The impact of conventional and heavy ion irradiation on tumor cell migration in vitro

PURPOSE: The influence of X-ray and (12)C heavy ion irradiation on tumor cell migration and of beta(3) and beta(1) integrin expression was investigated. MATERIAL AND METHODS: Two different tumor cell lines (U87 glioma and HCT116 colon carcinoma cells) were irradiated with 1, 3, or 10 Gy X-rays or (12)C heavy ions. 24 h after irradiation a standardized Boyden Chamber assay for migration analysis was performed and cells were lysed for Western blotting. RESULTS: Radiation-induced influences were cell line- and radiation type-dependent. X-rays decreased HCT116 migration at higher doses and appear to increase U87 migration after 3 Gy. Heavy ions decreased migration of both cell lines dose-dependently. A trend of increased beta(3) and beta(1) integrin expression in U87 cells after both radiation types was observed. beta(1) integrin expression in HCT116 cells was increased after X-rays but decreased after heavy ion irradiation. CONCLUSIONS: Results suggest that irradiation of tumor cells can modulate their migratory behavior. An increased migration, as shown with U87, leaves a higher probability of metastatic induction after irradiation of solid tumors in vivo, whereas an invariably reduced tumor cell migration, as shown after heavy ion treatment, could diminish the hazard of radiation-induced metastasis. As integrin expression and migration were only partially correlated, other migration-related surface molecules may be more relevant for radiation effects on tumor cell motility.     

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.     

Combination of EGFR/HER2 Tyrosine Kinase Inhibition by BIBW 2992 and BIBW 2669 with Irradiation in FaDu Human Squamous Cell Carcinoma

PURPOSE: To investigate the effect of the dual EGFR/HER2 (ErbB2) tyrosine kinase inhibitors BIBW 2992 and BIBW 2669 on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumor growth after single-dose irradiation in nude mice. MATERIAL AND METHODS: Cell proliferation, cell-cycle distribution and clonogenic cell survival after irradiation were assayed with and without BIBW 2992 or BIBW 2669 (3, 30, and 300 nM) in vitro. Tumor volume and tumor growth delay (GD(V2)) were determined in tumors growing in NMRI (nu/nu) nude mice, treated with (a) BIBW 2992 (20 mg kg(-1) body weight orally), BIBW 2669 (3-4 mg kg(-1) body weight orally) or carrier until a final tumor diameter of 15 mm, or, (b) 3 days before a 20-Gy single-dose irradiation or, (c) after a 20-Gy single-dose irradiation until reaching the final tumor diameter. RESULTS: BIBW 2992 and BIBW 2669 significantly increased the doubling time of FaDu cells in vitro. A marked dose-dependent antiproliferative effect with blockade of the cells in G0/G1-phase of the cell cycle was found. Incubation with BIBW 2669 or BIBW 2992 for 3 days marginally increased radiosensitivity of FaDu cells in vitro. For BIBW 2992, this effect was statistically significant (p = 0.006). Daily oral application of BIBW 2669 or BIBW 2992 in mice bearing unirradiated FaDu tumors showed a marked antiproliferative effect with a significant prolongation of tumor growth delay (p < 0.0001). After drug application for 3 days, followed by 20-Gy single-dose irradiation, a slight effect of both drugs on tumor growth delay was seen. For BIBW 2669, this effect was statistically significant (p = 0.007). However, this effect disappeared when tumor volumes were normalized to the time point of irradiation suggesting that both drugs showed no or only a slight radiosensitizing effect in vivo. Daily application of BIBW 2669 or BIBW 2992 after a single-dose irradiation showed a clear inhibition of tumor growth with a significantly longer tumor growth delay after drug treatment compared to control tumors (p < 0.002). Enhancement ratios were smaller for irradiated than for unirradiated tumors, suggesting an additive effect for combinations with radiotherapy. In all treatment arms, the effects of BIBW 2669 were not significantly different from BIBW 2992. CONCLUSION: BIBW 2669 and BIBW 2992 showed a clear antiproliferative effect in vitro, whereas radiosensitization was only marginal. The present data are the first to show an effect of combined irradiation and dual EGFR/ErbB2 inhibition on tumor growth delay in vivo. Further preclinical investigations using fractionated irradiation schedules and local tumor control as experimental endpoint are needed to evaluate a possible curative potential for the combination treatment.     

Human Glioblastoma and Carcinoma Xenograft Tumors Treated by Combined Radiation and Imatinib (Gleevec®)

BACKGROUND AND PURPOSE: Imatinib (Gleevec, Glivec) is an inhibitor of alpha- and beta-platelet-derived growth factor receptors and other tyrosine kinases, that are also associated with the function of growth factors. Imatinib has been approved for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors and is under investigation for the therapy of several other malignant tumors. Since radiotherapy is an important treatment option in many tumors, combined effects of imatinib and radiation were analyzed here. MATERIAL AND METHODS: In vitro, U87 cells (human glioblastoma), A431 cells (human epidermoid carcinoma), and HUVECs (human umbilical venous endothelial cells) were treated with imatinib alone and in combination with radiation. Clonogenic survival and cell proliferation were determined with and without additional radiation (0-10 Gy). In vivo, U87 and A431 cells (5 x 10(6)) were subcutaneously injected into hind limbs of balb c nu/u mice. Drug and radiation treatments started on day 0 when tumor volumes were approximately 400-500 mm(3). Tumors were treated with 5 x 5 Gy (U87) or 6 x 5 Gy (A431) on consecutive days from day 0. Imatinib was administered orally via the mouse diet starting on day 0 until the end of observation. Tumor growth and microvessel density (CD31 IHC) were analyzed. RESULTS: In vitro, imatinib increased radiosensitivity of U87 and A431 tumor cells as well as HUVECs in both clonogenic and cell number/proliferation assays. The enhancement of radiosensitivity in HUVECs was comparable to that observed in the tumor cells. In vivo, the concurrent and continuous administration of imatinib increased tumor growth delay of fractionated radiotherapy in the carcinoma and the glioblastoma models at reduced microvessel densities. No apparent additional toxicity by the combination of radiation and imatinib versus monotherapies was observed in terms of weight, skin, or general behavior. CONCLUSION: Imatinib (Gleevec), a "molecular targeted" approved drug for human malignancies, can enhance the tumor growth reduction induced by fractionated radiotherapy in glioblastoma and carcinoma models. The data provides a rationale to further investigate the combination.     

Impact of carbon ion irradiation on epidermal growth factor receptor signaling and glioma cell migration in comparison to conventional photon irradiation

Abstract

Purpose: Radiotherapy of malignant gliomas may be limited by an interference of radiation with the migratory potential of tumor cells. Therefore, the influence of conventional photon and modern carbon ion (¹²C) irradiation on glioblastoma cell migration and on epidermal growth factor receptor-related (EGFR) signaling was investigated in vitro.

Materials and methods: EGFR overexpressing glioblastoma cell lines U87 EGFR++ and LN229 EGFR++ were irradiated with 0, 2 or 6 Gy photons or ¹²C heavy ions. Migration was analyzed 24 h after treatment in a standardized Boyden Chamber assay. At different time points EGFR, protein kinase B (PKB/AKT) and extracellular signal-related kinases (ERK1/2) were analyzed by Western blotting.

Results: 2 Gy photon irradiation increased U87 EGFR++ migration and decreased motility of LN229 EGFR++ cells. Heavy ions decreased migration of both cell lines as a function of dose. There was a time-dependent increase of phosphorylation of EGFR, AKT and ERK1/2 in U87 EGFR++ after 2 Gy photon irradiation. After heavy ion irradiation EGFR, AKT or ERK1/2 remained unchanged.

Conclusions: Results suggest that the impact of irradiation on tumor cell migration depends on radiation type and cell line. Photons, but not heavy ions, potentially contribute to treatment failure by increasing EGFR-related tumor cell migration.     

Effects of three modifiers of glycolysis on ATP, lactate, hypoxia, and growth in human tumor cell lines in vivo

Background

High pretreatment tumor lactate content is associated with poor outcome after fractionated irradiation in human squamous cell carcinoma (hSCC) xenografts. Therefore, decreasing lactate content might be a promising approach for increasing tumor radiosensitivity. As the basis for such experiments, the effects of the biochemical inhibitors pyruvate dehydrogenase kinase dichloroacetate (DCA), lactate dehydrogenase oxamate, and monocarboxylic acid transporter-1 α-cyano-4-hydroxycinnamate (CHC) on tumor micromilieu and growth were investigated.

Materials and methods

Oxygen consumption (OCR) and extracellular acidification rates (ECAR) were measured in FaDu and UT-SCC-5 hSCC in response to DCA in vitro. Mice bearing FaDu, UT-SCC-5, and WiDr colorectal adenocarcinoma received either DCA in drinking water or DCA injected twice a day, or CHC injected daily. WiDr was also treated daily with oxamate. FaDu and UT-SCC-5 were either excised 8 days after treatment for histology or tumor growth was monitored. WiDr tumors were excised at 8?mm. Effect of inhibitors on ATP, lactate, hypoxia, and Ki67 labeling index (LI) was evaluated.

Results

DCA increased OCR and decreased ECAR in vitro. None of the treatments with inhibitors significantly changed lactate content, hypoxia levels, and Ki67 LI in the three tumor lines in vivo. ATP concentration significantly decreased after only daily twice injections of DCA in FaDu accompanied by a significant increase in necrotic fraction. Tumor growth was not affected by any of the treatments.

Conclusion

Overall, tumor micromilieu and tumor growth could not be changed by glycolysis modifiers in the three tumor cell lines in vivo. Further studies are necessary to explore the impact of metabolic targets on radiation response.     

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.     

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.     

Dichloroacetate induces tumor-specific radiosensitivity in vitro but attenuates radiation-induced tumor growth delay in vivo

Background

Inhibition of pyruvate dehydrogenase kinase (PDK) by dichloroacetate (DCA) can shift tumor cell metabolism from anaerobic glycolysis to glucose oxidation, with activation of mitochondrial activity and chemotherapy-dependent apoptosis. In radiotherapy, DCA could thus potentially enhance the frequently moderate apoptotic response of cancer cells that results from their mitochondrial dysfunction. The aim of this study was to investigate tumor-specific radiosensitization by DCA in vitro and in a human tumor xenograft mouse model in vivo.

Materials and methods

The interaction of DCA with photon beam radiation was investigated in the human tumor cell lines WIDR (colorectal) and LN18 (glioma), as well as in the human normal tissue cell lines HUVEC (endothelial), MRC5 (lung fibroblasts) and TK6 (lymphoblastoid). Apoptosis induction in vitro was assessed by DAPI staining and sub-G1 flow cytometry; cell survival was quantified by clonogenic assay. The effect of DCA in vivo was investigated in WIDR xenograft tumors growing subcutaneously on BALB/c-nu/nu mice, with and without fractionated irradiation. Histological examination included TUNEL and Ki67 staining for apoptosis and proliferation, respectively, as well as pinomidazole labeling for hypoxia.

Results

DCA treatment led to decreased clonogenic survival and increased specific apoptosis rates in tumor cell lines (LN18, WIDR) but not in normal tissue cells (HUVEC, MRC5, TK6). However, this significant tumor-specific radiosensitization by DCA in vitro was not reflected by the situation in vivo: The growth suppression of WIDR xenograft tumors after irradiation was reduced upon additional DCA treatment (reflected by Ki67 expression levels), although early tumor cell apoptosis rates were significantly increased by DCA. This apparently paradoxical effect was accompanied by a marked DCA-dependent induction of hypoxia in tumor-tissue.

Conclusion

DCA induced tumor-specific radiosensitization in vitro but not in vivo. DCA also induced development of hypoxia in tumor tissue in vivo. Further investigations relating to the interplay between tumor cell metabolism and tumor microenvironment are necessary to explain the limited success of metabolic targeting in radiotherapy.     

18F-FLT体外监测结肠癌细胞早期放射反应

目的 评价18F-脱氧胸腺嘧啶核苷（FLT）监测结肠癌细胞早期放射反应的作用.方法 应用四甲基偶氮唑蓝（MTT）检测并绘制SW480细胞受X线照射后生长曲线,光学显微镜下观察细胞形态变化.流式细胞仪检测照射后肿瘤细胞增殖周期的重新分布.分别于体外细胞以及肿瘤内检测照射前、后细胞摄取18F-FLT的变化.将18F-FLT（0.05±0.01）MBq加入培养液孵育细胞,分别于30,60,90,120 min测定细胞摄取18F-FLT的放射性.经荷瘤裸鼠尾静脉注射18F-FLT（1.90±0.85）MBq（0.25 ml）,60 min后处死动物,切除肿瘤与肌肉、肺、肝等,测定肿瘤与其他脏器摄取放射性比值变化.应用单因素方差分析进行统计学处理.结果 X线照射后,SW480细胞增殖受到明显的抑制,呈剂量依赖性.细胞形态随照射剂量的不同发生不同的变化.照射后细胞周期发生重新分布.10 Gy组,S期细胞百分比24 h从33.23%降至15.19%,72 h后降至12.44%.20 Gy组,S期细胞百分比24 h后从33.23%降至9.24%,72 h后降至5.43%.体外摄取实验发现,注射后60 min,SW480细胞摄取18F-FLT百分比为（5.21±1.60）%,10 Gy照射24 h后下降至（4.27±0.48）%,72 h降至（3.39±0.59）%.20 Gy组：照射后24 h,SW480摄取的放射性百分比下降至（3.41±0.58）%,72 h后降至（1.63±0.49）%.两照射组在72 h内分别下降了34.94%,69.72%（24 h∶F=8.253,P=0.009;72 h∶F=36.715,P＜0.001）.单位质量肿瘤组织摄取的18F-FLT随照射剂量的增加而逐渐降低（10Gy组：F=12.388,P=0.007;20 Gy组：F=16.744,P=0.004）.结论 18F-FLT在结肠癌细胞内的摄取可以快速反映照射治疗后的细胞变化,18F-FLT可能用于检测结肠癌放射治疗早期反应.     

Effect of the Hypoxic Cell Sensitizer Isometronidazole on Local Control of Two Human Squamous Cell Carcinomas after Fractionated Irradiation

BACKGROUND AND PURPOSE: Hypoxia of clonogenic tumor cells is a major reason for radioresistance and hence local failure in radiotherapy. The objective of the present study was to test the efficacy of the hypoxic cell sensitizer isometronidazole (ISO) during fractionated irradiation in two different human squamous cell carcinomas. MATERIAL AND METHODS: Local control was evaluated for FaDu (radiobiological hypoxic fraction [rHF] 7%) and GL tumors (rHF 0.1%) after single-dose (SD) irradiation under ambient conditions and after 30 fractions within 6 weeks (30 f/6 w). ISO was applied 60 min before SD irradiation at a concentration of 100 mg/kg body weight (b.w.) or 750 mg/kg b.w. in both tumors. During fractionated irradiation, ISO was applied daily 60 min before each fraction (100 mg/kg b.w., in FaDu also 750 mg/kg b.w.). RESULTS: 100 mg/kg b. w. ISO did not improve local control after SD irradiation or 30 f/6 w in both tumor models. Application of 750 mg/kg b. w. ISO significantly decreased the SD-TCD(50) in FaDu tumors (dose-modifying factor [DMF] = 1.2; p = 0.01) but not in the better oxygenated GL tumor. ISO at 750 mg/kg b.w. also significantly improved local control of FaDu tumors after 30 fractions in 6 weeks (DMF = 1.2; p = 0.01), indicating that hypoxic clonogenic cells in FaDu tumors are not only present before start of irradiation but also limit the efficacy of treatment during a fractionated course of radiotherapy. CONCLUSION: ISO at a concentration of 750 mg/kg b.w. shows an efficacy as a hypoxic cell sensitizer in severely hypoxic FaDu tumors but not in less hypoxic GL tumors. This supports the principle of hypoxic cell sensitization and improvement of local control of hypoxic tumors by nitroimidazole derivatives. However, doses of 750 mg/kg b. w. before each fraction may be difficult to achieve in the clinical situation. This, in light of the fact that other well-tolerable hypoxic cell sensitizers such as nimorazole with clinically proven efficacy at daily oral doses of < 3 g are available, limits the potential usefulness of ISO for radiation oncology.     

DEGRO-ÖGRO 2008

BACKGROUND AND PURPOSE: Tumor-induced anemia often occurs in cancer patients, and is corrected by recombinant human erythropoietins (rHuEPOs). Recent studies indicated that, besides erythroid progenitor cells, tumor and endothelial cells express erythropoietin receptor (EPOR) as well; therefore, rHuEPO may affect their functions. Here, the effect of rHuEPOalpha on irradiation in EPOR-positive human squamous cell carcinoma xenograft was tested. MATERIAL AND METHODS: A431 tumor-bearing SCID mice were treated from the tumor implantation with rHuEPOalpha at human-equivalent dose. Xenografts were irradiated (5 Gy) on day 14, and the final tumor mass was measured on day 22. The systemic effects of rHuEPOalpha on the hemoglobin level, on tumor-associated blood vessels and on hypoxia-inducible factor-(HIF-)1alpha expression of the tumor xenografts were monitored. The proliferation, apoptosis and clonogenic capacity of A431 cancer cells treated with rHuEPOalpha and irradiation were also tested in vitro. RESULTS: In vitro, rHuEPOalpha treatment alone did not modify the proliferation of EPOR-positive A431 tumor cells but enhanced the effect of irradiation on proliferation, apoptosis and clonogenic capacity. In vivo, rHuEPOalpha administration compensated the tumor-induced anemia in SCID mice and decreased tumoral HIF-1alpha expression but had no effect on tumor growth. At the same time rHuEPOalpha treatment significantly increased the efficacy of radiotherapy in vivo (tumor weight of 23.9 +/- 4.7 mg and 34.9 +/- 4.6 mg, respectively), mediated by increased tumoral blood vessel destruction. CONCLUSION: rHuEPOalpha treatment may modulate the efficacy of cancer radiotherapy not only by reducing systemic hypoxia and tumoral HIF-1alpha expression, but also by destroying tumoral vessels.     

In vivo and in vitro evaluation of Cy5.5 conjugated epidermal growth factor receptor binding peptide

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor and plays an important role in carcinogenesis. In this study, the epidermal growth factor receptor binding peptide (EGBP) was identified using a phage display method and evaluated in U87MG cells in order to investigate the possibility to target the EGFR using an optical imaging system. Cyanine dye 5.5 (Cy5.5) was conjugated with EGBP-GGG-SC, EGBP-AOC-SC, and EGBP-AM2BA-SC. Cellular binding study of EGBP-Linker-Cy5.5 conjugates or ¹²⁵I-EGBP-Linker compounds was performed in U87MG cells. Optical imaging studies were performed in U87MG bearing mice. Three of seven clones from the 12-mer peptide library showed a specific binding affinity to rhEGFR, and they encoded the same 12 amino acid peptide sequence, FPMFNHWEQWPP. Confocal images show that the fluorescent signal of EGBP-Linker-Cy5.5 conjugates was decreased in the order: EGBP-AOC-Cy5.5?EGBP-AM2BA-Cy5.5>EGBP-GGG-Cy5.5. EGBP-AOC-Cy5.5 appeared in cell cytoplasm and surface, and it was inhibited by free EGBP apparently. The cellular binding of EGBP-AOC-Cy5.5 exhibited a higher average radiance value than EGBP-GGG-Cy5.5 and EGBP-AM2BA-Cy5.5. Among various ¹²⁵I-EGBP-Linker compounds, EGBP-GGG showed a higher binding than other compounds. However, uptake of ¹²⁵I-EGBP-AOC was clearly inhibited by free EGBP in inhibition study. In an in vivo study, the fluorescent signal of EGBP-AOC-Cy5.5 treated mouse was mainly detected in the tumor and kidney. Among the three derivatives, EGBP-AOC-Cy5.5 was the optimized optical imaging agent for U87MG EGFR positive tumors in the animal model. This study demonstrated the EGBP-Linker-Cy5.5 conjugates may be useful as a potential EGFR target optical probe.     

Evaluation of (76)Br-FBAU as a PET reporter probe for HSV1-tk gene expression imaging using mouse models of human glioma.

The utility of 5-(76)Br-bromo-2'-fluoro-2'-deoxyuridine ((76)Br-FBAU), a uracil analog, as a PET reporter probe for use with the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene system for gene expression imaging was evaluated in vivo and in vitro using human and rat glioma cells. METHODS: Human glioma cell lines U87 and U251 were transduced with replication-defective adenovirus constitutively expressing HSV1-tk (Ad.TK) or a control expressing green fluorescent protein (Ad.GFP). These cells were incubated with (76)Br-FBAU for 20-120 min to determine the percentage of total dose uptake. In vitro uptake of equimolar concentrations (1.8 x 10(-8) mol/L) of (76)Br-FBAU and 2'-fluoro-2'-deoxy-5-iodouracil-beta-d-arabinofuranoside ((14)C-FIAU) was also determined in RG2-TK rat glioma cells stably expressing HSV1-tk and in control RG2 cells at 30-120 min. In vivo uptake of (76)Br-FBAU was determined in subcutaneous U87 tumor intratumorally transduced with Ad.TK by ex vivo biodistribution. Uptake in intracranial U87 tumors transduced with Ad.TK expressing HSV1-tk was measured by brain autoradiography. In vivo PET was performed on subcutaneous and intracranial U87 tumors transduced with Ad.TK and on subcutaneous and intracranial stably expressing RG2-TK tumors. RESULTS: U87 and U251 cells transduced with Ad.TK had significantly increased uptake of (76)Br-FBAU compared with cells transduced with Ad.GFP over 20-120 min. In stably expressing cells at 120 min, (14)C-FIAU uptake in RG2-TK tumor cells was 11.3 %ID (percentage injected dose) and in RG2 control cells was 1.7 %ID, and (76)Br-FBAU uptake in RG2-TK tumor cells was 14.2 %ID and in RG2 control cells was 1.5 %ID. Ex vivo biodistribution of subcutaneous U87 tumors transduced with Ad.TK accumulated (76)Br-FBAU significantly more than in the control Ad.GFP transduced tumor and normal tissue, with the lowest uptake in brain. Autoradiography showed localized uptake in intracranial U87 and U251 cells transduced with Ad.TK. PET image analyses of mice with RG2-TK tumors resulted in an increased tumor-to-background ratio of 13 and 26 from 2 to 6 h after injection, respectively, in intracranial tumors. CONCLUSION: (76)Br-FBAU accumulates in glioma cells constitutively expressing HSV1-tk by either adenoviral transduction or in stably expressing cell lines both in vitro and in vivo. (76)Br-FBAU shows promise as a PET reporter probe for use with the HSV1-tk in vivo gene expression imaging system.     

Nachweis einer strahleninduzierten Produktion von Tumor-Nekrose-Faktor alpha im Ewing-Sarkom RM 82 in vitro und in vivo

Aim

The expression of cytokines plays an important role in the transmission of the effects of ionizing radiation to tumor cells and normal tissue. Tumor necrosis factor alpha (TNF α), a pleiotropic monokine, is of special interest because of its cytotoxic effect on tumor cells and the induction of hemorrhagic necrosis in tumors. We examined the influence of ionizing radiation on TNF α production in a human Ewing’s sarcoma cell line in vitro and in vivo.

Methods

The protein and mRNA levels of the Ewing’s sarcoma cell line RM 82 were examined in vitro with ?Enhanced Amplified Sensitivity Immunoassay” (EASIA) and semiquantitative RT-PCR before and after treatment with single doses of 2 to 40 Gy, 1 to 72 hours after irradiation. After successful transplantation to nude mice, the time and dose correlation of TNF α mRNA production was examined in vivo.

Results

In vitro, RM 82 had a basal protein level of TNF α of 20.1±4.3 pg/ml/10⁶ cells. We observed a time- and dose-dependent increase of TNF α expression with a maximum of 125 pg/ml/10⁶ (5.9fold) 24 hours after irradiation with 20 Gy. At the mRNA level, the maximal up-regulation occurred 6 to 12 hours after 10 Gy. In vivo, the xenograft tumor maintained the capacity of TNF α expression. Time-and dose-dependency in mRNA production showed a maximum increase 6 hours after treatment with 10 Gy.

Conclusions

The presented experiments show in vitro a dose- and time-dependent up-regulation of TNF α in the Ewing’s sarcoma cell line RM 82 on protein and mRNA level. For the first time this phenomenon was also observed in vivo in a human xenograft tumor. This tumor model could be used for further experiments to examine the role of TNF α as a biologic radiation response modifier in human tumors.     

In-vivo visualization of radiation-induced apoptosis using (125)I-annexin V

BACKGROUND: As apoptosis occurs in tumors within a short time after irradiation, the detection of the frequency of apoptosis may be useful as an indicator of the effect of treatment. For the evaluation of apoptosis under these conditions, tissue extraction from patients is indispensable. AIM: To develop a noninvasive imaging technique to measure and monitor apoptosis in tumor cells caused by X-irradiation using (125)I-radiolabeled annexin V. METHODS: The tumors used were human ependymoblastomas, which were transplanted into nude mice. The tumors were irradiated at 2, 5 or 10 Gy. (125)I-annexin V was administered intravenously 6 h after irradiation. In the 5 Gy irradiation group, the isotope was injected at various time intervals (3, 6 and 12 h) after irradiation. Three hours after the injection, the mice were sacrificed, the tumors were quickly removed and frozen sections were prepared at 6 and 40 microm thickness using a cryomicrotome. In autoradiographic imaging, the tumor-to-muscle ratios were compared in the respective irradiated groups. In addition, apoptosis detection by the in-situ end-labeling (Klenow) assay was conducted on the same sections. The number of Klenow-positive cells was counted in 100 x fields for each section. RESULTS: Both autoradiography and immunohistochemical staining showed a significantly higher frequency of apoptosis in the neoplasms in all irradiated groups than in the control group (P<0.05). Although immunohistochemical staining revealed a peak apoptosis frequency in the 5 Gy irradiated group, autoradiography revealed a peak in the group receiving a lower dose than 5 Gy. When the time from irradiation to annexin injection was varied, both imaging methods showed a peak apoptosis frequency in the group receiving the injection 6 h after irradiation. CONCLUSION: It is possible to predict the effect of treatment in cancer in a noninvasive manner by apoptosis imaging in vivo after radiotherapy.     

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.     

Cell loss from viable and necrotic tumor regions after local gamma irradiation measured by 125I-UdR

Using a tracer technique, loss of cells from perivascular and average tumor cells of the syngeneic mammary adenocarcinoma EO 771 in male C57Bl/6J mice may be measured in the living animal, by the use of 125-labelled 5-iodo-2'-deoxyuridine (125I-UdR). It was the purpose of this paper to compare measurements in vivo with those made in vitro following local 60Co-gamma irradiation in the absorbed dose range from 10 to 27.5 Gy, incorporation of radioactivity into DNA of tumor cells and activity loss from labelled tumor cells were measured externally by a special scintillation counter device. In addition, by injecting the vital dye "light green" into the mice the I-125-activity of the stained viable and unstained necrotic regions were separately measured for loss of activity following gamma irradiation. A comparison was made between radiation induced growth delay and the depression of 125I-UdR incorporation into DNA of the proliferating tumor cells. After local tumor irradiation with a dose of 27.5 Gy 60Co gamma rays an enhancement of the activity loss by 0.5% per hour was externally observed for the perivascular tumor cell population. A lower enhancement of 0.4% per hour was externally registered in the average tumor cell population. Both values were evaluated relative to sham-irradiated control tumors. The measurements on isolated tumors were in comparatively good agreement with the external values. The activity loss rate from the viable, euoxic tissue increased by 0.4% per hour after 27.5 Gy 60Co gamma rays and by 0.3% per hour in the average cell population, the latter representing a mixture of euoxic and hypoxic cells. The results demonstrate, that the external measurements are a good indicator for radiation effects under in vivo-conditions.