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At least 253 records · Page 14Linked to original sources
PubMed1981

A system for determining the pharmacology of indirect radiation sensitizer drugs on multicellular spheroids.

We have characterized some of the physiology of multicellular spheroids of different sizes grown from Chinese hamster lung fibroblast (V79) cells. Among the parameters studied were oxygen tension distributions within the spheroid. This was achieved using ultramicroelectrodes with tip diameters of 1-5 mu and a perfusion system whereby environmental conditions such as flow, temperature, and chemical makeup of the milieu could be measured and controlled. Plateau pO2 values of less than 10 mm Hg were consistently obtained from spheroids under various conditions. We were able to modify these distributions by use of indirect radiation sensitizer drugs such as mechlorethamine HCl (mustargen) at nontoxic doses. We have also made determinations of the inhibitory capacities of several other drugs on the respiration rate of constituent cells of multicellular spheroids in single-cell suspensions. We have concluded that there are indeed hypoxic cells in spheroids whose radioresistance may be modified by essentially nontoxic levels of indirect radiosensitizer drugs and that the system described shows great promise for screening agents which may modify radiation response.

Animals↗
PubMed1981

Multicellular tumor spheroids of human colon carcinoma origin. Kinetic analysis of infiltration and in situ destruction in a xenogeneic (murine) host.

The relationship between destruction and concomitant host cell infiltration of human tumor xenografts has been quantitatively investigated by using the multicellular tumor spheroid model. Multicellular tumor spheroids of HT-29 human colon carcinoma cells were grown in vitro and subsequently implanted in the peritoneal cavity of BALB/c mice. At various times thereafter, spheroids were recovered and dissociated and their viability was quantitatively assessed by using a clonogenic assay. Little damage to spheroids was observed during the initial 4 days after implantation, but essentially complete destruction (greater than 99% reduction in clonogenic tumor cells) occurred between days 4 and 7. In parallel studies, host cell infiltration was assessed by light and electron microscopy both in situ on sections and on dissociated suspensions of spheroid cells. The data demonstrate the value of utilizing a model system in which both functional and morphological techniques can be combined in a quantitative assessment of the relationship between host cell infiltration and graft destruction in situ.

Animals↗
PubMed1994

Size-regulation and biochemical activities of the multicellular spheroid composed of rat liver cells.

It was reported that cell-sheets composed of rat liver cells could be obtained by using a collagen-conjugated thermo-responsive polymer, poly-N-isopropyl acrylamide, as a cell substratum and they were then able to transform into multicellular spheroids in the hydrophobic dish. In this study, we succeeded in easily obtaining the cell-sheets with the intended size by using the etched substratum. Biochemical analyses of spheroids derived from cell-sheets with areas of 0.2, 0.5 and 2.5 cm2 were carried out. Each cell-sheet rapidly aggregated at the same rate and finally turned into a multicellular spheroid within 5 days after the detachment. However, both decreases of DNA and LDH contents in cell-sheets with an area of 2.5 cm2 within 24 hr after the detachment were more obvious than in the others, respectively. It is tempting to speculate from these data that the vital spheroids with the desired size can be easily obtained by using our new method.

Acrylamides↗
PubMedDate not supplied

Growth characteristics of murine B16 melanoma multicellular spheroids: a model for invasion and effects of doxorubicin treatments.

Multicellular spheroids, derived from murine B16 melanoma cells, showed unique growth characteristics: when they reached about 500 microns in diameter, their morphology changed rapidly and they became amoeba-like irregular-shaped aggregates. This morphological characteristic closely resembled that of invasive cancer, and may serve as a model for local invasion. To test the possibility that the changes mentioned above can be inhibited by a drug, spheroids were treated with 0.8 microgram/ml of doxorubicin for one hour and their morphology was observed temporally. Although this concentration of the drug decreased the survival of the melanoma cells in monolayer to about 10(-3), the growth was not delayed nor were the "invasion"-like changes inhibited in the spheroids. We believe this system of multicellular spheroids is a useful model to study the mechanisms of tumour invasion, although doxorubicin could not inhibit "invasion"-like changes in this system.

Animals↗
PubMed1995

Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker.

We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide], a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitroimidazole and produce immunologically detectable adducts of the EF5 compound. Northern blotting analyses demonstrated that VEGF165 is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression. This study demonstrates that VEGF expression is up-regulated in response to hypoxia and in the microenvironments found in human multicellular tumor spheroids. This investigation also illustrates the utility of the EF5 binding in multi-cellular tumor spheroids as a means of studying the expression and regulation of hypoxia-inducible genes.

Carcinoma↗
PubMedDate not supplied

Induction of cell death by Doxorubicin in multicellular spheroids as studied by confocal laser scanning microscopy.

In the present study the effects of the anticancer drug Doxorubicin (Dox) on necrosis development and cell lethality of multicellular DU-145 spheroids (MCS) were examined. Multicellular spheroids consist of a peripheral rim of proliferating cells, a inner shell of nonproliferating, quiescent cells and a central core of dead cells. After the application of Dox for different time periods dead cell areas and single dead cells in MCS of different size classes were identified using a set of lethal fluorescence dyes, and a confocal laser scanning microscope (CLSM). The distribution of Dox within MCS was examined by determining Dox fluorescence in single cells and cell areas. Outgrowth experiments were performed to show the effects of Dox on cancer cell migration and cell proliferation. The application of low (400 nM) concentrations of Dox over a time period of 2hours resulted in distinct Dox fluorescence staining of the most peripheral cell layers of the MCS. After long term incubation (48hours) cell lethality was most prominent in large spheroids (diameter between 350 and 800 micron) which possess a dead cell core and single dead cells at the periphery. These MCS showed an approximately 120 microm +/- 30 microm increased dead cell core as compared to control MCS. The cytotoxic effect of Dox was lower in MCS of a diameter between 150-350 microm and nearly no cytotoxic effects were found in spheroids smaller than 150 microm in diameter. Dox fluorescence persisted in dead cells for at least three days. During this time the cytotoxic agent leaked slowly from dead cells and penetrated into the layers of quiescent cells and proliferating cells mediating a prolonged cytotoxicity. In conclusion, the most efficient cytotoxic effect on MCS larger than 150 microm in diameter, can be achieved using a Dox concentration of 400 nM, and applying the drug for long incubation periods to allow its accumulation and storage in the dead cell core and in the single dead cells within vital cell layers. Dox is gradually delivered from these storage sites and kills proliferating and quiescent cells when no Dox is present in the external medium.

Antibiotics, Antineoplastic↗
PubMed1996

Multicellular membranes as an in vitro model for extravascular diffusion in tumours.

Efficient extravascular diffusion is a critical requirement for hypoxic cell radiosensitisers, bioreductive drugs and hypoxic cell markers that must reach cells distant from functional blood vessels in tumours. The diffusion of simple nitroimidazoles with neutral (misonidazole, miso) and basic (pimonidazole, pimo) side chains, as well as 2-nitroimidazoles with acridine (NLA-1) and phenanthridine (2-NLP-3) DNA intercalating moieties was investigated using multicellular membranes (MMs), a new in vitro model for the extravascular compartment of tumours. The diffusion of miso through V79 MMs was concentration independent over the range 0.1-10 mM. Mathematical modelling of the flux kinetics provided a diffusion coefficient in MMs (DMM) of 5.5 x 10(-7) cm2 s-1 which was approximately 13-fold lower than in culture medium. Flux was little affected by the extent of hypoxia in MMs, indicating that hypoxic metabolism does not compromise diffusion of miso over distances in the order of 200 microns. The DMM for pimo was similar to miso, while those for 2-NLP-3 and NLA-1 were both lower. The results demonstrate compromised extravascular diffusion for DNA-intercalating nitroimidazoles, but indicate that this problem is more severe for the basic acridine derivative, NLA-1, than for the phenanthridine, 2-NLP-3. The MM model appears to be well suited to quantitative determination of drug diffusion in a multicellular environment.

Acridines↗
PubMed1996

Apoptosis regulated by growth factors in multicellular spheroids of trabecular cells.

Bovine and porcine trabecular cells were incubated up to one month in a polystyrene dish nonadherent environment for suspension culture, and their morphological changes were examined by light and electron microscopy. The trabecular cells formed multicellular spheroids during the first day. Apoptosis occurred selectively and continuously in the spheroids, which had initially been filled with cells. After one month, the final structure of the spheroids had become a meshwork of cells separated by large extracellular spaces. Epidermal and basic fibroblast growth factors prevented apoptosis of the trabecular cells in the spheroids which remained filled with cells for the entire observation period. The trabecular multicellular spheroids may represent the in vivo process of trabecular meshwork formation and can be used as a system for identifying regulatory factors involved in this process.

Animals↗
PubMed1996

Non-specific pinocytosis by human endothelial cells cultured as multicellular aggregates: uptake of lucifer yellow and horse radish peroxidase.

We have analyzed the pattern of time-dependent and concentration-dependent incorporation of Lucifer Yellow CH (LY) and Horseradish Peroxidase (HRP) by human umbilical vein endothelial cells cultured on a non-adhesive substratum, where they they become organized into stable, multicellular aggregates. The data were compared with those previously obtained from low-density cultures of non-growing endothelial cells adherent to plastic. While the linear trend of the incorporation kinetics is preserved, the rate of uptake with both time and concentrations is highly dependent on the culture conditions, namely typology of cell-cell and cell-substrate interactions. An at least two-fold increase of the rate of uptake was observed with both markers in the aggregated cells. The extracellular concentration of LY required to saturate the binding capacity of the cell surface shifts from approximately 0.25 mg/ml, with the adherent cells, to approximately 0.5 mg/ml in the aggregated cells; the rate of uptake of three different forms of HRP shows, besides a sharp quantitative increase, also qualitative variations, testified by differential changes of their incorporation rates. These results are entirely consistent with the assumption that the association of the endothelial cells into multicellular aggregates increases the rate of pinocytic uptake by modifying the physicochemical properties of the cell surface, thereby increasing its differential affinity for the extracellular markers.

Biological Transport↗
PubMed1996

[Biochemistry of space and time (telomeres, telomerases, and longevity of cell populations and multicellular organisms].

Analysis of recent data on telomeres and telomerases, limitations of proliferative potential of untransformed cells (Hayflick's limit), kinetics and molecular mechanism of cell proliferation and differentiation, and factors that determine the longevity of multicellular organisms suggests that: (i) chromosome end shortening during consecutive divisions of cells that lack telomerase limits cell proliferation beyond Hayflick's limit; (ii) Hayflick's limit can be a manifestation of mechanisms that determine the size of the living organism (it correlates with longevity) but not the longevity of multicellular organisms.

Animals↗
PubMed1977

A simplified method for production and growth of multicellular tumor spheroids.

A new technique, based on the growth of tumor cells in liquid media over an agar base, has been developed for the formation and growth of multicellular tumor spheroids. All of the 11 transformed cell lines tested formed multicellular tumor spheroids, while none of the 8 normal cell types tested did so. The advantages of the present technique over older methods include its simplicity, generality, and experimental flexibility.

Agar↗
PubMed1998

von Hippel-Lindau gene-mediated growth suppression and induction of differentiation in renal cell carcinoma cells grown as multicellular tumor spheroids.

Previous results using gene transfection methods have shown that the wild-type (WT) von Hippel-Lindau (VHL) gene can function as a potent tumor suppressor gene in vivo for renal cell carcinoma (RCC) cells in the absence of any suppressive effect on cell growth in monolayer cell culture under serum-rich conditions. Because we had previously found that the function of some oncogenes, such as mutant ras, can be influenced by three-dimensional growth as multicellular spheroids (J. Rak et aL, J. Cell Biol., 131: 1587-1598, 1995), we reasoned the same might be true for suppressor genes as well. We, therefore, decided to compare and study the effects of the WT VHL gene in monolayer versus three-dimensional culture systems of the RCC cell line 786-0, which contains an inactivated VHL gene. We found that the reintroduction of the WT VHL gene into mutant VHL RCC cells resulted in growth suppression in vitro, but only when the cells were grown as spheroid cultures. This decrease in cell proliferation was associated with several features of cell differentiation/morphogenesis, as shown by light and electron microscopy. Thus, in contrast to cultures of mutant VHL RCC cells, which formed very compact and cohesive spheroids, the WT VHL transfectants were loosely arranged and formed a network of tubular and trabecular structures within the spheroids. The morphological changes of the WT VHL spheroids were associated with the deposition of fibronectin in the extracellular space, a feature that was absent in the mutant and inactivated VHL gene-expressing spheroids. The results suggest the VHL gene may be involved in the maintenance of the epithelial phenotype of renal tubular cells, ie., it may act as a differentiation/morphogenetic factor. Moreover, this effect in tumors cells appears to be highly dependent on multicellular growth conditions that mimic the basic nature of solid tumors, such as RCC.

Animals↗
PubMedDate not supplied

Cell culture as spheroids: an approach to multicellular resistance.

Cells cultured as spheroids present an heterogeneity similar to that of tumours in vivo. In the spheroid peripheral layers, cells are proliferating, deeper cells are non-cycling, when in the aggregate centre, cells form often a necrotic core. A multicellular resistance is related on the cell contact to other cells or to the extracellular matrix. The mechanism of this resistance remains unknown. It seems to be linked to the spheroid centre hypoxia, quiescence of a large fraction of the cell population and to the apoptose inhibition. The "classical" or "unicellular" mechanisms of resistance, as mdr1, MRP, can coexist but are not responsible of this type of resistance. This culture model is a good opportunity to study a resistance which looks close to the patient tumour resistance. A new class of therapeutic molecules appears that can reverse multicellular resistance, inhibit tumours growth and preclude metastases. The mechanism of action of this new pharmacological class is the disruption of the cell adhesion forces.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2026

Field strains of the unicellular alga Chlamydomonas reinhardtii exhibit multicellular characteristics that shape their interactions.

Chlamydomonas reinhardtii is a unicellular green alga long studied as a biological model system but rarely considered from the perspective of its own ecology, thus epitomizing the disconnection between reductionist biology in the laboratory and life in nature. Here, we present insights into its ecology, understood from field strains. We examined bacterial communities that coenriched with C. reinhardtii from the field, revealing specific associations. We then compared the biology of C. reinhardtii field strains to laboratory strains, illuminating strain-level heterogeneity and adaptations to life in the field vs. the laboratory. Field strains exhibited more robust photosynthesis, higher abundances of pherophorin proteins, a propensity for palmelloid formation, and high cell wall permeability. Finally, we phenotyped cocultures of C. reinhardtii with a coenriched bacterial partner, demonstrating how differences between field and laboratory strains manifest in biotic interactions. Although the organisms in question are classically understood as unicellular, our observations of field strains highlighted their participation in multicellular units, challenging the utility of unicellular frameworks in extending our knowledge of model organism biology in the laboratory towards understanding microbial ecology.

Chlamydomonas reinhardtii↗
PubMed1988

Effects of cisplatin plus fluorouracil vs cisplatin plus cytarabine on head and neck squamous multicellular tumor spheroids.

We compared the efficacy of cisplatin plus fluorouracil vs cisplatin plus cytarabine against HEp-2 head and neck carcinoma cells in monolayer and multicellular tumor spheroid (MTS) systems. Increases in exposure time to cisplatin and fluorouracil from one to 24 hours resulted in approximately tenfold and 1000-fold increases, respectively, in cell lethality for both monolayer and MTS cells. Dose-response curves for cisplatin or fluorouracil on MTS cells closely followed those from monolayer cells, indicating good drug penetration into the MTS core. In contrast, dose-response curves on MTS cells after 24-hour exposure to cisplatin and cytarabine showed progressively lesser efficacy at higher drug concentrations. For monolayer cells, cisplatin plus fluorouracil and cisplatin plus cytarabine were both synergistic, the latter combination more synergistic than the former. For MTS cells, both combinations again showed synergistic interaction at moderate to high effect levels. Heightened synergistic interaction was demonstrated especially with the cisplatin plus cytarabine combination. Thus, the cisplatin plus cytarabine combination was always more synergistic than cisplatin plus fluorouracil. These data may serve as a basis for additional clinical trials of cisplatin plus cytarabine in the treatment of patients with head and neck carcinoma.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

Doxorubicin distribution in multicellular prostate cancer spheroids evaluated by confocal laser scanning microscopy and the "optical probe technique".

Multicell-mediated drug resistance is a major impediment for the effectiveness of chemotherapeutic approaches and has been shown to be a feature of many solid tumors. We used confocal laser scanning microscopy to evaluate the depth distribution of the fluorescent cytostatic drug doxorubicin (Dox) in two size classes of multicellular cancer spheroids (MCS) (psi150+/-50 microm and 350+/-50 microm). MCS (psi150+/-50 microm) solely consist of proliferating cells, whereas in MCS (psi350+/-50 microm) peripheral proliferating cell layers are followed in the depth of the tissue by drug resistant quiescent cell areas. A technique was developed which allows noninvasively to trace fluorescence distributions down to a depth of approximately 180 microm in living MCS. This was achieved by confocal radial recordings of the mean Dox fluorescence in 600 microm2 regions of interest (ROI), equidistantly spaced (10 microm) from the center of MCS towards their periphery. The resulting fluorescence intensity profiles were subsequently corrected for absorbtion and light scattering in the depth of the tissue by a convenient algorithm. A 10 min incubation of MCS (psi150+/-50 microm) with Dox (10 microM) led to a peripheral accumulation, after 2 h Dox was homogeneously distributed within the whole MCS. In contrast, after Dox treatment of MCS (psi350+/-50 microm) for 2 h, the drug was accumulated within the peripheral proliferating cell rim of 78+/-8 microm, whereas deeper, quiescent cell layers remained unstained. When MCS were incubated with verapamil, cyclosporin A, orthovanadate, and quinidine, which are known to reverse P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), Dox accumulated also in deeper cell layers. Genistein and indometacin which reverse multidrug resistance mediated by the multidrug resistance-associated protein (MRP) were without effects. The optical probe technique proved to be well suited to study MDR in a living three dimensional tissue context.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Decreased mitochondrial function in quiescent cells isolated from multicellular tumor spheroids.

Cells in the inner region of multicellular spheroids markedly reduce their oxygen consumption rate, presumably in response to their stressful microenvironment. To determine the mechanism behind this metabolic adaptation, we have investigated relative mitochondrial mass and mitochondrial function in cells isolated from different regions of tumor spheroids by using a combination of mitochondrial-specific fluorescent stains and flow cytometric analysis. Uptake of rhodamine 123 (R123) is driven by the mitochondrial membrane potential and thus reflects mitochondrial activity. Uptake of 10-nonyl-acridine orange (NAO) reflects total mitochondrial mass independently of activity because this compound binds to cardiolipin in the inner mitochondrial membrane. NAO fluorescence per unit cell volume only decreased 10-20% for cells from the inner spheroid region compared with those near the surface. There was greater than a twofold reduction in R123 fluorescence in the inner region cells, however. Thus, tumor cells in spheroids alter their rate of respiration predominately by downregulating mitochondrial function as opposed to degradation of mitochondria. There was a correlation between R123 staining per unit cell volume and the growth fraction of the cells from spheroids, but not for monolayer cultures. We also show a linear correlation between R123 staining and the rate of oxygen consumption for both monolayer- and spheroid-derived cells. After separating the inner region cells from the spheroid and replating them in monolayer culture, the R123 uptake recovered to normal levels prior to entry of the cells into S-phase. This reduction in mitochondrial function in quiescent cells from spheroids can explain the long period required for these cells to re-enter the cell cycle and may have important implications for the regulation of tumor cell oxygenation in vivo.

Aminoacridines↗
PubMed2006

Three-dimensional microcomputed tomography imaging of basic multicellular unit-related resorption spaces in human cortical bone.

This study employed microcomputed tomography (micro-CT) as a novel means for visualizing the morphology and quantifying the range (length) of basic multicellular unit (BMU)-related resorption spaces in human cortical bone. We tested the hypotheses that the density and range of spaces vary with age and sex. The sample included 82 human (18-92 years) anterior femoral midshaft samples. The morphology of the spaces (n = 99) was varied, including unidirectional, bidirectional, branched, and even highly clustered forms. The density of resorption spaces was negatively correlated with age for the combined sexes and females, with Spearman's rho values of -0.355 (P < 0.001) and -0.522 (P = 0.002), respectively. The density of spaces did not differ significantly between the sexes (P = 0.735). Mean range +/- SD for the combined sexes, females, and males was 2,706 +/- 1,177, 2,681 +/- 1,247, and 2,718 +/- 1,150 microm, respectively. Numerical simulation of the effect of the 7,000 microm scan field of view suggested that the actual mean range of the spaces for the pooled sample was actually on the order of 3,770 microm. Range did not correlate significantly with age for the combined sexes (P = 0.587) or females (P = 0.345) and males (P = 0.896) considered separately and was not significantly different (P = 0.883) between the sexes. These results suggest that the range of BMUs is not affected by age. The age-dependent decrease in resorption space density for the females and pooled sexes was most likely a consequence of cortical rarefaction, leading to difficulty detecting resorption spaces with micro-CT, rather than a decrease in overall remodeling activity.

Adolescent↗