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923: Cellular motility properties after X and proton beam irradiation
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
are downregulated quickly in response to ionizing radiation (IR) enabling the cells to initiate apoptosis. Here, we address the question whether a deregulated Mcl-1 stability in prostate cancer cells could increase survival in response to IR. Material and Methods: LnCaP and PC3 prostate cancer cell lines were irradiated with IR (0−10 Gy). Apoptosis was analyzed by flow cytometry and by quantifying DNA fragmentation as well as mitochondrial dissipation. Long-term toxicity was analyzed by a colony formation assay. Protein levels were analyzed by western blotting. Mcl-1 half life time was calculated after a densitometric analysis of the declining Mcl-1 levels induced by the translation inhibitor cycloheximide (1 mM). Knock-down of Mcl-1 and the Mcl-1 deubiquitylating enzyme USP9x was achieved by transfecting the cells with siRNA. Results: Although LnCaP and PC3 cells have similar Mcl-1 levels, Mcl-1 half life time was longer in LnCaP than in PC3 cells. In response to IR, PC3 cells were able to downregulate Mcl-1 levels whereas LnCaP cells even increased Mcl-1 levels. The ability to decrease Mcl-1 level correlated with apoptosis induction. Knock-down of Mcl-1 by siRNA resulted in concentration-dependent apoptosis induction in LnCaP cells whereas it hardly showed any toxicity in PC3 cells suggesting that LnCaP cells depend on Mcl-1 expression for survival. In response to IR, Mcl-1 knock-down increased apoptosis rate in both cell lines. Knock-down of USP9x, a deubiquitinase recognizing Mcl-1, increased apoptosis only slightly in non-irradiated prostate cells but clearly enhanced apoptosis in response to IR. Furthermore, downregulation of USP9x by siRNA resulted in increased clonogenic cell death in LnCaP cells. Conclusions: Our results suggest that targeting enzymes that control Mcl-1 stability (e.g. USP9x) can improve sensitivity to IR. No conflict of interest. 922 Relevance of 3D-microtissues to investigate the therapeutic oncogenes HER2 and PTK6 in breast cancer I. Hoefig1 , N. Falkenberg2 , M. Rosemann1 , J. Szumielewski2 , S. Richter1 , M.J. Atkinson1 , M. Aubele2 , N. Anastasov1 . 1 Helmholtz Zentrum Muenchen, Institute for Radiation Biology, Munich, Germany, 2 Helmholtz Zentrum Muenchen, Institute for Pathology, Munich, Germany Background: Traditionally, tumour cells are cultivated in two-dimensional (2D) monolayers on plastic surfaces. But in vivo, three-dimensional (3D) tumour tissues can differ vastly, e.g. by forming cell-to-cell-contacts and by adapting to oxygen, nutrient and drug gradients. Material and Methods: To resemble tissue-like conditions in vitro for studying oncogene function, we established a method to generate 3D-microtissues with HER2 and PTK6 RNAi knockdown using a lentiviral approach in breast cancer cells. We compared growth rates after knockdown and therapeutic treatments with 2D-monolayers or in vivo xenografts. Results: Comparison of HER2 with PTK6 knockdown revealed dominant growth impairment due to HER2 knockdown in T47D 2D-monolayers (48 hours), 3D-microtissues (12 days) and xenografts (40 days), in contrast to dominant growth impairment due to PTK6 knockdown in JIMT-1 2D-monolayers, 3D-microtissues and xenografts. Modeling therapeutic interventions based on HER2-signalling, we treated T47D cells grown in 2D-monolayers and 3D-microtissues with anti-HER2 trastuzumab and investigated combined effects with radiotherapy. Administration of both treatments significantly enhanced growth inhibition especially under 3D conditions. Conclusions: 3D-microtissues are a reliable tool for studying long-term effects of therapeutic HER2 and PTK6 oncogene knockdown and HER2-targeted treatments. Including 3D-microtissues in in vitro studies can predict therapeutic outcomes more accurately reducing cohort sizes and costs for in vivo studies. No conflict of interest. 923 Cellular motility properties after X and proton beam irradiation K. Jasinska1 , A. Borkowska1 , P. Koczurkiewicz2 , M. Michalik2 , Z. Madeja2 , P. Olko3 , B. Romanowska-Dixon4 , M. Elas1 , K. Urbanska1 . 1 Biochemistry Biophysics and Biotechnology, Biophysics, Krakow, Poland, 2 Biochemistry Biophysics and Biotechnology, Cell Biology, Krakow, Poland, 3 Institute of Nuclear Physics PAS, Radiation Protection and Dosimetry, Krakow, Poland, 4 Jagiellonian University Medical College, Ophthalmology and Ocular Oncology, Krakow, Poland Background: All malignant tumors are characterized by active invasion and destruction of surrounding tissue and while in metastatic stage they show poor response to different therapies. Cellular motility is one of the factors influencing migration and metastasis formation. Matrix metalloproteinases can degrade the basement membrane, resulting in promotion of tumor invasion. The goal of this study was to compare the cellular motility after a low dose irradiation of X-ray versus proton beam irradiation. Methods: BLM human melanoma and omm1.3 human uveal melanoma were irradiated with 1−5 Gy of X ray (300 kVp Phillips, 1 Gy/min) or proton beam (58 MeV) from AIC-144 cyclotron. Immediately after irradiation migratory and
motility properties were studied using wound test, time-lapse monitoring of individual cell movements and Western Blots for MMP2 and -9. Results: Proliferation of cells was slightly inhibited after 1−3 Gy, and strongly inhibited after 5 Gy. Wound test showed a decrease in wound regrowth by BLM cells after 3 Gy of proton beam irradiation. Strong intercellular interaction was observed in BLM cell culture, influencing their motility. In high density cultures, BLM cells exhibited lower speed, cellular displacement, and speed displacement after 4 Gy. In contrast, uveal melanoma omm1.3 cells showed increased displacement and speed displacement after 5 Gy of proton beam irradiation. Conclusion: Low doses of proton beam irradiation affect the migratory properties of melanoma cells. No conflict of interest. 924 Comparative study of chondrosarcomas response to DNA damage: Impact of HIF2 expression E. Lhuissier1 , N. Girard1 , O. Cauvard1 , C. Bazille2 , H. Benateau3 , A. Batalla4 , A. Llombart-bosch5 , C. Bauge1 , K. Boumediene1 . 1 Universite´ de Caen Basse-Normandie, Ea4652 Milpat, Caen, France, 2 CHU, Service d’anatomie Pathologique, Caen, France, 3 CHU, Service de Chirurgie Maxillo-faciale, Caen, France, 4 CLCC Baclesse, Service de Radiophysique, Caen, France, 5 University of Valencia, Pathology Department, Valencia, Spain Objective: Chondrosarcomas are rare tumors and potentially fatal. No effective treatment exists, chondrosarcomas are chemoresistant and radioresistant. Current treatment consists of resection of the tumor up to amputation. The aim of our work was to study the resistance of chondrosarcoma in response to different DNA damaging agents. Besides, HIF factors are known to play important role in cartilaginous tissues and treatment resistance of tumors. In addition, chondrosarcomas highly express HIF2 contrary to chondrocytes. The role of HIF2 in chondrosarcomas response to DNA damage was also investigated in this study. Methods: Five different chondrosarcoma cell lines with various grade and origin underwent irradiations with X rays (0 to 10 Gy), or treated with cisplatine (0 to 50 mM) in the presence or not of DMOG, an inductor of HIF factors that mimicks hypoxia. Cell growth and survival were evaluated by counting adherent cells periodically. Apoptosis was assayed by cell cycle analysis, Apo2.7 expression using flow cytometry, and by PARP cleavage using westernblot. Cell migration was assessed by wound healing assay. Results: Chondrosarcoma cell lines differently responded to DNA damage. They could be separated according to their sensitivity to X-rays (sensitive group: D10 = 3 Gy, intermediate group: D10 = 5 Gy and resistant group: D10 = 7 Gy) or cisplatin (sensitive group: LD50 Gy = 2.5 mM and resistant group: LD50 = 5 mM). Furthermore, we showed that cisplatin and X-rays induce apoptosis in some but not all cell lines. In addition, we showed that DMOG induced HIF2 expression in all chondrosarcoma cell lines. However, it differently affected their sensibility to treatments. Conclusion: Chondrosarcoma cell lines have various responses to DNA damaging agents and to HIF2. Cell death mechanisms involved are also dependent on cell line and type of treatment used. It is therefore essential to use multiple chondrosarcoma lines for studies on the response of these tumors to treatments, notably for the development of new therapeutic strategies such as hadrontherapy. No conflict of interest. 925 Multiparametric MRI evaluation of radiotherapy effects on the vascular compartment and white matter for glioblastoma treatment A. Gerault1 , A. Corroyer-Dulmont1 , A. Chakhoyan1 , D. Divoux1 , J. Toutain1 , M. Bernaudin1 , S. Valable1 , E. Petit1 . 1 CNRS UMR-6301 ISTCT − Universite´ de Caen Basse-Normandie UMR6301-ISTCT CERVOxy team. CEA DSV/I2BM UMR6301-ISTCT CERVOxy team − Normandie Univ, GIP CYCERON, Caen, France Background: Currently, patients with glioblastoma (GBM) have a median life expectancy lower than 15 months despite optimized conventional treatment combining surgery with chemo- and radiotherapy (RT). A pathophysiological hallmark for these tumors is hypoxia leading to a highly abnormal vasculature, contributing to limit the efficacy of RT. In response to RT, it has been proposed that white matter (WM) disturbances, at the origin of cognitive disorders in these patients, could be also a result of vascular alterations. However, radiation-induced changes in tumor blood vessels as well as tumor stroma sensitivity are known to be highly variable depending on RT protocols (fractionated or not) and relationships between the cellular responses of the neurovascular unit are complex and difficult to study. Accordingly, the aim of this project was to provide, with the use of multiparametric MRI, a longitudinal preclinical study enabling the evaluation of changes of tumor growth, tumoral endothelium and WM in response to RT. Material and Methods: An orthotopic human GBM model developed on athymic rats by an intrastriatal injection of U251 cells (5×104 cells/3 mL) was
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
are downregulated quickly in response to ionizing radiation (IR) enabling the cells to initiate apoptosis. Here, we address the question whether a deregulated Mcl-1 stability in prostate cancer cells could increase survival in response to IR. Material and Methods: LnCaP and PC3 prostate cancer cell lines were irradiated with IR (0−10 Gy). Apoptosis was analyzed by flow cytometry and by quantifying DNA fragmentation as well as mitochondrial dissipation. Long-term toxicity was analyzed by a colony formation assay. Protein levels were analyzed by western blotting. Mcl-1 half life time was calculated after a densitometric analysis of the declining Mcl-1 levels induced by the translation inhibitor cycloheximide (1 mM). Knock-down of Mcl-1 and the Mcl-1 deubiquitylating enzyme USP9x was achieved by transfecting the cells with siRNA. Results: Although LnCaP and PC3 cells have similar Mcl-1 levels, Mcl-1 half life time was longer in LnCaP than in PC3 cells. In response to IR, PC3 cells were able to downregulate Mcl-1 levels whereas LnCaP cells even increased Mcl-1 levels. The ability to decrease Mcl-1 level correlated with apoptosis induction. Knock-down of Mcl-1 by siRNA resulted in concentration-dependent apoptosis induction in LnCaP cells whereas it hardly showed any toxicity in PC3 cells suggesting that LnCaP cells depend on Mcl-1 expression for survival. In response to IR, Mcl-1 knock-down increased apoptosis rate in both cell lines. Knock-down of USP9x, a deubiquitinase recognizing Mcl-1, increased apoptosis only slightly in non-irradiated prostate cells but clearly enhanced apoptosis in response to IR. Furthermore, downregulation of USP9x by siRNA resulted in increased clonogenic cell death in LnCaP cells. Conclusions: Our results suggest that targeting enzymes that control Mcl-1 stability (e.g. USP9x) can improve sensitivity to IR. No conflict of interest. 922 Relevance of 3D-microtissues to investigate the therapeutic oncogenes HER2 and PTK6 in breast cancer I. Hoefig1 , N. Falkenberg2 , M. Rosemann1 , J. Szumielewski2 , S. Richter1 , M.J. Atkinson1 , M. Aubele2 , N. Anastasov1 . 1 Helmholtz Zentrum Muenchen, Institute for Radiation Biology, Munich, Germany, 2 Helmholtz Zentrum Muenchen, Institute for Pathology, Munich, Germany Background: Traditionally, tumour cells are cultivated in two-dimensional (2D) monolayers on plastic surfaces. But in vivo, three-dimensional (3D) tumour tissues can differ vastly, e.g. by forming cell-to-cell-contacts and by adapting to oxygen, nutrient and drug gradients. Material and Methods: To resemble tissue-like conditions in vitro for studying oncogene function, we established a method to generate 3D-microtissues with HER2 and PTK6 RNAi knockdown using a lentiviral approach in breast cancer cells. We compared growth rates after knockdown and therapeutic treatments with 2D-monolayers or in vivo xenografts. Results: Comparison of HER2 with PTK6 knockdown revealed dominant growth impairment due to HER2 knockdown in T47D 2D-monolayers (48 hours), 3D-microtissues (12 days) and xenografts (40 days), in contrast to dominant growth impairment due to PTK6 knockdown in JIMT-1 2D-monolayers, 3D-microtissues and xenografts. Modeling therapeutic interventions based on HER2-signalling, we treated T47D cells grown in 2D-monolayers and 3D-microtissues with anti-HER2 trastuzumab and investigated combined effects with radiotherapy. Administration of both treatments significantly enhanced growth inhibition especially under 3D conditions. Conclusions: 3D-microtissues are a reliable tool for studying long-term effects of therapeutic HER2 and PTK6 oncogene knockdown and HER2-targeted treatments. Including 3D-microtissues in in vitro studies can predict therapeutic outcomes more accurately reducing cohort sizes and costs for in vivo studies. No conflict of interest. 923 Cellular motility properties after X and proton beam irradiation K. Jasinska1 , A. Borkowska1 , P. Koczurkiewicz2 , M. Michalik2 , Z. Madeja2 , P. Olko3 , B. Romanowska-Dixon4 , M. Elas1 , K. Urbanska1 . 1 Biochemistry Biophysics and Biotechnology, Biophysics, Krakow, Poland, 2 Biochemistry Biophysics and Biotechnology, Cell Biology, Krakow, Poland, 3 Institute of Nuclear Physics PAS, Radiation Protection and Dosimetry, Krakow, Poland, 4 Jagiellonian University Medical College, Ophthalmology and Ocular Oncology, Krakow, Poland Background: All malignant tumors are characterized by active invasion and destruction of surrounding tissue and while in metastatic stage they show poor response to different therapies. Cellular motility is one of the factors influencing migration and metastasis formation. Matrix metalloproteinases can degrade the basement membrane, resulting in promotion of tumor invasion. The goal of this study was to compare the cellular motility after a low dose irradiation of X-ray versus proton beam irradiation. Methods: BLM human melanoma and omm1.3 human uveal melanoma were irradiated with 1−5 Gy of X ray (300 kVp Phillips, 1 Gy/min) or proton beam (58 MeV) from AIC-144 cyclotron. Immediately after irradiation migratory and
motility properties were studied using wound test, time-lapse monitoring of individual cell movements and Western Blots for MMP2 and -9. Results: Proliferation of cells was slightly inhibited after 1−3 Gy, and strongly inhibited after 5 Gy. Wound test showed a decrease in wound regrowth by BLM cells after 3 Gy of proton beam irradiation. Strong intercellular interaction was observed in BLM cell culture, influencing their motility. In high density cultures, BLM cells exhibited lower speed, cellular displacement, and speed displacement after 4 Gy. In contrast, uveal melanoma omm1.3 cells showed increased displacement and speed displacement after 5 Gy of proton beam irradiation. Conclusion: Low doses of proton beam irradiation affect the migratory properties of melanoma cells. No conflict of interest. 924 Comparative study of chondrosarcomas response to DNA damage: Impact of HIF2 expression E. Lhuissier1 , N. Girard1 , O. Cauvard1 , C. Bazille2 , H. Benateau3 , A. Batalla4 , A. Llombart-bosch5 , C. Bauge1 , K. Boumediene1 . 1 Universite´ de Caen Basse-Normandie, Ea4652 Milpat, Caen, France, 2 CHU, Service d’anatomie Pathologique, Caen, France, 3 CHU, Service de Chirurgie Maxillo-faciale, Caen, France, 4 CLCC Baclesse, Service de Radiophysique, Caen, France, 5 University of Valencia, Pathology Department, Valencia, Spain Objective: Chondrosarcomas are rare tumors and potentially fatal. No effective treatment exists, chondrosarcomas are chemoresistant and radioresistant. Current treatment consists of resection of the tumor up to amputation. The aim of our work was to study the resistance of chondrosarcoma in response to different DNA damaging agents. Besides, HIF factors are known to play important role in cartilaginous tissues and treatment resistance of tumors. In addition, chondrosarcomas highly express HIF2 contrary to chondrocytes. The role of HIF2 in chondrosarcomas response to DNA damage was also investigated in this study. Methods: Five different chondrosarcoma cell lines with various grade and origin underwent irradiations with X rays (0 to 10 Gy), or treated with cisplatine (0 to 50 mM) in the presence or not of DMOG, an inductor of HIF factors that mimicks hypoxia. Cell growth and survival were evaluated by counting adherent cells periodically. Apoptosis was assayed by cell cycle analysis, Apo2.7 expression using flow cytometry, and by PARP cleavage using westernblot. Cell migration was assessed by wound healing assay. Results: Chondrosarcoma cell lines differently responded to DNA damage. They could be separated according to their sensitivity to X-rays (sensitive group: D10 = 3 Gy, intermediate group: D10 = 5 Gy and resistant group: D10 = 7 Gy) or cisplatin (sensitive group: LD50 Gy = 2.5 mM and resistant group: LD50 = 5 mM). Furthermore, we showed that cisplatin and X-rays induce apoptosis in some but not all cell lines. In addition, we showed that DMOG induced HIF2 expression in all chondrosarcoma cell lines. However, it differently affected their sensibility to treatments. Conclusion: Chondrosarcoma cell lines have various responses to DNA damaging agents and to HIF2. Cell death mechanisms involved are also dependent on cell line and type of treatment used. It is therefore essential to use multiple chondrosarcoma lines for studies on the response of these tumors to treatments, notably for the development of new therapeutic strategies such as hadrontherapy. No conflict of interest. 925 Multiparametric MRI evaluation of radiotherapy effects on the vascular compartment and white matter for glioblastoma treatment A. Gerault1 , A. Corroyer-Dulmont1 , A. Chakhoyan1 , D. Divoux1 , J. Toutain1 , M. Bernaudin1 , S. Valable1 , E. Petit1 . 1 CNRS UMR-6301 ISTCT − Universite´ de Caen Basse-Normandie UMR6301-ISTCT CERVOxy team. CEA DSV/I2BM UMR6301-ISTCT CERVOxy team − Normandie Univ, GIP CYCERON, Caen, France Background: Currently, patients with glioblastoma (GBM) have a median life expectancy lower than 15 months despite optimized conventional treatment combining surgery with chemo- and radiotherapy (RT). A pathophysiological hallmark for these tumors is hypoxia leading to a highly abnormal vasculature, contributing to limit the efficacy of RT. In response to RT, it has been proposed that white matter (WM) disturbances, at the origin of cognitive disorders in these patients, could be also a result of vascular alterations. However, radiation-induced changes in tumor blood vessels as well as tumor stroma sensitivity are known to be highly variable depending on RT protocols (fractionated or not) and relationships between the cellular responses of the neurovascular unit are complex and difficult to study. Accordingly, the aim of this project was to provide, with the use of multiparametric MRI, a longitudinal preclinical study enabling the evaluation of changes of tumor growth, tumoral endothelium and WM in response to RT. Material and Methods: An orthotopic human GBM model developed on athymic rats by an intrastriatal injection of U251 cells (5×104 cells/3 mL) was