Franziska Hessel

Enhanced Susceptibility of Irradiated Tumor Vessels to Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibition

Previous experiments with PTK787/ZK222584, a specific inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, using irradiated human FaDu squamous cell carcinoma in nude mice, suggested that radiation-damaged tumor vessels are more sensitive to VEGFR inhibition. To test this hypothesis, the tumor transplantation site (i.e., the right hind leg of nude mice) was irradiated 10 days before transplantation of FaDu to induce radiation damage in the host tissue. FaDu tumors vascularized by radiation-damaged blood vessels appeared later, grew at a slower rate, and showed more necrosis and a smaller vessel area per central tumor section than controls. PTK787/ZK222584 at a daily dose of 50 mg/kg body weight had no impact on growth of control tumors. In contrast, tumors vascularized by radiation-damaged vessels responded to PTK787/ZK222584 with longer latency and slower growth rate than controls, and a trend toward further increase in necrosis, indicating that irradiated tumor vessels are more susceptible to VEGFR inhibition than unirradiated vessels. Although not proving causality, expression analysis of VEGF and VEGFR2 shows that enhanced sensitivity of irradiated vessels to a specific inhibitor of VEGFR tyrosine kinases correlates with increased expression of the molecular target.

Low-dose hyperradiosensitivity of human glioblastoma cell lines in vitro does not translate into improved outcome of ultrafractionated radiotherapy in vivo

Purpose: Low dose hyperradiosensitivity (HRS) has been observed in HGL21- and T98G human glioblastoma cells in vitro. The present study investigates whether these effects translate into improved outcome of ultrafractionated irradiation (UF) in vivo. Material and methods: T98G or HGL21 were transplanted on the hind leg of nude mice. Tumours were irradiated with UF (3 fractions of 0.4 Gy per day, interval 4 h, 7 days per week) or with conventional fractionation (CF; 1 fraction of 1.68 Gy per day, 5 days per week) over 2 or 4 weeks in HGL21 and 2,4 or 6 weeks in T98G. In HGL21, graded top-up doses under clamped hypoxia were applied after 4 weeks of fractionated irradiation. Additional groups of animals were irradiated with single doses under clamp hypoxic conditions with or without whole body irradiation (WBI) before tumour transplantation. Experimental endpoints were growth delay (time to 5-fold starting volume, GDV5) and local tumour control. Results: In T98G tumours median relative GDV5 was 1.2 [95%C.I. 0.96; ∞] in the CF and 0.8 [0.7; 1.02] in the UF arm (p = 0.009) indicating that ultrafractionation is less efficient than conventional fractionation. The TCD50 value of 33.5 Gy [22; 45] after UF was higher than TCD50 of 23.6 Gy [16; 31] after CF (p = 0.15). In HGL21 the median relative GDV5 was not significantly different between CF and UF. The top-up TCD50 value of 16.1 Gy [95% C.I. 9; 23 Gy] after CF was significantly lower than the corresponding value of 33.2 Gy [23; 44] after UF irradiation (p = 0.007), indicating a higher efficacy of CF compared to UF. Conclusion: The results on human T98G and HGL21 glioblastoma do not support the hypothesis that HRS in vitro translates into improved outcome of ultrafractionated irradiation in vivo.

Effect of [18F]FMISO stratified dose-escalation on local control in FaDu hSCC in nude mice

OBJECTIVE To investigate the effect of radiation dose-escalation on local control in hypoxic versus non-hypoxic hypoxic tumours defined using [(18)F]fluoromisonidazole ([(18)F]FMISO) PET. MATERIALS AND METHODS FaDu human squamous cell carcinomas (hSCCs) growing subcutaneously in nude mice were subjected to [(18)F]FMISO PET before irradiation with single doses of 25 or 35Gy under normal blood flow conditions. [(18)F]FMISO hypoxic volume (HV) and maximum standardised uptake value (SUVmax) were used to quantify tracer uptake. The animals were followed up for at least 120days after irradiation. The endpoints were permanent local tumour control and time to local recurrence. RESULTS HV varied between 38 and 291mm(3) (median 105mm(3)). Non-hypoxic tumours (HV below median) showed significantly better local control after single dose irradiation than hypoxic tumours (HV above median) (p=0.046). The effect of dose was significant and not different in non-hypoxic and in hypoxic tumours (HR=0.82 [95% CI 0.71; 0.93], p=0.002 and HR=0.86 [0.78; 0.95], p=0.001, respectively). Dose escalation resulted in an incremental increase of local tumour control from low-dose hypoxic, over low-dose non-hypoxic and high-dose hypoxic to high-dose non-hypoxic tumours. SUVmax did not reveal significant association with local control at any dose level. CONCLUSIONS The negative effect of [(18)F]FMISO HV on permanent local tumour control supports the prognostic value of the pre-treatment [(18)F]FMISO HV. Making the assumption that variable [(18)F]FMISO uptake in different FaDu tumours which all have the same genetic background may serve as an experimental model of intratumoural heterogeneity, the data support the concept of dose-escalation with inhomogeneous dose distribution based on pre-treatment [(18)F]FMISO uptake. This result needs to be confirmed in other tumour models and using fractionated radiotherapy schedules.

Ultrafractionation in A7 human malignant glioma in nude mice

Purpose: Low‐dose hyperradiosensitivity (HRS) has been demonstrated in numerous cell lines in vitro, including a number of radioresistant human malignant glioma cell lines such as A7. The aim of our experiment was to show whether HRS can be exploited by using ultrafractionated irradiation (UF) to improve local control of A7 tumours growing in nude mice. Extrapolation of the in vitro results predict a 3.7‐fold difference in the efficacy of UF compared with conventional fractionation (CF). Material and methods: Subcutaneuously growing A7 tumours were irradiated either with UF (126 fractions in 6 weeks, 0.4 Gy per fraction) or CF (30 fractions in 6 weeks, 1.68 Gy per fraction). The total dose was 50.4 Gy in both experimental arms. Fractionated irradiations were given under ambient conditions and followed by graded top‐up doses under clamp hypoxia. Endpoints were tumour growth delay and local tumour control 180 days after the end of treatment. Results: UF resulted in a significant decrease of tumour growth delay and in a significant increase of the top‐up TCD50 compared with CF (40.0 Gy [95% CI 29; 61 Gy] versus 28.3 Gy [24; 35 Gy], p=0.047). Conclusions: Despite a pronounced HRS phenomenon in vitro, UF was significantly less effective than CF in A7 human malignant glioma in nude mice. These results neither disprove the existence of HRS nor do they exclude a possible clinical value of UF. The findings rather indicate that simplistic extrapolation from results obtained after single‐dose exposure or few fractions in vitro is not sufficient to predict outcome of UF in vivo and that comprehensive evaluation of novel treatment options in animal models continues to be an essential requirement for clinical translation.

Effects of Lovastatin Alone or Combined with Irradiation on Tumor Cells in Vitro and in Vivo

Purpose:To evaluate the effect of lovastatin alone or combined with radiation on U87MG and FaDu cells in vitro and U87MG tumors in vivo.Material and Methods:Cell number, p21WAF1 expression, apoptosis, reproductive cell death, and cell-cycle distribution were investigated after incubation of U87MG and FaDu cells in vitro. The effect of lovastatin (50 mg/kg/day) on tumor growth and on tumor growth delay after single-dose irradiation with 20 Gy was investigated using U87MG tumors in nude mice.Results:Lovastatin dose dependently decreased cell number and proliferation of U87MG and FaDu cells. The proportion of cells in G0/G1 phase, apoptosis and p21 protein expression increased after lovastatin alone or combined with 4-Gy irradiation in both cell lines. Effects of lovastatin on cell cycle and cell number were more pronounced in U87MG compared to FaDu. No radiosensitization of clonogenic cells by lovastatin could be demonstrated in both cells lines, but the colony-forming ability after lovastatin alone was decreased in FaDu cells. In vivo, lovastatin decreased tumor volume over time but did not increase growth delay after irradiation of U87MG tumors with 20 Gy.Conclusion:The data support effects of lovastatin on proliferation, apoptosis and colony-forming ability in vitro and tumor volume in vivo. At the drug concentration achievable, lovastatin did not improve the effects of radiation on U87MG tumors in vivo.Ziel:Der Effekt von Lovastatin allein und in Kombination mit Bestrahlung auf U87MG- und FaDu-Zellen in vitro und U87MG-Tumoren in vivo wurde untersucht.Material und Methodik:Zellzahl, p21WAF1-Expression, Apoptose, reproduktiver Zelltod und Zellzyklusverteilung wurden nach Inkubation von U87MG- and FaDu-Zellen in vitro evaluiert. Der Effekt von Lovastatin (50 mg/kg/Tag) auf Tumorwachstum und Tumorwachstumsverzögerung nach Einzeitbestrahlung mit 20 Gy wurden an U87MG-Tumoren in Nacktmäusen untersucht.Ergebnisse:Lovastatin reduzierte dosisabhängig die Zahl und Proliferation von U87MG- und FaDu-Zellen in vitro. Der Anteil von Zellen in G0/G1, der Anteil apoptischer Zellen und die p21WAF1-Expression stiegen in beiden Zelllinien nach Lovastatin allein oder in Kombination mit 4-Gy-Bestrahlung. Die Effekte von Lovastatin auf Zellzahl und Zellzyklus waren bei U87MG ausgeprägter als bei FaDu. Ein strahlensensibilisierender Effekt von Lovastatin bezüglich des klonogenen Zelltods wurde bei keiner der Zelllinien gefunden, Lovastatin allein reduzierte jedoch die Koloniebildungsfähigkeit von FaDu-Zellen. In vivo reduzierte Lovastatin das Volumen von U87MG-Tumoren, führte aber zu keiner Verlängerung der Tumorwachstumsverzögerung nach Einzeitbestrahlung mit 20 Gy.Schlussfolgerung:Lovastatin zeigt Effekte auf die Proliferation, Apoptose und Koloniebildungsfähigkeit in vitro und das Tumorvolumen in vivo. In der erreichten Dosierung führt Lovastatin nicht zu einer Verstärkung des Effekts einer Bestrahlung von U87MG-Tumoren in vivo.

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.

Combined treatment of the immunoconjugate bivatuzumab mertansine and fractionated irradiation improves local tumour control in vivo

BACKGROUND AND PURPOSE To test whether BIWI 1 (bivatuzumab mertansine), an immunoconjugate of the humanized anti-CD44v6 monoclonal antibody BIWA 4 and the maytansinoid DM1, given simultaneously to fractionated irradiation improves local tumour control in vivo compared with irradiation alone. MATERIAL AND METHODS For growth delay, FaDu tumours were treated with 5 intravenous injections (daily) of phosphate buffered saline (PBS, control), BIWA 4 (monoclonal antibody against CD44v6) or BIWI 1 (bivatuzumab mertansine) at two different dose levels (50 μg/kg DM1 and 100 μg/kg DM1). For local tumour control, FaDu tumours received fractionated irradiation (5f/5d) with simultaneous PBS, BIWA 4 or BIWI 1 (two dose levels). RESULTS BIWI 1 significantly improved local tumour control after irradiation with 5 fractions already in the lower concentration. The dose modifying factor of 1.9 is substantial compared to the majority of other modifiers of radiation response. CONCLUSION Because of the magnitude of the curative effect, this approach is highly promising and should be further evaluated using similar combinations with improved tumour-specificity.

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.

Ultrafractionation does not improve the results of radiotherapy in radioresistant murine DDL1 lymphoma

Background and Purpose:Low-dose hyperradiosensitivity (HRS), i.e., a relatively higher efficacy of doses ≤ 0.5 Gy compared to doses > 1 Gy, has been shown in a number of tumor cell lines in vitro. Therefore ultrafractionated irradiation, i.e., application of very low doses per fraction, has been proposed to improve the effects of radiotherapy. The present study investigates ultrafractionation (UF) in radioresistant murine DDL1 T-cell lymphoma in mice.Material and Methods:UF was performed with 0.4 Gy per fraction, three fractions per day at 7 days per week, and conventional fractionation (CF) with 1.68 Gy per fraction, one fraction per day at 5 days per week. Tumor growth delay was evaluated for 2, 4 and 6 weeks of irradiation as time that tumors needed to reach fivefold the starting volume (GDV5).Results:GDV5 was not significantly different between UF and CF. The composite median relative GDV5 calculated for all tumors irradiated in the present study was 1.00 [95% confidence interval 0.99; 1.08] in the CF and 0.99 [0.92; 1.01] in the UF arm (p = 0.24).Conclusion:UF was not more efficient than CF in DDL1 tumors. Taken together with previous experiments on human A7 glioblastoma, which showed a negative effect of UF on local tumor control, the preclinical data obtained in this laboratory so far do not support the use of ultrafractionated schedules in radiotherapy.Hintergrund und Ziel:„Low-dose hyperradiosensitivity“ (HRS), d.h. eine relativ höhere Effektivität von Strahlendosen ≤ 0,5 Gy im Vergleich zu Dosen > 1 Gy, wurde für eine Reihe von Tumorzelllinien in vitro gezeigt. Um diesen Effekt zur Erhöhung der Effizienz einer Strahlentherapie auszunutzen, wurde die Ultrafraktionierung (UF), d.h. die Applikation von sehr niedrigen Dosen pro Fraktion, vorgeschlagen. Die vorliegende Studie untersucht die Wirksamkeit der UF an murinen strahlenresistenten DDL1-T-Zell-Lymphomen in Mäusen.Material und Methodik:Die Bestrahlungen im UF-Arm wurden mit 0,4 Gy pro Fraktion, drei Fraktionen pro Tag an 7 Tagen pro Woche, und im konventionell fraktionierten (CF) Arm mit 1,68 Gy pro Fraktion, einer Fraktion pro Tag an 5 Tagen pro Woche, durchgeführt. Die Dosis pro Woche betrug somit in beiden Armen 8,4 Gy. Die Tumorwachstumsverzögerung wurde nach 2, 4 und 6 Wochen als Zeit bis zum Erreichen des fünffachen Ausgangsvolumens (GDV5) ausgewertet.Ergebnisse:Die GDV5-Werte nach UF- und CF-Bestrahlung waren nicht signifikant unterschiedlich. Die zusammenfassende Analyse der medianen relativen GDV5 für alle Tumoren dieser Untersuchung ergab Werte von 1,00 [95%-Vertrauensbereich 0,99; 1,08] im CF- und 0,99 [0,92; 1,01] im UF-Arm (p = 0,24).Schlussfolgerung:Eine UF ist in DDL1-Tumoren nicht effizienter als eine CF. Unter Berücksichtigung früherer Experimente am humanen Glioblastom A7, die einen negativen Effekt der UF-Bestrahlung auf die lokale Tumorkontrolle ergaben, sprechen die bisher im eigenen Labor erzielten Resultate gegen den Einsatz ultrafraktionierter Bestrahlungsschemata in der Klinik.

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 (SUVmax) 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 SUVmax determined by PET. These findings suggest that the SUVmax does not necessarily reflect changes in tumour biology after irradiation.