20 research outputs found
Contribution des effets ciblés et non ciblés en radioimmunothérapie alpha et Auger de carcinoses péritonéales
We investigated in vitro and in vivo the relative contribution of targeted and non-targeted effects in the therapeutic efficacy against tumors of antibodies radiolabeled with alpha particle (212Pb, 213Bi) or Auger electron (125I) emitters. Targeted effects occurs in cells directly crossed by ionising particles while non-targeted effects are measured in cells neighbouring irradiated cells. Targeted effects were measured in vitro in cells exposed to antibodies radiolabeled with alpha or Auger emitters (donor cells) while non-targeted effects were investigated in recipient cells. Recipient cells consisted of cells not exposed to radiolabeled-mAbs, but grown in medium previously incubated for 2h with donor cells. We showed that the relative contribution of targeted effects versus non-targeted effects was higher during alpha RIT than Auger RIT. Alpha particles produced 53BP1 and gamma-H2AX foci in donor cells that could be differentiated in large, medium and small foci, while only small foci were observed in recipient cells. We assumed that large foci would correspond to locally multiply damage sites in DNA. Conversely, Auger RIT led predominantly to non-targeted effects compared with targeted effects. Use of radical scavengers showed that oxidative stress was involved in non-targeted effects. In vivo, we showed in athymic nude mice bearing tumor xenograft that non-targeted effects were also involved and participated to therapeutic efficacy of radiolabeled antibodies. These results indicate that although producing single DNA lesion, non-targeted effects can contribute to the therapeutic efficacy of mAbs radiolabeled with alpha particle or Auger electron emitters. These findings are particularly relevant for targeted therapy in which vectors cannot gain access to every tumor cell.LâefficacitĂ© dâune radioimmunothĂ©rapie (RIT) peut impliquer la coexistence des effets ciblĂ©s et des effets dit « non ciblĂ©s ». Les effets ciblĂ©s regroupent les effets biologiques observĂ©s dans les cellules ou tissus traversĂ©s par les particules ionisantes alors que les effets non-ciblĂ©s (ou bystander) sont observĂ©s dans des cellules qui nâont pas Ă©tĂ© irradiĂ©es mais qui sont au proche voisinage des cellules exposĂ©es. Nous avons au cours de cette Ă©tude Ă©valuĂ© in vitro et in vivo, la contribution des effets ciblĂ©s et non ciblĂ©s dans lâefficacitĂ© obtenue lors de la RIT alpha (212Pb, 213Bi) et de la RIT Auger (125I). Les effets ciblĂ©s ont Ă©tĂ© mesurĂ©s in vitro sur les cellules irradiĂ©es (cellules donneuses) alors que les effets bystander sont mesurĂ©s sur les cellules non irradiĂ©es (cellules receveuses) par une mĂ©thode de transfert de milieu. Elle consiste, Ă traiter les cellules receveuses dans un milieu de culture prĂ©-incubĂ© pendant 2h avec les cellules donneuses. Nos rĂ©sultats montrent que la contribution des effets ciblĂ©s est nettement plus importante quâen RIT alpha quâAuger. En RIT alpha, on observe que les lĂ©sions de lâADN (foci 53BP1et Îł-H2AX) pourraient ĂȘtre diffĂ©renciĂ©es en lĂ©sions complexes (sites multilĂ©sĂ©s = observation de gros foci) ou simples lĂ©sions (petit foci). Par contre en RIT Auger, ce sont les effets non ciblĂ©s qui prĂ©dominent sur les effets ciblĂ©s. Lâutilisation dâinhibiteurs pharmacologiques des ROS montre lâimplication du stress oxydatif dans ces effets non ciblĂ©s observĂ©s en RIT alpha et Auger. Ces effets non ciblĂ©s ont Ă©tĂ© observĂ©s Ă©galement in vivo sur des souris athymiques porteuses de carcinoses pĂ©ritonĂ©ales de petites tailles ; dĂ©montrant ainsi leur contribution dans lâefficacitĂ© thĂ©rapeutique finale observĂ©e aprĂšs la RIT alpha et Auger. Lâensemble de ces rĂ©sultats indiquent que mĂȘme si des lĂ©sions de lâADN sont produites aprĂšs irradiation, que les effets non ciblĂ©s pourraient aussi contribuer Ă lâefficacitĂ© thĂ©rapeutique finale observĂ©e avec les anticorps couplĂ©s aux Ă©metteurs de particules alpha ou dâĂ©lectrons Auger. Ces rĂ©sultats sont particuliĂšrement intĂ©ressants pour la thĂ©rapie ciblĂ©e car les vecteurs utilisĂ©s nâont pas souvent accĂšs Ă lâensemble des cellules constituant la tumeur
Contribution of targeted and non-targeted effects in alpha and Auger radioimmunotherapy of peritoneal carcinomatosis.
LâefficacitĂ© dâune radioimmunothĂ©rapie (RIT) peut impliquer la coexistence des effets ciblĂ©s et des effets dit « non ciblĂ©s ». Les effets ciblĂ©s regroupent les effets biologiques observĂ©s dans les cellules ou tissus traversĂ©s par les particules ionisantes alors que les effets non-ciblĂ©s (ou bystander) sont observĂ©s dans des cellules qui nâont pas Ă©tĂ© irradiĂ©es mais qui sont au proche voisinage des cellules exposĂ©es. Nous avons au cours de cette Ă©tude Ă©valuĂ© in vitro et in vivo, la contribution des effets ciblĂ©s et non ciblĂ©s dans lâefficacitĂ© obtenue lors de la RIT alpha (212Pb, 213Bi) et de la RIT Auger (125I). Les effets ciblĂ©s ont Ă©tĂ© mesurĂ©s in vitro sur les cellules irradiĂ©es (cellules donneuses) alors que les effets bystander sont mesurĂ©s sur les cellules non irradiĂ©es (cellules receveuses) par une mĂ©thode de transfert de milieu. Elle consiste, Ă traiter les cellules receveuses dans un milieu de culture prĂ©-incubĂ© pendant 2h avec les cellules donneuses. Nos rĂ©sultats montrent que la contribution des effets ciblĂ©s est nettement plus importante quâen RIT alpha quâAuger. En RIT alpha, on observe que les lĂ©sions de lâADN (foci 53BP1et Îł-H2AX) pourraient ĂȘtre diffĂ©renciĂ©es en lĂ©sions complexes (sites multilĂ©sĂ©s = observation de gros foci) ou simples lĂ©sions (petit foci). Par contre en RIT Auger, ce sont les effets non ciblĂ©s qui prĂ©dominent sur les effets ciblĂ©s. Lâutilisation dâinhibiteurs pharmacologiques des ROS montre lâimplication du stress oxydatif dans ces effets non ciblĂ©s observĂ©s en RIT alpha et Auger. Ces effets non ciblĂ©s ont Ă©tĂ© observĂ©s Ă©galement in vivo sur des souris athymiques porteuses de carcinoses pĂ©ritonĂ©ales de petites tailles ; dĂ©montrant ainsi leur contribution dans lâefficacitĂ© thĂ©rapeutique finale observĂ©e aprĂšs la RIT alpha et Auger. Lâensemble de ces rĂ©sultats indiquent que mĂȘme si des lĂ©sions de lâADN sont produites aprĂšs irradiation, que les effets non ciblĂ©s pourraient aussi contribuer Ă lâefficacitĂ© thĂ©rapeutique finale observĂ©e avec les anticorps couplĂ©s aux Ă©metteurs de particules alpha ou dâĂ©lectrons Auger. Ces rĂ©sultats sont particuliĂšrement intĂ©ressants pour la thĂ©rapie ciblĂ©e car les vecteurs utilisĂ©s nâont pas souvent accĂšs Ă lâensemble des cellules constituant la tumeur.We investigated in vitro and in vivo the relative contribution of targeted and non-targeted effects in the therapeutic efficacy against tumors of antibodies radiolabeled with alpha particle (212Pb, 213Bi) or Auger electron (125I) emitters. Targeted effects occurs in cells directly crossed by ionising particles while non-targeted effects are measured in cells neighbouring irradiated cells. Targeted effects were measured in vitro in cells exposed to antibodies radiolabeled with alpha or Auger emitters (donor cells) while non-targeted effects were investigated in recipient cells. Recipient cells consisted of cells not exposed to radiolabeled-mAbs, but grown in medium previously incubated for 2h with donor cells. We showed that the relative contribution of targeted effects versus non-targeted effects was higher during alpha RIT than Auger RIT. Alpha particles produced 53BP1 and gamma-H2AX foci in donor cells that could be differentiated in large, medium and small foci, while only small foci were observed in recipient cells. We assumed that large foci would correspond to locally multiply damage sites in DNA. Conversely, Auger RIT led predominantly to non-targeted effects compared with targeted effects. Use of radical scavengers showed that oxidative stress was involved in non-targeted effects. In vivo, we showed in athymic nude mice bearing tumor xenograft that non-targeted effects were also involved and participated to therapeutic efficacy of radiolabeled antibodies. These results indicate that although producing single DNA lesion, non-targeted effects can contribute to the therapeutic efficacy of mAbs radiolabeled with alpha particle or Auger electron emitters. These findings are particularly relevant for targeted therapy in which vectors cannot gain access to every tumor cell
Contribution des effets ciblés et non ciblés en radiothérapie vectorisée alpha/Auger et en radiothérapie externe
International audienc
RÎle des effets non-ciblés dans la réponse des cellules de cancer ORL à la radiothérapie
International audienceObjectifs : Ăvaluer in vitro, la contribution des effets ciblĂ©s et non-ciblĂ©s (effet bystander) des rayonnements ionisants ainsi que les mĂ©canismes molĂ©culaires impliquĂ©s.MĂ©thodes : 2 lignĂ©es cellulaires de cancers ORL ayant des radiosensibilitĂ©s opposĂ©es ont Ă©tĂ© utilisĂ©es. Les effets non-ciblĂ©s ont Ă©tĂ© Ă©valuĂ©s en utilisant un protocole de transfert de milieu. Celui-ci consiste Ă transfĂ©rer le milieu des cellules irradiĂ©es (cellules donneuses) 2 heures aprĂšs irradiation sur des cellules non irradiĂ©es (cellules receveuses). Les effets cytotoxiques produits tels que les lĂ©sions de lâADN et la mort cellulaire ont Ă©tĂ© Ă©valuĂ©s.RĂ©sultats : Les cellules donneuses SCC61 prĂ©sentent une forte diminution de la survie cellulaire en rĂ©ponse Ă lâirradiation. Lorsque le milieu de ces cellules irradiĂ©es est transfĂ©rĂ© Ă des cellules SCC61 receveuses, une cytotoxicitĂ© significative est Ă©galement observĂ©e, tĂ©moignant de la prĂ©sence des effets non-ciblĂ©s. La prĂ©sence de lĂ©sions de lâADN dans les cellules receveuses, mesurĂ©es par la formation de cassures double brin (foci H2AX) et des micronoyaux, a permis de confirmer la prĂ©sence dâeffets non-ciblĂ©s. Le transfert de milieu entre les cellules SCC61 et les cellules radiorĂ©sistantes SQ20B a permis dâĂ©tablir que les cellules SQ20B pouvaient transmettre la signalisation bystander mais quâelles ne pouvaient pas rĂ©pondre aux signaux des cellules radiosensibles.La prĂ©sence de radeaux lipidiques, plateformes enrichies en cĂ©ramide, jouent un rĂŽle essentiel dans les effets non ciblĂ©s. En effet, dans les cellules SCC61, lâinhibition de la formation des radeaux lipidiques avec du MBCD (mĂ©thyl-bĂ©ta-cyclodextrine) annule lâeffet bystander.Conclusions : Ces rĂ©sultats permettent dâĂ©tablir que les effets non-ciblĂ©s contribuent Ă la mort cellulaire des cellules radiosensibles alors que leur absence rĂ©duirait lâefficacitĂ© dâun traitement par radiothĂ©rapie. De plus, la membrane cellulaire jouerait un rĂŽle majeur dans la transmission de ces effets
Involvement of ceramide enriched domains in the targeted and non-targeted effects of HNSCC cancer cells
International audienceHypothesis: We investigated the relative contribution of targeted and non-targeted effects in the response of human head and neck cancer (HNSCC) cells to photon irradiation as well as the molecular mechanisms involved. Methods: The SCC61 (radiosensitive) and SQ20B (radioresistant) cell lines were used. Targeted effects were measured in cells exposed to increased radiation doses (donor cells) while non-targeted effects were investigated in recipient cells. Recipient cells consisted of cells not exposed to X-rays, but grown in medium previously incubated for 2h with donor cells (conditioned medium). DNA double strand breaks (DNA DSBs) were measured in donor and recipient cells, using immunofluorescent detection of gamma-H2AX
Ceramide-Enriched Membrane Domains Contribute to Targeted and Nontargeted Effects of Radiation through Modulation of PI3K/AKT Signaling in HNSCC Cells
International audienceWe investigated the potential involvement of ceramide-enriched membrane domains in radiation-induced targeted and nontargeted effects using head and neck squamous cell carcinoma with opposite radiosensitivities. In radiosensitive SCC61 cells, the proportion of targeted effects was 34% and nontargeted effects killed 32% of cells. In contrast, only targeted effects (30%) are involved in the overall death of radioresistant SQ20B cells. We then demonstrated in SCC61 cells that nontargeted cell response was driven by the formation of the radiation-induced ceramide-enriched domain. By contrast, the existence of these platforms in SQ20B cells confers a permissive region for phosphatidylinositol-3-kinase (PI3K)/AKT activation. The disruption of lipid raft results in strong inhibition of PI3K/AKT signaling, leading to radiosensitization and apparition of nontargeted effects. These results suggest that ceramide-enriched platforms play a significant role in targeted and nontargeted effects during radiotherapy and that drugs modulating cholesterol levels may be a good alternative for improving radiotherapy effectivenes
Therapeutic efficacy of brief intraperitoneal radioimmunotherapy of ovarian cancer using 213Bi-anti MISRII antibodies
Hypothesis: We assessed in in vitro and in vivo models of ovarian cancer the therapeutic efficacy of 16F12 mAbs directed against Mullerian Inhibiting Substance type II receptor (MISRII) radiolabeled with 213Bi Methods: In vitro, both direct and bystander cytotoxic effects were measured using clonogenic assay and standard medium transfer protocol. Typically, Clonogenic survival was assessed in SK-OV-3 donor cells expressing MISRII and exposed for 90 min to 0.06-0.5MBq/mL of 16F12 213Bi-mAbs. Bystander cytotoxicity was measured in recipient cells grown in non-radioactive culture medium preconditioned for 2 hours in the presence of donor cells. DNA double strand breaks (DSBs) were measured in both donor and recipients cells using immunofluorescent detection of gamma-H2AX and of 53BP1. In vivo we explored in athymic nude mice bearing intraperitoneal (IP) MISRII-expressing AN3CA tumor the therapeutic efficacy of brief-intraperitoneal radioimmunotherapy (BIP-RIT, 12.95 - 37 MBq; 37MBq/mg) or of intraperitoneal RIT (IP-RIT; 2.96-12.95 MBq; 37MBq/mg) using 213Bi-16F12. BIP-RIT mimics hyperthermic intraperitoneal chemotherapy as used in clinic. It consists of intraperitoneal injection of high activities of radiolabeled mAbs followed 30 min later by wash of the peritoneal cavity with saline solution to remove unbound radioactivity. The biodistribution of radiolabeled antibodies following IP-RIT (12.95 MBq; 37MBq/mg) or BIP-RIT (37 MBq; 37MBq/mg) was assessed. Results: In vitro we showed in donor cells a strong direct cytotoxicity of 16F12 213Bi-mAbs. A significant bystander cytotoxicity was also measured in recipient cells. Genotoxic effects were also demonstrated as measured by the formation of DNA DSBs in both donor and recipient cells. In vivo, results of biodistribution indicated that tumour uptake of 213Bi-16F12 during BIP RIT was higher than after IP RIT. The tumour-to-blood uptake ratio was 9 versus 3, respectively, one hour post RIT while it decreased down to 3 and 1, respectively, three hours post-RIT. Finally, a similar delay in tumor growth was observed in mice treated with 12.95 MBq of 213Bi-16F12 following IP-RIT or treated with 37 MBq using BIP-RIT. Conclusions: We confirmed in vitro the therapeutic efficacy of newly developed 16F12 213Bi-mAbs. in vivo results indicate that similar therapeutic efficacy and lower toxicity could be obtained with BIP-RIT compared with IP-RIT. BIP-RIT could be a new tool in the therapy of peritoneal carcinomatosis.JRC.G.I.5-Advanced Nuclear Knowledg
Differential Formation of Stress Granules in Radiosensitive and Radioresistant Head and Neck Squamous Cell Carcinoma Cells
International audiencePurpose: Stress granules (SGs) are cytoplasmic aggregates in which mRNAs and specific proteins are trapped in response to a variety of damaging agents. They participate in the cellular defense mechanisms. Currently, their mechanism of formation in response to ionizing radiation and their role in tumor-cell radiosensitivity remain elusive.Methods and materials: The kinetics of SG formation was investigated after the delivery of photon irradiation at different doses to head and neck squamous cell carcinoma cell lines with different radiosensitivities and the HeLa cervical cancer cell line (used as reference). In parallel, the response to a canonical inducer of SGs, sodium arsenite, was also studied. Immunolabeling of SG-specific proteins and mRNA fluorescence in situ hybridization enabled SG detection and quantification. Furthermore, a ribopuromycylation assay was used to assess the cell translational status. To determine whether reactive oxygen species were involved in SG formation, their scavenging or production was induced by pharmacologic pretreatment in both SCC61 and SQ20B cells.Results: Photon irradiation at different doses led to the formation of cytoplasmic foci that were positive for different SG markers. The presence of SGs gradually increased from 30 minutes to 2 hours postexposure in HeLa, SCC61, and Cal60 radiosensitive cells. In turn, the SQ20B and FaDu radioresistant cells did not form SGs. These results indicated a correlation between sensitivity to photon irradiation and SG formation. Moreover, SG formation was significantly reduced by reactive oxygen species scavenging using dimethyl sulfoxide in SCC61 cells, which supported their role in SG formation. However, a reciprocal experiment in SQ20B cells that depleted glutathione using buthionine sulfoximide did not restore SG formation in these cells.Conclusions: SGs are formed in response to irradiation in radiosensitive, but not in radioresistant, head and neck squamous cell carcinoma cells. Interestingly, compared with sodium arsenite-induced SGs, photon-induced SGs exhibited a different morphology and cellular localization. Moreover, photon-induced SGs were not associated with the inhibition of translation; rather, they depended on oxidative stress
Deciphering the role of stress granules in the cellular response to radiation of HNSCC cancer cells
International audienceBackground: Stress granules (SG) are cytoplasmic aggregates to which translationally stalled mRNAs are localized in response to various cellular stress, including oxidative stress, UV irradiation and chemotherapeutic agents. These cytoplasmic scaffolds are required to cope with stress. So far, little is known regarding the assembly of these structures in the context of ionizing radiation, especially whether they contribute to radioresistance/radiosensitivity of tumor cells.Aim: We conducted a comparative study in HNSCC cell lines with the aim of characterizing SG in response to photon and carbon ion irradiation
Involvement of direct and indirect (bystander) cytotoxic effects in alpha-RIT of small volume peritoneal carcinomatosis using 213Bi- and ÂČÂčÂČPb-labeled mAbs
We investigated in vitro and in vivo the relative contribution of direct and indirect (bystander) effects in the therapeutic efficacy of 213Bi- and 212Pb-labeled mAbs used for treating small volume peritoneal carcinomatosis.JRC.G.I.5-Advanced Nuclear Knowledg