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PubMed1996

Radiotoxic effect and dosimetry of 67GA in multicellular spheroids as compared to single cells of the lymphoma cell line U715.

PURPOSE: The purpose of the present study was to investigate if there were differences between U715 spheroids and single cells in the radiotoxic effect of 67Ga on cell growth and clonogenic capacity in vitro and to generate dosimetric approaches for the multicellular tumor model. METHODS AND MATERIALS: Human lymphoma U715 cells were cultured in vitro as single cells and multicellular spheroids, grown with the use of a combination of fibrin clot technique, spinner flasks, and liquid-overlay culture. Cells were incubated with 2.96-8.88 MBq/ml 67Gallium for 4 days. Spheroids were dispersed to single cells by treatment with plasmin. Residual proliferative and clonogenic capacity after 67Ga incubation were assayed using the MTT-test and clonogenic test, respectively. Autoradiography was performed with 1 microm sections and Ilford L4 liquid photographic emulsion. Dosimetric approaches were made, based on the MIRD-approach. RESULTS: During 67Ga incubation proliferation was inhibited. The residual proliferative or clonogenic capacity was inhibited by 8.88 MBq/ml for 39 and 88%, respectively. For single cells with 6.66 MBq/ml these inhibitions were 64 and 96%, respectively. Autoradiography showed an homogeneous distribution of 67Ga in spheroids and single cells. In single cells a 2.1-3.5 times higher 67Ga uptake/cell than in spheroids produced an equitoxic effect. The uptake parameters were implemented in new dosimetric approaches and showed that the efficacy of intracellular 67Ga was two times higher in spheroid clusters than in single cells due to energy deposition of internal conversion electrons within the cell clusters with a mean diameter size of nine cells. Both for proliferative and clonogenic capacity the exponential survival curves were superimposed. CONCLUSIONS: With the new approaches made in our dosimetric model the discrepancy found between 67Ga accumulation and radiotoxic effect in spheroids as compared to single cells can be explained by additional effects of the crossfire of internal conversion electrons within clusters of about nine cells in diameter in spheroids. Only twice as much 67Ga was needed to reach equitoxic absorbed doses in spheroids than was needed in single cells. Such might be important for the use of 67Ga treatment of small metastasis of malignant lymphoma.

Autoradiography↗
PubMed2001

Cytochrome P450 activity in control and induced long-term cultures of rat hepatocyte spheroids.

Long-term events such as enzyme induction or chronic toxicity require long-term liver culture models that maintain activity of xenobiotic metabolising enzymes. The levels of these enzyme activities and their responsiveness to chemical induction was studied in rat hepatocyte spheroids, a potential long-term hepatocyte culture model. In comparison with other long-term liver culture models, the basal metabolic activity of spheroids has not been well studied. Additionally, no existing data on the induction of CYP3A activity in spheroids could be found. The basal xenobiotic metabolising activity of rat hepatocyte spheroids was monitored over 14 days in culture, using testosterone as a probe substrate. When spheroids from days 2-14 in culture were compared to 24-h control spheroids, there was a differential maintenance of basal CYP activity. CYP2A and CYP3A activities were maintained over the culture period, while there were time-related decreases in CYP2C11 and CYP2C/CYP2B1/2 activities. The responsiveness of rat hepatocyte spheroids to chemical induction was studied following treatment with phenobarbitone (PB) or dexamethasone (DEX). PB treatment induced CYP2A, CYP2C, CYP2B1/2 and CYP3A activities. DEX treatment resulted in an induction of CYP3A and CYP2C11 activities. The results demonstrate that rat hepatocyte spheroids retained some of the liver-specific functions essential in a long-term hepatocyte culture model, thus making spheroids comparable to other long-term culture models available.

Animal Testing Alternatives↗
PubMed2002

Identification of genes differentially expressed in V79 cells grown as multicell spheroids.

PURPOSE: Growth of Chinese hamster V79 cells as multicell spheroids leads to an increase in resistance to killing by ionizing radiation and etoposide. Differential display was used to identify changes in gene expression that occur when cells are grown as spheroids. MATERIALS AND METHODS: Differential display was performed using exponentially growing Chinese hamster V79 cells and the outer cell layer of V79 spheroids. Using six different pairs of primers, 20 altered bands were selected. Eight genes, confirmed using reverse Northerns, showed a match in a GenBank search. Antibodies against a calcium-binding protein, mts1, confirmed differential expression of this protein. Intracellular free calcium levels were measured using fluo-3 fluorescence, and the effect of a calcium-binding agent on etoposide resistance was examined using the comet assay. RESULTS: Genes upregulated in the outer cell layer of spheroids relative to monolayers included: (1) mts1 (S100A4), a calcium binding protein implicated in proliferation, metastasis, cell adhesion, and angiogenesis; (2) cytochrome c oxidase II; (3) B-ind1, a mediator of Rac-1 signaling; (4) TRAM, an endoplasmic reticulum protein. Genes downregulated in spheroids were: (5) phosphoglycerate kinase; (6) ARL-3, a ras-related GTP binding protein; (7) MHC class III complement 4A; and (8) 2,4-dienoyl-CoA. Immunohistochemistry confirmed overexpression of mts1 and another calcium-binding protein, calreticulin, in V79 outer spheroid cells relative to monolayers. C6 rat glioma and SiHa human cervical carcinoma cells that demonstrate a contact effect also showed upregulation of mts1 or calreticulin, while WiDr colon carcinoma cells that lack contact resistance showed no change in expression of either calcium binding protein. Intracellular free calcium levels were found to be almost two times lower in the outer cells of V79 spheroids compared to monolayers. V79 monolayer and outer spheroid cells treated with the calcium chelating agent BAPTA-AM showed a similar level of DNA damage by etoposide. CONCLUSIONS: Expression of genes involved in calcium binding, signaling and metabolism are differentially expressed when V79 cells are grown as spheroids. Differences in the levels of intracellular calcium may underlie the contact effect.

Animals↗
PubMed2003

Dynamic assessment of angiogenesis in renal cell carcinoma spheroids by intravital microscopy.

PURPOSE: Renal cell cancer represents a suitable tumor model for in vivo observation of neo-angiogenesis. We used intravital microscopy and the well established dorsal skin fold chamber model to characterize neo-angiogenesis in freely implanted renal cell cancer spheroids. MATERIAL AND METHODS: Tumor spheroids were implanted into dorsal skin fold chambers of 8 nude mice. At days 3, 6, 10 and 14 after implantation the newly vascularized spheroid area, density of perfused microvessels in the spheroid versus the periphery, capillary center erythrocyte velocity and capillary diameter were recorded by intravital microscopy. Video images were analyzed by a computer assisted image analysis device. After the experiments the chambers were analyzed morphologically. RESULTS: The model enabled quantitative analysis of microcirculation and angiogenesis in the renal cell cancer spheroids during 14 days of observation. Mean spheroid center perfused microvessel density +/- SEM increased from 3 +/- 2 to 269 +/- 21 cm.-1 on days 3 to 10 and subsequently decreased to 189 +/- 38 cm.-1 on day 14. Spheroid periphery perfused microvessel density was significantly higher throughout the experiments, attaining a mean maximum of 522 +/- 34 cm. on day 14. Mean capillary diameter decreased continuously from 14.2 +/- 0.9 to 8.4 +/- 0.4 microm. on days 3 to 14. In contrast, mean capillary center erythrocyte velocity significantly increased during 14 days of observation from 0.09 + 0.02 mm. per second on day 3 to 0.24 +/- 0.08 mm. per second on day 14. Histological analysis after 14 days revealed the spheroids as cell clusters in the upper layers of the dorsal skin fold chamber. CONCLUSIONS: The model is suitable for the analysis of renal cell cancer angiogenesis. Although it is heterotopic, angiogenesis in renal cell cancer spheroids mimics important characteristics of human renal cell cancer.

Animals↗
PubMed2004

Microenvironmental regulation of proliferation in multicellular spheroids is mediated through differential expression of cyclin-dependent kinase inhibitors.

Multicellular spheroids composed of transformed cells are known to mimic the growth characteristics of tumors and to develop gradients in proliferation with increasing size. This progressive accumulation of quiescent cells is presumably an active process that occurs in response to the microenvironmental stresses that develop within the three-dimensional structure, and, yet, little is known regarding either the signals that induce the cell cycle arrest or the molecular basis for the halt in proliferation. We have previously reported that regulation of cyclin-dependent kinase (CDK) inhibitors (CKIs) differs in monolayer versus spheroid cell culture. In this study, we have examined the expression of three CKIs in EMT6 mouse mammary carcinoma and MEL28 human melanoma spheroids, as a function both of spheroid size and of location within the spheroid. We report that expression of the CKIs p18(INK4c), p21(waf1/cip1), and p27(Kip1) all increase as the spheroid grows and develops a quiescent cell fraction. However, by examining protein expression in discrete regions of the spheroid, we have found that only p18(INK4c) and p27(Kip1) expression positively correlate with growth arrest, whereas p21(waf1/cip1) is expressed predominantly in proliferating cells. Further analysis indicated that, in the quiescent cells, p18(INK4c) is found in increasing association with CDK6, whereas p27(Kip1) associates predominantly with CDK2. In MEL28 cells, CDK2 activity is completely abrogated in the inner regions of the spheroid, whereas in EMT6 cells, CDK2 activity decreases in accordance with a decrease in expression. We also observed a decrease in all cell cycle regulatory proteins in the innermost spheroid fraction, including CDKs, CKIs, and cyclins. Induction of CKIs from separate families, as well as their association with distinct target CDKs, suggests that there may be multiple checkpoints activated to ensure cell cycle arrest in non-growth-conducive environments. Furthermore, because very similar observations were made in both a human melanoma cell line and a mouse mammary carcinoma cell line, our results indicate that these checkpoints, as well as the signal transduction pathways that activate them, are highly conserved.

Animals↗
PubMed2003

Cryopreservation of organotypic brain spheroid cultures.

The cryopreservation of hen and rat brain spheroids was investigated. Brain spheroid cultures were prepared from 7-day-old hen embryos or 16-day-old rat embryos, by using a rotation-mediated culture system. The spheroids were cryopreserved in medium containing 5-15% dimethyl sulphoxide (DMSO) and stored in liquid nitrogen, by using a two-stage cooling procedure. The results show that the viability, as indicated by the total protein content of hen embryo brain spheroids at 24 hours, and at 3, 7 and 28 days after thawing, ranged from 45.5% to 64.2% of control values. It took 3 days for the post-thaw brain spheroids to stabilise, as indicated by their morphology and selected neural markers of functionality. These functions were maintained over a 28-day observation period. Spheroids cultured for 12-15 days in vitro before cryopreservation survived better than those that were cryopreserved after 5-7 days in vitro. The viability and biochemical functionality of spheroids after long-term (up to 6 months) storage were similar to those following short-term storage. The viability of rat brain spheroids cryopreserved in 15% DMSO, as indicated by total protein content, at 24 hours, and at 3 or 7 days after thawing, ranged from 23.1% to 32.1% of control values. This study shows for the first time that brain spheroids prepared from primary tissue can be successfully cryopreserved.

Acetylcholinesterase↗
PubMed2002

The DC electrical-field-induced Ca(2+) response and growth stimulation of multicellular tumor spheroids are mediated by ATP release and purinergic receptor stimulation.

It has been demonstrated that adenosine 5'-triphosphate (ATP) is actively secreted by cells, thereby eliciting Ca(2+)-dependent signal transduction cascades in an autocrine and paracrine manner. In the present study the effects of direct current (DC) electrical fields on ATP release, the intracellular Ca(2+) concentration [Ca(2+)](i) and growth of multicellular prostate tumor spheroids were investigated. Treatment of multicellular tumor spheroids by a single DC electrical field pulse with a field strength of 750 Vm(-1) for 60 seconds resulted in a transient Ca(2+) response, activation of c-Fos and growth stimulation. The initial [Ca(2+)](i) signal was elicited at the anode-facing side of the spheroid and spread with a velocity of approximately 12 microm per second across the spheroid surface. The electrical-field-evoked Ca(2+) response as well as c-Fos activation and growth stimulation of tumor spheroids were inhibited by pretreatment with the anion channel blockers NPPB, niflumic acid and tamoxifen. Furthermore, the Ca(2+) response elicited by electrical field treatment was abolished following purinergic receptor desensitivation by repetitive treatment of tumor spheroids with ATP and pretreatment with the purinergic receptor antagonist suramin as well as with apyrase. Electrical field treatment of tumor spheroids resulted in release of ATP into the supernatant as evaluated by luciferin/luciferase bioluminescence. ATP release was efficiently inhibited in the presence of anion channel blockers. Our data suggest that electrical field treatment of multicellular tumor spheroids results in ATP release, which concomitantly activates purinergic receptors, elicits a Ca(2+) wave spreading through the tumor spheroid tissue and stimulates tumor growth.

Adenosine Triphosphate↗
PubMedDate not supplied

Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers.

Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.

Animals↗
PubMed1984

Multicellular spheroids grown directly from human tumour material.

Human tumour cells from surgical material were grown as multicellular spheroids. In 16 out of 20 cultures spheroids with a diameter of more than 250 microns could be observed. In 6 out of 20 cultures more than 30 spheroids with a diameter of at least 300 microns were obtained, i.e. 30% of the cultures fulfilled the criterion for a possible chemosensitivity test on primary cell spheroids. A study of stained sections from the spheroids and the respective tumours, showed that the morphology of the spheroid was very similar that of the tumour from which the cells were derived. Samples from 9 malignant melanomas, 4 bladder carcinomas, 2 renal-cell carcinomas, 2 ovarian carcinomas, 1 lymphoma, 1 colon carcinoma, and 1 schwannoma were tested for spheroid growth. Spheroids were obtained from at least one representative of all these tumour types. However, investigations involving larger numbers of tumours are needed to find out which tumour types are most suitable for further biological characterization of primary cell spheroids and tests for therapy response.

Adult↗
PubMed1995

Calibration of misonidazole labeling by simultaneous measurement of oxygen tension and labeling density in multicellular spheroids.

To correlate misonidazole concentrations and oxygen pressures (Po2) at identical locations within EMT6/Ro multi-cell spheroids (mean diameters +/- SD: 867 +/- 20 microns), Po2 measurements were performed with oxygen-sensitive microelectrodes during incubation of these spheroids with tritiated misonidazole (10 mg/I; 445 microCi/mg). In each individual spheroid, Po2 profiles were correlated with the corresponding spatial distribution of misonidazole as quantified by conventional autoradiography and grain counting. To compare the oxygenation status of spheroids in the measuring chamber with that of spheroids in spinner culture, misonidazole labeling was performed in both environments following the same protocol. All experiments were conducted in 20% oxygen and BME or in 5% oxygen and DMEM to obtain spheroids with different degrees of oxygenation. Labeled misonidazole was fairly evenly distributed in the outer, better oxygenated regions of EMT6 spheroids. In contrast, there was an accumulation of the labeled substance near central necrosis where low oxygen tensions were measured. Grain densities were similar at corresponding oxygen pressures under both environmental conditions. Except for some scatter, grain density as a function of oxygen pressure showed little variation in the Po2 range of 20-60 mm Hg, but exhibited a steep increase below 10 mm Hg. The findings imply that a substantial rise in local misonidazole labeling indicates a metabolically active tissue region at low Po2 that is not necessarily identical with the radiobiologically hypoxic cell fraction. A comparison of the labeling densities of spheroids in spinner flasks and in the Po2 measuring chamber indicates that oxygenation of spheroids is better in rotation culture than during microelectrode measurements.

Animals↗
PubMed1989

Homogeneous penetration but heterogeneous binding of antibodies to carcinoembryonic antigen in human colon carcinoma HT-29 spheroids.

The monoclonal antibodies 38S1, directed against the carcinoembryonic antigen (CEA), were tested for penetration and binding in human colon carcinoma HT-29 spheroids. Penetration was studied with a method which has not previously been used in immunological investigations. The method, which allows unbound substances to be visualized, is based on freeze drying, vapour fixation, dry sectioning and dry autoradiography. The antibodies penetrated easily and all parts of the HT-29 spheroids seemed to be reached within 15 min. The penetration was even faster than in control glioma U-118MG spheroids that did not express CEA. Binding of the 38S1 antibodies was demonstrated after processing with conventional histology and autoradiography. The binding in the HT-29 spheroids was, after a 1-h incubation period, extremely heterogeneous and occurred mainly in the peripheral parts. More cells were binding the antibodies after 8-h and 32-h incubations and these cells were arranged in peripheral clusters. No binding at all was seen in the CEA-negative glioma spheroids. The distribution of CEA antigens in monolayers and in frozen sections of spheroids of HT-29 cells was analysed with immunohistochemical staining using polyclonal CEA antibodies. The CEA antigens were heterogeneously distributed in both spheroids and monolayers and were as heterogenous as the binding of the monoclonal antibodies in the living spheroids. Thus, the heterogeneous binding in the living spheroids was not due to penetration barriers, but instead to the heterogeneity in the CEA antigen expression.

Adenocarcinoma↗
PubMed1985

The growth and morphology of FRTL-5 thyroid epithelial cells grown as multicellular spheroids in vitro.

FRTL-5 cells, a diploid line of differentiated rat thyroid epithelial cells, have been grown as multicellular spheroids in spinner culture. Spheroids were initiated by seeding FRTL-5 cells either into Lab-Tek dishes or culture flasks with a 0.5% agar base. Thyroid stimulating hormone (TSH, greater than 1.0 mU/ml) was required for initial cell aggregation and spheroid growth. After 1 wk cellular aggregates were transferred to suspension culture in spinner flasks. As with FRTL-5 monolayer cultures, continued spheroid growth required the addition of TSH to the culture medium. The most unique characteristic of the FRTL-5 spheroids was the development of central lumina similar to thyroid follicles in vivo. Follicular structures were absent from spheroids not stimulated with TSH. In the presence of TSH epithelial cells seem metabolically active with morphological evidence of biosynthesis of thyroglobulin-like material and basal laminar-like components. In contrast, all evidence of cellular metabolic activity is absent from cells in spheroids maintained in the absence of TSH. Thus, nontransformed FRTL-5 cells grown as three-dimensional multicellular spheroids responded to hormonal manipulation in a manner comparable to follicular epithelial cells in vivo. This spheroid model might therefore prove to be a very effective tool for investigating aspects of thyroid physiology and pathology in vitro.

Animals↗
PubMed2005

Overexpression of heat shock proteins in pallido-nigral axonal spheroids of nonhuman aged primates.

The occurrence of spheroids has been described in the globus pallidus (GP) and substantia nigra pars reticulata (SNr) of aged rhesus monkeys. Opinions vary as to the origin of spheroids. Ultrastructural and immunohistochemical analysis suggested that spheroids originate from degenerating axons or astroglia. In the present study, we have investigated the GP and SNr of aged monkeys (Macaca fascicularis and Macaca mulatta). Although immunoreactive for microtubule-associated protein (MAP) 1A, tau, amyloid precursor protein, synaptophysin and phosphorylated neurofilament, spheroids were not immunoreactive for MAP1B and MAP2. We confirmed the axonal nature of pallido-nigral spheroids in aged rhesus monkeys. Pallido-nigral spheroids have been reported to overexpress stress proteins, such as ubiquitin, alphaB-crystallin, and heat shock protein (Hsp) 27. We further evaluated the expression of Hsps in pallido-nigral spheroids. As well as being intensely immunoreactive for ubiquitin, alphaB-crystallin, Hsp27, and Hsp70, spheroids were immunoreactive for Hsp32 (heme oxygenase-1), Hsp40, Hsp60, and Hsp90. On the basis of these findings, we speculate that Hsp32-immunoreactive spheroids might be expressed as an oxidative stress response. Induction of other Hsps might play a role in protection of axons from the aggregation of neurofilament, MAPs and other proteins, and failure to protect degenerating axons might result in their proteolysis by the ubiquitin-proteasome system.

Age Factors↗
PubMed1992

Activity of the human metallothionein promoter (hMT-IIA) in cell populations isolated from varying depths in multicell spheroids following exposure to cadmium.

In order to evaluate whether tumor microenvironment might influence the response of the metallothionein promoter to heavy-metal exposure, we transfected HT-29 colon carcinoma cells with the vector phMTIIA-CAT-neo, containing a fusion gene consisting of 426 bp of the human metallothionein-IIa (hMT-IIA) promoter immediately upstream of the bacterial chloramphenicol acetyl transferase (CAT) gene. We grew one of the stable transfectants (clone 20) as three-dimensional multicell tumor spheroids, exposed them to CdCl2 and measured CAT expression in cells isolated from various depths into the spheroids. Cellular populations were isolated by flow cytometry on the basis of Hoescht 33342 fluorescence intensity, taking advantage of the dye's diffusion gradient to isolate cells from inner (dim) and outer (bright) regions of stained spheroids. When intact spheroids were incubated for 18 h in the presence of 5 microM CdCl2, CAT activity was induced in all cell fractions isolated from the spheroids, but induction was 10-fold greater in cells in the outermost fraction (fraction 10) than inner fraction (fraction 2). When spheroids were dissociated, sorted into individual fractions and then incubated with cadmium, CAT expression was maximized in all fractions. Exposure of intact spheroids to 30 microM CdCl2 resulted in increased CAT induction in cells isolated from the internal fractions of the spheroids. The data suggest that limited diffusion of cadmium through cells organized in a tissue-like arrangement may account for the lower levels of hMT-IIA promoter activity observed in cells collected from increasing depths into the spheroids.

Cadmium↗
PubMed1990

Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments.

Adult rat hepatocytes formed floating multicellular spheroids in primary culture in an uncoated plastic dish with a positively charged surface. Cells in the spheroids formed in such a simple way were similar to those formed in dishes coated with proteoglycan fraction isolated from rat liver reticulin fibers; in both cases, cells maintained high ability to produce albumin and poor ability to proliferate in response to epidermal growth factor. Coating dishes with albumin was also helpful in spheroid formation; coating with 2-hydroxymethyl methacrylate resulted in formation of incomplete spheroids. Elimination of serum factors was essential for the formation of spheroids; when cells were washed with serum-containing medium before seeding or if the medium was replaced with a serum-containing medium, spheroid formation was completely inhibited. Collagens, fibronectin, and laminin, all of which promote the adhesion and spreading of hepatocytes on substrates, inhibited spheroid formation. Furthermore, collagens disintegrated spheroids, and cells in the monolayer initiated proliferation. Thus, two distinct, mutually exclusive features of primary culture of adult hepatocytes apparently exist; monolayer culture with proliferative activity in an adherent environment and spheroid culture with poor proliferative activity and high albumin-producing ability in a nonadherent environment.

Albumins↗
PubMedDate not supplied

Human choriocarcinoma (JAr) cells grown as multicellular spheroids.

JAr choriocarcinoma cells grew as multicellular spheroids, and exhibited a logarithmic pattern of growth, reaching geometric mean diameters of at least 1200 microns after 21 days in culture. HCG, E2 and P4 were secreted into the culture medium throughout the entire culture period, in proportion to spheroid size. This, along with the presence of lipid droplets, non-staining glycogen, Golgi apparatus and microvilli on the spheroid surface and in intercellular spaces indicated that the JAr cells within spheroids were functionally active. While spheroids of less than 800 microns GMD attached to culture dishes and produced cellular outgrowth, larger spheroids showed impaired attachment, and a delay in the subsequent production of outgrowth, which was not correlated with the development of a necrotic core. This outgrowth contained more multinucleated cells and cellular projections than that of smaller spheroids, suggesting that the cells in larger spheroids had undergone early differentiative changes. The spheroid culture system will be applied to the study of processes, such as implantation, which may require a three-dimensional arrangement of placental cells.

Choriocarcinoma↗
PubMed1989

Radiosensitivity of different human tumor cells lines grown as multicellular spheroids determined from growth curves and survival data.

Five human tumor cell lines were grown as multicellular tumor spheroids (MTS) to determine whether multicellular tumor spheroids derived from different types of tumors would show tumor-type dependent differences in response to single-dose irradiation, and whether these differences paralleled clinical behavior. Multicellular tumor spheroids of two neuroblastoma, one lung adenocarcinoma, one melanoma, and a squamous cell carcinoma of the oral tongue, were studied in terms of growth delay, calculated cell survival, and spheroid control dose50 (SCD50). Growth delay and cell survival analysis for the tumor cell lines showed sensitivities that correlated well with clinical behavior of the tumor types of origin. Similar to other studies on melanoma multicellular tumor spheroids our spheroid control dose50 results for the melanoma cell line deviated from the general pattern of sensitivity. This might be due to the location of surviving cells, which prohibits proliferation of surviving cells and hence growth of melanoma multicellular tumor spheroids. This study demonstrates that radiosensitivity of human tumor cell lines can be evaluated in terms of growth delay, calculated cell survival, and spheroid control dose50 when grown as multicellular tumor spheroids. The sensitivity established from these evaluations parallels clinical behavior, thus offering a unique tool for the in vitro analysis of human tumor radiosensitivity.

Cell Division↗
PubMed1992

Radioresponsiveness, sublethal damage repair and stem cell rate in spheroids from three human tumor lines: comparison with xenograft data.

Dose-control curves after fractionated irradiation were generated for small oxic spheroids from the two human glioma cell lines, U87 and A7, as well as the squamous cell carcinoma line FaDu. These data were fitted by the linear quadratic model assuming Poisson statistics. The alpha/beta values of A7, U87, and FaDu spheroids, respectively were 10.3 (8.1-12.9) Gy, 17.8 (15.1-21.1) Gy, and 37.9 (29.1-51.5) Gy. These data were compared with those previously published by Suit et al. (31) and Zietman et al. (40) for 6 mm xenografts of U87 and FaDu after fractionated irradiation and for A7 after single dose irradiation under clamped conditions. A good agreement in the alpha/beta values was observed for U87 and Fadu xenografts and spheroids assuming an oxygen enhancement ratio (OER) of 2.7. In addition, the ranking according to the single doses needed to control 50% of the tumors agreed for xenografts and spheroids from the three cell lines. U87 was the most resistant line in both model systems, followed by A7 and FaDu. However, the absolute values of alpha and beta, obtained from the direct fit to the dose-control data were only about half as high for U87 and FaDu xenografts than for the spheroids. Monte Carlo simulations showed that this discrepancy can be explained by a greater tumor heterogeneity of the xenografts. While the number of critical stem cells or spheroid rescuing units equaled the number of cells per spheroid for the three cell lines, the percentage of tumor rescuing units for Fadu and U87 xenografts was estimated to be below 1%. In a next step, survival curves were generated for exponentially growing cells of the three lines. A7 cells were significantly more radioresistant when plated on tissue plastic than in soft agar. Using the most resistance-promoting colony assay conditions for each cell line, a good agreement was observed for the alpha and SF2Gy values calculated from the colony and spheroid control data. This study shows that the spheroid model can quantitatively predict the repair capacity of sublethal damage as well as the rank order of radiation sensitivity of in vivo tumors.

Animals↗