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Targeted radiotherapy:   microGray doses and the bystander effect

Targeted radiotherapy:   microGray doses and the bystander effect Robert J. Mairs , 1 Natasha E. Fullerton 1 , Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted Therapy Group, CRUK Beatson Labs, Glasgow, Scotland & 2 Dept of Radiology, Duke University, North Carolina, USA.

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Targeted radiotherapy:   microGray doses and the bystander effect

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  1. Targeted radiotherapy:   microGray doses and the bystander effect Robert J. Mairs,1 Natasha E. Fullerton1, Michael R. Zalutsky2 and Marie Boyd1 1Targeted Therapy Group, CRUK Beatson Labs, Glasgow, Scotland & 2Dept of Radiology, Duke University, North Carolina, USA

  2. Neuroblastoma • 2nd commonest solid • tumour in children • peak age < 2 years • mostly disseminated at • 1st presentation: poor • prognosis • rapid growth • chemo/radiosensitive

  3. OH HO NH2 HO NH2 HO HO Adrenaline Noradrenaline transporter NH Noradrenaline NH NH2 HN NH2 N I NH Meta-iodobenzylguanidine(MIBG) Guanethidine Catecholamines, adrenergic neurone blockers and MIBG

  4. For mets but.. Preoperative [131I]MIBG treatment3 year old neuroblastoma stage IV patient(PA Voute, 2001) • 3 consecutive treatments; interval = 4 weeks • 1st scan: bone and bone marrow invasion + large retroperitoneal tumour • 2nd scan: decreased uptake in bone, bone marrow and primary tumour • 3rd scan: bone and bone marrow cleared; primary tumour resectable

  5. Killing/damage of un-irradiated cells due to irradiation of adjacent cells Physical: radiation cross-fire, i.e., direct traversal Biological: direct traversal not required Bystander Effects

  6. Biological effects of ionising radiation: the traditional perspective

  7. Manifestation of radiation-induced biological bystander effects (RIBBE) Transfer of medium cell death chromosomal aberrations mutations genomic instability unirradiated cells Transfer of serum Esp low dose & low dose rate

  8. Are RIBBEs significant in targeted radiotherapy? How do we investigate RIBBE in our gene therapy/targeted radiotherapy scheme? • - transfectant mosaic spheroids • - media transfer experiments p53

  9. Non-mosaic multicellular spheroids 100% GFP-expressing cells 100% GFP-non-expressing cells

  10. Transfected mosaic spheroids derived from the human glioma cell line UVW The spheroids, ranging in size from 100 to 500 m diameter, are composed of mixtures of cells transfected with the GFP gene (green) and cellstransfected with the NAT gene (red).

  11. 3 types of therapeutic decay particle Radionuclidedecay particle rangeUse 123I Auger electrons 10 nm imaging 131I beta particles 800 m therapy 211At alpha particles 60 m therapy

  12. NH NH N H2 211At [211At]astatine NH NH NH2 131I [131I]MIBG meta-iodobenzylguanidine [211At]MABG meta-astatobenzylguanidine

  13. Survival of NAT gene-transfected UVW spheroids after treatment with [211At]MABG 1 % NAT expressing cells surviving fraction 0.1 0 1 2 3 0.01 4 5 0.001 20 5 10 15 0 [211At]MABG conc. (kBq/ml) Greater TMS killing than attributable to physical cross fire, implicating RIBBE

  14. 24h Clonogenic Assay Media transfer protocol After 1hr incubation, gamma count the transferred medium and add activity, equivalent to that of radiopharmaceutical which leaked from cells, to a third flask - [= Activity control] Transfer of medium ACTIVITY CONTROL RECIPIENT DONOR Remove targeted radionuclide Replace medium and Incubate for 1hr Non-irradiated cells

  15. Groups evaluated Direct + Indirect: incubate with MIBG 2h; wash; incubate to allow generation of “bystander poisons” (direct + bystander effect) Donor: as Direct + Indirect but remove medium then replace with fresh medium (~ direct effect only) Recipient: receive medium from Donor (~ bystander effect only) Activity control: to correct for small amount of [*I]MIBG leaked from Donor cells and transferred to Recipients Untransfected: wild-type UVW cells (do not express NAT) recipients of medium from MIBG-treated wild-type UVW donors

  16. RIBBE after -irradiation of UVW/NAT cells 1.1 1.0 1.0 0.9 0.8 surviving fraction 0.7 0.1 surviving fraction 0.6 0.5 Direct + indirect 0.4 0.01 Donor 0.3 0 1 2 3 4 5 6 7 8 9 0.2 Recipient dose(Gy) 0.1 0 7 8 9 0 2 3 4 5 6 1 dose (Gy) • This cell line demonstrates RIBBE after external beam irradiation • Direct irradiation is more cytotoxic than RIBBE at high dose • - RIBBE are most significant at low doses

  17. Direct + indirect Donor Recipient Activity control Untransfected cells RIBBE after treatment with -emitter [131I]MIBG 1.1 1.0 0.9 0.8 0.7 0.6 surviving fraction 0.5 0.4 0.3 0.2 0.1 0 0 1 2 3 4 5 6 7 8 [131I]MIBG activity conc. (MBq/ml) • Medium from irradiated cells very cytotoxic - dose response; • significant effect at high doses; RIBBE cell kill almost as potent as direct kill in cells treated with radiopharmaceutical D/R

  18. RIBBE elicited by treatment with Auger electron emitter ([123I]MIBG) and -emitter ([211At]MABG) - high LET 1.1 1.1 1.0 1.0 0.9 0.9 Direct + Indirect 0.8 0.8 surviving fraction 0.7 0.7 Donor surviving fraction 0.6 0.6 Recipient 0.5 0.5 Activity control 0.4 0.4 0.3 Untransfected 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 35 30 25 0 1 2 3 4 5 6 7 8 [211At]MABG activity conc (kBq/ml) [123I]MIBG activity conc (MBq/ml) • U-shaped survival curves for RIBBE-kill - i.e. dose-related cytotoxicity at low activity • concentration and diminishing bystander kill at higher activity concentration • RIBBE elicited by high LET targeted radionuclides result in cell kill of magnitude similar • to that caused by direct irradiation - hence potentially powerful therapeutic effect

  19. Direct + indirect Donor Recipient RIBBE in a second cell line - EJ138 bladder carcinoma RIBBE after -irradiation 1.0 1.1 1.0 0.9 surviving fraction 0.1 0.8 0.7 0.6 surviving fraction 0.5 0.01 0 1 2 3 4 5 6 7 8 9 0.4 Dose (Gy) 0.3 0.2 0.1 0 7 8 0 2 3 4 5 6 9 1 Dose (Gy) - Cell line shows RIBBE in response to external beam irradiation - Recipient cells - dose response at low doses then plateau

  20. Direct + indirect Donor Recipient Activity control Untransfected cells 10 11 0 1 2 3 4 5 6 7 8 9 RIBBE following exposure of EJ138 cells to -emitter [131I]MIBG 1.1 1.0 0.9 0.8 0.7 0.6 0.5 surviving fraction 0.4 0.3 0.2 0.1 0 [131I]MIBG activity conc. (MBq/ml) Effect similar to that observed in UVW cell line: - no U-shaped survival curve - RIBBE cell kill less than in donor cells

  21. RIBBE following exposure of EJ138 cells to Auger electron emitter ([123I]MIBG) and -emitter ([211At]MABG) - high LET 1.1 1.1 1.0 1.0 0.9 0.9 0.8 0.8 Direct + Indirect 0.7 0.7 surviving fraction Donor 0.6 0.6 surviving fraction 0.5 Recipient 0.5 0.4 0.4 Activity control 0.3 0.3 EJ138 parental 0.2 0.2 0.1 0.1 0 0 0 5 10 15 40 20 35 45 25 30 0 1 2 3 4 5 6 7 8 9 10 11 [211At]MABG activity conc (kBq/ml) [123I]MIBG activity conc (MBq/ml) RIBBE elicited by treatment with -emitter ([211At]MABG) and Auger electron emitter ([123I]MIBG) - high LET Similar to effect observed in UVW cell line: - U-shaped survival curves for RIBBE-kill

  22. Conclusion • RIBBE from targeted radionuclides appear distinct from external beam irradiation • dose response • LET dependent • U shaped survival curves • RIBBE from high LET radionuclides at low doses are more • cytotoxic than direct irradiation • Only cells which take up radiopharmaceutical produce RIBBE

  23. Acknowledgements Glasgow University Marie Boyd Rob Mairs Susan Ross Tony McCluskey Ker Wei Tan Natasha Fullerton Duke University Mike Zalutsky Phil Welsh Ganesan Vaidyanathan Jennifer Dorrens Collaborators Heinz Bonish John Babich Kevin Prise Lez Fairbairn Nicol Keith Sally Pilmott Mike Zalutsky Funders Cancer Research UK NIH Neuroblastoma Soc. Glasgow University Ian Sunter Res Trust Glasgow Cancer School

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