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.

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.

Three-dimensional culture of human tumor cells under standardized medium conditions.

The 3-dimensional culture of human tumor spheroids under standardized medium conditions may reveal information on specific biological parameters that could be masked in serum-supplemented media. Spheroids derived from human tumor cells are growth retarded in media free of serum. Ex-Cyte IV is a substance derived from human blood that can be used to improve growth in tissue culture. In this study the growth of spheroids from four different human tumor cell lines was studied when grown in medium free of serum, medium supplemented with varying concentrations Ex-Cyte IV, and medium supplemented with foetal calf serum (FCS). The parameters used for comparisons were growth rate, growth enhancement, clonogenicity and cell cycle distribution. The four cell lines showed different growth rates in serum-free medium, which were increased to different extents when Ex-Cyte IV or FCS were added. The growth enhancing effect induced by Ex-Cyte IV was differently concentration dependent for each cell line. The clonogenicity of cells grown as spheroids in serum-free medium was lower than in spheroids grown in supplemented media. There was no difference in clonogenicity between the differently supplemented media. All four cell lines responded to growth in serum-free medium with a drop in the S-phase and G2M phase. The present study provides a novel approach to the study of human tumor cells in 3-dimensional culture under defined conditions. The human serum derived substance Ex-Cyte IV may provide a method to obtain information on specific biological parameters that could be masked in serum-supplemented media.

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.

Differences in repair of radiation induced damage in two human tumor cell lines as measured by cell survival and alkaline DNA unwinding.

We studied the relationship between the repair of radiation induced DNA strand breaks and cellular repair kinetics in two human tumor cell lines, NB-100 (neuroblastoma) and HN-1 (squamous cell carcinoma). Damage was quantified using the fluorometric analysis of DNA unwiding (FADU) for DNA damage, and cell survival was assessed using a clonogenic assay. In plateau phase cells repair of sublethal damage was virtually absent in NB-100 after 4 Gy (recovery ratio 1.0), whereas HN-1 cells did show sublethal damage repair (recovery ratio 1.4). Repair of potentially lethal damage was more pronounced in NB-100 cells (recovery ratio 2.3) than in HN-1 cells (recovery ratio 1.7) after 4 Gy. Graded doses of X-rays induced comparable levels of DNA damage in both tumor cell lines. However, in HN-1 cells more DNA strand breaks were repaired after 4 Gy, leaving about 25% of the initial damage unrepaired, whereas in NB-100 about 50% was unrepaired. This higher fraction of unrepaired DNA damage correlated well with the degree of sublethal damage repair which was lower in NB-100 than in HN-1 cell, but it did not correlate with the repair of potentially lethal damage, which was higher in NB-100 than in HN-1. Since the level of damage remaining post-irradiation may be the critical variable for survival, the FADU technique can contribute in elucidating the relationship between radiosensitivity and DNA damage repair capacity.

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.

The influence of pre-irradiation on the survival of two head and neck squamous cell carcinoma lines.

Induction of radioresistant tumor cells by previous irradiations may be a problem because the chance to treat a patient presenting with a recurrence with radiotherapy may be lost if a resistant subpopulation was induced by the initial treatment. We here report on experiments with two squamous carcinoma cell lines where cultures were initially irradiated with graded single doses (2-6 Gy) and were re-irradiated after 4 days with single and split doses (2-6 Gy). Both cell lines showed no alteration in radiosensitivity. Also, the split-dose recovery was not changed by the previous irradiations. Flow cytometric analysis showed no significant differences in cell cycle distribution, so that a possible influence of cell cycle redistribution could be excluded. Although preliminary, the results from this study give no indication that previously irradiated cells become more resistant to radiation. In view of the clinical importance of radioresistant clones in tumours, it seems important to perform similar experiments to those reported upon in this study with several radiation fractions as pre-irradiation treatment, to impose a stronger selection pressure on the surviving cells.

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.

Combined effects of tumor necrosis factor alpha and radiation in the treatment of renal cell carcinoma grown as radia spheroids.

We have investigated the antiproliferative effects of Tumor Necrosis Factor Alpha (TNF) and radiation on a recently described rat renal cell tumor line grown as multicellular tumor spheroids (MTS). Treatment commenced when the spheroids had reached a diameter of 250 microns. TNF was diluted in the tissue culture medium in different concentrations, ranging from 250-1000 ng/ml. TNF monotherapy had a dose-dependent inhibiting effect on spheroid growth. Single-dose irradiation with 2, 4 or 6 Gy also retarded spheroids significantly in their growth. In the combination treatment the highest dose of TNF (1000 ng/ml) was added 4 hours prior to radiation. TNF could not induce a potentiation of the radiation injury at 2 Gy. The combination with 4 Gy, however, had additive and the combination with 6 Gy synergistic antiproliferative effects; in these treatment regimens respectively 2 and 5 out of 24 spheroids were controlled, i.e. cured. These experiments suggest that TNF in combination with radiotherapy may be beneficial for the treatment of renal cell carcinoma or cancer in general.

Repair of radiation induced damage in two human tumour cell lines grown as spheroids and monolayers.

Growth curves of two human tumour cell lines grown as multicellular tumour spheroid (MTS) were used to obtain survival estimates by back-extrapolation after split and single dose irradiation. Neuroblastoma (NB-100) and squamous cell carcinoma (HN-1) single cells from monolayer culture were assessed for repair of sublethal and potentially lethal damage. The extent of repair was calculated on an iso-effect basis. When grown as spheroids squamous cell carcinoma cells showed a higher capacity to repair sublethal damage than neuroblastoma cells. Repair of potentially lethal damage did not contribute to this higher capacity of HN-1 cells, since this cell line was found to be deficient for this type of repair. Using the recovery ratio to estimate the beta-component of the survival curves, it was found that differences in repair capacity were determined by the alpha-component of the equation. Our results show the feasibility of back-extrapolating multicellular tumour spheroid growth curves to obtain survival estimates that can be applied to establish sublethal damage repair capacity.

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.

Radiosensitivity of different human tumor lines grown as xenografts determined from growth delay and survival data.

Four human tumor lines were grown as xenografts in nude mice to determine whether xenografts 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. Xenografts from a neuroblastoma, a squamous cell carcinoma, a melanoma and a lung adenocarcinoma were studied in terms of growth delay and tumor control dose (TCD50). To exclude an immunoreaction of the host in the radiation response of the tumor xenografts, the tumor lines were tested for their growth in immunosuppressed Wistar rats. No differences in growth of xenografts in either immunodeficient mice or immunosuppressed rats were observed. Both growth delay and local tumor control as expressed by cure correlated well with clinical behavior of the tumor types of origin. This study demonstrates that radiosensitivity of different human tumor lines can be evaluated in terms of growth delay and tumor control dose50 when they are grown as xenografts. To exclude immune reactions, proper controls should be included. The sensitivities established from these evaluations parallel clinical behavior, thus offering a tool for analysis of human tumor radiosensitivity of histologically different tumor types.

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.

Cell lines of human oral squamous-cell carcinomas retaining their differentiated phenotype.

Two cell lines from head-and-neck squamous-cell carcinomas (SCC) have been established and characterized. Cell line R105 was derived from a xenografted SCC of the floor of the mouth and cell line T87/rc from a SCC of the epiglottis. Identification of individual cytokeratins 4, 5, 7, 8, 10, 13, 14, 18 and 19 led to the conclusion that both cell types had squamous characteristics and that keratinization occurred in xenografts. Ultrastructurally, junctional complexes were observed in both cell lines. Characteristic marker chromosomes were found and although both cell lines were derived from male patients, the Y chromosome was missing from all examined cells. The basic biological parameters of both cell lines were modal chromosome numbers of 59 (R105) and 60 (T87/rc), a doubling time of 60 (R105) and 45 hr (T87/rc) and a DNA index of 1.54 (R105) and 1.31 (T87/rc). The tumorigenicity of the 2 cell lines was proved by the ability to form colonies on a plastic substratum, as well as in a soft agar assay. Furthermore, the cells could produce multi-cellular tumour spheroids, and formed tumour nodules after subcutaneous inoculation into nude mice. The R105 tumour cells appeared to be better differentiated than the T87/rc as observed by histology and immuno(histo)chemistry. Both cell lines appear to retain SCC differentiation after being xenografted into nude mice, cultured for more than 40 passages in vitro and thereafter again xenografted into nude mice.

Sublethal damage repair and radiosensitivity of human squamous cell carcinoma cells grown with different culture techniques.

In this study, cells of a human squamous cell carcinoma line, HN-1, were grown in monolayers and as multicellular tumour spheroids (MTS). Repair of radiation-induced damage was studied by irradiation with single and split doses of X rays (4-8 Gy). It was shown that the amount of sublethal damage that was repaired in this dose range was equal in cells growing in monolayers and as MTS. The radiosensitivity of spheroids, as expressed by spheroid "cure" dose, increased with increasing MTS diameter. It is postulated that, in MTS with no signs of hypoxia, radioresistance diminishes when MTS increase in diameter.

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.

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.

Multicellular aggregates from human tumor cell lines for radiation studies.

Aggregates of human tumor cells are widely used in experimental studies on tumor responses to treatment. Only a limited number of human tumor cell lines are capable of forming spheroids. In this study cellular characteristics of 7 lung cancer and 4 bladder cancer cell lines are described with respect to their spheroid forming capacity. Comparisons were made with four reference lines known for their propensity to form growing aggregates. In the absence of vimentin expression no spherical aggregates were formed. Spherical aggregates were formed by one bladder and one lung cancer cell line, of which only the latter exhibited growth. Cellular factors influencing the ability of spheroids to increase in volume after spherical aggregation are not yet defined. Viability and clonogenicity of cells in aggregates are not the determinant of growth capacity. The growth rate of cell lines that exhibited growth is determined by tissue culture conditions and additives. Type of medium, percentage of foetal bovine serum and glucose concentration influenced the growth rate of spheroids. Since the response to radiation may be influenced by the growth rate of the tumors, manipulation of tissue culture medium composition offers the possibility of testing the influence of growth rate on the radiation response of one type of spheroids.