Multicell Spheroids Research Articles

Ultrasonicarly induced nuclear aberrations in anin vitromulticellular tumour system

Chinese hamster V-79 cells, attached to an acoustically transparent membrane and EMT6/Ro mouse mammary sarcoma cells as small multicellular spheroids or in suspension, were exposed to therapeutic intensities of 1 MHz ultrasound (0-10 W/cm2) and examined for the production of atypical nuclear or chromosomal aberrations. In the monolayer configuration no abnormalities occurred. In multicell spheroids or suspension cells 3 classes of abnormalities were found: (1) pyknotic/irregular nuclei; (2) lysed nuclei with extruding nucleoplasm; and (3) agglomerations which have an irregular shape and "fused" morphology similar to that which has been reported for plant tissues (Cataldo et al, 1973).

Binding of 14C-misonidazole to hypoxic cells in V79 spheroids.

The metabolism-induced binding of 14C-labelled misonidazole (MISO) to hypoxic V79 cells in multicell spheroids has been quantitated using autoradiography. Hypoxia was shown to be the major determinant of the rate of binding. Maximally hypoxic cells bound MISO several times more rapidly than necrotic material in the centre of the spheroids, and up to 50 times more rapidly than well oxygenated cells. The rate of binding to chronically hypoxic cells at the edge of the necrotic centre was 20 times less than to similar cells in other spheroids made maximally hypoxic with N2. This difference is consistent with the greater radio-sensitivity of the chronically hypoxic cells, which is a consequence of their intermediate level of oxygenation. The results indicated that the ability to bind MISO might have considerable potential as a marker for hypoxic cells in tumours. However, some binding patterns cannot be explained by the simplest model of O2 diffusion. It may be necessary to invoke more complex models of O2 diffusion or metabolic gradients within the spheroid which affect the rate of binding.

Use of Hoechst 33342 for cell selection from multicell systems.

Hoechst 33342 staining of multicell spheroids, three-dimensional cell clusters grown in vitro, results in a marked gradient of cellular fluorescent intensities inward from the spheroid periphery. The penetration of the dye is concentration and time dependent, so staining can be coupled with fluorescence activated cell sorting techniques to allow disaggregated single cells to be sorted or selected according to their degree of staining and therefore their depth within the spheroid. We have found the staining procedure to be highly reproducible, and to result in minimal toxicity even to the more brightly staining external cells. Comparison of this technique with others for cell selection suggests that increased resolution is available with the Hoechst technique.

Oxygen tensions in multicell spheroids of two cell lines.

O2 tensions (Po2) were measured with microelectrodes in multicellular spheroids from EMT6/Ro and V-79-171-B cells. The measurements were performed in spheroids kept in flowing growth medium that was equilibrated with 5% CO2 and air at a temperature of 37 degrees C and contained 5.5 mM glucose. The recorded Po2 profiles are characterized by a diffusion-depleted zone surrounding the spheroids and by a steep drop in Po2 within the spheroids over mean distance of 220 and 188 micrometer from the surface of EMT6/Ro and V-79-171B spheroids over mean distance of 220 and 188 micrometer from the surface of EMT6/Ro and V-79-171B spheroids respectively. Smaller spheroid exhibit parabolic Po2 profiles, larger ones show a central plateau. The region of the steep decrease in Po2 corresponds to the thickness of the viable rim: the plateau region is created by the absence of O2 consumption in the central necrotic area. Po2 in the centre of EMT6/Ro spheroids decreased from 66 mmHg at a diameter of 400 micrometer to 13 mmHg at a diameter of 1000 micrometer. Under the present conditions during growth and in the experiments, values below 5 mmHg were recorded only in spheroids 1200 micrometer. Comparably low Po2 was recorded in V-79 spheroids with diameters of 650 micrometer +. In spheroids of this cell type with a diameter of 400 micrometer, Po2 was 42 mmHg. The findings provide evidence that necrosis may arise at average Po2 of 57 and 42 mmHg in EMT6/Ro and V-79-171B spheroids, respectively, grown under the conditions described.

Closing and opening of gap junction pores between two- and three dimensionally cultured tumor cells.

Intercellular signal transfer via gap junction pores in cultured multicell spheroids of BICR/M1R-K cells decreases with increasing spheroid age. In two days old spheroids the pores allow passage of Lucifer yellow molecules. Two days later, this fluorescent dye is retained in the injected cell even though the cells are still electrically coupled. Gap junction plaques of considerable size are still found in 9 days old spheroids, when the cells are completely uncoupled. The same cells growing as monolayer cultures do not exhibit such a gradual closing of their gap junction pores: Their coupling is established at first cell contact, probably by a gradual opening of the pores, which remain open even up to 9 days in culture.

Influence of growth, measuring conditions, and cell types on oxygen tensions in multicell tumor spheroids determined by microelectrodes

Influence of growth, measuring conditions, and cell types on oxygen tensions in multicell tumor spheroids determined by microelectrodes

Influences of Growth Conditions and Cell-Cell Contact on Responses of Tumor Cells to Ultrasound

The response of EMT6/Ro multicell spheroids at early stages of growth (1-3 days) to 1-MHz ultrasonic irradiation was compared to EMT6/Ro cells which were grown under suspension or as attached monolayers. The surviving fraction of cells was evaluated by measuring both direct cell lysis and clonogenicity of cells not lysed during sonication. Irradiation was for 1 min at 0, 1, 3, or $5\ {\rm W}/{\rm cm}^{2}$ (spatial peak, temporal peak) at 37°C. At 3 and $5\ {\rm W}/{\rm cm}^{2}$, there was a 9- to 10-fold enhancement in the surviving fraction of spheroids as compared to that of monolayer cells. Individual spheroid cells do not appear to be physiologically altered with respect to their response to ultrasound since cells from dissociated spheroids exhibit survival parameters which are similar to those of cells grown in monolayers. The basis for the increased survival of whole spheroids appears to be due to a physical protection of the cells in spheroids. Studies examining effects of trypsin treatment indicat...

Increased radioresistance of cells in cultured multicell spheroids. I. Dependence on cellular interaction

Monolayers of six different cell lines were investigated with respect to ionic coupling using micro-electrode techniques. In parallel, survival after Co-gamma-irradiation of monolayer- and spheroid cultures of these lines was compared. It was found that spheroids of coupled cell lines were more radioresistant than monolayers ("contact effect"). however, cell coupling did not enhance the survival of monolayers over single cells. This suggests that the contact effect is a "tissue phenomenon" requiring cellular interaction but is expressed only under conditions of three-dimensional growth.

Ultrasonic irradiation of multicellular tumor spheroids

In vitro multicell spheroids from a mouse mammary sarcoma (EMT6) were exposed to therapeutic levels of ultrasond (1 MHz, 1–5 W/cm 2, 1–5 min cw) in a polystyrene tube which contains standing waves and promotes cavitationally related damage. Spheroid sizes were varied from 1‐day old, 20‐micron‐diameter cell aggregations (∼11 cells/spheroid) to 25‐day, 800‐micron‐diameter multicell spheroids (∼80,000 cells/spheroid). Damage was assessed by histology, scanning electron microscopy (SEM) and growth studies on individual spheroids and by survival of individual component cells. Dye exclusion (0.05% eosin) and histology indicated that peripheral damage occurred; SEM revealed the presence of surface abnormalities (tears and holes in cell membranes) in exposed spheroids. Additionally, necrotic loci within spheroids were noted after a 3 W/cm2 1 min exposure. Growth studies and surviving fraction indicated that damage is inversely proportional to spheroid size. With initial spheroid diameters ⩾ 200–250 microns, a decrease in spheroid diameter occurred 24 h post‐sonication (3 W/ cm2, 5 min cw); the threshold was between 3–5 min of exposure. With initial diameters ⩽200 microns, reduction was produced by a 1‐min exposure. Ultrasonically induced surviving fractions of individual cells also increased with increase in spheroid size.

Uptake and mutagenicity of AF‐2 in chinese hamster V‐79 spheroids under aerobic and hypoxic conditions

AbstractChinese hamster V‐79 cells, when grown in suspension culture as multicell spheroids, display many of the features of organized tissue. AF‐2, a known mutagen, was used to examine the influence of some of these factors on mutagenesis. Since AF‐2 is fluorescent, FCM quantitation of intracellular drug content was performed after incubation of spheroids under either aerobic or hypoxic conditions. For the same level of toxicity, hypoxic spheroids accumulated twice as much AF‐2 as aerobic spheroids, but showed less than half the number of mutants resistant to 6‐thioguanine. Using velocity sedimentation techniques, a selective increase in the toxic and mutagenic effects of AF‐2 was found for internal vs external cells of the spheroids. However, in mutagenicity experiments using all the cells from spheroids, the response of the external cycling cells predominated over the response of the internal cells. Thus, in addition to the demonstration that cellular environment can modify mutagenesis, these results also suggest that the growth fraction of the target cells can be an important consideration in mutation experiments.

Shedding of mitotic cells from the surface of multicell spheroids during growth.

During the growth of EMT6/Ro mammary tumor multicell spheroids, a large number of cells are shed into the suspension medium. The rate of cell shedding was 218 cells per square millimeter of spheroid surface per hour, or up to 1.5% of the total spheroid cell content per hour. Shed cells had a clonogenic capacity equal to that of exponential monolayer cultures and were further characterized by volume distribution, mitotic index, flow cytofluorometry, and autoradiography. The results indicated that cells are released from the spheroid surface at mitosis, presumably due to a loosening of the cell-to-cell attachment during this cycle phase. These mitotic cells, when placed in monolayer culture, attached and grew synchronously with a cell cycle time of about 13 hours. Shed cells kept in suspension culture had a similar cell cycle time, but these cells reaggregated immediately after mitosis. The results indicated that cell shedding and reaggregation both occur near the time of mitosis and are intrinsic factors regulating the initiation and subsequent growth of multicell spheroids. Although these studies were done with spheroids cultured in vitro, shedding of mitotic cells may play an important role in the in vivo process of metastasis.

Different sensitivity to cytotoxic agents of internal and external cells of spheroids composed of thioguanine-resistant and sensitive cells.

When thioguanine-resistant V79 cells are introduced into spinner flasks containing V79 multicell spheroids, the mutant cells attach to the surface of the spheroids. The composite spheroids thus formed consist of external, thioguanine-resistant (TGr) and internal, thioguanine-sensitive cells. Cell sorting with a Becton Dickinson FACS II was used to determine the relative position of TGr cells and sensitivity to fluorescent drugs. After 2-4 days, the TGr cells are found internally as well as externally. The initial percentage of TGr cells varies from 1 to 50%, depending on the size of the single-cell inoculum, size of spheroids and frequency of addition of cells during a 24th period. Differential effects of drugs or radiation on external (cycling, oxic) vs internal (non-cycling, hypoxic) cells of composite spheroids can be assayed by simply plating cells trypsinized from spheroids into standard medium or medium with 2 mu g/ml 6-thioguanine, which allows growth of only TGr cells. For example, Adriamycin, which penetrates poorly into spheroids, is preferentially toxic to external cells, and causes a decrease in the percentage of TGr survivors. Irradiation of composite spheroids also causes preferential killing of external (oxic) TGr cells. However, AF-2, a nitrofuran which preferentially kills hypoxic cells, caused an increase in the 0/0 of TGr cells. This technique should prove useful in the evaluation of the preferential cytotoxicity of chemotherapeutic agents, alone or in combination.

Effects of lucanthone on chinese hamster V-79 cells I. Interaction with radiation in monolayers and spheroids

Lucanthone has previously been shown to reduce the capacity of cells to accumulate and repair sublethal radiation damage, which suggests its potential as an adjuvant to clinical radiotherapy. Present results indicate that lucanthone was effective even for V-79 cells having a markedly enhanced shoulder on the radiation survival curve as a result of growth as multicell spheroids. Lucanthone also proved to be an effective cytotoxic agent in large spheroids. Unlike radiation toxicity, however, lucanthone toxicity was equal for cells from asynchronous monolayers or small spheroids. Thus, for equal levels of drug toxicity, the radiation-modifying effect of lucanthone was greater in the spheroid; the “interaction” was larger in the system which had a greater capacity for accumulation and repair of sublethal radiation damage. Direct extrapolation of this result to the clinic would suggest that lucanthone be used judiciously, and that it might be of value as an adjuvant to radiotherapy only in the unusual circumstance where the malignant tissue has a larger capacity for accumulation and repair of sublethal radiation damage than the surrounding normal tissue.

Multicell Spheroids - An in Vitro Model of Normal and Malignant Tissues

Multicell Spheroids - An in Vitro Model of Normal and Malignant Tissues

Oxygen Diffusion Distance and Development of Necrosis in Multicell Spheroids

The multicell spheroid in vitro tumor model was used to study the effects of diffusion limitations of metabolites on the development of necrosis in a tumor-like environment. Chinese hamster V79-171b cells were grown in suspension as multicellular spheroids in different oxygen or glucose concentrations to diameters where they developed central necrosis surrounded by an outer rim of viable cells. The range of oxygen concentrations used in the gas phase was 1 to 20.3%, in equilibrium with complete nutrient medium. For each oxygen concentration the thickness of the rim of viable cells was determined from measurements of histological sections. The square of the thickness of the viable rims of spheroids grown at different oxygen concentrations between 5 and 20.3% increased linearly with the theoretically derived oxygen diffusion distance, showing that the oxygen diffusion distance is a main factor controlling, either directly or indirectly, the development of necrosis in this concentration range. The data were interpreted as meaning spheroids grown at more than 5% O/sub 2/ contain a very small proportion of cells which were severely hypoxic or anoxic. The viable rim thickness was relatively less affected during growth at less than 5% oxygen, implying that an unknown mechanism controls the formationmore » of necrosis when the oxygen diffusion distance is less than approximately 80 ..mu..m. Although alterations in the glucose concentration had some effect on viable rim thickness at these low oxygen concentrations, the effect was much less than expected if glucose alone were responsible for controlling viability in this outer region.« less

Cytotoxicity of glucose analogues in V79 multicell spheroids

2-Deoxy-D-glucose (2DG) and 5-thio-D-glucose (5TG) are glucose antimetabolites that are known to be selectively toxic to hypoxic cells grown as single cells or as monolayer cultures. These analogues were toxic to Chinese hamster V79 cells grown as multicell spheroids even under aerobic conditions. When spheroids, 500- to 600-microns diameter, were exposed to 7.5 mM of these chemicals for 3 days, the number of clonogenic cells per spheroid dropped to 50% for 5-thio-D-glucose and 20% for 2-deoxy-D-glucose, relative to control values. Survivals were reduced to less than 1% when the experiment was repeated in glucose-free medium. Scanning electron photomicrographs of spheroids treated with 7.5 mM of either analogue showed extensive damage to the outer cells. The cell killing observed was much more than could be predicted on the basis of the hypoxic fraction known to be present in these spheroids. The crowded tumor-like environment may make the cells vulnerable to the cytotoxic action of glucose analogues and other glycolytic inhibitors.

Study of the effect of vitamins C and E on the radiation response of multicell spheroids treated with adriamycin

Treatment with Adriamycin (0.6 kg/ml for 60 min) was not cytotoxic to multicell spheroids. At this concentration, the drug was not a sensitizer of hypoxic cells in V79 multicell spheroids, which were irradiated at 37°C in medium equilibrated with a mixture of 5% 02:3% C02:92% N 2. The addition of vitamins C and E did not increase the radiation sensitivity of Adriamycin-treated spheroids. In some experiments, catalase was included in the growth medium to overcome the toxic effect of hydrogen peroxide, which is known to be formed in solutions containing vitamin C and also in Adriamycin solutions treated with vitamin C or microsomal preparations. As a result of these experiments, it was found that catalase increased theradiation killing in multicell spheroids.

Density gradient centrifugation of cells separated from multicellular tumor spheroids.

Cells from a murine fibrosarcome (FSa) have been grown in vitro as multicell tumor spheroids (MTS). The growth rate of these MTS was determined. Following selected periods of growth, MTS were made into a single cell suspension and separated on linear density gradients of Renografin. While only 1 population of cells were separated from small spheriods (400 mum diameter), at least 3 subpopulations of tumor cells were separated and isolated from large spheroids (800 mum in diameter).

Radiation-Induced DNA Damage in V79 Spheroids and Monolayers

Chinese hamster V79 cells can be grown as monolayers or in suspension as multicell spheroids. When irradiated, cells from spheroids can accumulate more sublethal damage than cells from monolayers as evidenced by a wider shoulder on the survival curve. We have attempted to determine whether this increased resistance of spheroids to damage by ionizing radiation is reflected by DNA damage. The production and repair of single-strand breaks was quantified using alkaline sucrose gradient sedimentation and hydroxylapatite chromatography. Both methods indicated that equal amounts of radiation damage per rad were produced in cells grown as single cells or as spheroids, and that postirradiation rejoining of DNA single-strand breaks also occurred with similar efficiency. Thus, these results suggest that the enhanced survival of cells irradiated as spheroids is dependent on processes other than (or in addition to) initial radiation damage to DNA.

Rate of Death of Hypoxic Cells in Multicell Spheroids

The rate of death of hypoxic cells was measured in multicell spheroids, which are considered to model in vitro the microenvironments surrounding such cells within solid tumors. Two types of experim...