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PubMed1983

Frequency distribution histograms of oxygen tensions in multicell spheroids.

Recessed oxygen sensitive microelectrodes have been used to measure oxygen tensions in spheroids from EMT6/Ro mouse mammary carcinoma cells and from V-79-171B hamster lung fibroblasts. During the experiments the spheroids were exposed to conditions similar to those prevailing during growth and previous experiments with radiation and/or drugs. Frequency distribution curves were generated from 386 steady state readings in EMT6 (N = 20) spheroids with diameters ranging from 386 to 1900 micron and from 289 steady state readings in V79 spheroids (N = 20) with diameters between 376 and 1052 micron. Small spheroids exhibit higher pO2 values than spheroids of medium and large size. Oxygen tensions in small and medium size V79 spheroids are lower than in EMT6 spheroids of the equivalent size ranges. The pO2 histograms in large spheroids of both cell types are similar to pO2 frequency distributions in solid tumors. It is concluded that spheroids may represent a valuable model to provide evidence for the way pO2 histograms are influenced by the density of capillaries, by the cellular O2 consumption and by the sampling technique.

Animals↗
PubMed1992

Nitric oxide: its production in host-cell-infiltrated EMT6 spheroids and its role in tumour cell killing by flavone-8-acetic acid and 5,6-dimethylxanthenone-4-acetic acid.

Flavone-8-acetic acid (FAA) and its more dose-potent analogue 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA), appear to exert their antitumor effects through vascular and other host-mediated mechanisms and are known to induce the synthesis of nitric oxide by murine macrophages. We investigated the role of nitric oxide in the cytotoxic effects of these drugs in host-cell-infiltrated spheroids. EMT6 murine mammary adenocarcinoma cells were grown in culture to produce multicellular spheroids in vitro spheroids), which were then inoculated i.p. into mice. After 6 days the spheroids were removed ex vivo spheroids). Exposure to FAA (890 microM) and 5,6-MeXAA (80 microM) in vitro for 20 h increased nitrite concentrations to 6.7 and 9.7 nmol/spheroid, respectively, as compared with 0.7 nmol/spheroid in the absence of drug. FAA and 5,6-MeXAA did not increase nitrite production in in vitro spheroids in cells obtained by peritoneal lavage. However, mixed cultures of in vitro spheroids and peritoneal cells treated with 5,6-MeXAA produced nitrite (2.4 nmol/spheroid), indicating that interactions between host cells and tumour cells were important for induction. The effects of these drugs on ex vivo spheroids were prevented by co-incubation with NG-monomethyl-L-arginine, indicating that nitrite originated from the oxidation of L-arginine to nitric oxide. Cell sorting of disaggregated spheroids into EMT6 cells and Mac-1-positive macrophage populations indicated that both of these cell populations could be induced to synthesise nitric oxide by subsequent incubation with 5,6-MeXAA. Incubation of ex vivo spheroids with FAA and 5,6-MeXAA decreased the clonogenicity of EMT6 cells, and this effect was wholly (FAA) or partially (5,6-MeXAA) reversed by the presence of NG-monomethylarginine (250 microM). FAA and 5,6-MeXAA may therefore exert some of their cytotoxic effects on tumour cells through the production of nitric oxide.

Adenocarcinoma↗
PubMedDate not supplied

Binding of misonidazole to EMt6 and V79 spheroids.

Metabolism-induced binding of misonidazole was shown previously to be enhanced in hypoxic regions of V79 spheroids. Data are presented to show that the binding of misonidazole to EMT6 spheroids is similar to V79 in some respects and different in others. The binding rate in nitrogen was constant in all cells in EMT6 spheroids, whereas a 5-fold rise was found in binding rate across the outer 100 micrometers of V79 spheroids. A small enhancement of binding to the cells near the necrotic center in EMT6 spheroids in air was found, which was similar to the binding to chronically hypoxic cells in aerobic V79 spheroids. The binding rate in 1--5% oxygen was similar in EMT6 and V79 spheroids. Both showed a large rise in binding rate across the outer 120 micrometers and a constant binding rate to healthy cells interior to this. Since 1.5 oxygen should supply only the outer 40 micrometers of a typical spheroid, something beyond a simple oxygen diffusion limitation is required to explain the data. One possible explanation is the diffusion of some of the reactive product away from the hypoxic cells which produce it. This was tested by incubating a single cell suspension in misonidazole at 0.5% oxygen in the presence of absence of V79 spheroids. No evidence was found for an appreciable loss of reactive product from the spheroids.

Animals↗
PubMed1984

Method for the determination of oxygen consumption rates and diffusion coefficients in multicellular spheroids.

A method has been developed for the quantitative evaluation of oxygen tension (PO2) distributions in multicellular spheroids measured with O2-sensitive microelectrodes. The experimental data showed that multicellular tumor spheroids in stirred growth media were characterized by a diffusion-depleted zone surrounding the spheroids. This zone was elicited by an unstirred layer of medium next to the spheroid leading to a continuous decrease in the PO2 values from the bulk medium towards the spheroid surface. Theoretical considerations demonstrate that the volume-related O2 consumption rate, Q, in the spheroids can be assessed by measuring the PO2 gradient in the diffusion-depleted zone outside the spheroids. Accordingly, Krogh's diffusion constant, KS, in the spheroids can be determined through measuring the PO2 gradient within the spheroids. The results obtained suggest that multicellular spheroids represent useful in vitro tumor models for the experimental and theoretical analysis of the interrelationship among O2 supply to tumor cells, O2 metabolism in tumors tissue, and the responsiveness of cancer cells to treatment.

Animals↗
PubMed1985

The response to cytotoxic drugs of EMT6 cells treated either as intact or disaggregated spheroids.

We have compared the response to a number of cytotoxic drugs of cells treated either within intact multicellular spheroids or as isolated cells following spheroid disaggregation. The cells used were of the EMT6/Ca/VJAC mouse tumour line and spheroids were treated or disaggregated at a mean diameter of 250 micron. The response of cell to nitrogen mustard (HN2) or CCNU was similar under the two exposure conditions and we conclude that factors related to spheroid structure (i.e. drug penetrability, intercellular contact effect and microenvironment within the spheroid) do not influence the initial response to these agents. Recovery of potentially lethal damage occurring over 24 h, however, greatly modifies the level of cell killing in intact spheroids. EMT6 cells were found to be extremely resistant to vincristine under all exposure conditions. For adriamycin (ADM), cells were always initially more sensitive when exposed to the drug in suspension rather than in intact spheroids. When ADM exposure was prolonged beyond 1 h, however, delaying spheroid disaggregation for 24 h led to increased cell kill and reduced differential between the two conditions of exposure. The data suggest that both drug penetration problems and other factors related to spheroid structure are involved in determining the response of cells in small spheroids to ADM.

Animals↗
PubMed1987

Deriving cell survival curves from the overall responses of irradiated tumours: analysis of published data for tumour spheroids.

Curves of growth delay (GD) or 'cure' after graded doses of radiation have been analysed for 16 lines of human and animal tumours grown as multicellular spheroids in vitro. Dose-survival curves were derived for those cellular units from which spheroids regrow after unsuccessful irradiation (spheroid-regenerating cellular units, SRU). For 10 sets of data from 6 spheroid lines, the Do's and extrapolation numbers of the SRU derived by GD could be compared with the response of the clonogenic cells of the spheroids. For Do, a good correlation (r = 0.910) was found between the two; this was true also for Do derived from curves of spheroid 'cure' (7 sets of data from 6 spheroid lines) and clonogenic cells (r = 0.986). Using GD, the correlation of extrapolation numbers was less good (r = 0.682), the values for SRU commonly being higher than those for clonogenic cells. This may reflect features of the growth curves of spheroids after the lower range of doses of radiation. For human and animal tumour spheroids of 250 microns or less, derived Do ranged from 0.5 to 2.5 Gy. For spheroids of 350 microns or more, derived Do for animal tumour lines ranged from 3.4 to 4.2 Gy, for human lines from 1.5 to 2.1 Gy.

Animals↗
PubMed1988

The effect of combined heat and ultrasound on multicellular tumour spheroids.

The effect of combined ultrasound and heat treatments on Chinese hamster multicellular spheroids of varying size was investigated using growth rate, single cell survival and ultrastructural damage as endpoints. Ultrasonic irradiation at 37 degrees C had no effect on the growth rate of 200-730 microns spheroids. Similarly there was no effect on the growth rate of 350 microns spheroids when irradiated during a 60 min exposure to 41.5 degrees C. However, spheroids of 200-700 mm diameter showed growth delay when held at 43 degrees C for 1 h. The effect was enhanced with concomitant ultrasound irradiation but was not dependent on spheroid size. When 200 and 400 microns spheroids held at 43 degrees C for 60 min were irradiated with different ultrasonic intensities a dose-dependent decrease in surviving fraction and a dose-dependent increase in growth delay was obtained. When surviving fraction was plotted as a function of growth delay a good correlation was obtained, suggesting that the combination of heat and ultrasound irradiation does not produce cytostasis in the surviving cells of either 200 or 400 microns spheroids. At the ultrastructural level increased cytoplasmic vacuolation was the only result of ultrasonic irradiation at 37 degrees C. Exposure to 43 degrees C for 60 min was required to elicit thermal damage. This took the form of membrane evagination at the spheroid surface, vacuolation of the cytoplasm, grouping of organelles around the periphery of the nucleus, and fragmentation of the nucleolus. These effects were enhanced with concomitant ultrasonic irradiation but other features were also noted, viz. disaggregation of polyribosomes, dilation of the rough endoplasmic reticulum and blebbing of the nuclear membrane. Damage was independent of spheroid size. These results are in agreement with previous data obtained from single-cell studies. Indicating that there is a non-thermal, non-cavitational component to the cell killing in multicellular spheroids resulting from combined heat and ultrasound treatment.

Animals↗
PubMed1992

Linear quadratic model of radiocurability on multicellular spheroids of human lung adenocarcinoma LCT1 and mouse fibrosarcoma FSA.

The LCT1 cells derived from a human lung adenocarcinoma and the FSA cells from a mouse fibrosarcoma were found to form spheroids. The cure-dose relationship of spheroids and the survival curves of their component cells were analysed by using a linear-quadratic model for cell survival and a Poisson distribution for cure. The analysis resulted in three conclusions: (1) the double minus logarithm of cure probability was linearly related to radiation dose, (2) the critical cell number was constant at any given cure probability, and (3) cellular radiosensitivity was also constant. The experiments seem to meet these conditions for each of two kinds of spheroids. Control doses (50%) were 20 Gy for LCT1 spheroids and 21 Gy for FSA spheroids, both 400 microns in diameter. The analysis showed that the lower cellular radiosensitivity and the higher number of clonogenic cells made LCT1 spheroids more radioresistant than FSA spheroids and that the higher critical number of 130 cells made the LCT1 spheroids more sensitive than the FSA spheroids with 18 such cells. The overall radiocurability of spheroids was a result of these three opposing effects, indicating that the critical cell number can be one important factor in determining the radiocurability of multicellular systems.

Adenocarcinoma↗
PubMed1992

Mathematical modelling of microenvironment and growth in EMT6/Ro multicellular tumour spheroids.

In order to determine the role of micromilieu in tumour spheroid growth, a mathematical model was developed to predict EMT6/Ro spheroid growth and microenvironment based upon numerical solution of the diffusion/reaction equation for oxygen, glucose, lactate ion, carbon dioxide, bicarbonate ion, chlorine ion and hydrogen ion along with the equation of electroneutrality. This model takes into account the effects of oxygen concentration, glucose concentration and extracellular pH on cell growth and metabolism. Since independent measurements of EMT6/Ro single cell growth and metabolic rates, spheroid diffusion constants, and spinner flask mass transfer coefficients are available, model predictions using these parameters were compared with published data on EMT6/Ro spheroid growth and micro-environment. The model predictions of reduced spheroid growth due to reduced cell growth rates and cell shedding fit experimental spheroid growth data below 700 microns, but overestimated the spheroid growth rate at larger diameters. Predicted viable rim thicknesses based on predicted near zero glucose concentrations fit published viable rim thickness data for 1000 microns spheroids grown at medium glucose concentrations of 5.5 mM or less. However, the model did not accurately predict the onset of necrosis. Moreover, the model could not predict the observed decreases in oxygen and glucose metabolism seen in spheroids with time, nor could it predict the observed growth plateau. This suggests that other unknown factors, such as inhibitors or cell-cell contact effects, must also be important in affecting spheroid growth and cellular metabolism.

Animals↗
PubMed1997

Spheroid formation of hepatocytes using synthetic polymer.

It is known that hepatocytes show the different morphology depending on the surface condition of cultural substratum. The cells form a floating cell-aggregate called spheroid on proteoglycan-coated or positively charged dishes. The liver cell functions are generally high and maintained for the long-term in the spheroid culture. Since spheroid morphology is preferable for the construction of a bioartificial liver, it is important to develop an effective method of preparing spheroids. In this regard, we examined a preparation method of functional spheroid-like cell-aggregates, in which a synthetic polymer, Eudragit was added to culture medium for inducing liver cell-aggregation. The cell-cell attachment of the aggregate was loose at the beginning of the culture, but it became tight and spheroids were formed 2-3 days after inoculation. When 0.1% Eudragit was added to the medium, the liver functions such as albumin secretion, ammonia removal and urea synthesis were enhanced compared with monolayer and conventional spheroid cultures. The spheroid formation was also performed with suspension culture in a spinner flask. Approximately 80% of the cells inoculated formed spheroids by the addition of the polymer. Moreover, the polymer showed a protective effect from cell damage by agitation. Since this procedure does not require surface for cell attachment, a large amount of spheroids can be prepared in suspension culture.

Acrylic Resins↗
PubMed1990

Multicellular tumor spheroids from human gliomas maintained in organ culture.

Tumor tissue from seven human gliomas was maintained in long-term agar overlay culture as multicellular organotypic spheroids. Light microscopic and ultrastructural observation of the spheroids displayed morphological features similar to those of the original tumor tissue in vivo; in this respect they were different from spheroids obtained from permanent cell lines. The spheroids contained preserved vessels, connective tissue, and macrophages, revealing a close resemblance to the conditions in the original tumor. Flow cytometric deoxyribonucleic acid measurements of cells from the tumor spheroids and from biopsy material obtained directly from the operation revealed the same ploidy and the same amount of proliferating cells in the spheroids as in the original tumor. Fluorescence microscopy using bromodeoxyuridine (BUdR) incorporation and anti-BUdR monoclonal antibody confirmed the proliferative potential of tumor cells in the spheroids. Diameter measurements showed that the size of the spheroids from two of the tumors increased over time while in three other cases it decreased. Spheroids from the remaining two tumors showed no change in size, even after 80 days in culture. These growth data and the relatively high number of proliferating cells, as measured by flow cytometry, indicate that the degree of cell proliferation and cell loss from the spheroids are closely linked, as is the case for tumors in vivo. The culture system presented provides a valuable alternative to propagation of human tumors in animals.

Biopsy↗
PubMed1984

Effects of alpha-difluoromethylornithine on the growth of 9L rat brain tumor multicellular spheroids and their response to 1,3-bis(2-chloroethyl)-1-nitrosourea.

9L rat brain tumor cell spheroids were treated with alpha-difluoromethylornithine (DFMO) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone and in combination. In contrast to results obtained with 9L monolayer culture cells, very low concentrations of DFMO killed spheroid cell within 0.5 day after the start of treatment; cell kill was maximum within 2 to 3 days. DFMO cytotoxicity could be prevented by adding putrescine (to a final concentration of 1 mM) to the culture medium. DFMO also significantly slowed and eventually stopped the growth of spheroids in a dose-dependent manner. Cells in all regions of DFMO-pretreated spheroids were sensitized to BCNU as measured by colony-forming efficiency; this sensitization was prevented when putrescine was added to culture medium before BCNU treatment. When used as single agents, either a 3-day treatment with 10 mM DFMO or a 1-hr treatment with BCNU (1.5 micrograms/ml) produced similar growth delay, but used in combination, the two agents produced a much longer growth delay than produced by either agent alone. Growth of spheroids treated continuously for up to 28 days with 10 mM DFMO ceased at approximately 7 times the volume at the time of treatment. When spheroid cells were treated for 1 hr with BCNU (1.5 micrograms/ml) and then were treated continuously with DFMO, growth plateaued at approximately 3.5 times the volume at the time of treatment; when spheroids were treated first with DFMO for 3 days, then with BCNU (1.5 micrograms/ml) for 1 hr, and then treated continuously with DFMO, growth plateaued at approximately 1.5 times the volume at the time of treatment. The number of clonogenic cells per spheroid that survived combination treatment also reflected the cytotoxic effects of the two drugs. Thus, the combined drug treatment was very effective in inhibiting the growth of spheroids and in preventing an increase in the number of clonogenic cells per spheroid.

Animals↗
PubMed1995

Quantitative distinction of cisplatin-sensitive and -resistant mouse fibrosarcoma cells grown in multicell tumor spheroids.

As a suitable model to study the growth behavior and therapeutic response of drug-resistant and -sensitive cells in three-dimensional coculture we have established multicellular spheroids generated from both cisplatin-sensitive and -resistant cells of a murine fibrosarcoma cell line. A drug resistant clone was derived from the parent cisplatin-sensitive cells by intermittent drug exposure in vitro. As a prerequisite for analysis of differential growth and treatment response of spheroid subpopulations, two efficient methods to discriminate between the two morphologically indistinguishable subpopulations in mixed spheroids were established. In the cisplatin-resistant cell line chosen for the present study, resistance is mainly due to an increased cellular metallothionein content and is therefore associated with increased resistance to CdCl2. Exposure of colonies to high concentrations of CdCl2 thus allowed selective elimination of sensitive colonies. Permanent labeling of either resistant or sensitive cells was achieved by introduction of the Escherichia coli beta-galactosidase marker gene with a retroviral vector system. The transformation of an uncolored galactose derivative by this enzyme into an indigo stain allowed detection of cells carrying and expressing the marker gene. The marker gene and CdCl2 method led to identical results when used simultaneously to distinguish quantitatively between resistant and sensitive colonies grown from plated cells of untreated or irradiated mixed spheroids. The retroviral labeling method was also used successfully in the study of intact spheroids, showing that in 1:1 mixed spheroids, cisplatin-sensitive parent cells accumulate in the spheroid periphery, outgrowing resistant cells and displacing them into the metabolically restricted spheroid center. Only when sensitive and resistant cells are initially mixed at a ratio of 1:9 are the resulting spheroids composed of equal proportions of the 2 cell types throughout 10-20 days after spheroid initiation.

Animals↗
PubMed1999

Vascularization of human glioma spheroids implanted into rat cortex is conferred by two distinct mechanisms.

Aim of this study was to develop and characterize an applicable in vivo model to investigate angiogenesis of human gliomas. An established glioblastoma spheroid model was used to investigate the neovascularization of a standardized avascular solid tumor mass. Spheroids of two human glioma cell lines were labeled with an in vivo fluorescent dye. Single spheroids were implanted into the cortex of athymic rats. After 1, 3, 7, 14, and 21 days, brain sections containing the spheroid were immunostained for endothelial cells or vascular endothelial growth factor (VEGF). The dye-stained glioma spheroid and the endothelial cells were visualized by confocal microscopy. Two distinct mechanisms of tumor vascularization could be observed. (1) "Classical" angiogenesis with new vessels sprouting from existing host vessels into the spheroid was seen. (2) Individual endothelial cells were found to migrate towards and into the center of the spheroid where they coalesced to form new vessels. This process occurred as early as 24 hr after spheroid implantation. Spheroid vascularization was accompanied by an increase of VEGF expression, which peaked 7 days after implantation and returned to normal patterns by 14-21 days. Besides the "classical" angiogenesis by angiogenic blood vessels, the recruitment of individual endothelial cells seems to be an additional mechanism in early glioma vascularization. Our model proves to be a reliable, reproducible system to study in vivo angiogenesis of human gliomas.

Animals↗
PubMed2001

The role of actin filaments and microtubules in hepatocyte spheroid self-assembly.

Cultured rat hepatocytes self-assemble into three-dimensional structures or spheroids that exhibit ultrastructural characteristics of native hepatic tissue and enhanced liver-specific functions. The spheroid formation process involves cell translocation and changes in cell shape, indicative of the reorganization of the cytoskeletal elements. To elucidate the function of the cytoskeleton, hepatocytes undergoing spheroid formation were treated with drugs that disrupt the different cytoskeletal components. Cytochalasin D, which targets the actin filaments, caused inhibition of spheroid formation. The role of microtubules in this process was assessed by incubating the cells with taxol or nocodazole. Perturbation of microtubules had minimal effects on spheroid assembly. Scanning electron micrographs showed no morphological differences between spheroids formed in control cultures and those formed in the presence of taxol or nocodazole. In addition, the effects of those agents on hepatocyte functions were investigated. Albumin secretion and cytochrome P450 2B1/2 activities of hepatocytes were comparable in spheroids formed in the presence of taxol or nocodazole to those formed in control cultures. The levels of these liver-specific activities were lower in cytochalasin D--treated cultures where only dispersed cells or cell clumps were found but spheroids had not found. Thus, hepatocytes require an intact actin network to self-assemble efficiently into functional tissue-like structures. Perturbation of the microtubule lattice does not impair the formation process. Events that transpire during hepatocyte spheroid self-assembly exhibit striking similarities to processes commonly observed in tissue morphogenesis. The results provide insight into the mechanisms that cells employ to organize into tissues and can contribute to our understanding of how to control the cellular assembly in tissue engineering and clinical applications.

Actins↗
PubMed1995

1H NMR investigations of tumor spheroids grown from a human glioma biopsy or from a human malignant glioma cell line.

Three-dimensional spherical aggregates of cells, grown from a permanent human malignant glioma cell line (multicellular GaMG spheroids) and from a human glioma biopsy (fragment spheroids), were investigated by 1H NMR spectroscopy. In addition, 1H NMR spectra of biopsy specimens immediately after explantation and of cell monolayers from primary passage and passage 5 were acquired and compared to those of fragment spheroids. By allowing tumor cells to grow in a three-dimensional arrangement, many biological characteristics of the original tumor in vivo are preserved. A technical procedure was established, indicating that spheroids are particularly suited for NMR spectroscopic studies. Well-resolved proton NMR spectra were obtained from homogeneously reaggregated as well as from fragment spheroids which were immobilized in agar. We found that fragment spheroids closely resemble characteristic spectral patterns of the corresponding tumor tissue in vitro, thus making such tissue available for 1H NMR spectroscopic measurements under easy to standardize tissue-culture conditions. Furthermore, the effect of altering glucose supply on metabolism and growth was studied with multicellular GaMG spheroids. The spectroscopic differences found between cell suspensions and multicellular spheroids indicate that GaMG spheroids produce large amounts of lactate and that they can adapt their metabolism depending on glucose supply similar to tumors in vivo.

Biopsy↗
PubMed2005

Viable head and neck tumor spheroids stimulate in vitro autologous monocyte MCP-1 secretion through soluble substances and CD14/lectin-like receptors.

Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma (HNSCC) patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro augment their secretion of monocyte chemotactic protein-1 (MCP-1). Presently, the aims of the present work were to study whether the metabolic activity, secreted products and/or specific receptor/ligand on the surface of the F-spheroids and monocytes are necessary for stimulation of the monocyte MCP-1 secretion upon F-spheroid co-culture. Actinomycin D (1 mug/ml for 24 h) pre-treatment of the F-spheroids abolished the monocyte MCP-1 co-culture response. Co-culture of monocytes and F-spheroids separated by a semi-permeable membrane showed a decreased, but still present, monocyte MCP-1 co-culture response. Conditioned medium from F-spheroids stimulated allogenous monocytes to secrete MCP-1. The addition of glucose or galactose, but not mannose, to co-cultures partially inhibited the monocyte MCP-1 co-culture response. The addition of anti-CD14 antibody diminished the MCP-1 co-culture response. In conclusion, the monocyte MCP-1 co-culture response is dependent on metabolically active spheroids, secreted stimuli, and is augmented by direct contact with F-spheroids, possibly via lectin-like receptors and the CD14 receptor.

Autoantibodies↗
PubMed2004

Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.

OBJECTIVES: Ovarian carcinoma cells form multicellular aggregates, or spheroids, in the peritoneal cavity of patients with advanced disease. The current paradigm that ascites spheroids are non-adhesive leaves their contribution to ovarian carcinoma dissemination undefined. Here, spheroids obtained from ovarian carcinoma patients' ascites were characterized for their ability to adhere to molecules encountered in the peritoneal cavity, with the goal of establishing their potential to contribute to ovarian cancer spread. METHODS: Spheroids were recovered from the ascites fluid of 11 patients with stage III or stage IV ovarian carcinoma. Adhesion assays to extracellular matrix (ECM) proteins and human mesothelial cell monolayers were performed for each of the ascites spheroid samples. Subsequently, inhibition assays were performed to identify the cell receptors involved. RESULTS: Most ascites samples adhered moderately to fibronectin and type I collagen, with reduced adhesion to type IV collagen and laminin. Monoclonal antibodies against the beta1 integrin subunit partially inhibited this adhesion. Ascites spheroids also adhered to hyaluronan. Additionally, spheroids adhered to live, but not fixed, human mesothelial cell monolayers, and this adhesion was partially mediated by beta1 integrins. CONCLUSIONS: The cellular content of the ascites fluid has often been considered non-adhesive, but our findings are the first to suggest that patient-derived ascites spheroids can adhere to mesothelial extracellular matrix via beta1 integrins, indicating that spheroids should not be ignored in the dissemination of ovarian cancer.

Ascites↗