Repair of sublethal damage in two human tumor cell lines grown as multicellular spheroids

Multicellular tumor spheroids (MTS) provide a suitable in vitro model to study radiation sensitivity of tumor cells. Two cell lines of human origin, obtained from a neuroblastoma (NB-100) and a squamous cell carcinoma (HN-1), were exposed to graded doses (4-9 Gy) of radiation with 18 MV photons. Radiation was applied either as a single or as a split dose with an interval of 6 hr to determine the extent of sublethal damage repair. Treated spheroids regrew at approximately the same growth rate as control multicellular tumor spheroids, preceded by a static or regression phase. Radiation response was quantified in terms of regrowth delay, expressed as the time needed for treated spheroids to obtain an 8-fold increase of the initial volume at the time of irradiation. Data obtained from regrowth delay analysis were used to calculate the extent of sublethal damage repair, showing for the squamous cell carcinoma line a fractionally higher capacity to repair sublethal damage than the neuroblastoma line. Repair increased with larger dose fractions in both cell lines. Our results show that multicellular tumor spheroids from the two cell lines used in this study are best applicable at relatively high total radiation doses. This makes multicellular tumor spheroids a suitable model for the in vitro evaluation of clinical treatment rationales such as hyperfractionation.     

Sublethal damage repair in two radioresistant human tumor cell lines irradiated as multicellular spheroids

Melanoma and lung adenocarcinoma may be amenable for radiotherapy if it were possible to increase the presently used total dose. In order to investigate this, spheroids from two cell lines of human origin, one obtained from a BRO melanoma and one from an NCI-H125 lung adenocarcinoma were exposed to graded doses (3-9 Gy) of radiation with 18-MV photons. Radiation was applied either as a single dose or as split doses with an interval of 6 h to determine the extent of sublethal damage repair. Radiation response was quantified in terms of spheroid cure and specific growth delay. Both cells lines have previously been shown to be less sensitive than a neuroblastoma and a squamous cell carcinoma cell line grown as spheroids. Data obtained from the growth delay analysis were used to calculate the extent of split-dose recovery. Repaired dose for BRO spheroids did not increase after 7 Gy, whereas in NCI-H125, the repaired dose showed a steady increase. Recovery ratios did not differ between the two cell lines, but were lower than reported for normal tissues. Both cell lines revealed a low repair capacity was expressed by the beta-value of the linear-quadratic (LQ) equation. However, repair capacity for sublethal damage as expressed by the dose repaired and the beta-value of the LQ equation was not different from values reported earlier by us for neuroblastoma and squamous cell carcinoma when grown as spheroids. This indicates that the low radiosensitivity for the cell lines used in this study is determined by the alpha-value of the LQ equation. Our results support the clinical finding that the application of increased total radiation doses in the treatment of melanoma and lung adenocarcinoma may be feasible if radiation is applied in multiple small fractions to ensure normal tissue sparing.     

Oxygen tensions in two human tumor cell lines grown and irradiated as multicellular spheroids

Cells from two human cell lines were irradiated both as multicellular tumor spheroids (MTS) and in monolayer culture. Radiation response of MTS was quantified in terms of specific growth delay and proportion cured, and as clonogenic cell survival for monolayer cells. Radiation was applied either as a single or as a split dose with time intervals of 1, 2, and 4 h to determine the rate of sublethal damage repair. Using as endpoint the fraction of MTS cured at an iso-effect level, in MTS of NB-100 neuroblastoma cells repair of sublethal damage was complete within 1 h, whereas in MTS of HN-1 squamous cell carcinoma cells there was still some unrepaired damage left. At a larger dose for NB-100 MTS the repair curve showed a similar shape as for HN-1 spheroids. Using as endpoint specific growth delay, no difference in repair between the various time intervals was observed. In monolayer cells from both cell lines sublethal damage was not fully repaired in the time intervals used. Polarographic microelectrode measurements of oxygen tension inside MTS showed a marked difference in steepness of oxygen tension profiles between MTS from both cell lines. In HN-1 squamous cell carcinoma MTS with diameters up to 500 microns the central pO2 amounted to about 100 Torr, whereas in NB-100 neuroblastoma MTS with the same diameters central pO2-values lower than 30 Torr were observed. NB-100 MTS were irradiated with doses of 5 and 10 Gy gamma rays and subsequently the oxygen tension was measured 1 and 5 h after irradiation. A reoxygenation effect could not be observed, either after single dose or after split dose irradiation. If spheroids may be regarded as a suitable model for tumor responses in vivo, the results from these experiments indicate that reoxygenation is a process eluding polarographic measurements, or that no dramatic changes in oxygen tension are to be expected shortly after high single doses or early in a fractionation scheme.     

Pleiotropic effects of fenretinide in neuroblastoma cell lines and multicellular tumor spheroids

The efficacy and mechanism of action of fenretinide (4-HPR), a vitamin A analogue, was investigated in a panel of six neuroblastoma cell lines and multicellular tumor spheroids. The latter are three dimensional cell aggregates and as such, a model for micrometastases. In all cell lines, the production of reactive oxygen species (ROS) increased with 163-680% after 1 h of treatment with 4-HPR. In addition, a decrease of the mitochondrial membrane potential of 30-75% was observed after 4 h of incubation with 4-HPR. A 6-12-fold difference was observed between the IC50 values for cell proliferation and viability between the most sensitive (IMR32) and most resistant (NASS) cell line towards 4-HPR. Flow cytometric analysis showed an increased amount of apoptotic bodies and no cell-cycle arrest. The antioxidant Trolox completely inhibited the accumulation of 4HPR-induced ROS and prevented the 4HPR-associated cytotoxicity. In all neuroblastoma spheroids, 4-HPR induced a complete cytostasis at clinical relevant concentrations (3-10 microM). Immunohistochemical analysis of 4-HPR-treated spheroids showed a decreased staining for proliferation marker Ki-67 and an increased staining for cleaved-PARP, a marker of apoptosis. Our results suggest that 4-HPR might be a promising agent for the treatment of micrometastases and high-risk neuroblastoma.     

In vitro radiobiological parameters of human sarcoma cell lines

In vitro radiobiologic survival parameters have been determined for 7 human osteosarcoma, 5 human soft tissue and bone sarcomas, and 4 Ewing's sarcoma cell lines. The mean D0 values were 99.5 +/- 11.6 cGy, 90.5 +/- 7.7 cGy and 95.8 +/- 7.9 cGy for osteosarcomas, soft tissue and bone sarcomas and Ewing's sarcomas, respectively. These in vitro survival data do not predict the clinical radiation resistance generally attributed to osteosarcomas and soft tissue and bone sarcomas, and do not differ substantially from the results obtained with the clinically radioresponsive Ewing's sarcomas.     

Cytotoxicity of tetraplatin and cisplatin for human and rodent cell lines cultured as monolayers and multicellular spheroids

The cytotoxicity of tetraplatin (dl-trans), its d- and l-isomers, and cisplatin for four human tumor cell lines (myeloma 8226, ovarian 2008, A2780, and OVCAR-3), their cisplatin-resistant variants, and three rodent cell lines (V79, EMT6/Ro, and L1210) were compared. Tetraplatin was more, or equally as, potent as cisplatin for the human cell lines and for L1210 but was clearly less potent for V79 and EMT6/Ro. The d-trans tetraplatin was more potent than the l-trans. Cisplatin resistant human tumor cells were less resistant to tetraplatin. On comparing sensitivity of V79 and EMT6/Ro cells in two growth models, we observed that all of the platinum compounds were more cytotoxic to cells in multicellular spheroids than in exponentially growing monolayers. Uptake studies, however, showed that tetraplatin was more cytotoxic to spheroids because spheroids accumulated more drug than monolayers.     

Synergistic interaction between cisplatin and gemcitabine in neuroblastoma cell lines and multicellular tumor spheroids

The efficacy and mechanism of action of cisplatin and gemcitabine were investigated in a panel of neuroblastoma cell lines and multicellular tumor spheroids. In neuroblastoma spheroids, the combination of cisplatin and gemcitabine induced a complete cytostasis at clinical relevant concentrations. A synergistic effect was observed when cells were coincubated with both drugs or preincubated with gemcitabine first. These administration sequences resulted in NASS cells in decreased ERCC1 and XPA expression, two key proteins of the NER DNA repair system, and increased platinum adduct formation in DNA. Most of these phenomena were not observed in SJNB8 cells which might explain the lack of synergy between cisplatin and gemcitabine in SJNB8 cells. Our results showed favorable interactions between cisplatin and gemcitabine in 4 out of 5 cell lines. Therefore, we feel that inclusion of gemcitabine into cisplatin-containing regiments might be a promising new strategy for the treatment of neuroblastoma.     

紫杉醇对NCI-H520单层细胞和细胞球体中Survivin表达影响的研究

目的:研究紫杉醇(TAX)对NCI-H520细胞系单层细胞和细胞球体Survivin表达的影响以及与TAX细胞周期阻滞的关系.方法:用3000、300、30、3和O.3ng/mL的TAX溶液处理NCI-H520的单层细胞和细胞球体,分别用CCK-8试剂盒进行细胞毒性分析、流式细胞仪进行细胞周期分析、半定量RT-PCR检测Survivin mRNA水平的表达以及用Western blot检测蛋白水平的表达.结果:TAX能显著抑制NCI-H520单层细胞和细胞球体的生长,TAX对细胞球体的半数抑制浓度为(2118.51±93.76)ng/mL,对单层细胞的半数抑制浓度为(1708.24±58.95)ng/mL;300ng/mL的TAX作用4h后细胞周期开始向G2/M期阻滞,而且随着作用时间的延长,阻滞作用越明显;TAX作用4h后上调细胞内Survivin蛋白的表达,而对Survivin mRNA的表达无明显影响.结论:TAX通过影响单层细胞和细胞球体的细胞周期而产生生长抑制作用,这种抑制作用具有浓度依赖性;TAX可影响单层细胞和细胞球体的Survivin蛋白水平的表达,这种表达上调作用可能是依赖于TAX的细胞周期阻滞作用的.而对mRNA水平无明显影响,说明TAX对NCl-H520细胞内Survivin表达的影响可能不是通过改变Survivin从头合成途径进行的.     

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.     

Formation and growth of multicellular spheroids of human origin

Different types of human cells which normally grow as monolayers or suspension cultures were tested for their capacity to form and grow as spheroids. Sixteen out of the 27 tested tumour cell lines formed spheroids. Nearly all of these spheroids also grew. With only two exceptions the doubling times were longer when the tumour cells grew as spheroids than when they grew in conventional mass culture. Eleven out of 13 tested human non-tumour cells formed small spheroids but of these only the spheroids of lymphoid origin could grow. These lymphoid cells grew faster when aggregated to spheroids than when in single-cell suspension culture. None of the other non-tumour cells, which normally grew as monolayers, could grow as spheroids. The normally monolayer-cultured tumour cells formed symmetrical spheroids with smooth surfaces while the normally suspension-cultured cells formed irregular spheroids with rough surfaces. All large spheroids had a necrotic centre surrounded by a shell of viable cells. The thickness of the viable cell layer varied depending on cell type. The shape and organization of cells within the spheroids also varied largely. The results show that many types of human cells can be cultured as spheroids and that a wide spectrum of morphological appearances and growth rates can be obtained.     

Resistance to adriamycin in multicellular spheroids

Spheroids of EMT-6 mammary tumor cells were markedly more resistant to different exposure doses of Adriamycin (ADR) than monolayer cells in exponential or plateau growth phases. For example, after 1 hr exposures to 0.5 μ/ml, surviving fractions determined by colony formation assay were approximately 0.3 for cells from spheroids treated intact and 0.001 for single exponential phase cells. To evaluate whether this resistance was related to poor drug uptake the distribution of the natural fluorescence of ADR equivalents was determined fluorimetrically and by direct microscopic observations. A concentration gradient of fluorescence was observed from the outside to the centers of spheroids even after high concentrations (10 μ/ml) and long exposure times (2 hrs). Cells from dissociated spheroids took up more drug than intact spheroids further indicating the existence of a significant diffusion barrier. When the surviving fraction of cells was plotted versus absorbed ADR equivalents the cells in intact spheroids were still more resistant and both curves were bicomponent with the most resistant fraction comprising about 20% of the cells. By using a selective disaggregation technique after intact spheroids had been exposed to the drug it was possible to show directly that the inner spheroid cells were most resistant (D₀ = 0.25 μ/10⁶ cells). This resistance was not due to differences in the cell cycle state of these inner cells since separate experiments showed that both exponential and plateau phase monolayer cells were about equally sensitive when the surviving fraction was plotted vs absorbed drug (D₀, = 0.04 μ/10⁶ cells). Thus, other factors related to the metabolic state of the cells, the microenvironment, or the formation of different drug products must account for the observed resistance. Pretreatment of spheroids with misonidazole before ADR effectively reduced this resistant population of cells.     

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.     

In vitro cytotoxic drug sensitivity testing of human tumour xenografts grown as multicellular tumour spheroids.

Tumour cells from 7 patients with ovarian carcinoma and from 22 different human tumour xenografts representing a wide range of histological sub-types have been examined for multicellular spheroid forming ability. Spheroid formation was limited to cells derived from xenografts. Of the 22 lines tested, 5 formed spheroids capable of growth in isolation. There was no clear relationship between histological type and spheroid-forming ability. The plating efficiency of tumour cells obtained from spheroids was always greater than for the cells obtained from the dissociated tumour of origin and was in some cases as much as 6-fold greater. Spheroid growth was nearly exponential for 4 cell lines. Volume growth delay was used to investigate the activity of melphalan, adriamycin, the Vinca alkaloids, CCNU and cisplatin. Differences between lines in drug response broadly reflected patient and in vivo xenograft response.     

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.     

Photodynamic therapy of transitional cell carcinoma multicellular tumor spheroids with hypericin

The objective of the present study was to evaluate the sensitivity of the bladder transitional cell carcinoma (TCC) to hypericin PDT in a 3-D system using multicellular tumor spheroids. The photodynamic response in RT-112 human bladder TCC spheroids was also compared to 2-D cultured monolayer cells. Following a 2-4 h incubation with 8-30 microM hypericin, spheroids or monolayer cells were irradiated at the light dose of 12 J/cm2, delivered at a fluence rate of 10-100 mW/cm2. The PDT effects were evaluated using a clonogenic assay. The results show that compared with the cells in a monolayer, cells in spheroids were dramatically less sensitive to hypericin PDT (<2000-fold). Studies of fluorescence microphotographs of centrally cut frozen sections of hypericin-exposed spheroids showed a gradient in hypericin concentration from the peripheral to the central region of the spheroid. Although it can be suggested that heterogeneity of drug uptake might be responsible for the observed resistance of spheroid to hypericin PDT, hypericin sensitized spheroids that were dissociated prior to light irradiation were as sensitive as the monolayer cells to hypericin PDT, suggesting that other factors such as oxygen depletion might be responsible for the resistance of spheroids to hypericin PDT.     

Radiation-induced apoptosis in human sarcoma and glioma cell lines

Six human soft-tissue sarcoma and 14 glioma cell lines, exhibiting considerable differences in radioresponsiveness and histological grade of differentiation of the parental tumour, were examined with respect to apoptosis development after irradiation with ⁶⁰Co γ-rays. After test doses of 6 and 25 Gy, significant changes characteristic of apoptosis occurring within 6 to 30 hr were exhibited by only 2 differentiated sarcoma cell lines, EL7 and ESS2. The characteristic intemucleosomal fragmentation of DNA was detected as early as 6 hr after exposure of subconfluent monolayer cultures to 6 Gy. It was limited to cells that had detached from the culture plate, whereas adherent cells showed random degradation of DNA, namely after higher doses (25Gy) or longer incubation times (30 hr). As assessed by fluorescence microscopy of unfixed cultures stained with Hoechst 33342 and propidium iodide, the proportion of cells showing apoptotic bodies in non-irradiated controls was < 0.l% and 0.3% for EL7 and ESS2, respectively. The doseresponse relationship for apoptosis was determined at 9 hr post-irradiation. After 2 Gy, the percentage of apoptotic cells was elevated to 3.4% in EL7 and 4.5% in ESS2 cultures. Saturation was obtained above 6 Gy, with 8.4% apoptosis in EL7 and 15% in ESS2 after 25 Gy. Taken together, rapid ionizing-radiation-induced apoptosis seems to be limited to a subgroup of sarcomas and is unlikely to occur in gliomas. © 1995 Wiley-Liss Inc.     

Effects of physiological oxygen environment on drug-induced cell lethality of multicellular tumor spheroids from human lung cancer

Advanced malignant tumors of certain histological types contain a hypoxic and necrotic core. Multicellular tumor spheroids (MTS) have the characteristics of chronically hypoxic cells in the center. We studied the effects of physiological oxygen environment on MTS growth and the cell lethality produced by doxorubicin (DXR) and cisplatin (DDP). MTS were made from 2 human lung cancer cell lines; PC-6 small cell and PC-10 squamous cell carcinoma, and grown for 2, 3 or 4 weeks; either in 5% CO2/air or 5% 02/5% CO2/90% N2. They were exposed to graded concentrations of DXR for 1 hr and cell lethality was determined by clonogenic assay. In the physiological oxygen environment MTS growth was retarded for both cell lines. PC-6 MTS grown in physiological oxygen environment were more sensitive to DXR than those developed in air. The differential sensitivity was most pronounced with the 2 week old MTS and gradually narrowed with increasing MTS size. In contrast, PC-10 MTS developed in the physiological oxygen environment were more resistant to DXR than those in air; the differences were again most pronounced in 2 week old MTS. There were little differences in cell kill effects of DDP, irrespective of cells being in monolayer or in MTS and growing in air or in physiological oxygen environment. These observations are consistent with the interpretation that cells in PC-6 MTS are scarcely affected by the physiological oxygen environment but easily affected by DXR, whereas cells in PC-10 MTS responded vice versa.     

卵巢癌多细胞球体对紫杉醇耐药及其机制的探讨

背景和目的:多细胞球体(multicellularspheroids,MCS)对传统细胞毒化疗药物的敏感性明显低于单层细胞。本实验旨在探讨卵巢癌MCS耐药的分子机制。方法:以单层细胞为对照,以三维培养方法获得的人卵巢癌A2780MSC和CAOV3MCS为模型,采用台盼蓝拒染法比较紫杉醇对单层细胞和MCS生长的抑制作用,流式细胞仪比较单层细胞和MCS细胞周期的分布和细胞凋亡率;采用流式细胞仪、蛋白质免疫印迹法、激光共聚焦显微镜检测单层细胞及MCS的P-糖蛋白(P-glycoprotein,P-gp)表达及亚细胞分布;采用RT-PCR法检测mdr1mRNA表达水平。结果:(1)不同浓度的紫杉醇(0.2、2.0、10.0、20.0μmol/L)作用后,MCS细胞生长抑制率明显低于单层细胞(PA2780=0.003,PCAOV3=0.015);经20.0μmol/L的紫杉醇作用后,单层细胞的细胞凋亡率明显高于MCS,差异有统计学意义(PA2780=0.034,PCAOV3=0.032)。(2)流式细胞仪、蛋白质免疫印迹法、激光共聚焦显微镜检测提示P-gp在单层细胞中不表达,在MCS中表达明显升高(P均<0.05);RT-PCR证实MCS中有mdr1mRNA表达,而单层细胞中未检出其表达。(3)流式细胞仪检测提示将单层细胞培养成MCS时,G0/G1期细胞比率增加,S期和G2/M期细胞比率降低(P均<0.05)。结论:卵巢癌MCS对紫杉醇化疗耐药性增加,其高表达P-gp,并且与G0/G1期细     

Swelling of multicellular spheroids induced by hyperthermia

EMT6 multicellular spheroids invariably swell by 10 to 50 per cent after incubation at 43 to 45 degrees C for 1 h. Both scanning electron and optical microscopy reveal morphological alterations particularly in the outer region of the spheroids. While the control cells are contiguous to one another and tightly held to the spheroid body, the heated spheroids exhibit partially disrupted contacts among cells. Measurements of intercellular volume and water volume of spheroids with labelled water and inulin show that changes in the spheroid volume are not due to an increase in cell volume, but that they can be explained by a 60-100 per cent increase in the intercellular space within a spheroid. Continuous observation of individual spheroids heated to 43-45 degrees C shows loss of adhesion of cells in the outer region and even detachment of a few surface cells. This 'melting' of the spheroid surface appears to result from a disorder in the extracellular material. Treatment with cell swelling agents such as hypotonic solution, ouabain, excess extracellular potassium ions, or ionophore nigericin, K+/H+ exchanger, each separately causes the spheroids to swell at the control temperature. On the other hand, A23187, Ca2+ ionophore, causes shrinkage of the spheroids. Thus, under hyperthermia, the volume of spheroids increases due to the disruption in the cell organization in their outer region.