The effect of a single fraction compared to multiple fractions on painful bone metastases: a global analysis of the Dutch Bone Metastasis Study.

PURPOSE: To answer the question whether a single fraction of radiotherapy that is considered more convenient to the patient is as effective as a dose of multiple fractions for palliation of painful bone metastases. PATIENTS: 1171 patients were randomised to receive either 8 Gy x 1 (n = 585) or 4 Gy x 6 (n = 586). The primary tumour was in the breast in 39% of the patients, in the prostate in 23%, in the lung in 25% and in other locations in 13%. Bone metastases were located in the spine (30%), pelvis (36%), femur (10%), ribs (8%), humerus (6%) and other sites (10%). METHOD: Questionnaires were mailed to collect information on pain, analgesics consumption, quality of life and side effects during treatment. The main endpoint was pain measured on a pain scale from 0 (no pain at all) to 10 (worst imaginable pain). Costs per treatment schedule were estimated. RESULTS: On average, patients participated in the study for 4 months. Median survival was 7 months. Response was defined as a decrease of at least two points as compared to the initial pain score. The difference in response between the two treatment groups proved not significant and stayed well within the margin of 10%. Overall, 71% experienced a response at some time during the first year. An analysis of repeated measures confirmed that the two treatment schedules were equivalent in terms of palliation. With regard to pain medication, quality of life and side effects no differences between the two treatment groups were found. The total number of retreatments was 188 (16%). This number was 147 (25%) in the 8 Gy x 1 irradiation group and 41 (7%) in the 4 Gy x 6 group. It was shown that the level of pain was an important reason to retreat. There were also indications that doctors were more willing to retreat patients in the single fraction group because time to retreatment was substantially shorter in this group and the preceding pain score was lower. Unexpectedly, more pathological fractures were observed in the single fraction group, but the absolute percentage was low. In a cost-analysis, the costs of the 4 Gy x 6 and the 8 Gy x 1 treatment schedules were calculated at 2305 and 1734 Euro respectively. Including the costs of retreatment reduced this 25% cost difference to only 8%. The saving of radiotherapy capacity, however, was considered the major economic advantage of the single dose schedule. CONCLUSION: The global analysis of the Dutch study indicates the equality of a single fraction as compared to a 6 fraction treatment in patients with painful bone metastases provided that 4 times more retreatments are accepted in the single dose group. This equality is also shown in long term survivors. A more detailed analysis of the study is in progress.

Late effects of intraoperative radiation therapy in anastomotic rat colon.

PURPOSE: to determine whether intraoperative radiotherapy causes long-term negative effects on the healing of colonic anastomoses in the rat. METHODS AND MATERIALS: 175 rats were divided into seven equal groups. One group served as sham-irradiated control group. In the others, following a colonic resection, 1 or 2 cm of the distal bowel limb was irradiated with a single dose of 10, 15, or 20 Gy (groups 10/1, 15/1, 20/1, 10/2, 15/2, and 20/2, respectively). Subsequently, an anastomosis was constructed. The animals were killed after 6 (n = 10 in each group) or 12 (n = 15) months. The abdomen was inspected for abnormalities and the colonic diameter was measured. The anastomotic segment was analyzed biochemically (hydroxyproline) and histologically. RESULTS: During the experimental period, 1 rat (group 15/1) died because of anastomotic leakage and 3 others died from unknown causes. There was no difference in colonic diameter between groups. Altogether 17 rats developed an adenocarcinoma in the irradiated area: 11 of these had received a dose of 20 Gy. Histological observation indicated that fibrosis was present only in a limited number of animals, mostly after irradiation with a dose of 15 or 20 Gy. All anastomoses were functional and showed normal histology. The hydroxyproline content of the anastomotic segment was increased--with respect to the control group--only in the 20/2 group after 6 months. After 12 months, the hydroxyproline concentration in the (irradiated) segment distal to the anastomosis proper was higher in the 10/1 and 15/1 groups than in the control group. Otherwise, there were no differences between groups. CONCLUSION: Intraoperative irradiation with a single dose of 10-20 Gy, delivered to the distal limb used for anastomotic construction, does not appear to constitute a threat to anastomotic integrity. Dose-related changes included formation of adenocarcinomas and fibrosis, but function and histology of the anastomosis proper remained unaffected.

Moderate doses of intraoperative radiation severely suppress early strength of anastomoses in the rat colon.

Intraoperative irradiation appears to be a valuable addition to the modalities available to treat patients with large bowel cancer. However, its potential effect on healing of anastomoses has not been investigated extensively. For this purpose, male Wistar rats underwent colonic resection. Subsequently, 1 cm of each bowel end was irradiated with doses of 10, 15, 20 or 25 Gy and intestinal continuity was restored. After 3 or 7 days, animals were killed and the anastomoses were analyzed for bursting pressure (intraluminal force), breaking strength (longitudinal force) and hydroxyproline content. Intraoperative irradiation led to a massive (40-70%) and significant (P < 0.025) reduction in bursting pressure 3 days after operation compared to the control group for every dose used. After 7 days, the bursting site was outside the area of the anastomosis in all groups. The breaking strength at day 3 was also reduced, even after 10 Gy. At day 7, when tearing still occurred in the wound area, the breaking strength was still significantly lower in the 15- and 25-Gy groups than in the control group. The hydroxyproline content of the anastomoses was significantly reduced only after irradiation with the higher doses. Thus intraoperative irradiation constitutes a threat to early strength of anastomoses in the rat colon, and even at moderate doses it may threaten the integrity of the anastomosis.

A semiquantitative histological analysis of repair of anastomoses in the rat colon after combined preoperative irradiation and local hyperthermia.

Hyperthermia is a promising method for increasing the efficacy of radiation therapy of colorectal cancer. To study the histological aspects of healing of an anastomosis in the colon, after combined preoperative (sham) irradiation and (sham) hyperthermia treatment, 48 male Wistar rats were divided randomly into four groups. In each animal, a segment of the colon was treated successively by (sham) irradiation (single dose of 25 Gy X rays) and/or (sham) hyperthermia (44 degrees C, 30 min). After 5 days, a resection of the colon was performed by construction of an anastomosis: The distal limb consisted of (sham-) irradiated and/or (sham-) hyperthermia-treated bowel. Rats were killed 3 or 7 days after the surgical procedure. Evaluation of healing of the anastomosis was made by: (1) histological analysis of sections stained with hematoxylin and eosin, (2) semiquantitative measurement of collagen in the area of the anastomosis and (3) semiquantitative analysis of the number of macrophages by immunocytochemistry. Healing of the anastomoses in animals receiving irradiation or hyperthermia alone and in control animals was relatively uneventful. There were no differences between groups in formation of collagen or infiltration by macrophages in the area of the anastomosis. Animals treated with both radiation and hyperthermia showed marked necrosis, infiltration by polymorphonuclear leukocytes and rupture of the anastomosis. It is concluded that preoperative irradiation with a single dose of 25 Gy in combination with local hyperthermia at 44 degrees C for 30 min leads to disturbed repair of anastomoses.

Intra-operative irradiation prolongs the presence of matrix metalloproteinase activity in large bowel anastomoses of the rat.

There exists a growing interest in intra-operative radiation therapy as a treatment modality for large bowel cancer. In a previous experimental study we showed that high-dose intra-operative irradiation delays the healing of colonic anastomoses. However, the contribution of proteases is unknown. In the present study, the gelatinolytic and collagenolytic activity in the healing anastomoses is investigated. After a resection of a 1-cm length of colon (uninjured colon), the rats were irradiated with a single dose of 25 Gy, either to the proximal limb, referred to as the proximal group, or to both proximal and distal limbs of the bowel, referred to as the combined group, before anastomotic construction. Both groups were compared to a control group with anastomoses which were sham-irradiated. The animals were killed 1, 3 or 7 days after operation. The gelatinolytic activity in uninjured and anastomotic tissue was quantified by gelatin zymography and the collagenolytic activity by an assay using a fibrillar rat collagen substrate. Compared with resected uninjured colon, most of the gelatinolytic activities were markedly increased in anastomotic tissue of all groups during the first postoperative week, and new additional activities were detected. The additional metalloproteinases (the 95-kDa family) of both irradiated groups were significantly elevated compared to the anastomoses of the sham-irradiated control group at 7 days after operation. In anastomotic tissue of all groups, the collagenolytic activity of the tissue was also significantly increased at 1 and 3 days after construction with respect to the resected, uninjured colon. After 7 days this effect had disappeared for the sham-irradiated anastomoses, but the activity in the anastomoses in both the proximal and combined groups remained significantly elevated. The findings provide evidence that intra-operative irradiation prolongs the presence of elevated gelatinolytic and collagenolytic activities in colon anastomoses. It may contribute to a reduced or delayed accumulation of collagen and other matrix proteins that supply anastomotic strength.

High-dose preoperative irradiation without detrimental effect on early repair of anastomoses in the colon of the rat.

Preoperative radiotherapy as an adjunct to surgery for rectal carcinoma is generally thought to impair the healing of colorectal anastomoses. To delineate the presumed hazards of preoperative irradiation, we investigated this effect in a new model where, in contrast to experiments reported so far, anastomoses were constructed using normal tissue for the proximal limb and irradiated tissue for the distal limb. A group of 120 male Wistar rats, divided randomly into 12 groups of 10 each, were used. In 60 animals, a colonic segment of 2.2 cm was irradiated with a single dose of 25 Gy X rays administered 28 or 5 days or 3 or 1 day(s) before colonic resection. For each experimental group, a control group was included which was sham-irradiated on the same preoperative day. The animals were sacrificed on the third or the seventh postoperative day, and healing of the anastomosis was evaluated by measurement of bursting pressure, breaking strength and hydroxyproline concentration and content. Comparison between each experimental group and its control group showed that preoperative irradiation did not reduce the strength of the anastomoses. Also, the concentration and content of hydroxyproline in the tissue of the anastomoses were unchanged. These data indicate that construction of a colonic anastomosis consisting of one irradiated bowel end in rats is not by definition detrimental to the development of early wound strength.

Combined preoperative irradiation and direct postoperative 5-fluorouracil without negative effects on early anastomotic healing in the rat colon.

BACKGROUND AND PURPOSE: Preoperative irradiation with direct postoperative chemotherapy could benefit patients undergoing surgery for colorectal cancer. This study was designed to examine, in an experimental model, if such treatment is feasible without detrimental effects on early anastomotic healing. MATERIAL AND METHODS: A colonic segment was irradiated (25 Gy) in 3 groups (n = 10 each) of male Wistar rats. After 5 days, a colonic resection was performed with anastomotic construction; only the distal limb consisted of irradiated bowel. Postoperatively, animals received daily intraperitoneal 5-fluorouracil (5-FU, group I/CH: 17.5 mg/kg; group I/CL: 12.5 mg/kg) or saline (group I). Three additional groups were treated similarly, but with sham-irradiation: CH, CL and C, respectively. All rats were killed 7 days postoperatively. Parameters measured were: weight, serum albumin and protein, and anastomotic bursting pressure, breaking strength and hydroxyproline content. RESULTS: Body weight was diminished significantly in rats receiving chemotherapy. Serum albumin and protein was significantly lower in irradiated groups. At sacrifice, 40% of I/CH rats had functional rectal stenosis. The average bursting pressure (P = 0.0005) and the average breaking strength (P = 0.012) were only reduced significantly in the CH group. The anastomotic hydroxyproline content was significantly higher in the I/CH and I/CL groups vs. the control group. CONCLUSION: High-dose direct postoperative 5-FU leads to reduced anastomotic strength. Although the combination of preoperative irradiation (25 Gy) and direct postoperative high-dose 5-FU does not reduce early anastomotic strength, some stenosis may occur. The combination of preoperative irradiation and low-dose 5-FU has no such effect.

Combined preoperative irradiation and local hyperthermia delays early healing of experimental colonic anastomoses.

OBJECTIVE: To determine if a combination of preoperative irradiation and local hyperthermia of a colonic segment is detrimental to subsequent early anastomotic healing. DESIGN: A prospective randomized experimental trial. SETTING: An animal research laboratory. INTERVENTIONS: Eighty male Wistar rats were randomly divided into 4 groups. In each animal, a segment of the colon was treated successively by (sham) irradiation and (sham) hyperthermia. After 5 days, a colonic resection was performed and an anastomosis was constructed; the distal limb consisted of (sham) irradiated, (sham) hyperthermia-treated bowel. The rats were killed 3 or 7 days after surgery. MAIN OUTCOME MEASURES: Body weight, serum albumin and protein levels, anastomotic bursting pressure, breaking strength, and hydroxyproline content. RESULTS: All animals tolerated (sham) treatment well. Weight was diminished, though not notably, in treated animals vs the control group. After combined preoperative irradiation and hyperthermia, the frequency of local anastomotic complications increased: 4 of 20 animals had a covered perforation when they were killed. In this group, the bursting pressure was lower 3 days after the operation (P = .008). The breaking strength was also lower but not notably. The serum albumin level was significantly lower in this group vs the control group (P = .006); the serum protein level was not decreased. After 7 days, no differences existed between the groups. The hydroxyproline content of the anastomotic tissue was notably higher in rats treated with radiation plus hyperthermia vs control rats (in both the 3- and 7-day groups). The anastomotic hydroxyproline concentration did not differ between the groups. CONCLUSIONS: The combination of preoperative irradiation and hyperthermia results in increased local anastomotic complications. Anastomotic strength is at risk in the first days after the anastomotic reconstruction. Preoperative irradiation or hyperthermia alone does not lead to impaired anastomotic healing in the early phase.

Ewing's sarcoma of bone: oncologic and functional results.

This paper describes 29 patients with Ewing's sarcoma of bone treated between 1975 and 1990 at the University of Nijmegen Hospital, Nijmegen, The Netherlands. Osteomyelitis was the primary diagnosis in 24%. Treatment consisted of chemotherapy in combination with surgery and/or radiotherapy. Nine patients received radiotherapy only; five of them died of disease. Five patients underwent an intralesional excision; four of them died of disease. Twelve patients underwent a wide excision; there is no evidence of disease in any of them. Three patients underwent a radical disarticulation; all died of disease. The disease-free survival at 1.5 years was 66%. This figure at 5 years was 55%. After wide excision and reconstruction in tumors of expendable, femoral or radial bones good functional results were obtained in all cases.

Intraoperative irradiation delays anastomotic repair in rat colon.

BACKGROUND: There exists a growing interest in intraoperative radiation therapy as a treatment modality for large-bowel cancer. Since such therapy could interfere with wound repair, we investigated its effects on early healing of colonic anastomoses. METHODS: After resection of 1 cm of colon, rats were irradiated with a single dose of 25 Gy, either to the proximal limb (P group) or to both proximal and distal limbs of the bowel (PD group) before anastomotic construction. Both groups were compared with a sham-irradiated control group. Animals were killed 3, 7, or 14 days after operation, and healing was assessed by mechanical and biochemical (collagen) parameters. RESULTS: Three days after operation, bursting pressure was significantly lowered in the P group, whereas in the PD group both bursting pressure and breaking strength were strongly reduced. At day 7, the breaking strength was still reduced in the PD group, but not significantly so in the P group. The collagen synthetic capacity of the anastomotic segments was significantly lowered in both irradiated groups at day 3, resulting in a diminished collagen concentration in the actual wound area after 7 days. At 14 days after operation, no differences in strength were found between control and irradiated groups, while anastomotic hydroxyproline levels were significantly higher in both the P and PD groups than in the control group. CONCLUSIONS: High-dose intraoperative radiation therapy delays the healing of colonic anastomoses; it transiently reduces strength, probably as a result of a diminished accumulation of collagen.

Radiosensitization by cytotoxic drugs. The EORTC experience by the Radiotherapy and Lung Cancer Cooperative Groups.

UNLABELLED: A three-arm randomized trial was performed to assess the acute and late toxicity and the impact on survival of the combination high-dose, split-course radiotherapy with 30 mg/m2 cisplatin (cDDP) weekly, with 6 mg/m2 cisplatin daily compared to radiotherapy alone in patients with non-small cell lung cancer (NSCLC). The study started in May 1984 and was closed in May 1989 after 331 patients were randomised. The analysis was performed after a minimum follow-up period of 22 months. Radiotherapy (RT) consisted of 30 Gy, 10 fractions, five fractions a week; then a 3-week split followed by 25 Gy in 10 fractions. Nausea and vomiting were increased for a majority of the patients in the combined treatment arms during treatment. There was no addition of bone marrow suppression, renal dysfunction or esophagitis. Increase of late radiation damage was not observed. Local control (= absence of local progression) was improved for patients treated according to the daily cisplatin arm. This has lead to an improvement in overall survival. There was no effect in time to distant metastasis due to the combined modality. The treatment influence was confirmed in the multivariate analysis. CONCLUSION: local control and survival can be improved by combining radiotherapy with daily low-dose cisplatin in patients with inoperable NSCLC.

T3 laryngeal cancer, primary surgery vs planned combined radiotherapy and surgery.

The Dutch Co-operative Head and Neck Oncology Group performed a retrospective, nationwide study of laryngeal cancer between 1975 and 1984. The results for T3 laryngeal cancer treated with primary laryngectomy (n = 137) with post-operative radiotherapy when indicated or planned combined (pre-operative) radiotherapy with laryngectomy (n = 113) are analysed. The disease-free survival independent prognostic factors were treatment modality (planned combined treatment fared better, P = 0.001), incomplete resection of disease (P = 0.006), positive lymph nodes in the neck dissection specimen (P = 0.03) and poor differentiation (P = 0.04). Local control (95% vs. 85%, P = 0.01) as well as regional control (96% vs. 79%, P = 0.0001) was improved in the combined group compared with the primary laryngectomy group. Regional control was 69% for N0 patients if the neck nodes were not treated electively, compared with 98% for the planned combined treatment group. It is concluded that elective treatment of the neck nodes in T3 laryngeal cancer is mandatory. Radiotherapy is preferred, since as well as regional control, local control will also improve.

Effect of irradiation on healing of newly made colonic anastomoses in the rat.

PURPOSE: Short-term effects of radiotherapy on the healing process of newly made colonic anastomoses are investigated by measuring the anastomotic strength in a rat model. METHODS AND MATERIALS: Four groups of Wistar rats were used. In all groups, rats underwent a 1 cm sigmoid resection with end-to-end anastomosis. Group I served as a control group. In group II the anastomosis was irradiated after closure of the abdominal wall with a single dose of 20 Gy of 250 kV x rays. Group III was irradiated with a single dose of 20 Gy while the abdominal wall was not closed, and the surrounding tissues were carefully covered by a lead plate, simulating intra-operative radiotherapy. Group IV was treated as group III, but a larger dose of 25 Gy was applied. Animals were sacrificed 3 or 7 days after the operation. General condition of the rats was determined by observation, weight loss, serum protein and albumin at sacrifice. Anastomotic healing was evaluated by inspection, bursting pressure, hydroxyproline and protein contents of the anastomotic segment. RESULTS: Direct postoperative externally irradiated rats (group II) showed a marked weight loss, hypoproteinaemia and hypo-albuminaemia because of involvement of small bowel in the irradiated volume. With respect to anastomotic healing there were no significant differences between control and irradiated groups. CONCLUSION: These data suggest that the application of a single dose of irradiation (20 and 25 Gy) on colonic anastomoses given in a direct postoperative or intraoperative model has no measurable side effect on the early healing of newly made colonic anastomoses. Direct postoperative external irradiation results in unwanted side effects in the adjacent bowel.

Effects of concomitant cisplatin and radiotherapy on inoperable non-small-cell lung cancer.

BACKGROUND AND METHODS: Cisplatin (cis-diamminedichloroplatinum) has been reported to enhance the cell-killing effect of radiation, an effect whose intensity varies with the schedule of administration. We randomly assigned 331 patients with nonmetastatic inoperable non-small-cell lung cancer to one of three treatments: radiotherapy for two weeks (3 Gy given 10 times, in five fractions a week), followed by a three-week rest period and then radiotherapy for two more weeks (2.5 Gy given 10 times, five fractions a week); radiotherapy on the same schedule, combined with 30 mg of cisplatin per square meter of body-surface area, given on the first day of each treatment week; or radiotherapy on the same schedule, combined with 6 mg of cisplatin per square meter, given daily before radiotherapy. RESULTS: Survival was significantly improved in the radiotherapy-daily-cisplatin group as compared with the radiotherapy group (P = 0.009): survival in the radiotherapy-daily-cisplatin group was 54 percent at one year, 26 percent at two years, and 16 percent at three years, as compared with 46 percent, 13 percent, and 2 percent, respectively, in the radiotherapy group. Survival in the radiotherapy-weekly-cisplatin group was intermediate (44 percent, 19 percent, and 13 percent) and not significantly different from survival in either of the other two groups. The survival benefit of daily combined treatment was due to improved control of local disease (P = 0.003). Survival without local recurrence was 59 percent at one year and 31 percent at two years in the radiotherapy-daily-cisplatin group; 42 percent and 30 percent, respectively, in the radiotherapy-weekly-cisplatin group; and 41 percent and 19 percent, respectively, in the radiotherapy group. Cisplatin induced nausea and vomiting in 86 percent of the patients given it weekly and in 78 percent of those given it daily; these effects were severe in 26 percent and 28 percent, respectively. CONCLUSIONS: Cisplatin, given daily in combination with the radiotherapy described here to patients with nonmetastatic but inoperable non-small-cell lung cancer, improved rates of survival and control of local disease at the price of substantial side effects.

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.

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.