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PubMed1978

Growth fraction as the major determinant of multicellular tumor spheroid growth rates.

Multicellular tumor spheroids (MTS) from seven murine solid tumors have been compared in terms of their growth rate and the fraction of the cells that are in cycle. Growth rates ranged from a low of 19 micron/day through a maximum of 106 micron/day, demonstrating a far wider range of growth rates than is observed when the same tumors are grown as monolayers. Through the use of an [125I]iodouridine deoxyribose incorporation assay, it has been possible to demonstrate that cells within the MTS that are in cycle double at a slower rate than they do in monolayers and that the depth of the dividing shell ranges from less than 20 to more than 100 micron. The depth of the dividing shell or the growth fraction is highly correlated with the MTS growth rate (r = 0.97). We conclude that the major factor that determines the wide range of MTS growth rates, in spite of relatively uniform doubling times in monolayer, is the fraction of the cells that are in cycle.

Cell Cycle↗
PubMed1978

Multicellular tumor spheroid formation by breast cancer cells isolated from different sites.

Fourteen breast cancer lines (8 human, 5 rat, and 1 mouse) have been studied in terms of their ability to form multicellular tumor spheroids (MTS) with the agar-base method. Only 8 of the lines formed MTS in contrast to a 100% efficiency in a series of 11 varied tumors reported in the initial studies with this method. We have compared the lines that do and do not form MTS in terms of a variety of characteristics (e.g., estrogen receptors, time in serial passage, growth in nude mice, etc.), and only one characteristic, the source of the original tumor cells, was predictive of MTS-forming ability. All 8 of the breast cancer lines (and the original 11 lines) that formed MTS had been obtained from solid growths (primaries or metastases), while the 6 breast cancer lines that did not form MTS were all derived from pleural effusions. Similarly, artificial selection for an ascites variant of the MTS-forming rat 13762 adenocarcinoma line produced the 13762-A line, which could no longer form MTS. These results suggest that breast cancer cells derived from pleural effusions are genetically different from the bulk of the tumor cells in solid breast cancer samples, that they are unable to grow in true solid form, and that these differences persist in spite of prolonged propagation in tissue culture.

Adaptation, Physiological↗
PubMed1984

Response of 9L rat brain tumor multicellular spheroids to single and fractionated doses of 1,3-bis(2-chloroethyl)-1-nitrosourea.

This study was designed to examine the relative effect of each of four fractions of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) against 9L rat brain tumor multicellular spheroids and to compare the results of the cell survival and growth delay assays. Similar levels of cell kill resulted when BCNU was administered either as single fractions of 1.5, 3.0, 4.5, or 6.0 micrograms/ml for 1 hr or as one to four fractions of 1.5 micrograms/ml that were administered sequentially for 1 hr each. Survival was increased if the assay was delayed until 24 hr after drug treatment, which indicates that 9L cells in spheroids recover from BCNU-induced potentially lethal damage. When BCNU was administered in 1.5-micrograms/ml fractions, plating efficiencies depended markedly on the interval between the fractions. The 12-hr protocol produced an overall higher cell kill. Fractionation schedules of 24 and 36 hr produced less cell kill than did the other schedules. Survival plateaued for the last three treatments with BCNU in the 36-hr schedule. Cells in S phase at the time of administration of the initial 1.5-micrograms/ml fraction of BCNU moved into G1- and G2-M phases by 12 hr after treatment. For time periods longer than 12 hr, cells began to appear in the BCNU-resistant S phase. Thus, the movement of cells into the drug-sensitive and -resistant phases after the first fraction correlates well with the corresponding overall cytotoxic effect produced by treatment with the combined BCNU (1.5 micrograms/ml) fractions. For a higher concentration (3.0 micrograms/ml for 1 hr), maximum cell kill was reached within the 12- to 18-hr interval, after which cell kill plateaued. Cells were not found in the S-phase fraction 12 to 36 hr after the first treatment with 3.0 micrograms/ml; maximum cell kill for the fractionated protocols resulted at these times. Therefore, BCNU, which is classified as a cell cycle-nonspecific drug, can induce a partial synchrony in 9L spheroid cells, which determines the overall cytotoxicity produced by fractionated BCNU protocols. Although spheroids did not shrink during or after exposure to BCNU, growth was retarded by treatment with all doses and schedules. An optimum time point for growth delay measurement could not be determined from the data. However, correlations between cell survival and growth delay were obtained with arbitrarily chosen end point volumes of four and ten times the volume at the time of treatment.

Animals↗
PubMed1984

A possible bias in growth-kinetic estimates of the thickness of the growing layer in multicellular tumor spheroids.

Yuhas and Li (Cancer Res., 38: 1528-1532, 1978) have proposed a method for estimating the thickness of the growing layer in multicellular tumor spheroids. Their method assumes, however, that the thickness of the growing layer is independent of spheroid radius; this assumption seems implausible in view of the fact that, for purely geometrical reasons, oxygen diffusion distance is greater in small than in large spheroids. In this communication, theoretical growth rates are calculated for a growth kinetic model based on Burton's (Growth, 30: 157-176, 1966) model of oxygen diffusion, and it is shown that for this model the Yuhas-Li estimates of thickness of the growing layer are 10 to 30% below the true thickness of this layer in the larger of the two spheroids used for the estimation. To generalize beyond this particular model, it is shown that, for any model where the growing layer is thicker in small than in large spheroids, the Yuhas-Li method underestimates the thickness of the growing layer. However, for our particular model at least, the bias in the Yuhas-Li estimates is fairly constant and relatively small, so that these estimates may be quite serviceable, especially for purely comparative studies.

Cell Division↗
PubMed1980

Comparison of in situ and peripheral host immunity to syngeneic tumours employing the multicellular spheroid model.

The multicellular tumour spheroid was used as a model system to assess the in situ host immune response to the EMT6/Ro mammary tumour in syngeneic BALB/cKa mice. In sensitized mice the spheroids were rapidly infiltrated by host cells including macrophages, lymphocytes and granulocytes. Tumour cell killing was evident within 1 day and resulted in the eventual complete destruction of the spheroids. Host cells within the spheroids had a greater cytolytic capacity than the surrounding peritoneal cells and virtually no cytolytic activity was detectable in cells from the spleen. A similar discrepancy between in situ and peripheral immunity was found in mice bearing solid EMT6/Ro tumours.

Animals↗
PubMed1982

Delivery of lethal hits by cytotoxic T lymphocytes in multicellular conjugates occurs sequentially but at random times.

We propose a stochastic model to explain kinetic data on the delivery of lethal hits by cytotoxic T lymphocytes in multicellular conjugates. The comparison of our model with data of Zagury et al. suggests that cytotoxic T cells deliver lethal hits at random, and when conjugated to multiple target cels these cells attack undamaged target cells one at a time. The mean rate at which lethal hits are delivered per target cell is approximately the same for conjugates containing one or two target cells but slows substantially in conjugates containing three target cells. If our model is modified so that both previously hit and unhit target cells are susceptible to being lethally hit at the same rate, a very poor fit to Zagury et al's data is obtained. This leads us to conclude that previously hit cells are either not hit again or are hit at a substantially reduced rate.

Immunity, Cellular↗
PubMed1978

In vitro analysis of the response of multicellular tumor spheroids exposed to chemotherapeutic agents in vitro or in vivo.

Multicellular tumor spheroids (MTS) have been exposed to chemotherapeutic agents in vitro (nitrogen mustard) or in vivo (cyclophosphamide) and analyzed in vitro in terms of altered growth patterns. Whether the MTS were exposed in vitro or in vivo, the major effect of the drugs was to induce a dose-dependent lag period before the normal MTS growth rate resumed. Exposure of MTS in the peritoneal cavity to i.v. injection cyclophosphamide results in patterns similar to the in vitro exposure system, except that a host anti-MTS reaction was detected. In combination, these two methods allow the study of the responses of these complex tumor forms to chemotherapy.

Animals↗
PubMed1981

Activity of 4'-(9-acridinylamino)methanesulfon-m-anisidide against Chinese hamster cells in multicellular spheroids.

The cytotoxic activity of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), a novel acridine derivative with clinical antitumor activity, has been examined in multicellular spheroids grown from Chinese hamster V79-171b cells. m-AMSA is much less effective against cells within these tumor-like structures than it is against exponential-phase V79-171b cells in monolayer cultures, the initial D0 of the survival curve for the latter being approximately 10-fold lower than for the former following a 60-min exposure to the drug. The resistance of spheroid cells to m-AMSA appears to be at least partially a result of the noncycling or slowly cycling state of the majority of these cells, although they are more sensitive than cells in plateau-phase monolayers. A further component of resistance in spheroids requires the presence of an intact spheroid structure and may be due to drug transport limitations. The use of sequential trypsinization techniques to recover cells at varying depths within spheroids demonstrates that a 60-min m-AMSA treatment preferentially kills cells nearest the spheroid surface, suggesting that tumor cells at a distance from the vasculature may limit the efficacy of m-AMSA in vivo.

Aminoacridines↗
PubMed1982

Isolation of quiescent cells from multicellular tumor spheroids using centrifugal elutriation.

A quiescent (nonproliferating) subpopulation was identified by flow cytometric analysis using two-step acridine orange staining in the EMT6/Rochester, N. Y. subline multicellular tumor spheroid, an in vitro culture system which provides a cellular microenvironment which mimics that of many of in vivo tumors. To isolate a viable quiescent cell subpopulation, centrifugal elutriation which allows for cell separation mainly on the basis of size was used. This technique provided single cells of relatively homogeneous cell volume which varied over a wide range (approximately 100 to 5000 cu microgram). Though the relatively small cell volume fractions were the most enriched (82%) in quiescent cells, such cells were also observed in significant numbers (congruent to 20%) even in the largest cell fractions. The cell clonogenicity of the various elutriation constant in fractions was also assessed and shown to be lowest (plating efficiency congruent to 20%) in the small spheroid cells but relatively constant in fractions containing intermediate and large cells (plating efficiency congruent to 50%). Continuous [3H]thymidine labeling indicated a slower rate of accumulation of labeled cells in the small spheroid cells, which may result from the transition of proliferating spheroid cells to the quiescent compartment during the course of labeling. These finding indicate the utility of centrifugal elutriation for quiescent cell characterization in in vitro tumor systems.

Animals↗
PubMed1982

Factors that influence initiation and growth of 9L rat brain gliosarcoma multicellular spheroids.

This report details optimum conditions for initiating and growing 9L rat brain gliosarcoma multicellular spheroids. Best results were achieved when 0.5 to 2.0 x 10(6) cells were seeded initially into agarose (0.5 to 1.0%)-coated Petri dishes. Spheroids initiated in this manner appeared to grow in spinner culture at a rate slightly faster than spheroids initiated and grown in spinner culture. An optimum growth rate was obtained for individual spheroids grown in the well of cluster dishes coated with 0.5 to 0.75% agarose in growth medium containing 10% newborn calf serum and 0.9 g glucose per liter. Three methods for dissociation of spheroids at both room temperature and 37 degrees have been developed. The "race track" method of fractional dissociation produced a constant and similar number of cells for each disaggregation interval, and the plating efficiency of cells in each fraction was similar.

Animals↗
PubMed1995

Neovascularization induced growth of implanted C6 glioma multicellular spheroids: magnetic resonance microimaging.

Magnetic resonance imaging has been used to follow noninvasively tumor neovascularization and tumor growth in a model system of multicellular C6 rat glioma spheroids implanted s.c. in nude mice. By positioning a single spheroid approximately 1 cm from the site of incision both the vascularization of the tumor and the wound healing processes were spatially separated and could be simultaneously followed. The model proposed here provides defined initial conditions of tumor geometry and cell proliferative status and separation of initial tumor growth from neovascularization. Magnetic susceptibility relaxation provided an intrinsic marker for blood containing vessels. The implanted spheroid induced vessel growth within 4 days after implantation that was geometrically oriented toward the spheroid and distinct from wound healing at the site of incision. Volume measurements showed a corresponding 4-day lag in growth followed by Gompertz progression. Sham implantation of agarose beads of similar diameter showed no induction of vessel growth, ruling out a direct effect of wound healing. The new vessels penetrating the tumor were highly permeable to the contrast reagent gadolinium-diethylenetriaminepentaacetic acid. This permeability may be due to the action of vascular endothelial growth factor, a major angiogenic growth factor in this system, and a potent permeability factor.

Animals↗
PubMed1995

Cyclocreatine accumulation leads to cellular swelling in C6 glioma multicellular spheroids: diffusion and one-dimensional chemical shift nuclear magnetic resonance microscopy.

Cyclocreatine, an analogue of creatine, inhibits tumor cell proliferation in vitro and in vivo. The effects of cyclocreatine in large C6 glioma multicellular spheroids were mapped here by magnetic resonance microscopy. Diffusion-weighted images of C6 glioma spheroids resolved the bright viable rim and the dark necrotic center. Sequential sets of diffusion images, following cyclocreatine administration, showed increasing self-diffusion coefficients of the intracellular water in the viable rim (0.49 x 10(-5) cm2/s for untreated spheroids, 0.62 x 10(-5) cm2/s after 48 h perfusion with 20 mM cyclocreatine). This fact correlated with cellular swelling apparent in histological sections. The radial distribution of cyclocreatine and soluble lipids across perfused C6 spheroids was measured by one-dimensional chemical shift imaging. Cyclocreatine accumulation was prominent throughout the viable cell layer, with no cyclocreatine accumulation in the necrotic center. In both cyclocreatine-treated and control spheroids the lipid signal was highest in the necrotic center and lower in the inner viable cell layer.

Animals↗
PubMed1994

Cryopreservation of supernumerary multicellular human embryos obtained after intracytoplasmic sperm injection.

OBJECTIVE: To investigate the pregnancy rate of frozen-thawed human supernumerary multicellular embryos that were cryopreserved after intracytoplasmic sperm injection or IVF. DESIGN: A clinical study. SETTING: Consenting patients in an academic research environment. PATIENTS: Couples with severe male infertility, indicated by failed or sporadic fertilization after IVF or subzonal insemination or by < 500,000 progressively motile spermatozoa in the entire ejaculate, and couples for IVF during the same period. INTERVENTIONS: After microinjection or IVF, the three best-quality embryos were transferred, and 1,171 embryos from intracytoplasmic sperm injection compared with 2,495 embryos from IVF were frozen with dimethyl sulphoxide. Of these, 413 and 969 embryos were thawed, respectively. MAIN OUTCOME MEASURE: The survival rate, the total and clinical pregnancy rates, the delivery rate, and the preclinical abortion rate were calculated. RESULTS: Fifty-three percent of the thawed intracytoplasmic sperm injection embryos survived. Twenty-two pregnancies have been established in 101 transfers, corresponding to a total pregnancy rate of 21.8% per transfer. The clinical pregnancy rate was 12.9% per transfer and the delivery rate was 5.9% per transfer. Of the IVF embryos, 51% survived and 37 pregnancies have been established in 253 transfers. The total and clinical pregnancy rates and the delivery rate were 14.6%, 10.7%, and 7.1%, respectively. The preclinical abortion rate was 40.9% for cryopreserved intracytoplasmic sperm injection embryos and 27.0% for IVF embryos. CONCLUSIONS: The high incidence of preclinical abortions after transfer of human embryos cryopreserved after intracytoplasmic sperm injection requires extension of the series.

Abortion, Spontaneous↗
PubMed1995

Determination of the radiobiologically hypoxic fraction in multicellular spheroids from data on the uptake of [3H]fluoromisonidazole.

Fluoromisonidazole [1-(2-nitroimidazolyl)-2-hydroxy-3-fluoropropane, FMISO] shows promise as a hypoxia imaging agent: it binds preferentially to anoxic cells in monolayers in vitro and accumulates in radiobiologically hypoxic tumors in vivo. The multicellular spheroid model was used to determine if the radiobiologically hypoxic fraction could be predicted from data on the uptake of FMISO. Chinese hamster V79-171b spheroids approximately 500 microns in diameter were exposed to 50 mM [3H]FMISO for 1 to 6 h under aerobic (5% CO2 in air), hypoxic (5% CO2, 5% O2, in N2) or anoxic (5% CO2 in N2) conditions and FMISO uptake was measured. Uptake in anoxic spheroids was similar to that in anoxic cell monolayers, while there was virtually no uptake in aerobic spheroids. A mathematical model was developed to calculate the radiobiologically hypoxic fraction in the hypoxic spheroids from the data on FMISO uptake. A radiobiologically hypoxic fraction of 15% was obtained, consistent with that determined from radiation survival assays (17%) and measurements of oxygen consumption (22%). We conclude that the rate of FMISO uptake in V79-171b spheroids correlates with the radiobiologically hypoxic fraction. Furthermore, the radiobiologically hypoxic fraction can be calculated from data on FMISO uptake if the dependence of FMISO uptake on oxygen concentration is known for a given tumor cell type.

Aerobiosis↗
PubMedDate not supplied

Leukotriene and 5-lipoxygenase inhibitor induced variations in thymidine uptake in a human glioma cell line cultured as monolayers or as multicellular spheroids.

Effects of exogenous leukotrienes and/or treatment with the 5-lipoxygenase inhibitor, AA-861, on the 3H-thymidine uptake were investigated in a human glioma cell line, U-343MGa, growing as monolayers or multicellular spheroids. The spheroids contained about four times more endogenous leukotrienes than the monolayers. Administration of 0.1 microM exogenous leukotriene D4 increased the 3H-thymidine uptake in the spheroids while it gave a decrease in the monolayers. Inhibition of 3H-thymidine uptake was induced in the spheroids by 10 microM AA-861 and this inhibition was only seen during the period at which central necrosis develops in the spheroids. This drug showed an inhibitory effect on monolayers one day after subculture but gave no measurable effect on the monolayers two days later. It seemed that AA-861 exerted an inhibitory action at culture conditions associated with "cellular stress" such as subculture (trypsinization) of monolayers and induction of necrosis in spheroids. Induction of "cellular stress" with heat or exposure to a Ca(2+)-ionophore also gave an inhibitory action of AA-861 and the inhibition could be counteracted by administration of exogenous B4 leukotrienes. The observed effects are probably related to the activation of the arachidonic acid cascade and indicate that leukotrienes in some way interact with "cellular stress" and induce acute changes in 3H-thymidine uptake. Further research is necessary to reveal the detailed molecular mechanisms.

Antineoplastic Agents↗
PubMed1994

Increased activity and expression of NAD(P)H:quinone acceptor oxidoreductase in confluent cell cultures and within multicellular spheroids.

NAD(P)H:quinone acceptor oxidoreductase (NQO1, EC 1.6.99.2) is an enzyme that is believed to play a central role in the bioreductive activation of several compounds, particularly quinones. The results of this study demonstrate that the activity of NQO1 is significantly elevated (2.5-fold) in HT-29 human colon cells that are in the plateau phase of the growth curve as opposed to cells in the exponential phase. Analysis of gene expression using semiquantitative reverse transcription-polymerase chain reaction and Northern blot analysis demonstrates that the increased enzyme activity is associated with increased NQO1 mRNA levels. Sequential trypsinization of layers of cells from HT-29 multicellular spheroids and analysis of gene expression by reverse transcription-polymerase chain reaction demonstrate that NQO1 expression is elevated in cells close to the necrotic center. Maximum expression occurs at a depth of 90-110 microns, with reduced expression as the distance toward both the surface and the necrotic center decreases. HT-29 spheroids were significantly more responsive than monolayers (concentration producing 50% inhibition, 124.6 and 364 nM, respectively) to the experimental drug, 2,5-dimethyl-3,6 diaziridinyl-1,4 benzoquinone. While the environmental stimulus responsible for causing elevated NQO1 expression has not been identified, the fact that NQO1 expression is influenced by microenvironmental conditions will have important implications for those drugs that are activated by NQO1.

Aziridines↗
PubMed1994

Development and characterization of suramin-resistant Chinese hamster fibrosarcoma cells: drug-dependent formation of multicellular spheroids and a greatly enhanced metastatic potential.

Suramin-resistant DC-3F/SU 1,000 Chinese hamster fibrosarcoma cells were obtained by continuous exposure of parental DC-3F cells to increasing concentrations of suramin (1 mg/ml final concentration). These cells are 10-fold more resistant to suramin compared to the parental cell line as determined by colony formation in the continuous presence of drug; the 50% effective dose for DC-3F is 35 micrograms/ml whereas the 50% effective dose for DC-3F/SU 1,000 is 380 micrograms/ml. The resistance is not due to reduced drug accumulation and is stable for at least 10 months in the absence of drug. Sensitive and resistant cells show comparable growth rate, cell size, and DNA content. In the presence of suramin, DC-3F/SU 1,000 cells form big multicellular spheroids which regrow as monolayer cultures when the drug is removed. Similar morphological changes are not observed for sensitive DC-3F cells exposed to isotoxic doses of suramin but appeared early on during the development of resistance. Inoculation of DC-3F or DC-3F/SU 1,000 cells s.c. into nude mice results in 100% tumor take within 1 week for both groups. Although the tumor size increases at the same rate, only animals given injections of DC-3F/SU 1,000 cells show extensive and persistent s.c. hemorrhages around the tumor. By 3 weeks, 30% of DC-3F-injected mice (9 of 30) show approximately 5 metastases/lung compared to -262 metastases/lung in 100% of DC-3F/SU 1,000-inoculated mice (30 of 30). These findings have several important implications: (a) given the fact that suramin is currently used clinically, special precaution may be warranted in patients undergoing suramin treatment; (b) the drug may possess an unusual potential to interfere with processes essential to invasion and metastasis which, when properly used, may result in the development of antimetastatic therapies; and (c) suramin may serve as a model compound for other molecules of the antiangiogenic and/or antimetastatic type.

Animals↗
PubMed1993

Dynamic control of cell-cell adhesion for multicellular organization.

Cadherin cell-cell adhesion molecules are associated with cytoskeletal proteins, including alpha and beta catenin, and plakoglobin. This cadherin-catenin complex plays an indispensable role in construction of ordered multicellular structures such as polarized epithelium. alpha-catenin is crucial for the cell binding function of cadherins; without it, cells cannot use the cadherin adhesion system for their adhesion. beta-catenin and plakoglobin possibly play more regulatory roles, as it was shown that their tyrosine phosphorylation correlated with modified cadherin activities. The expression of some cadherin-associated proteins is controlled by the wingless/Wnt-1 signal in embryos. These regulatory mechanisms of cadherin function and expression may be involved in dynamic control of cell-cell contacts during morphogenesis, and even in certain processes of cell growth and differentiation.

Animals↗