Proliferation and micromilieu during fractionated irradiation of human FaDu squamous cell carcinoma in nude mice

Purpose: Previous functional radiobiological experiments demonstrated a significant acceleration of repopulation after 3 weeks and reoxygenation after 12 days of radiotherapy of FaDu tumours. Owing to the temporal coincidence between repopulation and reoxygenation, it was hypothesized that the improved oxygenation status during fractionated irradiation might be the preceding stimulus for increased proliferation. The study investigated whether these changes in repopulation and re‐oxygenation are reflected by histological parameters of proliferation and the tumour micromilieu.

Materials and methods: Human FaDu squamous cell carcinomas in nude mice were irradiated with three to 18 fractions of 3?Gy daily or every second day under normal blood flow and clamp hypoxia. At different time points, tumours were excised and stained for Ki67, BrdUrd, epidermal growth factor receptor (EGFR) and markers of the micromilieu (HOECHST 33342, pimonidazole, ER‐MP12).

Results: On average, Ki67 and BrdUrd labelling indices decreased initially and increased again at later times during the course of fractionated radiotherapy. A similar kinetic pattern was found for the staining intensity of the EGFR. The vascular density in the viable tumour area remained constant during the whole course of irradiation, while the perfused fraction of vessels decreased within the first week of irradiation and returned to baseline values after 2 weeks. There was a corresponding increase in perfusion and a decrease in cellular hypoxia.

Conclusions: The histological results were in surprisingly good agreement with the kinetics of clonogen repopulation and re‐oxygenation determined previously using functional assays. The results support that the kinetics of repopulation of FaDu squamous cell carcinoma in response to fractionated irradiation are determined not only by intracellular processes, but also by a complex interaction of proliferation parameters with a changing microenvironment.     

Impact of increased cell loss on the repopulation rate during fractionated irradiation in human FaDu squamous cell carcinoma growing in nude mice

Purpose: To determine the impact of increased necrotic cell loss on the repopulation rate of clonogenic cells during fractionated irradiation in human FaDu squamous cell carcinoma in nude mice.

Materials and methods: FaDu tumours were transplanted into pre‐irradiated subcutaneous tissues. This manoeuvre has previously been shown to result in a clear‐cut tumour bed effect, i.e. tumours grow at a slower rate compared with control tumours. This tumour bed effect was caused by an increased necrotic cell loss with a constant cell production rate. After increasing numbers of 3‐Gy fractions (time intervals 24 or 48?h), graded top‐up doses were given to determine the dose required to control 50% of the tumours (TCD₅₀). All irradiations were given under clamp hypoxia.

Results: With increasing numbers of daily fractions, the top‐up TCD₅₀ decreased from 37.9?Gy (95% CI: 31; 45) after single dose irradiation to 14.1?Gy (8; 20) after irradiation with 15 fractions in 15 days. Irradiation with 18 daily 3‐Gy fractions controlled more than 50% of the tumours without a top‐up dose. After irradiation with six fractions every second day, the top‐up TCD₅₀ decreased to 26.9?Gy (22; 32). No further decrease of the TCD₅₀ was observed after 12 and 18 irradiations every second day. Assuming a constant increase of TCD₅₀ with time, the calculated doubling time of the clonogenic tumour cells (T_clon) was 7.8 days (4.4; 11.3). The T_clon calculated for FaDu tumours growing in pre‐irradiated tissues was significantly longer (p=0.0004) than the T_clon of 5.1 days (3.7; 6.5) determined under the same assumptions in a previous study for FaDu tumours growing in normal subcutaneous tissues.

Conclusions: Increased necrotic cell loss by pre‐irradiation of the tumour bed resulted in longer clonogen doubling times during fractionated radiotherapy of human FaDu squamous cell carcinoma. This implies that a decreased necrotic cell loss might be the link between reoxygenation and repopulation demonstrated previously in the same tumour model.     

Impact of increased cell loss on the repopulation rate during fractionated irradiation in human FaDu squamous cell carcinoma growing in nude mice

PURPOSE: To determine the impact of increased necrotic cell loss on the repopulation rate of clonogenic cells during fractionated irradiation in human FaDu squamous cell carcinoma in nude mice. MATERIALS AND METHODS: FaDu tumours were transplanted into pre-irradiated subcutaneous tissues. This manoeuvre has previously been shown to result in a clear-cut tumour bed effect, i.e. tumours grow at a slower rate compared with control tumours. This tumour bed effect was caused by an increased necrotic cell loss with a constant cell production rate. After increasing numbers of 3-Gy fractions (time intervals 24 or 48 h), graded top-up doses were given to determine the dose required to control 50% of the tumours (TCD50). All irradiations were given under clamp hypoxia. RESULTS: With increasing numbers of daily fractions, the top-up TCD50 decreased from 37.9 Gy (95% CI: 31; 45) after single dose irradiation to 14.1 Gy (8; 20) after irradiation with 15 fractions in 15 days. Irradiation with 18 daily 3-Gy fractions controlled more than 50% of the tumours without a top-up dose. After irradiation with six fractions every second day, the top-up TCD50 decreased to 26.9 Gy (22; 32). No further decrease of the TCD50 was observed after 12 and 18 irradiations every second day. Assuming a constant increase of TCD50 with time, the calculated doubling time of the clonogenic tumour cells (Tclon) was 7.8 days (4.4; 11.3). The Tclon calculated for FaDu tumours growing in pre-irradiated tissues was significantly longer (p=0.0004) than the Tclon of 5.1 days (3.7; 6.5) determined under the same assumptions in a previous study for FaDu tumours growing in normal subcutaneous tissues. CONCLUSIONS: Increased necrotic cell loss by pre-irradiation of the tumour bed resulted in longer clonogen doubling times during fractionated radiotherapy of human FaDu squamous cell carcinoma. This implies that a decreased necrotic cell loss might be the link between reoxygenation and repopulation demonstrated previously in the same tumour model.     

Impact of preirradiation of the tumour bed on cell production rate and cell loss of human FaDu squamous cell carcinoma growing in nude mice

Purpose: To explore whether the tumour bed effect (TBE) in FaDu squamous cell carcinoma growing in nude mice is caused by a reduced tumour cell production rate and/or by increased tumour cell loss. Materials and methods: Human FaDu tumours were studied in NMRI nude mice. The volume doubling time (VDT) between 100 and 400mm3 was determined for tumours in unirradiated subcutaneous (sc) tissues (group 1), tumours in sc tissues preirradiated with 12.5Gy (group 2), tumours irradiated in situ with 12.5Gy (group 3), and tumours from group 3 re-transplanted into unirradiated sc tissues (group 4). Labelling index (LI), potential doubling time (T pot), relative necrotic area and apoptotic index (AI) were evaluated in tumours from groups 1 and 2. Results: The median VDT were 2.6 days (95% CI 2-4) in group 1 and 7.0 days (4-15) in group 2 (p < 0.001). The VDT were not significantly different between groups 2 and 3, and group 1 and 4. In groups 1 and 2, the T pot values (3.1 0.6 days (SD) versus 2.9 0.5 days) and the LI were identical (10 1.5%). The median relative necrotic area was significantly larger in group 2 (37% [23-42]) compared with group (6% [0.3-27]). The apoptotic index was low (0.2%) and did not differ between groups 1 and 2. Conclusions : The results indicate that the TBE in FaDu squamous cell carcinoma is not caused by a reduced cell production rate in the viable tumour compartment. Rather, the TBE reflects a decreased viable tumour cell compartment due to increased cell loss. Necrosis appears to be the major component of the tumour bed induced cell loss in FaDu tumours, whereas apoptosis has no impact on the TBE in this model.     

Impact of preirradiation of the tumour bed on cell production rate and cell loss of human FaDu squamous cell carcinoma growing in nude mice.

PURPOSE: To explore whether the tumour bed effect (TBE) in FaDu squamous cell carcinoma growing in nude mice is caused by a reduced tumour cell production rate and/or by increased tumour cell loss. MATERIALS AND METHODS: Human FaDu tumours were studied in NMRI nude mice. The volume doubling time (VDT) between 100 and 400 mm3 was determined for tumours in unirradiated subcutaneous (sc) tissues (group 1), tumours in sc tissues preirradiated with 12.5 Gy (group 2), tumours irradiated in situ with 12.5 Gy (group 3), and tumours from group 3 re-transplanted into unirradiated sc tissues (group 4). Labelling index (LI), potential doubling time (Tpot), relative necrotic area and apoptotic index (AI) were evaluated in tumours from groups 1 and 2. RESULTS: The median VDT were 2.6 days (95% CI 2-4) in group 1 and 7.0 days (4-15) in group 2 (p<0.001). The VDT were not significantly different between groups 2 and 3, and group 1 and 4. In groups 1 and 2, the Tpot values (3.1 +/- 0.6 days (SD) versus 2.9 +/- 0.5 days) and the LI were identical (10 +/- 1.5%). The median relative necrotic area was significantly larger in group 2 (37% [23-42]) compared with group (6% [0.3-27]). The apoptotic index was low (0.2%) and did not differ between groups 1 and 2. CONCLUSIONS: The results indicate that the TBE in FaDu squamous cell carcinoma is not caused by a reduced cell production rate in the viable tumour compartment. Rather, the TBE reflects a decreased viable tumour cell compartment due to increased cell loss. Necrosis appears to be the major component of the tumour bed induced cell loss in FaDu tumours, whereas apoptosis has no impact on the TBE in this model.     

Effect of changing the weekly dose intensity of fractionated irradiation on local control of two human squamous cell carcinomas in nude mice

Purpose : To compare the effect of fractionated irradiation with increasing, constant or decreasing weekly dose intensity on local tumour control. Materials and methods : Human squamous cell carcinomas, FaDu and GL, were grown in nude mice. Thirty fractions were applied under ambient conditions with increasing, constant or decreasing weekly dose intensity within a constant overall treatment time of 6 weeks. Dose intensity was changed every 2 weeks. Irradiations were terminated in some groups of animals after 20 fractions in 4 weeks. Endpoint was the tumour control dose 50% (TCD 50) at day 120 (FaDu) or day 180 (GL) after end of treatment. Results : In FaDu tumours the TCD 50 value of 60 Gy (95% CI 56; 63) for fractionated irradiation with decreasing dose intensity, i.e. high initial doses, was slightly but significantly lower than the TCD50 of 68 Gy (60; 81) after low initial doses (p =0.03). The TCD50 value of 62Gy (57; 68) after constant doses was intermediate (constant vs increasing p =0.30; constant vs decreasing p =0.15). The higher effecacy of high initial doses in FaDu tumours was explained by local control occurring already during the course of irradiation. In GL tumours the TCD50 values were 52Gy (43; 62) after high initial dose intensity, 50 Gy (43; 66) after constant doses, and 55 Gy (42; 89) after low initial dose intensity. These values were not statistically different (p -values 0.20-0.75). Conclusions : The data support the view that initial dose concentration during fractionated irradiation does not enhance radioresistance of FaDu and GL tumours, for instance by an earlier onset of clonogen repopulation.     

Effect of changing the weekly dose intensity of fractionated irradiation on local control of two human squamous cell carcinomas in nude mice

PURPOSE: To compare the effect of fractionated irradiation with increasing, constant or decreasing weekly dose intensity on local tumour control. MATERIALS AND METHODS: Human squamous cell carcinomas, FaDu and GL, were grown in nude mice. Thirty fractions were applied under ambient conditions with increasing, constant or decreasing weekly dose intensity within a constant overall treatment time of 6 weeks. Dose intensity was changed every 2 weeks. Irradiations were terminated in some groups of animals after 20 fractions in 4 weeks. Endpoint was the tumour control dose 50% (TCD50) at day 120 (FaDu) or day 180 (GL) after end of treatment. RESULTS: In FaDu tumours the TCD50 value of 60 Gy (95% CI 56; 63) for fractionated irradiation with decreasing dose intensity, i.e. high initial doses, was slightly but significantly lower than the TCD50, of 68 (60; 81) after low initial doses (p=0.03). The TCD50 value of 62 Gy (57; 68) after constant doses was intermediate (constant vs increasing p =0.30; constant vs decreasing p=0.15). The higher efficacy of high initial doses in FaDu tumours was explained by local control occurring already during the course of irradiation. In GL tumours the TCD50 values were 52 Gy (43; 62) after high initial dose intensity, 50 Gy (43; 66) after constant doses, and 55 Gy (42; 89) after low initial dose intensity. These values were not statistically different (p-values 0.20-0.75). CONCLUSIONS: The data support the view that initial dose concentration during fractionated irradiation does not enhance radioresistance of FaDu and GL tumours, for instance by an earlier onset of clonogen repopulation.     

Volumetrical and morphological responses of human laryngeal squamous cell carcinoma xenografts treated with fractionated irradiation

Xenografts of both primary human laryngeal squamous cell carcinoma and its metastases were irradiated with five daily fractions of 5.0 Gy. Tumor volume changes, morphology, mitotic index and mitotic figures were studied. Primary xenografts disappeared within 17 +/- 3 days. Grafts of metastases showed complete regression within 26 +/- 5 days, or regrowth after a delay period. Mitotic activity was higher in the grafts of metastases. The number of mitotic figures decreased and ceased within 14 days in the primary tumor, while some were still occasionally noted in the grafts of metastases. Telophase stages were practically absent after the first fraction. This study suggests that the response of metastases to radiation therapy is lower than the response of the primary tumor.     

Selective inhibition of the epidermal growth factor receptor tyrosine kinase by BIBX1382BS and the improvement of growth delay,but not local control,after fractionated irradiation in human FaDu squamous cell carcinoma in the nude mouse

Purpose: To investigate the effect of BIBX1382BS, an inhibitor of the epidermal growth factor receptor tyrosine kinase, on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumour growth and local tumour control after fractionated irradiation over 6 weeks in nude mice. FaDu human squamous cell carcinoma is epidermal growth factor receptor positive and significant repopulation during fractionated irradiation was demonstrated in previous experiments.

Materials and methods: Receptor status, receptor phosphorylation, cell cycle distribution, cell proliferation and clonogenic cell survival after irradiation were assayed with and without BIBX1382BS (5?µM) in vitro. Tumour volume doubling time, BrdUrd and Ki67 labelling indices and apoptosis were investigated in unirradiated tumours growing in NMRI nude mice treated daily with BIBX1382BS (50?mg?kg^?1 body weight orally) or carrier. Tumour growth delay and dose–response curves for local tumour control were determined after irradiation with 30 fractions within 6 weeks.

Results: BIBX1382BS blocked radiation‐induced phosphorylation of the epidermal growth factor receptor and reduced the doubling time of FaDu cells growing in vitro by a factor of 4.9 (p=0.008). Radiosensitivity in vitro remained unchanged after incubation with BIBX1382BS for 3 days and decreased moderately after 6 days (p=0.001). BIBX1382BS significantly reduced the volume doubling time of established FaDu tumours in nude mice by factors of 2.6 when given over 15 days (p<0.001) and 3.7 when applied over 6 weeks (p<0.001). When given simultaneously to fractionated irradiation, growth delay was significantly prolonged by an average of 33 days (p=0.003). Local tumour control was not improved by BIBX1382BS. The radiation doses necessary to control 50% of the tumours locally were 63.6?Gy (95% confidence interval 55; 73) for irradiation alone and 67.8?Gy (60; 77) for the combined treatment (p=0.5).

Conclusions: Despite clear antiproliferative activity in rapidly repopulating FaDu human squamous cell carcinoma and significantly increased tumour growth delay when combined with fractionated irradiation, local tumour control was not improved by BIBX1382BS. The results do not disprove that epidermal growth factor receptor inhibition might enhance the results of radiotherapy. However, the results imply that further preclinical investigations using relevant treatment schedules and appropriate endpoints are necessary to explore the mechanisms of action and efficacy of such combinations.     

Selective inhibition of the epidermal growth factor receptor tyrosine kinase by BIBX1382BS and the improvement of growth delay, but not local control, after fractionated irradiation in human FaDu squamous cell carcinoma in the nude mouse

PURPOSE: To investigate the effect of BIBX1382BS, an inhibitor of the epidermal growth factor receptor tyrosine kinase, on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumour growth and local tumour control after fractionated irradiation over 6 weeks in nude mice. FaDu human squamous cell carcinoma is epidermal growth factor receptor positive and significant repopulation during fractionated irradiation was demonstrated in previous experiments. MATERIALS AND METHODS: Receptor status, receptor phosphorylation, cell cycle distribution, cell proliferation and clonogenic cell survival after irradiation were assayed with and without BIBX1382BS (5 microM) in vitro. Tumour volume doubling time, BrdUrd and Ki67 labelling indices and apoptosis were investigated in unirradiated tumours growing in NMRI nude mice treated daily with BIBX1382BS (50 mg kg(-1) body weight orally) or carrier. Tumour growth delay and dose-response curves for local tumour control were determined after irradiation with 30 fractions within 6 weeks. RESULTS: BIBX1382BS blocked radiation-induced phosphorylation of the epidermal growth factor receptor and reduced the doubling time of FaDu cells growing in vitro by a factor of 4.9 (p=0.008). Radiosensitivity in vitro remained unchanged after incubation with BIBX1382BS for 3 days and decreased moderately after 6 days (p=0.001). BIBX1382BS significantly reduced the volume doubling time of established FaDu tumours in nude mice by factors of 2.6 when given over 15 days (p<0.001) and 3.7 when applied over 6 weeks (p<0.001). When given simultaneously to fractionated irradiation, growth delay was significantly prolonged by an average of 33 days (p=0.003). Local tumour control was not improved by BIBX1382BS. The radiation doses necessary to control 50% of the tumours locally were 63.6 Gy (95% confidence interval 55; 73) for irradiation alone and 67.8 Gy (60; 77) for the combined treatment (p=0.5). CONCLUSIONS: Despite clear antiproliferative activity in rapidly repopulating FaDu human squamous cell carcinoma and significantly increased tumour growth delay when combined with fractionated irradiation, local tumour control was not improved by BIBX1382BS. The results do not disprove that epidermal growth factor receptor inhibition might enhance the results of radiotherapy. However, the results imply that further preclinical investigations using relevant treatment schedules and appropriate endpoints are necessary to explore the mechanisms of action and efficacy of such combinations.     

The effects of uneven fractionated irradiation on mouse squamous cell carcinoma and normal tissue

In uneven fractionated radiotherapy combining large doses and small doses, we studied the influence of the order of their combination on tumors and on normal tissues, using squamous cell carcinoma and skin reaction of mice. The number of fractions was 6, and we used various combinations of 4 small doses and 2 large doses (4 times larger). The antitumor effect was smallest when a large dose was given at the end of treatment, but in skin reactions, there was no difference in effect even when the order of the combination of large and small doses was changed.     

Differentiation status of human squamous cell carcinoma xenografts does not appear to correlate with the repopulation capacity of clonogenic tumour cells during fractionated irradiation

PURPOSE: To investigate the magnitude and kinetics of repopulation in a moderately well differentiated UT-SCC-14 human squamous cell carcinoma [hSCC] in nude mice. This question is of interest because clinical data indicate a higher repopulation capacity in those SCC that have preserved characteristics of differentiation, which appears to be in contrast to results on FaDu and GL hSCC previously reported from this laboratory. METHODS AND MATERIALS: UT-SCC-14 tumours were transplanted subcutaneously into the right hind leg of NMRI nu/nu mice. Fractionated radiation treatments were delivered, either under clamped hypoxia at 5.4 Gy/fraction or under ambient conditions (consistent with an OER of 2.7). Tumours were irradiated every day, every 2nd day, or every 3rd day with 6, 12 or 18 fractions. 1, 2 or 3 days after the last fraction, graded top-up-doses under clamped conditions were given for the purpose of estimating the 50% tumour control dose (TCD50). A total of 22 TCD50 assays were performed and analysed using maximum likelihood techniques. RESULTS: The data demonstrate a slow but significant repopulation of clonogenic cells during fractionated irradiation of UT-SCC-14 hSCC. The results under hypoxic conditions are consistent with a constant repopulation rate, with a clonogenic doubling time (Tclon) of 15.6 days (95% CI: 9.7, 21.4). This contrasts with ambient conditions where Tclon was 68.5 days (95% CI: 124, 161). Both Tclon values are longer than the 6-day volume doubling time of untreated tumours. CONCLUSIONS: Less pronounced repopulation for irradiation under ambient compared to clamped hypoxic conditions might be explained by preferential survival of hypoxic and therefore non-proliferating clonogenic cells. Taken together with previous studies on poorly differentiated FaDu and moderately well differentiated GL hSCC, the results are consistent with considerable variability in the magnitude and kinetics of repopulation in different experimental squamous cell carcinomas, and with a relationship between reoxygenation and repopulation during fractionated irradiation. The differentiation status of hSCC growing in nude mice does not to appear to correlate with the proliferative capacity of clonogenic tumour cells during treatment. The results do not support the hypothesis gained from clinical data of higher repopulation in well-differentiated tumours.     

Differentiation status of human squamous cell carcinoma xenografts does not appear to correlate with the repopulation capacity of clonogenic tumour cells during fractionated irradiation

Purpose: To investigate the magnitude and kinetics of repopulation in a moderately well differentiated UT‐SCC‐14 human squamous cell carcinoma [hSCC] in nude mice. This question is of interest because clinical data indicate a higher repopulation capacity in those SCC that have preserved characteristics of differentiation, which appears to be in contrast to results on FaDu and GL hSCC previously reported from this laboratory.

Methods and Materials: UT‐SCC‐14 tumours were transplanted subcutaneously into the right hind leg of NMRI nu/nu mice. Fractionated radiation treatments were delivered, either under clamped hypoxia at 5.4?Gy/fraction or under ambient conditions (consistent with an OER of 2.7). Tumours were irradiated every day, every 2nd day, or every 3rd day with 6, 12 or 18 fractions. 1, 2 or 3 days after the last fraction, graded top‐up‐doses under clamped conditions were given for the purpose of estimating the 50% tumour control dose (TCD₅₀). A total of 22 TCD₅₀ assays were performed and analysed using maximum likelihood techniques.

Results: The data demonstrate a slow but significant repopulation of clonogenic cells during fractionated irradiation of UT‐SCC‐14 hSCC. The results under hypoxic conditions are consistent with a constant repopulation rate, with a clonogenic doubling time (T_clon) of 15.6 days (95% CI: 9.7, 21.4). This contrasts with ambient conditions where T_clon was 68.5 days (95% CI: 124, 161). Both T_clon values are longer than the 6‐day volume doubling time of untreated tumours.

Conclusions: Less pronounced repopulation for irradiation under ambient compared to clamped hypoxic conditions might be explained by preferential survival of hypoxic and therefore non‐proliferating clonogenic cells. Taken together with previous studies on poorly differentiated FaDu and moderately well differentiated GL hSCC, the results are consistent with considerable variability in the magnitude and kinetics of repopulation in different experimental squamous cell carcinomas, and with a relationship between reoxygenation and repopulation during fractionated irradiation. The differentiation status of hSCC growing in nude mice does not to appear to correlate with the proliferative capacity of clonogenic tumour cells during treatment. The results do not support the hypothesis gained from clinical data of higher repopulation in well‐differentiated tumours.     

Intestinal cell proliferation during fractionated abdominal irradiation.

In the mouse, at normal steady state of cell proliferation, the compensatory proliferative response to intestinal irradiation is such that when radiation exposures totalling 1,000 R are concentrated over the first few days of the week, summated proliferative activity for the entire week is near control levels. Symmetrically distributed exposures over a five-day treatment week (200 R daily, and especially 333 R on Monday, Wednesday and Friday) result in depressed levels of overall weekly proliferation. In these instances, the weekend break is particularly crucial. Similar results were obtained when the one-week measurement period was inserted between the third and fifth week of abdominal therapy, except in this instance, 200 R per day did not result in sub-control levels of proliferation, whereas 333 R on M, W and F, continued to do so. The intestine seems able to maintain its barrier epithelium for extended periods of diminished cell input, provided such is not too severe and that it seems from decreased cell production rate per crypt rather than from crypt attrition. A partial explanation for this relative tolerance is given by the finding that the vast majority of proliferative cells, even those irradiated and rendered permanently incapable of further division, succeed in migrating up the villus and hence help to maintain a barrier epithelium. In that sense, nearly all cell divisions become useful, even in the face of repeated exposures.     

Impact of the tumour bed effect on microenvironment,radiobiological hypoxia and the outcome of fractionated radiotherapy of human FaDu squamous-cell carcinoma growing in the nude mouse

Purpose : To investigate the impact of the tumour bed effect (TBE) on histological parameters of the micromilieu, radiobiological hypoxic fraction and local control after fractionated irradiation in FaDu squamous-cell carcinoma in the nude mouse. This tumour has previously shown a clear-cut TBE caused by increased necrotic cell loss at a constant cell production rate in the viable tumour compartment. Materials and methods : Human FaDu tumours were studied in the NMRI nude mouse. Tumours were transplanted either into unirradiated subcutaneous (s.c.) tissues (controls) or s.c. tissues pre-irradiated with 12.5 Gy (TBE group). In both groups we measured the volume doubling time (VDT), potential doubling time (T pot) , relative necrotic area, and in the viable tumour compartment the relative vascular area (9F1 mAb), relative hypoxic area (NITP or pimonidazole), relative perfused area (Hoechst 33342), and the perfused fraction of vasculature. The tumour control dose 50% (TCD 50), radiobiological hypoxic fraction (rHF) and dose-modifying factors (DMF) for the comparison of tumours in the TBE and control groups were determined from local tumour control data after treatment with single doses under ambient conditions or under clamp hypoxia, and after irradiation with 30 fractions under ambient conditions within 6 weeks using maximum-likelihood analysis. Results : A clear-cut TBE (VDT = 4.0 days (95%CI 2.9;4.4) for the control group versus 7.2 days (6.4;8.9) for the TBE group; p <0.0001) caused by increased necrosis (mean relative necrotic area of 12% (5;20)) versus 33% (10;41); p = 0.07) at a constant cell production rate (T pot = 2.2 days (1.4;2.3) versus 2.2 days (1.7;2.6); p = 0.30) was confirmed. Histological analysis of the micromilieu within the vital subarea revealed no systematic differences between the TBE and control groups. The rHF of 2% (0.1;27) for control tumours was lower than the 15% (95% CI 2;91) for the TBE group, but this difference was nonsignificant (p = 0.12). Compared with control tumours, the TCD 50 for irradiation under clamped hypoxia was in a statistical trend lower for tumours in the TBE group (DMF 1.11 (0.98;1.28), p = 0.09). After fractionated irradiation, tumours of the TBE group were significantly more radiosensitive (TCD 50 56.6 Gy (46;70) versus 78.7 Gy (63;100); p = 0.003). Conclusions : The results on FaDu tumours growing in pre-irradiated tissues indicate that increased necrosis caused by impairment of the vascular supply may increase the radiosensitivity of tumours treated by fractioned irradiation.     

Impact of the tumour bed effect on microenvironment, radiobiological hypoxia and the outcome of fractionated radiotherapy of human FaDu squamous-cell carcinoma growing in the nude mouse.

PURPOSE: To investigate the impact of the tumour bed effect (TBE) on histological parameters of the micromilieu, radiobiological hypoxic fraction and local control after fractionated irradiation in FaDu squamous-cell carcinoma in the nude mouse. This tumour has previously shown a clear-cut TBE caused by increased necrotic cell loss at a constant cell production rate in the viable tumour compartment. MATERIALS AND METHODS: Human FaDu tumours were studied in the NMRI nude mouse. Tumours were transplanted either into unirradiated subcutaneous (s.c.) tissues (controls) or s.c. tissues pre-irradiated with 12.5 Gy (TBE group). In both groups we measured the volume doubling time (VDT), potential doubling time (T(pot)), relative necrotic area, and in the viable tumour compartment the relative vascular area (9F1 mAb), relative hypoxic area (NITP or pimonidazole), relative perfused area (Hoechst 33342), and the perfused fraction of vasculature. The tumour control dose 50% (TCD 50), radiobiological hypoxic fraction (rHF) and dose-modifying factors (DMF) for the comparison of tumours in the TBE and control groups were determined from local tumour control data after treatment with single doses under ambient conditions or under clamp hypoxia, and after irradiation with 30 fractions under ambient conditions within 6 weeks using maximum-likelihood analysis. RESULTS: A clear-cut TBE (VDT = 4.0 days (95%CI 2.9;4.4) for the control group versus 7.2 days (6.4;8.9) for the TBE group; p <0.0001) caused by increased necrosis (mean relative necrotic area of 12% (5;20)) versus 33% (10;41); p = 0.07) at a constant cell production rate (T(pot) = 2.2 days (1.4;2.3) versus 2.2 days (1.7;2.6); p = 0.30) was confirmed. Histological analysis of the micromilieu within the vital subarea revealed no systematic differences between the TBE and control groups. The rHF of 2% (0.1;27) for control tumours was lower than the 15% (95% CI 2;91) for the TBE group, but this difference was nonsignificant (p = 0.12). Compared with control tumours, the TCD50 for irradiation under clamped hypoxia was in a statistical trend lower for tumours in the TBE group (DMF 1.11 (0.98;1.28), p = 0.09). After fractionated irradiation, tumours of the TBE group were significantly more radiosensitive (TCD50 56.6 Gy (46;70) versus 78.7 Gy (63;100); p = 0.003). CONCLUSIONS: The results on FaDu tumours growing in pre-irradiated tissues indicate that increased necrosis caused by impairment of the vascular supply may increase the radiosensitivity of tumours treated by fractioned irradiation.     

Comparison of [18F]FDG uptake and distribution with hypoxia and proliferation in FaDu human squamous cell carcinoma (hSCC) xenografts after single dose irradiation

Purpose: This study investigated the uptake of [¹⁸F]2-fluoro-2-deoxy-glucose ([¹⁸F]FDG) in the human tumour xenograft FaDu at early time points after single dose irradiation with Positron-Emission-Tomography (PET), autoradiography and functional histology.

Materials and methods: [¹⁸F]FDG-PET of FaDu hSCC xenografts on nude mice was performed before 25 Gy or 35 Gy single dose irradiation and one, seven or 11 days post irradiation (p.irr.). Before the second PET, mice were injected with pimonidazole (pimo) and bromodeoxyuridine (BrdU). After the PET tumours were excised, sliced and subjected to autoradiography and functional histology staining (pimo, BrdU, Ki67). [¹⁸F]FDG tumour uptake was quantified in the PET scans by maximal standard uptake value (SUV_max) and in the autoradiography after co-registration to the histology slices.

Results: No differences in the overall [¹⁸F]FDG uptake between the two dose groups and time points were found with PET or autoradiography. Comparing autoradiography and histology, the [¹⁸F]FDG uptake was constant in tumour necrosis over time, while it decreased in vital tumour areas and particularly in hypoxic regions. No differences in the [¹⁸F]FDG uptake between positive and negative areas of Ki67 and BrdU were found.

Conclusions: The decline of [¹⁸F]FDG uptake in vital tumour and in pimopositive areas as seen in autoradiography, was not reflected by evaluation of SUV_max determined by PET. These findings suggest that the SUV_max does not necessarily reflect changes in tumour biology after irradiation.     

Remissions and acute toxicity during accelerated fractionated irradiation of non-small-cell bronchial carcinoma

Accelerated fractionation (total dose 66 Gy, single dose 1.8 to 2 Gy, 1 to 2 fractions daily, total treatment period 4 to 5.5 weeks) brings about more than 40% of complete remissions and more than 90% of objective remissions (assessed by radiology) of non-small cell bronchogenic carcinomas. Tumor-induced symptoms are improved in more than 90%. A treatment period of less than four weeks causes considerable acute (and persistent) oesophageal reactions. In case of a treatment period of more than five weeks there is no substantial increase of toxicity.     

Effects of boron neutron capture therapy on human oral squamous cell carcinoma in a nude mouse model

PURPOSE: The effect of boronophenylalanine (BPA)-mediated boron neutron capture therapy (BNCT) on human oral squamous cell carcinoma (SCC) xenografts in nude mice was examined. MATERIALS AND METHODS: Tumor-bearing mice were given BPA at a dose of 250 mg/kg body weight. The tumor (10)B concentration 2 h after an injection of BPA was higher than those 1 or 3 h after the injection. Neutron irradiation was performed beginning 1, 2 or 3 h after an injection of BPA and the effects on body weight of the animals, tumor growth, survival of tumor-bearing animals, and histology of tumor and normal tissue were examined. Fragmented nuclear DNA, 5-bromo-2'-deoxyuridine (BrdU), and von Willebrand Factor (vWF) were detected by immunohistochemical staining. RESULTS: Tumor volumes of untreated control animals increased continuously, whereas those of BNCT-treated animals were markedly decreased. Animals given neutron irradiation 2 h after the injection of BPA survived for a longer period as compared with those given neutron irradiation 1 or 3 h after the injection. BNCT reduced the incorporation of BrdU into tumor cells, and induced the enlargement and vacuolation of tumor cells. Disintegration of blood vessels and dense inflammatory cell infiltration were also observed in the stroma of the tumor, but not surrounding normal tissues. CONCLUSION: These results indicate that BPA-mediated BNCT can exert a curative effect on human oral SCC xenografts in nude mice, if an optimal 10B concentration in tumors is achieved and that the disintegration of blood vessels in tumor stroma may contribute to tumor remission by BNCT.