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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

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


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


Preoperative 131 i mibg treatment 3 year old neuroblastoma stage iv patient pa voute 2001

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


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

Bystander Effects


Biological effects of ionising radiation the traditional perspective
Biological effects of ionising radiation: adjacent cellsthe traditional perspective


Manifestation of radiation-induced adjacent cells

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


Are RIBBEs significant in targeted radiotherapy? adjacent cells

How do we investigate RIBBE in our gene

therapy/targeted radiotherapy scheme?

  • - transfectant mosaic spheroids

  • - media transfer experiments

p53


Non adjacent cells-mosaic multicellular spheroids

100% GFP-expressing cells

100% GFP-non-expressing cells


Transfected mosaic spheroids derived from the human glioma cell line uvw
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).


3 types of therapeutic decay particle cell line UVW

Radionuclidedecay particle rangeUse

123I Auger electrons 10 nm imaging

131I beta particles 800 m therapy

211At alpha particles 60 m therapy


NH cell line UVW

NH

N H2

211At

[211At]astatine

NH

NH

NH2

131I

[131I]MIBG

meta-iodobenzylguanidine

[211At]MABG

meta-astatobenzylguanidine


Survival of nat gene transfected uvw spheroids after treatment with 211 at mabg
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


24h treatment with [

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


Groups evaluated treatment with [

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


RIBBE after treatment with [-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


Direct + indirect treatment with [

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


RIBBE elicited by treatment with Auger electron emitter ([ treatment with [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


Direct + indirect treatment with [

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


Direct + indirect treatment with [

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


RIBBE following exposure of EJ138 cells to Auger electron treatment with [

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


Conclusion

Conclusion treatment with [

  • 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 treatment with [

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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