermoresistant than cells from monolayer cultures and nearly as thermoresistant as cells in spheroids. From these data we conclude that the greater thermoresistance of EMT/Ro cells in spheroids is the result of cellular physiological changes associated with growth in suspension and is not mediated by intercellular contact.

Animals↗
PubMed2003

Protein evolution: structure-function relationships of the oncogene beta-catenin in the evolution of multicellular animals.

Beta-catenin functions as a cytoskeletal linker protein in cadherin-mediated adhesion and as a signal mediator in wnt-signal transduction pathways. We use a novel integrative approach, combining evolutionary, genomic, and three-dimensional structural data to analyze and trace the structural and functional evolution of beta-catenin genes. This approach also enabled us to examine the effects of gene duplication on the structure and function of beta-catenin genes in Drosophila, C. elegans, and vertebrates. By sampling a large number of different taxa, we identified both ancestral and derived motifs and residues within the different regions of the beta-catenin proteins. Projecting amino acid substitutions onto the three- dimensional structure established for mouse beta-catenin, we identified specific domains that exhibit loss and gain of selective constraints during beta catenin evolution. Structural changes, changes in the amino acid substitution rate, and the appearance of novel functional domains in beta-catenin can be mapped to specific branches on the metazoan tree. Together, our analyses suggest that a single, beta-catenin gene fulfilled both adhesion and signaling functions in the last common ancestor of metazoans some 700 million years ago. In addition, gene duplications facilitated the evolution of beta-catenins with novel functions and allowed the evolution of multiple, single-function proteins (cell adhesion or wnt-signaling) from the ancestral, dual-function protein. Integrative methods such as those we have applied here, utilizing the 'natural experiments' present in animal diversity, can be employed to identify novel and shared functional motifs and residues in virtually any protein among the proteomes of model systems and humans.

Amino Acid Motifs↗
PubMed2001

Compartmentation of intracellular water in multicellular tumor spheroids: diffusion and relaxation NMR.

Diffusion and relaxation of water in C6 glioma and MLS human ovarian carcinoma spheroids were measured from 1D projections acquired using a 2D diffusion-relaxation correlation pulse sequence and processed by non-negative least-square (NNLS) analysis. Systematic underestimation of I(s) and ADC(s) were observed for I(s)/(I(s) + I(f)) < 0.001. In the presence of spheroids, two apparent diffusion coefficient (ADC) compartments were observed, where ADC(f), ADC(s), and I(f), I(s) are the respective ADCs and signal intensities of the fast and slow compartments. These compartments differed also in their T(2) relaxation (ADC(s) = 0.5-0.74 x 10(-5) cm(2)/s, T(2) = 36-45 ms; and ADC(f) = 2.2-2.8 x 10(-5) cm(2)/s, T(2) = 280-316 ms). The two ADC compartments and the slow T(2) compartment were consistent with slow exchange. The fast T(2) compartment showed a drift with diffusion weighting, suggesting that it represents water exchanging between compartments that differ in their ADC and T(2). Both ADC(s) and I(s) were markedly attenuated with increasing diffusion time (Delta) for Delta < 100 ms, and increased at longer Delta. These results are consistent with restricted diffusion and fast relaxation of intracellular water for short diffusion time (T(1)' = 46.6 ms), and with predominant extracellular contribution to ADC(s) at longer diffusion times. Magn Reson Med 46:68-77, 2001.

Animals↗
PubMed1995

Quantitative NMR microscopy of multicellular tumor spheroids and confrontation cultures.

In cancer research, tumor spheroids are a well established system to study tumor metabolism resembling the situation in vivo more closely cell monolayers. Spherical aggregates of malignant melanoma cells (MV3) and their invasion into rat brain aggregates have been investigated by quantitative NMR microscopy. Relaxation times (T1, T2) and diffusion parameter images were acquired with an in-plane resolution of 14 x 14 microns2. The authors were able to demonstrate that the morphology of the spheroids can be visualized on these NMR maps. The contrast was mainly manifested in relaxation maps, where average relaxation times T1 = 1.94 +/- 0.17 s and T2 = 42.8 +/- 6.3 ms were obtained for proliferating cells, and T1 = 2.49 +/- 0.31 s and T2 = 104.3 +/- 29.4 ms for the necrobiotic center. The mean diffusion coefficients were 0.59 +/- 0.12 micron2/ms and 0.85 +/- 0.14 micron2/ms, respectively. The authors could follow the dynamic process of tumor cell invasion in the investigated co-culture system. Knowledge about tumor cell migration and tumor cell invasion is essential for the understanding of cancer and its therapy. Quantitative NMR microscopy can study this dynamic process noninvasively and therefore may help to assess the influence of therapy on the micromilieu of these spheroids.

Animals↗