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PubMed2003

Proliferation, migration, and invasion of human glioma cells exposed to fractionated radiotherapy in vitro.

Radiotherapy is a well established treatment for malignant gliomas. This study describes the migration, proliferation, and invasion behaviour of two human glioma cell lines (GaMg and U-87 Mg) grown as multicellular tumour spheroids after radiotherapy. Migration and proliferation studies were performed using conventional and accelerated fractionation up to 60 Gy and 59.4 Gy, respectively. A dose-dependent growth and migratory response to irradiation independent of the type of fractionation was observed. A coculture system in which tumour spheroids were confronted with foetal rat brain aggregates was used for invasion studies. Marked invasion of the glioma spheroids into the brain aggregates occurred with or without radiotherapy. For the GaMg cells, flow cytometric DNA histograms after treatment with 10 Gy and 40 Gy showed an accumulation of cells in the G2/M phase of the cell cycle. Radiotherapy inhibits tumour cell growth and migration, but the invasiveness of the remaining tumour cells seems to be unaffected.

Animals↗
PubMed2003

Migration, proliferation, and invasion of human glioma cells following treatment with simvastatin.

This study describes the migration, proliferation, and invasion behaviour of two human glioma cell lines, GaMg and U-87 Mg, grown as multicellular tumour spheroids after 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase inhibitor (simvastatin) therapy. Migration and proliferation studies were performed using simvastatin in concentrations of 0.2-30 microg/ml(-1). A coculture system in which tumour spheroids were confronted with foetal rat brain aggregates was used for invasion studies. A dose-dependent growth and migratory inhibitory response to simvastatin treatment was observed. Marked invasion of the glioma spheroids into the brain aggregates could be seen in both treated and nontreated groups. Simvastatin therapy inhibits tumour cell growth and migration, but the invasiveness of the remaining tumour cells seems to be unaffected.

Animals↗
PubMed2006

Differential distribution of stem cells in the auditory and vestibular organs of the inner ear.

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.

Animals↗
PubMed2005

Microscopic images of intraspheroidal pH by 1H magnetic resonance chemical shift imaging of pH sensitive indicators.

OBJECTIVE: We investigate microscopic pH heterogeneity within tumor spheroids using a novel 1H NMR methodology that provides high resolution measurements of intraspheroidal pH. MATERIAL AND METHODS: High resolution microscopic images of intraspheroidal pH were obtained by 1H NMR using chemical shift selective excitation of the H2 resonance of imidazole added to the incubation medium. Imidazole accumulated in the intraspheroidal space in a pH dependent manner. Maps of intraspheroidal pH could be obtained by transforming pixel by pixel (32 x 32 micro) the regional variation of imidazole H2 intensity into a relative pH scale. RESULTS: Our analysis revealed drastic intraspheroidal pH alterations depending on the size of the spheroid, ca. 0.6 pH units more acidic in the necrotic core than in the periphery, for spheroids of 600 mum diameter. The presence of concentric regions having similar intraspheroidal pH was consistently observed. The thickness of these regions decreased from pH 7.2 to pH 6.8 and increased below the latter pH value. CONCLUSION: Our observations are compatible with the general model of spheroid growth where the more external layers of cells are in active growth and depict more alkaline pH values while the inner layers remain quiescent or evolve to a necrotic core, depicting more acidic pH values.

Animals↗
PubMed2004

Withaferin A is a potent inhibitor of angiogenesis.

The medicinal plant Withania somnifera is widely researched for its anti-inflammatory, cardioactive and central nervous system effects. In Ayurveda , the major Traditional Indian medicine system, extracts from W. somnifera are distinctively employed for the treatment of arthritis and menstrual disorders. Because these conditions involve angiogenic processes we hypothesized that the W. somnifera extracts might contain angiogenesis inhibitors. We employed an endothelial cell-sprouting assay to monitor the purification of substances from W. somnifera root extracts and isolated as the active principle the previously known natural product withaferin A. We show that withaferin A inhibits human umbilical vein endothelial cell (HUVEC) sprouting in three-dimensional collagen-I matrix at doses which are relevant to NF-kappa B-inhibitory activity. Withaferin A inhibits cell proliferation in HUVECs (IC50 =12 nM) at doses that are significantly lower than those required for tumor cell lines through a process associated with inhibition of cyclin D1 expression. We propose that the inhibition of NF-kappa B by withaferin A in HUVECs occurs by interference with the ubiquitin-mediated proteasome pathway as suggested by the increased levels of poly-ubiquitinated proteins. Finally, withaferin A is shown to exert potent anti-angiogenic activity in vivo at doses that are 500-fold lower than those previously reported to exert anti-tumor activity in vivo. In conclusion, our findings identify a novel mode of action of withaferin A, which highlights the potential use of this natural product for cancer treatment or prevention.

Angiogenesis Inhibitors↗
PubMed2004

Hanging-drop multicellular spheroids as a model of tumour angiogenesis.

The establishment of a vascular network within tumours is a key step in the progression towards an aggressive, metastatic state, with poor prognosis. We have developed a novel in vitro model to specifically capture the interaction between endothelial cells and solid tumours. Micro-vascularised in vitro tumour constructs were produced by introducing endothelial cells to multicellular spheroids formed in hanging drops. Upon introduction, the endothelial cells migrated into the tumour spheroid, establishing tubular networks and luminal structures. This system relies on the natural pro-angiogenic capacity of multicellular spheroids, and does not require the addition of exogenous angiogenic factors, or use of extracellular-matrix substitutes.

Cell Communication↗
PubMed2005

Predicting aggregation kinetics of DU 145 prostate cancer cells in liquid-overlay culture.

The predictive capacity of a novel population-balance model to simulate aggregation kinetics of attachment-dependent cells at the resolution of one-cell increments has been evaluated. Using spheroid assembly of DU 145 human prostate cancer cells as a representative system, the mathematical model proved to be robust in simulating aggregation over a 5-fold range of surface densities from 5 x 10(3) to 2.5 x 10(4) cells/cm(2) with a single matrix of rate constants. For cultures at 1 x 10(5) cells/cm(2), more than 75% of simulated aggregate concentrations are within the standard deviation of measured concentrations. For the two extreme densities, at least two-thirds of model predictions are within 35% of the mean for experimental data. Error in model predictions is attributed to uncertainty in measurements and intrinsic changes in aggregation. The model has application to the rational design of spheroids in tissue engineering and bioseparation processes in pharmaceutical manufacturing.

Cell Aggregation↗
PubMed2006

Direct self-assembly of hepatocytes spheroids within hollow fibers in presence of collagen.

Opposite to the established view that collagen is an extracellular substratum for only dispersed hepatocyte culture, hepatocyte spheroids were directly formed within hollow fibers by addition of moderate concentrations of soluble collagen. Morphologically, these spheroids indicated a close relationship with their in vivo structure of liver. The albumin and urea synthetic profiles confirmed that those spheroids maintained liver-specific functions for at least 8 days. Spheroid formation by addition of collagen not only presents a potential methodology for clinical use of spheroids in bioartificial liver device but also indicates a likely function of collagen for self-assembly of primary cells in tissue engineering.

Animals↗
PubMed2005

Role of E-cadherin molecules in spheroid formation of hepatocytes adhered on galactose-carrying polymer as an artificial asialoglycoprotein model.

The role of E-cadherin in the spheroid formation of hepatocytes adhered on the poly(N-p-vinylbenzyl-D-lactonamide) (PVLA) as a model ligand for asialoglycoprotein receptors (ASGP-R) of hepatocytes was studied. Expression of E-cadherin was increased in round hepatocytes adhered on a high-coating density of PVLA (100 microg/ml), and also in flat ones adhered on a low-coating density of PVLA (1 microg/ml) in the presence of epidermal growth factor (EGF). Hepatocyte spheroids formed on the high-coating density of PVLA in the presence of EGF after 48 h were inhibited by an anti-E-cadherin monoclonal antibody (ECCD-1). From immunofluorescence and confocal laser microscopy, E-cadherin was localized in the intercellular boundaries and concentrated at the inside surface of aggregated cells. As a result, E-cadherin could play an important role in hepatocyte assembly.

Asialoglycoproteins↗
PubMed2006

Imaging growth dynamics of tumour spheroids using optical coherence tomography.

Optical coherence tomography (OCT) was used to monitor the dynamics of tumour spheroid formation by the hanging drop method. In contrast to microscopy, the estimates obtained using OCT for the volume of the spheroid, were consistent with the measured changes in cell number as a function of time. The OCT images also revealed heterogeneous structures in the spheroids of approximately 200 microm diameter. These corresponded to the necrotic regions identified by fluorescence of propidium iodide stained cells.

Cell Count↗
PubMed2007

Three-dimensional modeling of transport of nutrients for multicellular tumor spheroid culture in a microchannel.

The growth dynamics of avascular tumors in a microchannel bioreactor is investigated. A three-dimensional flow and nutrient transport model, incorporating the multicellular tumor spheroid (MTS) growth model, has been developed to study the influence of nutrients (oxygen and glucose) supply and distribution on the MTS growth. Numerical simulations based on the EMT6/Ro tumor cells show that the continuous-flow perfusion is more efficient to deliver nutrients to the MTS than the diffusion-only static culture. It is further demonstrated that as long as there is bulk flow, the growth of a single tumor spheroid at the early stage is insensitive to the flow velocity and the channel size. For multiple tumor spheroids in the same microchannel, however, increasing the perfusion velocity can improve the nutrient environment for the disadvantageous downstream tumor spheroid. The flow shear stress exerting on the MTSs in the current microchannel bioreactor is estimated to be far below the critical value to affect the MTS growth, which means that there is still much room for increasing perfusion velocity to satisfy the higher nutrient requirement by the growing tumor spheroids.

Animals↗
PubMedDate not supplied

Vitamin D, tamoxifen and beta-estradiol modulate breast cancer cell growth and interleukin-6 and metalloproteinase-2 production in three-dimensional co-cultures of tumor cell spheroids with endothelium.

Reciprocal interactions between tumor cells and endothelial cells constitute the most important stage of tumor metastasis. There is growing evidence suggesting that beta-estradiol and vitamin D modulate the progression of steroid-sensitive breast cancers. In keeping with those results, the purpose of the study reported here was to determine the cytotoxic and antiproliferative activity of tamoxifen (TAM) in the T47D human breast cancer cell line depending on the cell culture model (three-dimensional (3D, spheroid) or two-dimensional (2D, monolayer)) and to estimate the antiproliferative activity of vitamin D in balanced TAM/beta-estradiol conditions. The study was also designed to investigate whether vitamin D might influence interleukin-6 (IL-6) and metalloproteinase-2 (MMP-2) production in a co-culture of T47D cell spheroids with an endothelial cell monolayer in the presence of beta-estradiol and TAM. Spectrophotometric analysis with MTT revealed that the cytotoxic and antiproliferative activity of TAM was dependent on the culture model, the density of cell culture, and culture medium supplements. In balanced TAM/beta-estradiol medium, vitamin D only slightly inhibited T47D cell proliferation in both 2D and 3D cultures. Direct contact of tumor cell spheroids with the endothelium induced production of MMP-2 and IL-6, which was significantly inhibited in TAM/beta-estradiol balanced medium. Addition of vitamin D further inhibited MMP-2 production, but enhanced the production of IL-6 as was shown by ELISA assay. Our co-culture model in TAM/beta-estradiol balanced medium proved to be useful for examining direct and paracrine interactions of tumor cells with the endothelium in conditions that were closer to in vivo conditions than in the standard 2D model.

Antineoplastic Agents↗
PubMed2004

Ovarian carcinoma spheroids disaggregate on type I collagen and invade live human mesothelial cell monolayers.

Ovarian carcinoma patients frequently develop malignant ascites containing single and aggregated tumor cells, or spheroids. Spheroids have been shown to be resistant to many therapies, but their contribution to ovarian cancer dissemination remains undetermined. We have previously shown that ascites spheroids adhere to extracellular matrix (ECM) proteins and live human mesothelial cells via beta1 integrin subunits. Here, we assessed the ability of spheroids that were generated from the human ovarian carcinoma cell line NIH:OVCAR5 to disseminate and invade in vitro. Spheroids were seeded on ECM proteins for 24 h. While laminin and type IV collagen stimulated some cell migration, spheroids completely disaggregated on type I collagen substrates. A monoclonal antibody against the beta1 integrin subunit significantly inhibited disaggregation on all proteins tested. To test their invasive ability, spheroids were added to monolayers of live human LP9 mesothelial cells. Within 24 h, the spheroids adhered and disaggregated on top of the monolayers, and within a week had established foci of invasion encompassing a 200-fold larger surface area. Addition of a monoclonal antibody against the beta1 integrin subunit drastically reduced spheroid invasion into the mesothelial cell monolayers. GM 6001, a broad-scale matrix metalloproteinase inhibitor, also significantly blocked spheroid invasion into the mesothelial cell monolayers. Epsilon-amino-N-caproic acid, a serine protease inhibitor, partially inhibited spheroid invasion. Based on their ability to attach to, disaggregate on, and invade into live human mesothelial cell monolayers, spheroids should thus be regarded as potential contributors to the dissemination of ovarian cancer.

Aminocaproates↗
PubMed2006

Hepatocyte spheroid formation on a titanium dioxide gel surface and hepatocyte long-term culture.

The cell morphology and expression of differentiated functions of primary rat hepatocytes on a titanium dioxide (TiO(2)) gel surface were investigated. Polystyrene culture dishes were coated with TiO(2) gel by spin-coating an ethanol solution of titanium n-butoxide, hydrolyzing in a humidity chamber and drying with nitrogen gas. The TiO(2) gel layer formed on the polystyrene dishes was transparent and robust, and its surface was quite flat. Rat hepatocytes inoculated on the TiO(2) gel-coated polystyrene dishes gradually accumulated with increasing culture time, and then spontaneously formed many hepatocyte spheroids at 90 +/- 21 microm diameter from about 3 days of culture. The diameter of the spheroids increased during the culture, and was 151 +/- 43 microm at 14 days of culture. Ammonia removal and albumin secretion by hepatocytes on the TiO(2) gel-coated polystyrene dishes were maintained at a high level for at least 14 days of culture compared with on a type I collagen-coated dish and a non-coated polystyrene dish. These results indicate that TiO(2) gel is an adequate material for hepatocyte spheroid formation and long-term culture of spheroids.

Animals↗
PubMed2003

Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges.

This study focuses on recent improvement in epithelial monolayer cultures originating from whole extirpated Botryllus schlosseri (Urochordata) buds. Buds (n = 2,000) were taken at different ('A' to 'D') blastogenic stages. We tested the suitability of 35 combinations of various substrates and media on attachment, cell spread, epithelial growth frequencies and on monolayer lifespans. Under favorable conditions, cultured buds at blastogenic stages 'B' to 'D' (but not stage 'A') started to attach to the substrates following a 3-day transient period that leads to formation of spheres and attached monolayers. Substrate type is important for the attachment and the development of monolayers. Under various culture conditions, some of stages 'B' and 'C' buds develop (3-20 days) one or more large (1 mm diameter) spheres. Stage 'D' buds develop monolayers (up to 20% of buds) without going through a sphere phase. Neither spheres nor attached monolayers of epithelium were observed in stage 'A' bud cultures. Spheres grew at a rate of 60 microm in diameter per day using specific medium types and did not attach unless the appropriate substrate was present. When attached, epithelial monolayers expanded at a rate of 200 microm in diameter per day, for 3-15 days, and subsequently detached and died. Sixteen types of media were tested. Medium and substrate combinations were found to determine epithelial lifespan. These results revealed significant improvements in the culture of epithelial monolayers from Botryllus palleal buds. However, an early senescence of the developed epithelial sheets (up to two weeks from onset of appearance) may indicate an internal ageing clock that should be taken into consideration in future approaches.

Animals↗
PubMed2004

Enhanced cytotoxic effects of 5-aminolevulinic acid-mediated photodynamic therapy by concurrent hyperthermia in glioma spheroids.

During photodynamic therapy (PDT) both normal and pathological brain tissue, in close proximity to the light source, can experience significant temperature increases. The purpose of this study was to investigate the anti-tumor effects of concurrent 5-aminolevulinic acid (ALA)-mediated PDT and hyperthermia (HT) in human and rat glioma spheroids. Human or rat glioma spheroids were subjected to PDT, HT, or a combination of the two treatments. Therapies were given concurrently to simulate the conditions that will occur during patient PDT. Predictions of diffusion theory suggest that brain tissue immediately adjacent to a spherical light applicator may experience temperature increases approaching 8 degrees C for laser input powers of 2 W. In the in vitro model employed here, HT had no effect on spheroid survival at temperatures below 49 degrees C, while sub-threshold fluence PDT results in only modest decrease in survival. HT (40-46 degrees C) and PDT interact in a synergistic manner if the two treatments are given concurrently. The degree of synergism increases with increasing temperature and light fluence. Apoptosis is the primary mode of cell death following both low-fluence rate PDT and combined HT + PDT.

Aminolevulinic Acid↗
PubMed2006

Human glioblastoma biopsy spheroids xenografted into the nude rat brain show growth inhibition after stereotactic radiosurgery.

BACKGROUND: The Gamma Knife is currently used to boost treatment of malignant gliomas. However, few experimental studies have focused on its radiobiological effects. In this work, the growth and invasiveness of human glioblastoma spheroids xenografted into nude rat brains were assessed after radiosurgery. Temporary in vitro as well as long-term in vivo radiation effects were studied. METHODS: Glioblastoma biopsy spheroids were irradiated with 12 or 24 Gy. Short-term in vitro spheroid viability and tumour cell migration was determined by microscopic techniques. Pre-irradiated glioblastoma spheroids were implanted into brains of immunosuppressed rats. Long-term tumour development was assessed by magnetic resonance (MR) imaging, and animal survival was recorded. An immunohistochemical analysis was performed on the sectioned rat brains. RESULTS: Both un-irradiated and irradiated spheroids remained viable during 2 months in culture, but a dose-dependent inhibition of tumour growth and migration was seen. MR imaging 4 weeks after implantation also showed a dose-dependent inhibition in tumour development. Median animal survival times were 25.5 days (control group), 43 days (12 Gy group) and 96 days (24 Gy group). The study of in vivo long-term radiation effects on the remaining viable tumour population showed no difference in Ki-67 labelling index and microvascular density before and after radiosurgery. CONCLUSIONS: A dose-dependent inhibition of tumour growth and invasion, as well as a dose-dependent increase in animal survival was observed. The model system described is well suited for assessing the radiobiological effects of Gamma Knife radiosurgery. The results indicate that radiosurgery of malignant gliomas might be effective in controlling tumour progression in selected glioblastoma patients.

Analysis of Variance↗
PubMed2006

Acute morphological sequelae of photodynamic therapy with 5-aminolevulinic acid in the C6 spheroid model.

OBJECTIVE: Aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) may represent a treatment option for malignant brain tumors. We used a three-dimensional cell culture system, the C6 glioma spheroid model, to study acute effects of PDT and how they might be influenced by treatment conditions. METHODS: Spheroids were incubated for 4 h in 100 microg/ml ALA in 5% CO(2) in room air or 95% O(2) with subsequent irradiation using a diode laser (lambda = 635 nm, 40 mW/cm(2), total fluence 25 J/cm(2)). Control groups were "laser only", "ALA only", and "no drug no light". Annexin V-FITC, a marker used for detection of apoptosis, propidium iodide (PI), a marker for necrotic cells and H 33342, a chromatin stain, were used for morphological characterization of PDT effects by confocal laser scanning and fluorescence microscopy. Hematoxylin-eosin staining and TdT-FragEL (TUNEL) assay were used on cryosections. Growth kinetics were followed for 8 days after PDT. RESULTS: PDT after incubation in 5% CO(2) provided incomplete cell death and growth delay in spheroids of >350 microm diameter. However, complete cell death and growth arrest occurred in smaller spheroids (<350 microm). Incubation in 95% O(2) with subsequent PDT resulted in complete cell death and growth arrest regardless of spheroid size. In incompletely damaged spheroids viable cells were restricted to spheroid centers. The rate of cell death in all control groups was negligible. Cell death was accompanied by annexin/PI costaining, but there was also evidence for annexin V-FITC staining without PI uptake. CONCLUSIONS: PDT of experimental glioma results in rapid and significant cell death that could be verified as acute necrosis immediately after irradiation. This effect depended on O(2) concentration and spheroid size.

Aminolevulinic Acid↗