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PubMed1990

[Infiltrative and cytolytic activities of lymphokine-activated killer (LAK) cells against a human glioma mass: ultrastructural analysis using a three-dimensional multicellular spheroid model].

In the present study, we have investigated not only the infiltrative and cytotoxic activities of lymphokine-activated killer (LAK) cells on a tumor mass, but also the ultrastructural cell-to-cell interaction between LAK effector cells and target tumor cells during the cytolytic process within a three-dimensional solid tumor. A multicellular tumor spheroid (MTS) of a human malignant glioma cell line (U-251MG) was utilized as a solid tumor model. LAK cells were generated from peripheral blood lymphocytes (PBL) of a healthy donor after 4-day culture in the presence of interleukin-2 (IL-2). MTSs of 500 microns diameter were cocultivated with either LAK cells or non-activated PBL, and then time-sequential kinetic, morphological, and ultrastructural examinations were carried out. It was demonstrated that the number of viable tumor cells present within MTSs gradually decreased in parallel with the increase in the number of LAK cells. Morphological analyses revealed that LAK cells directly infiltrated toward the inner areas of MTSs and caused a progressive tumor destruction. In contrast, PBL hardly exhibited such activities. Ultrastructurally, it was found that the infiltrating LAK effector cells were composed of heterogeneous subpopulations, T-like cells and large granular lymphocyte (LGL)-like cells, and that both types of lymphocytes tightly adhered to the tumor cells and extended their cytoplasmic extensions deeply into the targets which underwent a progressive degeneration. Concerning the cellular interaction, it was found that these two kinds of LAK cells displayed some distinct ultrastructural feature in the process of target cell killing. Particularly, it should be stressed that LGL-like LAK cells exhibited a significant development of the intracytoplasmic secretory granules, suggesting an association with the lethal hit of target cell lysis.

Brain Neoplasms↗
PubMed1990

Hematon, a multicellular functional unit in normal human bone marrow: structural organization, hemopoietic activity, and its relationship to myelodysplasia and myeloid leukemias.

An increasing amount of data provides strong evidence for the complex multifactorial control of primary hemopoietic functions. Here we present a new multicellular functional unit, the Hematon, isolated from the light-density floating fraction of normal human bone marrow (BM) aspirates. The Hematon is organized in a compact, three-dimensional spheroid complex from central adipocytes, fibroblastoid cells, and resident macrophages that compartmentalize myeloid, erythroid, and megakaryocyte progenitor cells and their progenies. The Hematon fraction is more than twofold more abundant in progenitor cells when compared to the mononuclear cell (MNC) fraction as gauged by cytological techniques and by analysis of granulocyte-macrophage colony-forming unit (GM-CFU) populations. Individual Hematons may produce, within 2-3 weeks, up to 50,000 hemopoietic cells of different cell lineages in organotypic microcultures. Recombinant human hematopoietic growth factors interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF) significantly stimulated the endogenous cell production of some but not all of the individually treated Hematons, indicating the heterogeneity of factor-responsive cells within the Hematon population. Comparative observations of 184 BM aspirates support the hypothesis that the presence of Hematons in a BM aspirate correlates positively with homeostatic blood cell production, because the Hematon was present in normal BM (31/40) and it was rare among patients with myelodysplastic syndromes (15/53), acute myeloblastic leukemia (7/39), and chronic myelocytic leukemia (5/52). We suggest that the Hematon represents a unifying model around which the variability of fundamental BM functions and dysfunctions can be explored.

Bone Marrow↗
PubMedDate not supplied

Effectiveness of cis-platin and carboplatin in the chemotherapy of squamous cell carcinoma grown as multicellular spheroids.

We compared cis-platin (DDP) and its analogue, carboplatin (JM8, CBDCA) in their ability to inhibit spheroid growth. The activities of DDP and JM8 were also compared in an antimetabolic assay for their ability to inhibit (3H)-thymidine incorporation in multicellular tumor spheroids. The spheroids were derived from a squamous cell carcinoma cell line HN-1, originally derived from a tumor of the tongue. To produce equal levels of growth delay in spheroids, carboplatin was required at concentrations approximately 16 times that of DDP. Carboplatin also required much longer incubation periods than DDP to produce equivalent growth delay and proportions of cured spheroids. Reflecting the initial response to chemotherapy, the antimetabolic assay showed that carboplatin was required at higher concentrations and longer exposure times to produce equal inhibition of the nucleotide precursor thymidine. These findings may have implications for the clinical use of these drugs and in particular would support a role for carboplatin in the treatment of squamous cell carcinoma of the head and neck, since total free-drug exposure of patients to carboplatin may be up to 16-fold greater than with DDP, and the clinical side effects of carboplatin have been shown to be well tolerated. However, one must be cautious about applying in vitro data to clinical situations.

Antineoplastic Agents↗
PubMedDate not supplied

Multicellular tumor spheroids in radiotherapy research (review).

Culturing of human tumor cells as multicellular spheroids can be a tool to study radiation responses. The degree of structural and functional differentiation in the primary tumor may be retained in spheroids rather than in conventional monolayer cultures. In the liquid overlay culture technique spheroids can be individually assessed for their responses to treatment, whereas in spinner flasks, large quantities of similarly sized spheroids can be produced. Studying the response of spheroids to irradiation can be performed on single cells obtained after disaggregation of these spheroids, or on intact spheroids, using cure and growth delay as endpoints. Clonogenic cell survival is especially difficult to perform on spheroids of human tumor cells. Modern calculation methods, however, may offer promising correlates between growth curves and single cell survival. Spheroids of human tumor cell lines show tumor type dependent radiation responses, offering an approach for comparison of radiosensitivity of tumor cell lines of different histologic origin. Contact effect, as a modifying factor of radiation response in spheroids, has especially been studied in murine cell lines. The use however, of human tumor cell lines, may offer new insight in this phenomenon. Radiobiologic hypoxia has been observed in spheroids of both murine and human origin. Reoxygenation after irradiation has also been described by radiobiologic parameters. So far, no physiologic reoxygenation processes after radiation treatment have been identified. In view of the clinical relevance of oxygen to radiation responses and treatment outcome, reoxygenation processes should be further elucidated in spheroids of human origin. Repair of potentially lethal damage in spheroids has been reported for only one murine cell line. In an indirect manner it has also been studied in spheroids of human origin. Sublethal damage repair has been studied rather extensively in murine cell line spheroids. However, only recently it has been reported in human tumor spheroids in relation to the clinical curability of the tumors of origin. Use of human tumor cell lines to study radiation responses of spheroids is necessary to determine tumor type dependent differences in several radiation related phenomena, such as reoxygenation, contact effect, and repair processes.

Animals↗
PubMed1990

Radioresistance of rat glioma cell lines cultured as multicellular spheroids. Correlation with electrical cell-to-cell-coupling.

Two chemically induced rat glioblastomas, RG2 and F98, were cultured as monolayers and as multicellular spheroids and subjected to Co-gamma-irradiation. In parallel, intercellular communication between cells was determined as electrical coupling between neighbouring cells using micro-electrode techniques. A third glioblastoma with known radiobiological response (9L) was assayed with respect to intercellular communication and included into this analysis. Electrical coupling was low for RG2, intermediate for F98, and high for 9L. Radioresistance of spheroids, as expressed in terms of the mean inactivation dose computed from the survival curves increased in the same direction (RG2: 2.4 Gy; F98: 5.1 Gy; 9L: 6.5 Gy). A comparison of these parameters demonstrates a correlation between solid tumor radioresistance and gap-junctional cell-to-cell communication, at least for the class of glioblastomas analysed in this study.

Animals↗
PubMed1988

Role of necrosis in regulating the growth saturation of multicellular spheroids.

Growth curves for multicellular spheroids of 15 different tumor and normal cell lines were characterized by doubling times which decreased with increasing growth until a stable saturation was attained. In spite of the identical and constant conditions during growth, the size at saturation varied by factors of 67 in spheroid volume and 75 in cell content. These saturation sizes showed no correlation with the monolayer doubling times or clonogenic efficiencies, the initial spheroid growth rate or clonogenic capacity at saturation, the cell packing density, or the species of origin and type of cell line. There was a strong correlation between the maximal spheroid size and the size at which necrosis initially developed, suggesting control by necrosis. Crude extracts prepared from spheroids with extensive necrosis showed dose-dependent cytostatic and cytotoxic activities against monolayer cultures, while similar extracts from spheroids without necrosis had little effect. This activity was also detected in the culture medium to which the large spheroids had been exposed prior to preparation of extracts, suggesting that the responsible factor(s) can diffuse through the spheroid. The extract from spheroids of one cell line inhibited the growth and clonogenicity of four other cell lines, including human diploid fibroblasts. DNA content profiles measured during exposure to this extract showed that the cytostatic effect was not due to the arrest of cells in a specific cell cycle phase. The cell volumes were increased during culture in medium containing the extract from spheroids with extensive necrosis. These data support the hypothesis that growth saturation in spheroids is regulated by factors produced, released, or activated during the process of necrosis and suggest that these toxic factors have potential therapeutic use.

Animals↗
PubMed1988

Cell contacts and surface features of three murine tumors grown as multicellular spheroids.

Ultrastructural observations using scanning and transmission electron microscopy were made on three murine tumors, line 1 lung carcinoma, fibrosarcoma (FSA) and mammary carcinoma MCa-11, grown in vitro as multicellular tumor spheroids (MTS). The cytology of these MTS revealed the presence of characteristic cellular organelles as well as varying amounts of intracisternal type-A viral particles. In line 1 and FSA, the occurrence of gap junctions in the outer shells of these MTS was correlated with the growth behavior of these spheroids. In FSA, extracellular collagen bundles were identified next to tumor cells and represent synthetic activity by these cells under these conditions. No specific cytological correlations were made with the slow growing MCa-11 spheroid.

Animals↗
PubMed1988

Glucose diffusivity in multicellular tumor spheroids.

In order to understand the role of glucose limitations in controlling multicellular tumor spheroid growth, knowledge of the glucose diffusion coefficient is essential. The effective diffusivity of glucose in spheroids of rodent and human tumor cell lines has been determined by measuring the efflux of tritium labeled L-glucose from spheroids with time. When the rapid and irreversible binding of L-glucose in spheroids is properly taken into account, measurements of the efflux of this diffusion tracer from spheroids into label-free medium can be correlated to the diffusion equation in order to obtain the effective glucose diffusivity in spheroids. Such measurements have been made in EMT6/Ro mouse mammary tumor spheroids as well as in spheroids derived from human colon carcinoma cells (HT29, CO112, and WiDr) and from human squamous carcinoma cells (CaSki and A431). EMT6/Ro spheroids have a glucose diffusivity of 1.1 x 10(-6) cm2/s, while glucose diffusion coefficients in the human cell spheroids studied vary from 5.5 x 10(-7) cm2/s to 2.3 x 10(-7) cm2/s. These values are low enough to suggest that significant gradients in glucose concentration may exist in spheroids and tumors. It is thus believed that these glucose diffusivities, as well as their variation with cell line, may have important implications for the role played by glucose in the growth and cellular heterogeneity of spheroids and tumors.

Algorithms↗
PubMed1987

Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular tumor spheroids.

The binding and penetration of two 125I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) and their F(ab')2 and Fab fragments were measured in multicellular spheroids of poorly (HT29) and moderately well differentiated (Co112) human colon adenocarcinomas which express different amounts of CEA. Spheroids cultured in vitro model tumor microenvironments where poor vascular supply may modulate antigen expression and accessibility. The two MAb studied, 202 and 35, were shown previously to react with different CEA epitopes and to have high affinities of 1.2 and 5.8 X 10(9) M-1, respectively. MAb 202 has also been shown to cross-react with antigens present on human granulocytes and normal epithelial cells from human lung and pancreas. Specific binding of intact MAb and fragments of both antibodies was demonstrated for both types of human colon carcinoma spheroids compared to mouse colon carcinoma (CL26) and mammary tumor (EMT6/Ro) spheroids. Total binding of MAb and fragments was greater (1.5- to 2.5-fold) after 4 h compared to 1 h of exposure; the amount of binding compared to control IgG1 was 5- to 30-fold greater after 1-h incubation and 15 to 200 times greater after 4 h. This binding was stable as demonstrated by short and long wash experiments at 37 degrees and 4 degrees C. The binding of F(ab')2 and Fab fragments of the anti-CEA MAb 35 to spheroids of human colon Co112 was almost 2-fold greater than that of the intact MAb. However, for MAb 202, the binding of intact MAb and F(ab')2 was greater than that of Fab fragments. In addition the binding of both intact and F(ab')2 fragments of MAb 202 was greater than that obtained with MAb 35. Specific binding of both antibodies to HT29 spheroids, which express less CEA, was decreased for MAb and fragments of both 202 and 35. Autoradiography and immunoperoxidase experiments were performed to determine the penetration of MAb and fragments after incubation with intact spheroids. Comparisons were made with labeled MAb directly applied to frozen sections of spheroids. F(ab')2 and Fab fragments of both antibodies were bound at the surface of intact spheroids and penetrated to eight to ten cells, but the intact MAb were localized mainly at the spheroid surface and the outer one to three cell layers. There was much less binding at the surfaces of HT29 compared to Co112 spheroids. An enzyme immunoassay using MAb 35 and 202 demonstrated that Co112 spheroids produced about 8-fold more CEA/mg of cell protein than did monolayer cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1977

A simple method for cloning and replica plating of mammalian cells using multicellular spheroids.

V 79/4 Chinese hamster cells or HeLa cells grow in Eagle's MEM supplemented with 25 microgram/ml dextran sulphate to form clonal multicellular spheroids. These cell clones, consisting of 5-10(2) cells, are easy to separate, to transfer from one culture vessel into another and grow as normal monolayer colonies on Dederon cloth circles after subculture in Eagle's MEM without dextran sulphate. A simple replica technique is described by which 500 clones can be transfered onto at least 3 replica cloth circles, 10 cm in diameter, with a replica plating efficiency of approximately 100%.

Animals↗
PubMed1987

Effects of anticancer agents on the shedding of cells from human multicellular tumor spheroids.

We studied the effects of anticancer agents on the inhibition of cell shedding from the surface of multicellular tumor spheroids (MTS). MTS were produced from 2 human tumor cell lines; one melanoma and the other squamous cell lung cancer, by using liquid overlay culture technique. The cell shedding from the melanoma MTS was approximately 10-fold higher than the squamous cell carcinoma MTS. In the melanoma MTS, all 3 drugs studied - vincristine (VCR), doxorubicin (ADR) and cisplatin (DDP)-inhibited cell shedding and the degree of inhibition of cell shedding was drug concentration related. In the squamous cell carcinoma MTS, VCR was as active in inhibiting cell shedding as in the melanoma MTS, but ADR and DDP were less efficacious. When effects on cell shedding were compared with those on cell lethality, VCR produced inhibition of cell shedding at much lower concentrations than those producing cell kill effects. ADR and DDP produced cell lethality as effective as, or more effective than, inhibition of cell shedding. These data seem to parallel known effects of these agents on cell kill and inhibition of metastases. MTS may serve as an in vitro model for the study of cell shedding and metastasis.

Carcinoma, Squamous Cell↗
PubMed1986

Proliferative and clonogenic heterogeneity of cells from EMT6/Ro multicellular spheroids induced by the glucose and oxygen supply.

The culture of EMT6/Ro multicellular tumor spheroids in a 20-fold range of glucose concentrations and a 4-fold range of oxygen tensions had profound effects on the cell subpopulations which developed in the spheroids. As the spheroids increased in diameter the rate of accumulation of cells with a G1-phase DNA content was much greater for those spheroids cultured in the lower oxygen and glucose concentrations. Growth fractions of 600- to 800-micron-diameter spheroids, calculated from either DNA content or [3H]thymidine labeling data, showed a direct correlation with the glucose concentrations in the culture medium. The cells from spheroids cultured in low glucose concentrations also showed a more rapid loss in clonogenicity as a function of increasing spheroid diameter. Lowering the oxygen tension enhanced these effects of reduced glucose concentrations. Selective dissociation of 600- to 800-micron-diameter spheroids demonstrated that both the proliferation index and the clonogenic capacity of the spheroid cells decreased with increasing depth into the spheroid cell rim. The rates of decrease of both of these parameters as a function of depth into the spheroid were greater for spheroids cultured in the lower oxygen and glucose concentrations. These results indicate that both the glucose and oxygen supply are critical in the development of nonproliferating and nonclonogenic cell subpopulations in spheroids.

Animals↗
PubMed1986

Oxygenation and differentiation in multicellular spheroids of human colon carcinoma.

Oxygenation and development of necrosis were evaluated in multicellular spheroids of poorly differentiated (HT29) and moderately well-differentiated (Co112) human adenocarcinoma of the colon. Spheroids were grown in vitro under well-controlled oxygen and nutrient conditions in spinner flasks up to sizes of 2800-micron diameter after 5 wk of culture. Morphological studies showed that the Co112 spheroids contained pseudoglandular structures with lumen, very similar to the characteristics of the original tumor specimen from the patient and to the cells when grown as xenograft tumors in nude mice. Microelectrodes were used to measure the oxygen tension (PO2) profile within individual spheroids at different stages of growth. Histological sections through the centers of spheroids were measured to determine the thickness of the viable rim of cells surrounding spheroid necrotic centers in order to estimate the size of the severely hypoxic zone of cells by comparison with the PO2 profiles of the same spheroids. The data demonstrate significant differences between these two human colon tumor spheroid systems. Both spheroid types exhibited steep PO2 gradients at relatively small sizes of less than 600-micron diameter, but for any given size in this range, the more differentiated Co112 spheroids were more hypoxic. Although severe hypoxia (PO2, less than 10 mm of Hg) was present in both spheroid types at larger sizes, there was a significant difference in the central PO2 values which were between 5 and 10 mm of Hg in large Co112 spheroids but remained at or close to 0 mm of Hg in large HT29 poorly differentiated human colon tumor spheroids. The presence of pseudoglandular structures and lumen in the Co112 spheroids was associated with changes in the shape of PO2 profiles. Such profiles have not previously been seen in other poorly differentiated human or rodent tumor spheroids. Furthermore, the PO2 profiles of both of these human tumor spheroid types were often continuously curving with a very shallow gradient in the inner edge of the viable rim of cells surrounding the necrotic center. Regulation of oxygen consumption and/or diffusion in these inner regions of human spheroids could produce these continuously curving PO2 gradients.

Adenocarcinoma↗
PubMed1985

[Studies on hyperthermia and radiation treatment of uterine cancer using multicellular tumor spheroid].

Anticancer effects of hyperthermia and radiation on uterine cancer cells were studied using multicellular tumor spheroids and monolayer cultured cells. Cell lines used were SKG-3a(uterine cervical epidermoid carcinoma), HeLa-S3 (uterine cervical adenocarcinoma) and HEC-59 (uterine corpus adenocarcinoma). All cell lines grown in monolayer culture have similar growth rates, but as spheroids, their growth rate are HeLa-S3 greater than HEC-59 greater than SKG-3a cells. The radiosensitivity of all cell lines in monolayer are as follows according to dose-survival relationships. :HEC-59 greater than HeLa-S3 greater than SKG-3a cells. Survival assay to hyperthermia on SKG-3a and HEC-59 cells resulted in no response from 37 degrees to 41 degrees C and in cytotoxicity over 42 degrees C. There is no difference in sensitivity to hyperthermia between the two types of cells. The responses of three cell lines grown as spheroids in hyperthermia and radiation demonstrated that the combination therapy is not effective on the SKG-3a cell, but induced a 1.3-fold effectiveness ratio in HEC-59 and 1.6-fold in HeLa-S3 cells, i.e. more cytotoxicity than with radiation alone. These results suggests that hyperthermia and radiation employed together may be more effective on radioresistant adenocarcinoma cells than on radiosensitive epidermoid carcinoma cells.

Adenocarcinoma↗
PubMed1985

Heat response of human melanoma multicellular spheroids: comparisons with single cells and xenografted tumors.

Multicellular spheroids were grown from cells derived directly from a human melanoma xenograft propagated in athymic mice. The histological appearance of the spheroids was similar to that of the parent xenograft. The spheroids were heated in culture medium (42.5-44.5 degrees C); growth delay and single cell survival measured in soft agar were used as end points. There was a good correlation between the results obtained with these two end points, indicating that growth delay depended mainly on cell survival. Large spheroids (200 +/- 12 microns in diameter) were found to be more heat sensitive than small ones (100 +/- 5 microns in diameter), probably because the physiological conditions in large spheroids were more favorable for cell inactivation. The cells were more resistant when heated as spheroids than as single cells. This effect was not a secondary effect of differences in cell-cycle distribution. Spheroids were also found to be more heat resistant than xenografted tumors. In the tumors, heat treatment caused vascular damage which resulted in delayed cell death due to hypoxia and/or nutrient deficiency. It is concluded that spheroids seem well suited for studies of primary heat-induced cytotoxic effects. However, they appear not to mirror the complex heat response of tumors since that response also includes secondary effects, related to heat-induced reduced perfusion.

Agar↗
PubMed1982

[Method of recording the linear growth and cell mass displacement of multicellular animals and plants].

A photoelectrical method has been developed of registration of the linear growth and cell mass movements in the actively zones (growing tips, edges, etc). The device proposed transforms to electrical signals the changes in lighting of microscopic visual field caused by shifting the growing structures in the area restricted by the aperture of the ocular field diaphragm of the "cat-eye" type. By means of this technique, oscillograms of growth of the number of multicellular organisms have been obtained for the first time. In many cases (blastodisc edge and tail bud of the salmon Oncorhynchus gorbusha, mantle edge of Mytilus edulis, osculum edge of the sponge Tethya, apical area of brown alga Fucus and of red algae Polyides and Ahnfeltsia), a pulsatory character of growth (with periods of some minutes) were registered. A similar growth character was also confirmed for several species of Thecaphora hydroids. No pulsations was registered during the linear growth of a brown alga Laminaria (which contrary to Fucus is growing intercalarly), of Nitella (Charophyta) and of several high plants.

Animals↗
PubMed1983

[Kinetics of multicellular spheroids developed from a human glioblastoma, and effect of 1,3-bis (2-chloroethyl)-1-nitrosourea].

Multicellular spheroids (MCS) were yielded in vitro by early stages of human glioblastoma cell cultures. The spheroids were grown in suspension in the culture medium in agarose-coated (0.65%) dishes. The volume doubling time during the exponential growth phase was longer (13 days) than the population doubling time of monolayer cultures (2-3 days). Pathological examination of MCS showed a thin proliferative outer layer and areas of necrosis in the center. In two experiments BCNU caused a growth delay of MCS which was dose-related. The cells were sensitive to BCNU in monolayer cultures (CFE's assay), whereas the tumor was resistant in vivo. The usefulness and pitfalls of studies using MCS cultured from early passages of human malignant gliomas are discussed.

Brain Neoplasms↗
PubMed1984

Demonstration of an extracellular matrix in multicellular tumor spheroids.

Multicellular spheroids of a human glioma cell line (U-118 MG) and a human thyroid cancer cell line ( HTh -7) were analyzed for the presence of extracellular matrix (ECM) using light microscopy, transmission electron microscopy, and indirect immunofluorescence staining for fibronectin, laminin, and collagen. These studies were supplemented by analyses of glycosaminoglycans using autoradiography or chemical methods after metabolic labeling with [35S]sulfate or [3H]glucosamine in conjunction with various extraction procedures. The results showed that both types of spheroids contained an ECM composed of fibronectin, laminin, collagen, and glycosaminoglycans. The organization of the ECM in the spheroids seemed to be similar to that of tumors in vivo. These findings help justify the use of the spheroid system as an in vitro model for the study of biological phenomena of human tumors in vivo. Furthermore, it is concluded that the formation of an ECM in vitro is not confined to normal cells but can be promoted in transformed cells using appropriate culture conditions.

Cell Line↗