A comparison of adriamycin and mAMSA

Summary Multicellular spheroids were used to compare the two chemotherapeutic agents adriamycin (ADM) and 4[(9-acridinyl)-amino] methanesulphon-m-anisidide (mAMSA). Chinese hamster cells, V79 379A, a human small cell lung carcinoma, designated ME/MAR, and a human melanoma xenograft, HX117, were grown as spheroids (200 or 400 m in diameter) and treated with either drug for 1 h, at 37°C, in air. Cytotoxicity was assayed using both cell survival and growth delay.Both drugs were highly toxic towards V79 but showed less activity toward the human tumour single cell suspensions; ADM was more effective towards HX117 and ME/MAR than mAMSA. When grown as spheroids, the cells developed marked resistance to both drugs. In all cases, cytotoxicity was drug dose and spheroid size dependent. The response of HX117 spheroids to both drugs was similar. In contrast, ADM was more effective toward 200 m diameter ME/MAR spheroids, and mAMSA showed greater activity than ADM against V79 spheroids. Both endpoints gave qualitatively equivalent results, and a comparison of the two showed relatively long growth delays for a given level of cell kill, for both drugs and with all three cell lines. The greater cytotoxicity of ADM toward ME/MAR spheroids is consistent with the clinical finding that ADM has a use in the treatment of small cell carcinoma of the lung, while mAMSA has not demonstrated any activity in the treatment of lung cancer.     

The radiobiology of human neuroblastoma

The radiation response of a human neuroblastoma xenograft HX138 has been studied in vitro and in vivo using single cells in suspension, multicellular spheroids, and xenografts in immune-suppressed mice. End-points used were growth delay and clonogenic cell survival. Growth delay experiments with spheroids and xenografts showed a high degree of radioresponsiveness. Cell survival curves obtained from all systems were characterised by the lack of a shoulder. An increase in Do of the cell survival curve was seen after irradiation of intact spheroids and xenografts, perhaps due to the presence of a contact effect. Cellular capacity for split-dose recovery in vitro was modest. Delayed assay experiments using spheroids and xenografts showed some potentially lethal damage (PLD) repair in vitro but not in vivo. The results show this human tumour line to be intrinsically highly radiosensitive, with a limited repair capacity.     

Characterization of radiation sensitivity of human squamous carcinoma A431 cells

Studies have been performed to investigate the radiosensitivity of human squamous carcinoma cells. A431 cells were grown in vitro as exponential and fed-plateau monolayer cultures or as multicellular spheroids. Radiobiological studies of various cultures showed that fed-plateau phase cells were more sensitive (D0 = 1.3 Gy) than exponentially growing cells (D0 = 1.5 Gy). After a single dose of 12 Gy or two doses of 6 Gy irradiation, A431 cultures exhibited a large capacity for potentially lethal damage (PLD) repair (PLD repair factor = 17), but a relatively small sublethal damage (SLD) repair. In order to measure the radiation sensitivity of proliferating (P) and quiescent (Q) cells, enriched populations of P- and Q-cells were isolated from A431 spheroids. Flow cytometric analysis with acridine orange (AO) staining demonstrated that there was a shift of the RNA histograms in fed-plateau and spheroid cultures towards lower values, suggesting the presence of a subpopulation of Q-cells. Centrifugal elutriation was used to isolate the Q-cells from dissociated spheroid cells. Coulter cell volume distributions and flow cytometric analysis showed that Q-cells had a small cell volume (approximately 1380 microns3), low RNA content and a G1-like DNA content. Continuous labelling experiments with tritiated thymidine confirmed the non-proliferating nature of the Q-cells. Irradiation of the Q-cells after isolation from spheroids with between 0 to 10 Gy showed that they were more radiosensitive (decreased D0) than the P-cells isolated from these spheroids. The latter were, however, similar in radiosensitivity to exponential G1 cells.     

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.     

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.     

Changes in subcellular distribution of topoisomerase IIalpha correlate with etoposide resistance in multicell spheroids and xenograft tumors

The outer cells of Chinese hamster V79 spheroids are about 10 times more resistant than monolayers to DNA damage and cell killing by the topoisomerase (topo) II inhibitor etoposide. Although the amount and catalytic activity of topo IIalpha are identical for monolayers or the outer cells of spheroids, and the cell proliferation rate is the same, our previous results indicated that phosphorylation of topo IIalpha is at least 10 times higher in V79 monolayers than in spheroids. Because phosphorylation of topo IIalpha has been associated with nuclear translocation, we examined subcellular distribution of Topo IIalpha in monolayers, spheroids, and xenograft tumors using immunohistochemistry. Topo IIalpha was located predominantly in the nucleus of V79, human SiHa, and rat C6 monolayers but was found mainly in the cytoplasm of the proliferating outer cells of spheroids formed from these cell lines. Conversely, the outer cells of WiDr human colon carcinoma spheroids showed predominantly nuclear localization of topo IIalpha, and only WiDr cells showed no increase in resistance to etoposide when grown as spheroids. Cells sorted from xenografts resembled the spheroids in terms of sensitivity to etoposide and location of topo IIalpha. When the outer cells of V79 spheroids were returned to monolayer growth, the rate of redistribution of topo IIalpha to the nucleus occurred with similar kinetics as the increase in sensitivity to killing by etoposide. Removal and return of individual outer V79 spheroid cells to suspension culture resulted in the translocation of topo IIalpha to the nucleus for the first 24 h, accompanied by an increase in sensitivity to DNA damage by etoposide. Therefore, the cytoplasmic topo IIalpha distribution in outer spheroid cells and tumors appears to correlate not with morphological changes associated with growth in suspension but rather with the presence of neighboring, noncycling cells.     

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.     

Growth and radiation sensitivity of the MLS human ovarian carcinoma cell line grown as multicellular spheroids and xenografted tumours

The growth characteristics and the radiation sensitivity of multicellular spheroids of the MLS human ovarian carcinoma cell line grown in spinner culture in atmospheres of 5% CO2 in air or 5% CO2, 5% O2 and 90% N2 were studied and compared to that of MLS xenografted tumours. The spheroids grew exponentially with a volume-doubling time of approximately 24 h up to a diameter of approximately 580 microns and then the growth rate tapered off, more for spheroids grown at the low than at the high oxygen tension. Thirty days after initiation, the spheroid diameters were approximately 1,500 microns at the low and 2,100 microns at the high oxygen tension. The tumour volume-doubling times were approximately 8 days (V less than 200 mm3) and 17 days (V = 1,000-4,000 mm3). The histological appearance of the spheroids and the tumours was remarkably similar; both developed large central necrosis and both were composed of epithelial cells and showed pseudoglandular structures with lumen. The spheroids were slightly less differentiated than the tumours. The intrinsic, cellular radiation sensitivity was independent of whether the cells were grown in vitro as spheroids or in vivo as tumours, as revealed by irradiating single cells from dissociated spheroids and tumours under aerobic conditions and intact spheroids and tumours under hypoxic conditions. Studies of 1,600 microns spheroids grown in 5% CO2 in air showed that the intrinsic radiation sensitivity of the chronically hypoxic cells was the same as that of acutely hypoxic cells. The fraction of radiobiologically hypoxic cells under these conditions was approximately 15% and similar to those of 9% (V = 200 mm3) and 28% (V = 2,000 mm3) found for the tumours. Spheroids with diameter of 1,200 microns did not show survival curves parallel to those for acutely hypoxic cells, i.e. they did not contain a measurable fraction of clonogenic cells at complete radiobiological hypoxia. The final portion of their survival curves represented partially hypoxic cells; the OERs were 1.6 and 1.3 for spheroids grown at the high and the low oxygen tension, respectively. The considerable similarity between the spheroids and the tumours suggests that MLS spheroids constitute a valuable in vitro model for studies of human tumour radiation biology and related physiological processes. MLS spheroids may be particularly useful in studies of therapeutic consequences of partial radiobiological hypoxia since complete hypoxia and different levels of partial hypoxia can be studied separately by varying spheroid size and the oxygen tension in the culture medium.     

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.     

Radiation repair in human endothelial cells

Damage to blood vessels caused by ionizing radiation is considered to be an important dose limiting factor for late effects in many organs. However, the radiation response of the endothelial cells which line the vasculature has not been well-documented, particularly for human endothelial cells. In the present study, human endothelial cells were obtained from fresh umbilical cords and cultured in monolayer. Immunofluorescent staining of factor VIII antigen was used to verify the endothelial nature of the cultured cells. The cells were irradiated with Cs-137 rays (1.35 Gy/min) in plateau phase to determine radiation sensitivity and ability to repair potentially lethal damage (PLD) and sub-lethal damage (SLD). The endothelial cells demonstrated moderate radiosensitivity that varied slightly between different cords. As demonstrated by delayed plating experiments, PLD repair ability was substantial, with repair factors of 0.70-0.80. SLD repair capability, as demonstrated by split dose experiments, was relatively modest. A survival enhancement of 2.0-2.2, for example, was observed when 8 Gy single dose survival was compared to two 4 Gy doses. In terms of PLD and SLD repair as well as initial slope (alpha) the endothelial cells were similar to normal human lung and skin fibroblasts previously studied. Compared to malignant cell lines, PLD repair was generally larger whereas the initial slope (alpha) was intermediate-steeper than the radioresistant tumor types but shallower than the more sensitive tumor derived cells.     

Use of a cell sorter for assays of cell clonogenicity

A fluorescence-activated cell sorter has the capability of recognizing, categorizing, counting, and sorting cells, and it thus provides a convenient and rapid method for "micromanipulation" of known numbers of cells into appropriate vessels for cell viability or clonogenicity studies. Subsequent microscopic observation of the sorted cells allows discrimination between the separate processes of cell attachment (plating efficiency) and clonogenic growth (viability). Examples are presented showing the power of these techniques for studying the low-dose regions of radiation survival curves for Chinese hamster V79 cells grown as monolayers or spheroids, for cells irradiated under aerobic versus hypoxic conditions, and for cells of large spheroids exposed to Adriamycin, where the slow penetration of the drug results in a differential exposure to the various cell subpopulations.     

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.     

The effect of lonidamine (LND) on radiation and thermal responses of human and rodent cell lines

Rodent and human cells were tested for response to Lonidamine (LND) (1-(2,4 dichlorobenzyl) 1-indazol-3-carboxylic acid) combined with radiation or hyperthermia. Lonidamine exposure before, during, and after irradiation caused varying degrees of inhibition of potentially lethal damage (PLD) repair which was cell line dependent. In human glioma, melanoma, squamous cell carcinoma, and fibroblasts, LND exposure did not inhibit or only partially inhibited repair of potentially lethal damage. LND up to 100 micrograms/ml produced only a low level of toxicity in these cells and only slightly inhibited glucose consumption at the maximum concentration. In human glioma cells, LND treatment alone did not inhibit PLD repair, but when combined with hyperthermia treatment at moderate levels easily achievable in the clinic, there was complete inhibition of potentially lethal damage repair. These data suggest that LND effectiveness is cell type dependent. Combinations of LND, hyperthermia, and radiation may be effective in cancer therapy especially in tumors such as glioma in which repair of potentially lethal damage may be extensive.     

Cellular radiosensitivity in V79 cells is linked to alterations in chromatin structure

V79 cells grown as spheroids are more radioresistant than those grown as monolayers. Viable cells from spheroid culture contain restraints to ethidium bromide driven rewinding of DNA supercoils that are absent in monolayer cells. Spheroid cells also contain a DNA-protein matrix that is more resistant to detergent-induced degradation. The increase in structural integrity may be related to a 55-60 kD protein in the nucleoids of spheroid, but not monolayer cells. Spheroid cell radioresistance may therefore be related to a more stable chromatin platform for high fidelity repair of DNA damage.     

The influence of once or twice daily irradiation regimens on growth of squamous cell carcinoma spheroids of different diameters

Large tumours are in general more difficult to cure by radiation treatment than small tumours. Several factors may be responsible for this phenomenon which evolves during tumour growth. In an earlier study using squamous cell carcinoma cell line HN-1, we have shown in split-dose recovery experiments that the amount of sublethal damage repair is equal in spheroids of different diameters. To elucidate this repair capacity further, we have employed other radiation regimens, calculated with the LQ-equation to be iso-effective, in spheroids of different sessions. Using specific growth delay to quantify radiation response after two to five fractions, it was shown that repair capacity was equal in spheroids of different sizes. For small spheroids the specific growth delay was smaller in once daily fractionation regimens than when radiation was administered in twice daily sessions. In large spheroids this advantage of accelerated fractionation was not observed. If spheroids from this squamous cell carcinoma cell line may be regarded as a relevant model system for their in vivo counterparts, then the results from the present study may indicate that accelerated fractionation of treatment is advantageous for small lesions, but not for larger tumours.     

Radiation response of human melanoma multicellular spheroids measured as single cell survival, growth delay, and spheroid cure: comparisons with the parent tumor xenograft

The radiation response of multicellular spheroids, initiated from a human melanoma xenograft (E.E.) propagated in athymic mice, was studied using cell survival, growth delay, and spheroid cure as endpoints. The relationship between these endpoints was analyzed, and the radiation response of the spheroids was compared with the parent xenograft. At irradiation, the spheroids were 100 +/- 5 micron in diameter and did not contain radiobiologically hypoxic cells. Growth delay of the spheroids mainly depended on the fraction of surviving cells as measured in soft agar, that is, there was a good correlation between these two endpoints. Moreover, Do-values calculated from spheroid cure curves were similar to those of the cell survival curves measured in soft agar. However, the number of stem cells per spheroid, calculated from SCD50-values (the doses required to cure 50% of the spheroids), was at least a factor of seven lower than the clonogenicity of cells from disaggregated spheroids would indicate. The cellular radiosensitivity of the spheroids was similar to the parent xenograft. An intercellular contact effect was not found for the spheroids, in agreement with observations from studies of xenografted tumors. Moreover, specific growth delays, as well as Do-values calculated from cure curves were similar for spheroids and tumors when the data for the latter were corrected for the presence of hypoxic cells. The high degree of conformity in the results indicates that multicellular spheroids and xenografted tumors may complement one another in studies of human tumor radiobiology.     

Deriving cell survival curves from the overall responses of irradiated tumours: analysis of published data for tumour spheroids

Curves of growth delay (GD) or 'cure' after graded doses of radiation have been analysed for 16 lines of human and animal tumours grown as multicellular spheroids in vitro. Dose-survival curves were derived for those cellular units from which spheroids regrow after unsuccessful irradiation (spheroid-regenerating cellular units, SRU). For 10 sets of data from 6 spheroid lines, the Do's and extrapolation numbers of the SRU derived by GD could be compared with the response of the clonogenic cells of the spheroids. For Do, a good correlation (r = 0.910) was found between the two; this was true also for Do derived from curves of spheroid 'cure' (7 sets of data from 6 spheroid lines) and clonogenic cells (r = 0.986). Using GD, the correlation of extrapolation numbers was less good (r = 0.682), the values for SRU commonly being higher than those for clonogenic cells. This may reflect features of the growth curves of spheroids after the lower range of doses of radiation. For human and animal tumour spheroids of 250 microns or less, derived Do ranged from 0.5 to 2.5 Gy. For spheroids of 350 microns or more, derived Do for animal tumour lines ranged from 3.4 to 4.2 Gy, for human lines from 1.5 to 2.1 Gy.     

Repair and proliferation: major determinants of the multifraction radiation response

An analysis of multifraction radiation survival data for V79 monolayers and spheroids, in which measured responses to each treatment were intercompared with the alpha/beta ratio derived using the common "reciprocal-dose" methodology at selected survival levels, resulted in quantitatively and qualitatively different conclusions. These discrepancies were the result of non-constant toxicity of each radiation dose in the multifraction scheme, which was obvious in the multifraction dose response curves, but not in the reciprocal dose plots. These data appear to indicate incomplete recovery between radiation exposures. However, under some circumstances, lack of repair was offset by cell proliferation, thus emphasizing the roles of repair, repopulation, and redistribution in the multifraction radiation response.     

Effectiveness in inhibition of recovery of cell survival by cisplatin and carboplatin: influence of treatment sequence

Clinical protocols have been designed to combine platinum-based drugs and radiation in the treatment of cancer. The rationale for this approach has been developed from preclinical studies demonstrating that platinum compounds can potentiate the cytotoxic effects of radiation toward cells. In the present study multicellular spheroids derived from squamous cell carcinoma cell line HN-1 have been used to study the effects of both cisplatin and carboplatin when administered prior to, concurrently, and after irradiation treatment. To study the influence of platinum compounds on sublethal damage repair, single and split doses of radiation were applied. Growth delay and proportion cured spheroids served as endpoints. Both cisplatin and carboplatin had no potentiating effect when administered 24 hr prior to irradiation. When administered 3 hr after completion of irradiation procedures, growth delay after single and split doses were enhanced to the same extent. The drug enhancement ratio for cisplatin was larger (1.5) than for carboplatin (1.2). Both single and split doses were enhanced by the same factor, which was interpreted as no effect on sublethal damage repair. When platinum compounds were present in the target cells at the time of irradiation, especially the split dose radiation response was strongly enhanced: the drug enhancement ratio was 3.9 for cisplatin and 3.2 for carboplatin. Recovery from sublethal damage was totally repressed. This study shows that platinum compounds can potentiate radiation and that for maximum effect the sequence of the two treatment modalities is of utmost importance. Moreover, these results may in part explain the heterogeneous outcomes of trials combining platinum compounds and radiation.     

Radiosensitivity of human melanoma spheroids influenced by growth rate

The radiosensitivity of human melanoma cell line BRO was investigated using the multicellular tumor spheroid system. By adding different concentrations of bovine serum to the tissue culture medium, two different growth rates could be obtained. Spheroids (200-250 microns) were irradiated with graded single doses of X rays (2-8 Gy). The radiation response was quantified using specific growth delay, clonogenic cell survival, and spheroid cure. All three assays showed a growth rate dependent radiation response. At both growth rates the spheroid growth fraction and critical cell number were of comparable magnitude. There was a strong correlation between the radiation response of spheroid regenerating units and clonogenic cells from dispersed spheroids. Cell survival curves indicated a decreased ability to accumulate sublethal damage in fast growing multicellular tumor spheroids. From this study it appears that the intrinsic radiosensitivity of human melanoma cell line BRO cells in multicellular spheroids is modulated by intratumoral conditions.