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PubMed2002

Rho family small GTPases control migration of hematopoietic progenitor cells into multicellular spheroids of bone marrow stroma cells.

Seeding of hematopoietic progenitor cells (HPC) into the bone marrow requires a complex interaction between cell membrane and adhesion systems and cell signaling pathways. We established a multicellular, spheroid coculture model to study HPC migration in a three-dimensional stromal environment. Here, entry of primary CD34(+) cells into stroma cell spheroids was independent of the integrins very late antigen (VLA)-4, VLA-5, lymphocyte function-associated antigen-1, and the chemokine receptor CXCR4. Experiments using a panel of bacterial toxins selectively targeting key regulators of cellular locomotion, the Rho family small GTPases Rho, Rac, and Cdc42, revealed a considerable reduction or even abrogation of TF-1 cell migration without an increase of apoptosis or impairment of proliferation. Pertussis toxin, an inhibitor of Galpha(i) proteins, showed a similar effect. In some in vitro invasion assays, phosphatidylinositol-3 kinase (PI-3K) was shown to mediate Rac- and Cdc42-induced cell motility and invasion. However, inhibition of the PI-3K pathway by LY294002 did not impair TF-1 cell migration in our three-dimensional model system.

Bacterial Proteins↗
PubMed2002

The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro.

The use of adenoviruses for gene transfer and as oncolytic agents is currently receiving widespread attention. As specific constraints to adenovirus distribution and spread cannot be studied in cell cultures, there is a need for an in vitro three-dimensional (3D) model mimicking the in vivo biology of tumors. We studied the interactions between tumor and adenoviruses using multicellular spheroids grown from primary brain tumor material. Using beta-galactosidase and luciferase reporter genes expressed by replication-defective adenoviruses, we showed that infection was restricted to the first layer of cells. Using a replication-competent adenovirus expressing the luciferase gene, we showed that transgene expression in the spheroid was considerably enhanced and that viral spreading deep into the 3D structure took place. In addition, a tetrazolium salt-based metabolic assay could be used to compare the oncolytic activity of different concentrations of replication-competent adenoviruses. We can conclude that organotypic spheroids offer a versatile in vitro system for studying distribution, spread, and oncolysis by adenoviruses in a clinically relevant model.

Adenoviridae↗
PubMedDate not supplied

Activated eosinophils infiltrate MCF-7 breast multicellular tumor spheroids.

BACKGROUND: Previous studies in our laboratory have shown that activated eosinophils and eosinophilic cell lines inhibit the in vitro growth of MCF-7 breast tumor cells. We have also shown that IL-4 and IL-5 partially inhibit MCF-7 growth in vitro. In this study MCF-7 multicellular tumor spheroids (MTS) were developed to study the effect of eosinophils and IL-4 on tumor growth. MATERIALS AND METHODS: Hypo- and hyperdense metrizamide density gradient fractions of eosinophils from peripheral blood of individuals with mild to moderate esoinophilia were co-cultured with 2-day-old MCF-7 MTS in medium containing bacto agar overlay at 37 degrees C. RESULTS: Light microscopic analyses revealed the attachment of eosinophil effector cells to the spheroid borders. Moreover, the culture media was greater than 90% devoid of effector cells. At six days post co-culture, very large spheroids were observed in both test and control dishes; however the necrotic cores in the co-cultures were more intense and larger than in the control. When MCF-7 tumor cells (1 x 10(6)) were pretreated with IL-4 at 0.5 ng/ml, there was a dramatic decrease in the number of spheroids formed. CONCLUSION: These data strongly indicate that cytokines like IL-4 and perhaps other eosinophil mediators are capable of killing and inhibiting tumor growth; they suggest that tumor infiltrating eosinophils can degranulate and release toxic inhibitory factors into the tumor milieu which destroy the surrounding tumor. These observations, along with the use of the eosinophil: MTS tumor model provide a unique model system for in depth studies of the role of eosinophils and the cytokines they produce in breast cancer and may offer potential therapeutic implications.

Breast Neoplasms↗
PubMed2003

[Impact of cyclin-dependent kinase inhibitor p27 on resistance of ovarian cancer multicellular spheroids to taxol].

OBJECTIVE: To examine the expression of the cyclin-dependent kinase inhibitor p27 in ovarian cancer multicellular spheroid (MCS) and explore the reversal effect of p27-antisense oligodeoxynucleotide (p27-ASON) on taxol resistance in ovarian cancer cell lines. METHODS: Three-dimensional culture was used to form MCS of human ovarian cancer cell lines A2780 and CAOV3. The MCSs were divided into 3 groups: MCSs transduced with p27-ASON or p27-sense oligodeoxynucleotide (p27-SON) by LipofectAMINE respectively and MCS without transduction. Monolayer A2780 and CAOV3 cells were cultured as controls. The cells were exposed to taxol of different concentrations (0.2, 2.0, 10.0, and 20.0 micro mol/L) for 24 hours. The expression of p27 in those cells was detected with Western blot and flow cytometry (FACS), and the subcellular distribution of p27 was detected by laser confocal microscopy before and after the experiment. The cell cycle profile and apoptosis were analyzed by FACS. The resistance to taxol was detected with trypan blue exclusion testing. RESULTS: Compared with that in monolayer cells, expression of p27 in MCS cells was significantly higher (P(A2780) = 0.011, P(CAOV3) = 0.024). The percentage of cells in G0-G1 phase was significantly higher in the MCS cells than in the monolayer cells, and the percentages of cells in S and G2-M phases were significantly lower in MCS cells than in monolayer cells. The apoptotic rate of the monolayer cells was significantly higher than those of MCS cells treated with taxol of the concentration of 20.0 micro mol/L (P(A2780) = 0.003, P(CAOV3) = 0.015). The apoptotic rate of p27-ASON/MCS was significantly higher than that of MCS (P(A2780) = 0.022, P(CAOV3) = 0.036). There was no significant difference of apoptotic rate between p27-SON/MCS and MCS (P(A2780) = 0.412, P(CAOV3) = 0.071). The monolayer A2780 and CAOV3 cells formed compact spheroids after three-dimensional culture. Cells transduced with p27-SON formed compact MCS too. However, cells tranduced with p27-ASON formed loose collections of cells easy to shatter. The expression of p27 in p27-ASON/MCSs was downregulated. CONCLUSION: The drug resistance of ovarian cancer MCS is related to upregulation of p27. P27-ASON reverses the resistance of ovarian cancer to taxol, thus increasing the chemotherapeutic sensitivity of ovarian cancer cells.

Antineoplastic Agents, Phytogenic↗
PubMedDate not supplied

Oxygenation and response to irradiation of organotypic multicellular spheroids of human glioma.

PURPOSE: Investigation of the oxygenation status of organotypic multicellular spheroids (OMS) and their response to irradiation. MATERIALS AND METHODS: Tumour specimens of glioblastoma multiforme patients (n = 16) were initiated as OMS. Following 20 Gy gamma-irradiation, the cell migratory capacity was evaluated. Spheroid oxygenation was determined by micro-electrode pO2 measurements and pimonidazole immunostaining. Spheroids prepared from established human glioma cell lines were used as a reference. RESULTS: Irradiation inhibited spheroid outgrowth by 12 to 88% relative to the non-irradiated controls. A large interpatient variation was noticed. Oxygen measurements revealed a gradual decrease in pO2 level from the periphery to the core of the spheroids, but the pO2 values remained within an oxygenated range. However, in the cell line spheroids an intermediate layer of hypoxia surrounding the central core was observed. CONCLUSION: Cell line spheroids with a hypoxic cell fraction and well-oxygenated OMS both show high resistance to irradiation, indicating that hypoxia may not be the biological factor determining the radioresistance of glioma spheroids in vitro.

Brain Neoplasms↗
PubMed1992

The RBE-LET relationship for rodent intestinal crypt cell survival, testes weight loss, and multicellular spheroid cell survival after heavy-ion irradiation.

This report presents data for survival of mouse intestinal crypt cells, mouse testes weight loss as an indicator of survival of spermatogonial stem cells, and survival of rat 9L spheroid cells after irradiation in the plateau region of unmodified particle beams ranging in mass from 4He to 139La. The LET values range from 1.6 to 953 keV/microns. These studies examine the RBE-LET relationship for two normal tissues and for an in vitro tissue model, multicellular spheroids. When the RBE values are plotted as a function of LET, the resulting curve is characterized by a region in which RBE increases with LET, a peak RBE at an LET value of 100 keV/microns, and a region of decreasing RBE at LETs greater than 100 keV/microns. Inactivation cross sections (sigma) for these three biological systems have been calculated from the exponential terminal slope of the dose-response relationship for each ion. For this determination the dose is expressed as particle fluence and the parameter sigma indicates effect per particle. A plot of sigma versus LET shows that the curve for testes weight loss is shifted to the left, indicating greater radiosensitivity at lower LETs than for crypt cell and spheroid cell survival. The curves for cross section versus LET for all three model systems show similar characteristics with a relatively linear portion below 100 keV/microns and a region of lessened slope in the LET range above 100 keV/microns for testes and spheroids. The data indicate that the effectiveness per particle increases as a function of LET and, to a limited extent, Z, at LET values greater than 100 keV/microns. Previously published results for spread Bragg peaks are also summarized, and they suggest that RBE is dependent on both the LET and the Z of the particle.

Animals↗
PubMed2003

Photodynamic therapy of transitional cell carcinoma multicellular tumor spheroids with hypericin.

The objective of the present study was to evaluate the sensitivity of the bladder transitional cell carcinoma (TCC) to hypericin PDT in a 3-D system using multicellular tumor spheroids. The photodynamic response in RT-112 human bladder TCC spheroids was also compared to 2-D cultured monolayer cells. Following a 2-4 h incubation with 8-30 microM hypericin, spheroids or monolayer cells were irradiated at the light dose of 12 J/cm2, delivered at a fluence rate of 10-100 mW/cm2. The PDT effects were evaluated using a clonogenic assay. The results show that compared with the cells in a monolayer, cells in spheroids were dramatically less sensitive to hypericin PDT (<2000-fold). Studies of fluorescence microphotographs of centrally cut frozen sections of hypericin-exposed spheroids showed a gradient in hypericin concentration from the peripheral to the central region of the spheroid. Although it can be suggested that heterogeneity of drug uptake might be responsible for the observed resistance of spheroid to hypericin PDT, hypericin sensitized spheroids that were dissociated prior to light irradiation were as sensitive as the monolayer cells to hypericin PDT, suggesting that other factors such as oxygen depletion might be responsible for the resistance of spheroids to hypericin PDT.

Anthracenes↗
PubMed2004

Multicellular tumor spheroids in gene therapy and oncolytic virus therapy.

Multicellular clusters of cells, or spheroids, can be grown and sustained in vitro, facilitating various cancer research and treatment approaches. As spheroids are three-dimensional and retain many of the cell types and features present in the original clinical tissues, they provide an important link between two-dimensional cell lines and in vivo xenograft studies. With regards to gene therapy and replication-competent oncolytic viruses in particular, spheroids allow sustained viability of primary tumor cells in vitro, a requirement for reliable analysis of replication. Analysis of clinical samples is of paramount importance for preclinical evaluation of candidate anticancer agents.

Cell Survival↗
PubMed2005

A multicellular spheroid-based drug sensitivity test by scanning electrochemical microscopy.

The respiratory activity of a multicellular spheroid was non-invasively monitored by scanning electrochemical microscopy (SECM) to investigate the anticancer drug sensitivity. The effects of the three anticancer drugs, cisplatin (CDDP), 5-fluorouracil (5-FU), and paclitaxel (TXL), were continuously evaluated based on respiratory activity for 5 days. The drug sensitivities obtained by SECM were higher compared to those evaluated by the spheroid volume and conformed to those evaluated by a conventional colorimetric assay. Our results show that the SECM-based assay directly correlates with the number of viable cells within the spheroid, whereas the spheroid volume does not necessarily correlate with the number of viable cells. Furthermore, the results obtained by spheroid culture (3-D) were compared to those of cells cultured in a flask (2-D) and within a collagen gel (3-D). The drug sensitivity of cells cultured in 2-D is more pronounced than that of the cells cultured in 3-D. Since the cellular proliferation status in a 3-D culture is similar to that in vivo, the drug sensitivity test performed in the spheroid culture will give meaningful results that can be extended to an in vivo application. A SECM-based assay is perfectly suitable to directly evaluate the drug sensitivity of the spheroid.

Antimetabolites, Antineoplastic↗
PubMed1992

[Combination chemotherapy of solid tumor--effects of hyaluronidase on doxorubicin (DXR) penetration into multicellular tumor spheroids (MTS)].

We have studied the effects of hyaluronidase (HYD) on the penetration and cell kill effect of doxorubicin (DXR) using multicellular tumor spheroids (MTS). MTS of approximately 500 microns in diameter were prepared by liquid over lay culture technique from PC-10 lung and HEp-2 laryngeal squamous carcinoma cell lines. Cells in MTS and monolayer were exposed for various durations to HYD, followed by 1 hr, rest interval, and by 1 hr. exposure to DXR. MTS and monolayer cells were then trypsinized to a single cell suspension and subjected to clonogenic assay. For PC-10 MTS, pretreatment with HYD for 24 hr. resulted in approximately 10-fold increases in DXR cell kill effects as compared to DXR alone. HEp-2 MTS were more sensitive to HYD pretreatment. Thus, 1 hr. exposure to HYD produced approximately 4-fold increases in DXR-induced cell lethality. Fluorescent microscopic study revealed that 1hr. exposure of MTS to DXR produced DXR fluorescence only 1-2 outer layer of MTS. When MTS were pretreated with HYD, there was an enhanced penetration of DXR fluorescence into the MTS core. HYD-induced enhancement of DXR penetration and its cell kill effect was dependent on the exposure time and tumor cell origin.

Carcinoma, Squamous Cell↗
PubMed1991

Cell shedding from x-irradiated multicellular spheroids of human lung carcinomas.

We studied the effect of radiation on cell shedding from the surface of multicellular spheroids. Spheroids were produced from two human lung cell lines, one adenocarcinoma (LCT1) and the other small cell carcinoma (LCT2), by using a liquid overlay culture technique. The number of cells shed from both kinds of spheroids did not change significantly when they were irradiated. The number of clonogenic cells shed from both kinds of irradiated spheroids decreased sharply as the dose of irradiation increases. There were no significant differences in clonogenic cell shedding per spheroid between LCT1 and LCT2 spheroids. 400 microns spheroids were more radioresistant to inhibition of clonogenic cell shedding than 250 microns spheroids. Shed cells were more radiosensitive than spheroid cells. In these experiments, we did not obtain any results indicating that radiation enhances metastasis.

Adenocarcinoma↗
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↗
PubMed1991

Self-diffusion of water in multicellular spheroids measured by magnetic resonance microimaging.

Nuclear magnetic resonance microimaging measurements of the self-diffusion coefficient of water in large (greater than 2 mm) EMT-6 multicellular spheroids were performed in order to elucidate diffusion mechanisms in tumors. Pulsed gradient spin echo-imaging methods were developed for measuring diffusion in an intravoxel multicompartment system. The self-diffusion coefficient (at 22 degrees C) for water in the medium (Dm) consisted of only a single diffusion compartment [Dm = 1.99 +/- 0.03 (SE) x 10(-5) cm2/s]. Similarly, the spheroid necrotic center showed a single water diffusion compartment with a self-diffusion coefficient (Dc) significantly lower than that of the medium (Dc = 1.54 +/- 0.05 x 10(-5) cm2/s). The spheroid viable rim region showed two distinct compartments of approximately equal volume, one with a large diffusion coefficient (1.70 +/- 0.12 x 10(-5) cm2/s) and a second with a significantly smaller diffusion coefficient (0.25 +/- 0.01 x 10(-5) cm2/s). We propose that these two experimentally distinguishable compartments correspond to the extra- and intracellular regions, respectively, of the viable rim of the spheroid. Although the diffusion coefficients were significantly different in the medium, the necrotic center, and the viable rim, the activation energy for diffusion was the same in the three regions (0.20 eV). Studies of perfused spheroids at 37 degrees C show the same dependence of the diffusion coefficients on the diffusion filter as observed for unperfused spheroids at 22 degrees C. These results demonstrate the ability of nuclear magnetic resonance microimaging to investigate diffusion at the cellular level, which will lead to a better understanding of microenvironmental regulation in tumors.

Animals↗
PubMed1991

Cytotoxicity of tetraplatin and cisplatin for human and rodent cell lines cultured as monolayers and multicellular spheroids.

The cytotoxicity of tetraplatin (dl-trans), its d- and l-isomers, and cisplatin for four human tumor cell lines (myeloma 8226, ovarian 2008, A2780, and OVCAR-3), their cisplatin-resistant variants, and three rodent cell lines (V79, EMT6/Ro, and L1210) were compared. Tetraplatin was more, or equally as, potent as cisplatin for the human cell lines and for L1210 but was clearly less potent for V79 and EMT6/Ro. The d-trans tetraplatin was more potent than the l-trans. Cisplatin resistant human tumor cells were less resistant to tetraplatin. On comparing sensitivity of V79 and EMT6/Ro cells in two growth models, we observed that all of the platinum compounds were more cytotoxic to cells in multicellular spheroids than in exponentially growing monolayers. Uptake studies, however, showed that tetraplatin was more cytotoxic to spheroids because spheroids accumulated more drug than monolayers.

Animals↗
PubMed1991

[Effects of split dose irradiation on monolayer cells and multicellular tumor spheroid].

Human neuroblastoma (GOTO) and squamous cell carcinoma (SQ5), grown as the monolayer cells or as the multicellular tumor spheroid (MTS) were irradiated. GOTO cells were radiosensitive, while SQ5 cells were moderately radioresistant. Cells were irradiated by the single or the split doses and the recovery between split doses were investigated. The recovery ratio (R.R.) of monolayer cells were calculated by the procedure A: the dose ratio at the same survival levels or by the procedure B: the survival rates ratio at the same doses. The therapeutic response of MTS was determined by the method of spheroid control dose-50 (SCD50) and R.R. of MTS was calculated by the ratio of the values of SCD50. R.R. of GOTO-monolayer cells was 1.1 (by the procedure A) and 1.6 (by the procedure B). GOTO-MTS did not show the significant difference between SCD50 of single dose irradiation and that of split doses one. When SQ5 cells were irradiated by the split doses, R.R.s obtained by the procedure A and B were 1.3 and 2.1, respectively. R.R. of SQ5-MTS was not different from that of monolayer cells obtained by the procedure A. MTS with two different sizes were irradiated by the single or split doses, and there was no remarkable difference with respect to the radiocurability and R.R. Although the number of cell lines were limited, their growth condition did not affect the capacity of recovery from radiation damage.

Carcinoma, Squamous Cell↗
PubMedDate not supplied

Oxygen tensions in two human tumor cell lines grown and irradiated as multicellular spheroids.

Cells from two human cell lines were irradiated both as multicellular tumor spheroids (MTS) and in monolayer culture. Radiation response of MTS was quantified in terms of specific growth delay and proportion cured, and as clonogenic cell survival for monolayer cells. Radiation was applied either as a single or as a split dose with time intervals of 1, 2, and 4 h to determine the rate of sublethal damage repair. Using as endpoint the fraction of MTS cured at an iso-effect level, in MTS of NB-100 neuroblastoma cells repair of sublethal damage was complete within 1 h, whereas in MTS of HN-1 squamous cell carcinoma cells there was still some unrepaired damage left. At a larger dose for NB-100 MTS the repair curve showed a similar shape as for HN-1 spheroids. Using as endpoint specific growth delay, no difference in repair between the various time intervals was observed. In monolayer cells from both cell lines sublethal damage was not fully repaired in the time intervals used. Polarographic microelectrode measurements of oxygen tension inside MTS showed a marked difference in steepness of oxygen tension profiles between MTS from both cell lines. In HN-1 squamous cell carcinoma MTS with diameters up to 500 microns the central pO2 amounted to about 100 Torr, whereas in NB-100 neuroblastoma MTS with the same diameters central pO2-values lower than 30 Torr were observed. NB-100 MTS were irradiated with doses of 5 and 10 Gy gamma rays and subsequently the oxygen tension was measured 1 and 5 h after irradiation. A reoxygenation effect could not be observed, either after single dose or after split dose irradiation. If spheroids may be regarded as a suitable model for tumor responses in vivo, the results from these experiments indicate that reoxygenation is a process eluding polarographic measurements, or that no dramatic changes in oxygen tension are to be expected shortly after high single doses or early in a fractionation scheme.

Carcinoma, Squamous Cell↗
PubMed1991

Differential penetration of targeting agents into multicellular spheroids derived from human neuroblastoma.

We have used a multicellular tumour spheroid model for determination of the penetration of various targeting agents of potential use in the treatment of neuroblastoma. Both the radiopharmaceutical meta-iodobenzylguanidine (mIBG) and the beta subunit of nerve growth factor (beta-NGF) distributed uniformly throughout spheroids, though the latter was poorly concentrated relative to mIBG. In contrast, the anti-neuroectodermal monoclonal antibody. UJ13A bound only to peripheral cell layers with little accumulation in the spheroid interior. Differential penetration of targeting agents may influence the choice of conjugated radionuclide which is likely to achieve maximum therapeutic benefit.

3-Iodobenzylguanidine↗
PubMed1990

ATP concentrations in multicellular tumor spheroids assessed by single photon imaging and quantitative bioluminescence.

To evaluate the interrelationship among the cellular energy status and the development of necrosis in tumor microregions, local ATP concentrations and the extent of necrosis were determined in multicellular tumor spheroids, i.e., in spherical tumor cell aggregates. The spheroids were grown in rotated suspension cultures using EMT6 cells that were derived from a murine mammary sarcoma. The distribution of viable and necrotic cell areas was assessed by histological investigations. The regional distribution of ATP concentrations was measured with a novel technique using quantitative bioluminescence and single photon imaging. This method makes it possible to determine ATP concentrations in absolute terms with a spatial resolution at the level of a single cell. The results show that ATP concentrations in the center of EMT6 spheroids decrease from values of 1.0 to 1.5 mM in small spheroids with 300 microns in diameter to values close to or at the background level in 750 microns spheroids. Necrosis was detectable in spheroids larger than 300 microns, and virtually no spheroid without necrosis was found at sizes larger than 600 microns. Since the emergence of central necrosis precedes the drop in ATP to undetectably low values, the data suggest that energy metabolism is not or not directly involved in the development of necrosis in tumor spheroids under the growth conditions investigated.

Adenosine Triphosphate↗