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PubMed1995

Abnormal hepatic copper accumulation of spheroid composed of liver cells from LEC rats in vitro.

The LEC rat is a mutant strain displaying hereditary hepatitis, and shows abnormal accumulation of copper (Cu) similar to that occurring in Wilson's disease. We prepared a multicellular spheroid composed of LEC rat liver cells to investigate the mechanism for abnormal accumulation of Cu. These multicellular spheroids were prepared by detaching the monolayer on the collagen-conjugated thermo-responsive polymer coated culture dish at a temperature below the critical solution temperature and culturing on the non-adhesive substratum. Long-term cultured spheroids of LEC rat liver cells as well as SD rat liver cells were attempted. Non-parenchymal cells obtained by collagenase perfusion from the LEC liver were fewer than those from the SD liver. Cells from the LEC rat, over 11 weeks of age, did not form a cell sheet; however, a mixture of parenchymal cells from LEC rats over aged 11 weeks and non-parenchymal cells from SD rats of any age yielded intact spheroids. We examined the toxicity, the accumulation and distribution of Cu in spheroids. The accumulation of Cu in LEC spheroids was higher than that in SD spheroids. Results suggest that spheroids consisting of LEC liver cells are useful as an alternative model to in vivo tests to investigate the mechanism for abnormal accumulation of Cu in liver.

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↗
PubMed1998

Differential regulation of cyclin-dependent kinase inhibitors in monolayer and spheroid cultures of tumorigenic and nontumorigenic fibroblasts.

The Ras proto-oncogene has been implicated in the in vivo development of tumors and in the in vitro transformation of cultured cell lines. In both of these conditions, Ras-mediated disruption of cell cycle-regulatory mechanisms leads to unregulated cellular proliferation, although the exact mechanisms by which Ras accomplishes this are not clear. Using as a model the M1 and MR1 rat fibroblast cell lines, which differ in the expression of a regulated Ras (M1 cells) versus a constitutively active Ras (MR1 cells), we examined the role of Ras in the control of cellular proliferation in two-dimensional (monolayer) and three-dimensional (spheroid) cell cultures. These cell lines are very similar in their monolayer growth characteristics, but M1 cells will arrest their cell cycle progression in aggregate culture, whereas MR1 cells proliferate normally as small spheroids. We report here that G1-phase arrest in plateau-phase monolayer cultures of both M1 and MR1 cells correlates with up-regulated expression of the cyclin-dependent kinase (CDK) inhibitor p18INK4c. Enhanced p18INK4c expression was also observed in G1-arrested M1 cells cultured as multicellular spheroids but was not induced in small proliferating MR1 multicellular spheroids. The kinetics of G1 arrest in M1 cells after inoculation into aggregate culture correlated well with the induction of p18INK4c expression. Conversely, resumption of proliferation in monolayer culture of arrested M1 cells isolated from spheroids coincided with the loss of expression of p18INK4c. After extended culture, cells in the inner region of MR1 spheroids arrested in the G1 phase without any up-regulation of p18INK4c expression. In this case, the CDK inhibitor p21(Cip1/Waf1) was selectively induced in the inner regions of large MR1 spheroids, concomitant with a decrease in cyclin and CDK expression. Thus, Ras-dependent regulation of p18INK4c expression seems to control the ability of rat embryo fibroblasts to proliferate as small multicellular aggregates, whereas p21(Cip1/Waf1) expression seems to regulate the G1-phase arrest induced by the stressful microenvironment found within the inner region of large spheroids.

Animals↗
PubMed2000

Continuum Field Charging of Dielectric Spheroids.

Classical continuum theory for field charging is applied in an analysis of the ionic charging of spheroidal dielectrics. Assuming that the particle orientation is fixed during the charging process, the saturation charge and charging rate are determined as functions of the orientation and aspect ratio of spheroids. For spheroids of small dielectric constants the saturation charge becomes the largest when the electric field is directed perpendicular to the major axis of the spheroid. For an ensemble of randomly oriented spheroids the average saturation charge can be approximated as the arithmetic average of the saturation charges for the spheroid with the electric field directed along the three principal axes of the spheroid. In addition, the ensemble average of the dimensionless charging rate of randomly oriented spheroids of moderate axial ratio approximates the dimensionless charging rate of a sphere. Copyright 2000 Academic Press.

Journal Article↗
PubMed2002

Inhibition of vascular endothelial growth factor-associated tyrosine kinase activity with SU5416 blocks sprouting in the microvascular endothelial cell spheroid model of angiogenesis.

The angiogenic vascular endothelial growth factor (VEGF) is believed to play a critical role in endothelial cell proliferation, differentiation, and sprouting. Small molecules that selectively inhibit the VEGF receptor-associated tyrosine kinase activities of Flk-1 (KDR) and Flt-1 have been developed. These agents, a prototype being SU5416, have effects on the proliferation of cultured endothelial cells, constrain angiogenesis in vivo, and have been proposed as antitumor drugs. Although SU5416 inhibits in vivo angiogenesis, it is not clear which of the complex processes leading to angiogenesis are impacted by VEGF receptor-associated tyrosine kinase inhibition. We utilized SU5416 and a microvascular endothelial cell line derived from mouse heart (SMHEC4) to specifically examine the role of VEGF receptor-associated tyrosine kinase activity on in vitro models of angiogenesis. We characterized spheroid formation and sprouting, a new model of angiogenesis, in this stable cell line. SU5416 inhibits (approximately 50%) VEGF (50 ng/ml) stimulated and basal DNA synthesis of SMHEC4 cultured in monolayer. SU5416 does not prevent the aggregation and organization of SMHEC4 into tri-dimensional spheroids. CD31, a marker of differentiated endothelial cells, is negligibly expressed in monolayer cultures but highly expressed in SMHEC4 spheroids. The content and biochemical characteristics of spheroidal CD31 are unaltered by SU5416. SU5416 also does not prevent the spontaneous and rapid (approximately 3-h) alignment into cords by SMHEC4 on Matrigel. These two models suggest that the organization and differentiation of endothelial cells is independent of VEGF receptor-associated tyrosine kinase signaling. SMHEC4 spheroids embedded in collagen gels spontaneously and rapidly (approximately 6 h) sprout capillary-like projections and subsequently (1-2 days) form complex self-anastomosing networks. In addition, VEGF (50 ng/ml) markedly stimulates sprouting of capillary-like projections from SMHEC4 spheroids. Both the spontaneous and the VEGF-stimulated sprouting are nearly eliminated by SU5416. This demonstrates that VEGF receptor-associated tyrosine kinase activity is essential to the formation of capillary-like structures from SMHEC4 spheroids. Overall, these observations demonstrate that (a) the spheroid sprouting model is appropriate for the study of angiogenesis since it appears to recapitulate many of its steps and (b) SU5416 can inhibit endothelial cell proliferation and sprouting without impacting the organization and differentiation of endothelial cells.

Angiogenesis Inhibitors↗
PubMed1987

A comparison of adriamycin and mAMSA. II. Studies with V79 and human tumour multicellular spheroids.

Multicellular spheroids were used to compare the two chemotherapeutic agents adriamycin (ADM) and 4'[(9-acridinyl)-amino] methanesulphon-m-anisidide (mAMSA). Chinese hamster cells, V79 379A, a human small cell lung carcinoma, designated ME/MAR, and a human melanoma xenograft, HX117, were grown as spheroids (200 or 400 micron in diameter) and treated with either drug for 1 h, at 37 degrees C, in air. Cytotoxicity was assayed using both cell survival and growth delay. Both drugs were highly toxic towards V79 but showed less activity toward the human tumour single cell suspensions; ADM was more effective towards HX117 and ME/MAR than mAMSA. When grown as spheroids, the cells developed marked resistance to both drugs. In all cases, cytotoxicity was drug dose and spheroid size dependent. The response of HX117 spheroids to both drugs was similar. In contrast, ADM was more effective toward 200 micron diameter ME/MAR spheroids, and mAMSA showed greater activity than ADM against V79 spheroids. Both endpoints gave qualitatively equivalent results, and a comparison of the two showed relatively long growth delays for a given level of cell kill, for both drugs and with all three cell lines. The greater cytotoxicity of ADM toward ME/MAR spheroids is consistent with the clinical finding that ADM has a use in the treatment of small cell carcinoma of the lung, while mAMSA has not demonstrated any activity in the treatment of lung cancer.

Amsacrine↗
PubMed1994

Destruction of WiDr multicellular tumor spheroids with the novel thymidylate synthase inhibitor 1843U89 at physiological thymidine concentrations.

The activity of a novel thymidylate synthase inhibitor, 1843U89, against WiDr human colon carcinoma multicellular tumor spheroids was investigated. Continuous exposure of the spheroids to 3 nM 1843U89 for 10 days resulted in spheroid disruption, whereas 100 nM methotrexate (MTX) was required for similar effects. Short-term treatment experiments demonstrated that a 3-day exposure to 100 nM 1843U89 caused spheroid disruption 9 days after drug removal. A 4-day exposure to 10 nM 1843U89 caused spheroid disruption 8 days after drug removal. In contrast, treatment with 10 or 100 nM 1843U89 for 6-48 h or treatment with 1 nM 1843U89 for up to 5 days caused only growth delay. Continuous exposure of spheroids to 30 nM 1843U89 in the presence of 0.05-0.3 microM thymidine was as effective in causing spheroid disruption as treatment in the absence of thymidine, but treatment in the presence of 0.7-3.0 microM thymidine caused partial reversal of spheroid disruption. The results of these experiments suggest that 1843U89 should have potent solid tumor activity in humans but should be less effective in mice due to differences in circulating thymidine levels (0.1 vs 1 microM, respectively).

Antineoplastic Agents↗
PubMedDate not supplied

A simplified method for the culturing of primary adult rat and human hepatocytes as multicellular spheroids.

A simple and highly reproducible method was established for the culturing of adult rat and human hepatocytes as multicellular aggregates (spheroids). Purified rat and human liver parenchymal cells were cultured on nontissue culture (bacteriological) polystyrene petri dishes on a rotating platform. After an overnight incubation, the cells were found to form multicellular aggregates. The aggregates became spheroidal in shape after several days in culture. Histological sections of the spheroids showed an organized structure consisting of squamated cells on the outermost layer and cuboidal cells in the interior. Cellular structures characteristic of hepatocytes in the liver in vivo including bile canaliculi, peroxisomes, Golgi bodies, abundant mitochondria, and rough and smooth endoplasmic reticulum were observed with electron microscopy. The spheroids were found to be viable up to the longest time studied of approx. 1 month in culture as demonstrated by their adherence and growth on collagen-coated substratum. The morphological resemblance between hepatocytes cultured as spheroids and the liver in vivo suggests that the spheroids may be a useful in vitro experimental model of the liver. Our simple method should allow hepatocytes to be cultured as spheroids easily in any laboratory equipped for cell culture. Our study here also is the first to report the culturing of human hepatocytes as spheroids.

Animals↗
PubMed2000

Adenovirus-mediated viral IL-10 gene transfer prolongs survival of xenogeneic spheroidal aggregate-cultured hepatocytes.

Xenotransplantation of hepatocytes appears to be a novel promising therapy for some forms of liver disease, and may well overcome the problem of donor shortage. We have previously reported that hepatocytes with a spheroidal shape (spheroids) are ideal for cell transplantation. The application of gene transfer techniques to this hepatocyte transplantation could possibly regulate the xenogeneic rejection reaction and, therefore, result in prolongation of the survival of the transplanted hepatocytes. In this study, we chose the adenovirus as a vector and an immunosuppressive cytokine named viral IL-10 (vIL-10) for transfection. A series of experiments was performed to elucidate the efficacy of transfection to the spheroids with adenovirus vectors and the effect of transfected vIL-10 on the survival of xenogeneic hepatocytes. We examined the cell survival quantitatively by evaluating beta-galactosidase (beta-gal) activity, which was transfected into the hepatocytes in the xenogeneic spleen, and semiquantitatively by the histological findings. The results of in-vitro studies identified an efficient expression of the beta-gal gene within the spheroids infected with Ad-CMVLacZ (LacZ-encoding adenovirus vector with CMV promotor) and the presence of BCRF1 mRNA within the spheroids transfected with AdCMVvIL-10 (vIL-10-expressing adenovirus vector with CMV promotor) under the condition of 1 MOI, for 1 h. Xenogeneic hepatocytes with a spheroidal shape showed comparable survival to syngeneic hepatocytes for up to 4 days after transplantation with co-transplantation of the vIL-10-transfected hepatocytes. From this study, we concluded that adenovirus-mediated vIL-10 gene transfer prolongs the survival of xenogeneic hepatocyte spheroids. Furthermore, spheroids possess ideal properties for gene transfection, as well as cell transplantation.

Adenoviridae↗
PubMed1990

Long-term culture of rat liver cell spheroids in hormonally defined media.

Liver cells of new-born rats, which were found to be able to form spheroidal aggregates when cultured on a nonadherent plastic substratum, were studied under various conditions of culture, mainly by adding different nutrients and growth factors to the culture medium. Analysis of hepatocyte-specific functions was carried out by immunoprecipitation to detect specific proteins newly secreted by liver cell spheroids on different days of culture. When no supplement was added to culture medium, the secretion of albumin and transferrin by liver cell spheroids was no longer detectable after 2 weeks of culture. When dexamethasone, glucagon, insulin, and EGF were added to culture medium, the secretion of albumin and transferrin remained detectable at least until 60 days of culture. This was even more striking when trace elements were added in addition to the three hormones and EGF. The effects of addition of these various factors to culture medium were also detectable with respect to alpha-FP secretion. Even after 54 days of culture in total supplemented medium, these liver cell spheroids could be transferred on a collagen-coated plastic substratum to form a monolayer of uniform liver parenchyma-like cells. The presence of extracellular matrix-like material was observed on the surface of cell spheroids. This could be responsible for attachment and fusion between cell spheroids. Thus, liver cell spheroids cultured in total supplemented medium ensured cell attachment to a biological matrix and cell-cell contact, which is thought to help maintain cell differentiation. Liver cell spheroids offer the possibility of toxicological and pharmacological studies as well as cultures in biomatrix and coculture systems. In addition these liver cells can be used for experiments in liver cell transplantation.

Animals↗
PubMed1989

Misonidazole chemosensitization of EMT6 spheroids to melphalan.

Nitroaromatic radiosensitizers are effective chemosensitizers in vitro and in vivo. We have used EMT6 tumour cells grown as multicellular spheroids to try and further understand the role that hypoxia plays in this process. Our results show that the cell killing produced in whole spheroids following a 1-h exposure to melphalan (L-PAM) was enhanced by a 3-h pre-exposure to 5 mM misonidazole (MISO), an enhancement ratio of 1.3-1.7 being obtained. Sequentially disaggregating spheroids we also found that both L-PAM toxicity and MISO chemosensitization were relatively constant as a function of depth within the spheroid. The binding of 14C-MISO to spheroid cells, measured by scintillation counting of disaggregated cells and by autoradiography analysis of sectioned spheroids, demonstrated that binding increased with depth. However, cells in the outer layers of the spheroid bound more 14C-MISO than expected with fully aerobic cells, while in the innermost viable cells the binding was less than that measured in cells which were fully radiobiologically hypoxic. This suggests that the majority of viable spheroid cells were at oxygen tensions intermediate between those found in either fully aerobic or radiobiologically hypoxic cells, yet their levels of oxygenation were sufficiently low for MISO chemosensitization to occur.

Animals↗
PubMed1988

Effects of changes in oxygen tension, pH, and glucose concentration on the response to CCNU of EMT6 mouse tumor monolayer cells and multicellular spheroids.

We have studied the effect of individually changing medium glucose content, pH and oxygen tension upon the response to CCNU (1-(2-chloroethyl)3-cyclohexyl-1-nitrosourea) of EMT6/Ca/VJAC cells grown as early plateau phase monolayer cultures or small (200 microns diameter) spheroids. The effect of changing all three factors together has also been studied in the spheroid model. All the changes in medium conditions (except for 4 hr hypoxia) were maintained for 24 hr prior to drug exposure. Plating efficiency (PE) of monolayer cells was decreased by reduced medium pH (below 6.5) or oxygen tension while no change in PE was brought about by reduced medium glucose content. In small spheroids reduction in PE caused by low pH was similar to that seen in monolayer, there was again no effect of reduced glucose, and the effect of hypoxia was clearly less than in monolayer. Combination treatment of spheroids (pH 6.5, 120 mg/l glucose and hypoxia) reduced the PE of spheroid cells to 50% of control. Reducing medium glucose content from 920 to 0 mg/l, or oxygen tension from 20% to near zero (for either 4 or 24 hr) reduced the sensitivity of monolayer cells to CCNU. A similar pattern was seen for reducing medium pH from 7.2 to 6.1 during the 24 hr pre-incubation period and 1 hr drug exposure period. A reverse trend was, however, seen if medium pH during the drug exposure period was maintained at 7.2 following reduced pH pre-incubation. Reduced sensitivity to CCNU was seen for cells within small spheroids pre-incubated in medium at low pH (for both schedules) or under hypoxia (for either 4 or 24 hr) whereas reduced medium glucose content appeared to have no such effect. Cells in small spheroids after 24 hr combination treatment were also less sensitive to CCNU than cells from control spheroids.

Anaerobiosis↗
PubMed1990

Radiosensitivity of human melanoma spheroids influenced by growth rate.

The radiosensitivity of human melanoma cell line BRO was investigated using the multicellular tumor spheroid system. By adding different concentrations of bovine serum to the tissue culture medium, two different growth rates could be obtained. Spheroids (200-250 microns) were irradiated with graded single doses of X rays (2-8 Gy). The radiation response was quantified using specific growth delay, clonogenic cell survival, and spheroid cure. All three assays showed a growth rate dependent radiation response. At both growth rates the spheroid growth fraction and critical cell number were of comparable magnitude. There was a strong correlation between the radiation response of spheroid regenerating units and clonogenic cells from dispersed spheroids. Cell survival curves indicated a decreased ability to accumulate sublethal damage in fast growing multicellular tumor spheroids. From this study it appears that the intrinsic radiosensitivity of human melanoma cell line BRO cells in multicellular spheroids is modulated by intratumoral conditions.

Cell Aggregation↗
PubMed1993

Radioresponsiveness of human glioma, sarcoma, and breast cancer spheroids depends on tumor differentiation.

PURPOSE: Differences in the intrinsic radiosensitivity within and between different tumor classes have been noticed for human tumor cell lines using the clonogenic assay. By far, most of the cell lines studied up to now were derived from poorly differentiated tumors. In this study, the influence of tumor differentiation on the radiation doses necessary to control 50% of small oxic spheroids (SCD50) was determined. Evidence of a distinct dependence of radioresponsiveness on tumor progression provides a background for an investigation of the underlying mechanisms. METHODS AND MATERIALS: Spheroids were aggregated from 1000-1500 cells in agarose coated 24 multi-well plates. Their diameters ranged from 156 to 405 microns, depending on the cell line. Spheroids were irradiated with graded 60Co single doses using spheroid control as end point and a minimum follow-up period of 3 months. RESULTS: Cell lines from three low grade gliomas and 10 malignant gliomas were studied in the spheroid control assay. The group mean SCD50 values were 6.1 +/- 1.6 Gy and 13.1 +/- 3.3 Gy, respectively. Four cell lines from grade 2 soft tissue sarcomas had a mean SCD50 value of 6.2 +/- 0.5 Gy and one undifferentiated sarcoma line of 11.0 Gy. Three well-differentiated breast cancer lines expressed the cell adhesion molecule E-cadherin, had an epithelioid morphology in monolayer culture, were estrogen receptor positive or contact inhibited in multicellular spheroids. Two undifferentiated breast cancer lines had a fibroblastoid morphology and were marker negative. The mean SCD50 value of the former was 10.5 +/- 1.0 Gy while that of the undifferentiated lines was 14.8 +/- 2.8 Gy. Analysis of variance revealed a significant effect of the tumor type as well as the grade of dedifferentiation on the SCD50 after irradiation with one fraction or 2Gy/fraction. The surviving fractions at 2 Gy (SF2), obtained from the spheroid control rates after different fractionation schedules by approximation of the linear quadratic model assuming Poisson statistics were significantly dependent on tumor type (p = 0.001, ANOVA F-test) but not on tumor differentiation (p = 0.27). The alpha/beta ratios did not depend on tumor type (p = 0.08, ANOVA F-Test) but significantly increased with the grade of tumor cell dedifferentiation (p = 0.03). CONCLUSION: The spheroid model is suitable for measuring the radioresponsiveness of differentiated cell lines with very low colony forming efficiencies. Tumor cell differentiation is an important factor for the radioresponsiveness and recovery capacity of human tumor cells.

Breast Neoplasms↗
PubMed2000

Analytical description of transmembrane voltage induced by electric fields on spheroidal cells.

An analytical description of transmembrane voltage induced on spherical cells was determined in the 1950s, and the tools for numerical assessment of transmembrane voltage induced on spheroidal cells were developed in the 1970s. However, it has often been claimed that an analytical description is unattainable for spheroidal cells, while others have asserted that even if attainable, it does not befit the reality due to the nonuniform membrane thickness, which is unrealistic but inevitable in spheroidal geometry. In this paper we show that for all spheroidal cells, membrane thickness is irrelevant to the induced transmembrane voltage under the assumption of a nonconductive membrane, which was also applied in the derivation of Schwan's equation. We then derive the analytical description of transmembrane voltage induced on prolate and oblate spheroidal cells. The final result, which we cast from spheroidal into more familiar spherical coordinates, represents a generalization of Schwan's equation to all spheroidal cells (of which spherical cells are a special case). The obtained expression is easy to apply, and we give a simple example of such application. We conclude the study by analyzing the variation of induced transmembrane voltage as a spheroidal cell is stretched by the field, performing one study at a constant membrane surface area, and another at a constant cell volume.

Cell Membrane↗
PubMed1999

A polarization model overcoming the geometric restrictions of the laplace solution for spheroidal cells: obtaining new equations for field-induced forces and transmembrane potential.

We present a new model for a variety of electric polarization effects on oblate and prolate homogeneous and single-shell spheroids. For homogeneous spheroids the model is identical to the Laplace model. For single-shell spheres of cell-like geometry the calculated difference of the induced dipole moments is in the thousandths range. To solve Laplace's equation for nonspherical single-shell objects it is necessary to assume a confocal shell, which results in different cell membrane properties in the pole and equator regions, respectively. Our alternative model addresses this drawback. It assumes that the disturbance of the external field due to polarization may project into the medium to a characteristic distance, the influential radius. This parameter is related to the axis ratio of the spheroid over the depolarizing factors and allows us to determine the geometry for a finite resistor-capacitor model. From this model the potential at the spheroid's surface is obtained and, consequently, the local field inside a homogeneous spheroid is determined. In the single-shell case, this is the effective local field of an equivalent homogeneous spheroid. Finally, integration over the volume yields the frequency-dependent induced dipole moment. The resistor-capacitor approach allowed us to find simple equations for the critical and characteristic frequencies, force plateaus and peak heights of deformation, dielectrophoresis and electrorotation for homogeneous and single-shell spheroids, and a more generalized equation for the induced transmembrane potential of spheroidal cells.

Cell Membrane↗
PubMed1999

Redox-regulation of intrinsic prion expression in multicellular prostate tumor spheroids.

The cellular function of the intrinsic prion protein (PrPc) remains largely unknown. In the present study PrPc expression was investigated in multicellular prostate tumor spheroids and was correlated to the intracellular redox state as evaluated using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In small tumor spheroids (diameter 100 +/- 20 microm) reactive oxygen species (ROS) levels were increased as compared with large (diameter 250 +/- 50 microm) spheroids. ROS generation was mediated by the mitochondrial respiratory chain and a NADPH oxidaselike enzyme, because carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, and diphenylene iodonium chloride (DPI) significantly reduced ROS levels. The elevated ROS were correlated to an increased expression of PrPc, Cu/Zn superoxide dismutase (SOD-1), and catalase in small as compared with large spheroids. In large tumor spheroids, PrPc was predominantly expressed in the peripheral cell layers and colocalized with SOD-1 and catalase. Raising intracellular ROS in large tumor spheroids by hydrogen peroxide, menadione, buthionine sulfoximine (BSO), and incubation in glutamine-reduced medium increased PrPc expression. In small spheroids PrPc was downregulated after incubation with the radical scavengers dehydroascorbate (DHA) and vitamin E. Our data indicate that PrPc expression in tumor spheroids is related to the intracellular redox state and may participate in antioxidative defense.

Antioxidants↗
PubMed1981

Different sensitivity to cytotoxic agents of internal and external cells of spheroids composed of thioguanine-resistant and sensitive cells.

When thioguanine-resistant V79 cells are introduced into spinner flasks containing V79 multicell spheroids, the mutant cells attach to the surface of the spheroids. The composite spheroids thus formed consist of external, thioguanine-resistant (TGr) and internal, thioguanine-sensitive cells. Cell sorting with a Becton Dickinson FACS II was used to determine the relative position of TGr cells and sensitivity to fluorescent drugs. After 2-4 days, the TGr cells are found internally as well as externally. The initial percentage of TGr cells varies from 1 to 50%, depending on the size of the single-cell inoculum, size of spheroids and frequency of addition of cells during a 24th period. Differential effects of drugs or radiation on external (cycling, oxic) vs internal (non-cycling, hypoxic) cells of composite spheroids can be assayed by simply plating cells trypsinized from spheroids into standard medium or medium with 2 mu g/ml 6-thioguanine, which allows growth of only TGr cells. For example, Adriamycin, which penetrates poorly into spheroids, is preferentially toxic to external cells, and causes a decrease in the percentage of TGr survivors. Irradiation of composite spheroids also causes preferential killing of external (oxic) TGr cells. However, AF-2, a nitrofuran which preferentially kills hypoxic cells, caused an increase in the 0/0 of TGr cells. This technique should prove useful in the evaluation of the preferential cytotoxicity of chemotherapeutic agents, alone or in combination.

Animals↗