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
Neuroblastoma
• 2nd commonest solid
tumour in children
• peak age < 2 years
• mostly disseminated at
1st presentation: poor
prognosis
• rapid growth
• chemo/radiosensitive
Catecholamines, adrenergic neurone blockers
and MIBG
NH
HO
NH2
N
H
HO
Adrenaline
I
OH
HO
NH2
NH2
Meta-iodobenzylguanidine
(MIBG)
HO
Noradrenaline
N
H
N NH2
NH
Guanethidine
Noradrenaline transporter
For mets but..
Preoperative [131I]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
Bystander Effects
• Killing/damage of unirradiated 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:
the traditional perspective
Manifestation of radiation-induced
biological bystander effects (RIBBE)
Transfer of medium
cell death
unirradiated
cells
chromosomal
aberrations
mutations
genomic
instability
Transfer of serum
Esp low dose
& low dose
rate
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
Non-mosaic multicellular spheroids
100% GFP-expressing cells
100% GFP-non-expressing cells
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 cells
transfected with the NAT gene
(red).
3 types of therapeutic decay particle
Radionuclide
123I
131I
211At
decay particle
range
Use
Auger electrons
10 nm
imaging
beta particles
800 m
therapy
60 m
therapy
alpha particles
[211At]astatine
NH
NH
N
H
N
H
N
H2
131I
[131I]MIBG
meta-iodobenzylguanidine
N
H2
211At
[211At]MABG
meta-astatobenzylguanidine
surviving fraction
Survival of NAT gene-transfected UVW spheroids after
treatment with [211At]MABG
1
% NAT expressing cells
0.1
0
1
2
3
4
5
0.01
0.001
0
5
10
15
[211At]MABG conc. (kBq/ml)
Greater TMS killing than attributable to
physical cross fire, implicating RIBBE
20
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
DONOR
RECIPIENT
Remove targeted radionuclide Non-irradiated cells
Replace medium and
Incubate for 1hr
24h
Clonogenic Assay
ACTIVITY
CONTROL
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
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Direct + indirect
Donor
surviving fraction
surviving fraction
RIBBE after -irradiation of UVW/NAT cells
1.0
0.1
0.01
0 1 2 3 4 5 6 7 8 9
Recipient
0
1
2
3
4
5
6
7
8
dose(Gy)
9
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
RIBBE after treatment with -emitter [131I]MIBG
1.1
1.0
surviving fraction
0.9
0.8
Direct + indirect
0.7
Donor
0.6
Recipient
0.5
0.4
Activity control
0.3
Untransfected cells
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
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Direct + Indirect
Donor
Recipient
Activity control
Untransfected
0
0
1
2
[123I]MIBG
3
4
5
6
7
8
activity conc (MBq/ml)
surviving fraction
surviving fraction
RIBBE elicited by treatment with Auger electron emitter ([123I]MIBG)
and -emitter ([211At]MABG) - high LET
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
20 25
30
35
[211At]MABG activity conc (kBq/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
RIBBE in a second cell line - EJ138 bladder carcinoma
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Direct + indirect
Donor
Recipient
0
1
2
3
4
5
6
7
8
surviving fraction
surviving fraction
RIBBE after -irradiation
1.0
0.1
0.01
0 1 2 3 4 5 6 7 8 9
Dose (Gy)
9
Dose (Gy)
- Cell line shows RIBBE in response to external beam irradiation
- Recipient cells - dose response at low doses then plateau
RIBBE following exposure of EJ138 cells to -emitter [131I]MIBG
1.1
1.0
0.9
surviving fraction
0.8
Direct + indirect
0.7
Donor
0.6
Recipient
0.5
0.4
Activity control
0.3
Untransfected cells
0.2
0.1
0
0
1
2
3
4
5
6
7
8
9
10 11
[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
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Direct + Indirect
Donor
Recipient
Activity control
EJ138 parental
surviving fraction
RIBBE following exposure of EJ138 cells to Auger electron
emitter ([123I]MIBG) and -emitter ([211At]MABG) - high LET
0 1 2 3 4 5 6 7 8 9 10 11
[123I]MIBG activity conc (MBq/ml)
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5 10 15 20 25 30 35 40 45
[211At]MABG activity conc (kBq/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
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 University
Marie Boyd
Rob Mairs
Susan Ross
Tony McCluskey
Ker Wei Tan
Natasha Fullerton
Collaborators
Heinz Bonish
John Babich
Kevin Prise
Lez Fairbairn
Nicol Keith
Sally Pilmott
Mike Zalutsky
Duke University
Mike Zalutsky
Phil Welsh
Ganesan Vaidyanathan
Jennifer Dorrens
Funders
Cancer Research UK
NIH
Neuroblastoma Soc.
Glasgow University
Ian Sunter Res Trust
Glasgow Cancer
School