targeted radiotherapy: microgray doses and the bystander effect robert j. mairs, 1 natasha e....
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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
NeuroblastomaNeuroblastoma
• 2nd commonest solid tumour in children
• peak age < 2 years
• mostly disseminated at 1st presentation: poor prognosis
• rapid growth
• chemo/radiosensitive
Meta-iodobenzylguanidine(MIBG)
NH
NH
NH2
I
HO NH2
AdrenalineHO
Noradrenaline
HO NH2
HO
OH
Guanethidine
NH2
HN
NHN
Catecholamines, adrenergic neurone blockers Catecholamines, adrenergic neurone blockers and MIBG and MIBG
Noradrenaline transporter
Preoperative [Preoperative [131131I]MIBG treatmentI]MIBG treatment
3 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
For mets but..
Bystander Effects
• 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
Biological effects of ionising radiation: Biological effects of ionising radiation: the traditional perspectivethe traditional perspective
Transfer of medium
Transfer of serum
cell death
chromosomal aberrations
mutations
genomicinstability
Manifestation of radiation-induced biological bystander effects (RIBBE)
unirradiateunirradiateddcellscells
Esp low dose & low dose rate
p53
Are RIBBEs significant in targeted radiotherapy?
- transfectant mosaic spheroids
- media transfer experiments
How do we investigate RIBBE in our gene therapy/targeted radiotherapy scheme?
NonNon-mosaic multicellular spheroids-mosaic multicellular spheroids
100% GFP-expressing cells 100% GFP-non-expressing cells
Transfected mosaic Transfected mosaic spheroids derived from spheroids derived from the human glioma cell the human glioma cell line UVWline 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).
3 types of therapeutic decay particle3 types of therapeutic decay particle
Radionuclide decay particle range Use
123I Auger electrons 10 nm imaging
131I beta particles 800 m therapy
211At alpha particles 60 m therapy
[211At]astatine
[211At]MABGmeta-astatobenzylguanidine
[131I]MIBGmeta-iodobenzylguanidine
NH
NH
NH2
131I
NH
NH
N H2
211At
0.001
0.01
0.1
1
surv
ivin
g f
ract
ion
0 5 10 15 20
[211At]MABG conc. (kBq/ml)
543210
% NAT expressing cells% NAT expressing cells
Survival of NAT gene-transfected UVW spheroids after treatment with [211At]MABG
Greater TMS killing than attributable to physical cross fire, implicating RIBBE
Transfer of mediumTransfer of medium
Non-irradiated cellsNon-irradiated cellsRemove targeted radionuclideRemove targeted radionuclideReplace medium andReplace medium andIncubate for Incubate for 1hr1hr
After 1hr incubation, gamma count thetransferred medium and add activity,equivalent to that of radiopharmaceutical which leaked from cells, to a third flask -[= Activity control]
Clonogenic Assay
24h24h
DONOR RECIPIENT ACTIVITY CONTROL
Media transfer protocol
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
Groups evaluated
RIBBE after RIBBE after -irradiation of UVW/NAT cells
- 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
Recipient
Donor
Direct + indirect
su
rviv
ing
fra
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on
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0 1 2 3 4 5 6 7 8 9
dose (Gy)
0.01
0.1
1.0
su
rviv
ing
fra
cti
on
0 1 2 3 4 5 6 7 8 9
dose(Gy)
RIBBE after treatment with RIBBE after treatment with -emitter [-emitter [131131I]MIBG I]MIBG
Untransfected cells
Activity control
Direct + indirect
Donor
Recipient
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
su
rviv
ing
fra
cti
on
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0 1 2 3 4 5 6 7 8
[131I]MIBG activity conc. (MBq/ml)
RIBBE elicited by treatment with Auger electron emitter ([RIBBE elicited by treatment with Auger electron emitter ([123123I]MIBG) I]MIBG) and and -emitter ([-emitter ([211211At]MABG) - At]MABG) - high LEThigh LET
- 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
su
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on
0
0.10.20.30.40.50.60.70.80.91.01.1
0 1 2 3 4 5 6 7 8
Untransfected
Activity control
Recipient
Donor
Direct + Indirect
[123I]MIBG activity conc (MBq/ml)
su
rviv
ing
fra
cti
on
[211At]MABG activity conc (kBq/ml)
35302520151050
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0
RIBBE in a second cell line - EJ138 bladder carcinoma
- Cell line shows RIBBE in response to external beam irradiation- Recipient cells - dose response at low doses then plateau
0.01
0.1
1.0
su
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fra
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0 1 2 3 4 5 6 7 8 9Dose (Gy)
00.1
0.20.3
0.40.50.6
0.70.80.91.01.1
su
rviv
ing
fra
cti
on
0 1 2 3 4 5 6 7 8 9
Dose (Gy)
RIBBE after RIBBE after -irradiation
Recipient
Donor
Direct + indirect
RIBBE following exposure of EJ138 cells to -emitter -emitter [131I]MIBG
Effect similar to that observed in UVW cell line: - no U-shaped survival curve - RIBBE cell kill less than in donor cells
su
rviv
ing
fra
cti
on
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
[131I]MIBG activity conc. (MBq/ml)
0 1 2 3 4 5 6 7 8 9 10 11
Untransfected cells
Activity control
Direct + indirect
Donor
Recipient
RIBBE following exposure of EJ138 cells to Auger electron emitter ([123I]MIBG) and -emitter ([211At]MABG) - high LET
Similar to effect observed in UVW cell line: - U-shaped survival curves for RIBBE-kill
su
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ing
fra
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00.10.20.30.40.50.60.70.80.91.01.1
0 40
su
rviv
ing
fra
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[123I]MIBG activity conc (MBq/ml)
EJ138 parental
Activity control
Recipient
Donor
Direct + Indirect
00.10.20.30.40.50.60.70.80.91.0
1.1
0 1 2 3 4 5 6 7 8 9 10 11 453530252015105
[211At]MABG activity conc (kBq/ml)
RIBBE elicited by treatment with RIBBE elicited by treatment with -emitter ([-emitter ([211211At]MABG) At]MABG) and Auger electron emitter ([and Auger electron emitter ([123123I]MIBG) - I]MIBG) - high LEThigh LET
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
Acknowledgements
Glasgow UniversityMarie BoydRob MairsSusan Ross Tony McCluskeyKer Wei TanNatasha Fullerton
Duke UniversityMike ZalutskyPhil WelshGanesan VaidyanathanJennifer Dorrens
FundersCancer Research UKNIHNeuroblastoma Soc.Glasgow UniversityIan Sunter Res TrustGlasgow Cancer School
CollaboratorsHeinz BonishJohn BabichKevin PriseLez FairbairnNicol KeithSally PilmottMike Zalutsky