Transcript Misure di radiosensibilizzazione di gliomi per adroterapia

Radiation Biology Task
Radiosensibilization of gliomas
for hadron therapy
INFN Sections of Milan and Naples
TPS Status report–
November 2009
Turin
Rationale
•
Glioblastoma multiforme (GBM) is the most aggressive of the gliomas, a collection of tumors arising
from glia or their precursors within the CNS
•
GBM is also the most common in humans
– unfavourable prognosis
– marked radioresistance
•
•
Current approach: alkylating agent temozolomide (TMZ) in combination with conventional RT
Alkylating agents work by 3 different mechanisms all of which achieve the same end result disruption of DNA function and cell death
– Alkyl groups are attached to DNA bases. This results in the DNA being fragmented by repair
enzymes as they attempt to replace the alkylated bases. Alkylated bases prevent DNA synthesis
and RNA transcription from the affected DNA.
– Formation of cross-bridges: Cross-linking prevents DNA from being separated for synthesis or
transcription
– Induction of mispairing of the nucleotides leading to mutations
TMZ is usually dissolved in DMSO, (CH3)2SO
– Crosses membranes leaving cells unharmed.
– Known cryopreservant and radioprotector (radical scavanger)
•
•
Suitable for hadron therapy
Working hypothesis
Possible enhancement of cell killing by
TMZ as a result of high-LET
irradiation (12C e 1H)
Cell lines
•
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T98G: GBM, fibroblast morphology
In vitro growth parameters characterised
Genotype: mut. PTEN, p53, p16, p14ARF
Data on x-rays and TMZ (fewer on a e b)
No data on ions
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LN229: GBM, epithelial morphology
Unsatisfactory in vitro characterisation
Genotype: PTEN wt; mut. p53, p16, p14ARF
Known response to x-rays and TMZ (scant on a/b)
Preliminary studies on combined TMZ and 12C
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U87 MG: glioblastoma-astrocytoma, epithelial morphology
In vitro characterisation
Genotype: p53 wt; mut. PTEN, p53, p16, p14ARF
Datia on response to low LET and neutrons
Preliminary studies on combined action of TMZ and12C
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U373: glioblastoma-astrocytoma, epithelial morphology
Characterised in vitro
Genotype: p53 wt; mut. PTEN, p53, p16, p14ARF
Data on low-LET radioresponse
Work done (march-november 2009)
• In vitro characterisation of GBM cell lines
– Growth curves
– Cellular response toTMZ and vehicle (DMSO) alone
• Cellular radiosensitivity following x-rays alone or
combined withTMZ
– Dose-response curves (clonogenic assay)
Cellular growth parameters in vitro
• Initial lag phase
• Exponential growth phase
• Plateau
Growth curves (Naples)
U87 MG growth curve
LN229 growth curve
106
2
Cell density (cells/cm )
2
Cell density (cells/cm )
106
105
104
103
104
103
0
50
100
150
200
250
Time (h)
105
2
104
103
0
20
40
60
80
Time (h)
0
50
100
150
Time (h)
T98G growth curve
Cell density (cells/cm )
105
100
120
140
160
180
200
250
300
Growth curves (Milan)
5
10
Cell density (cells/cm2)
2
Cell density (cell/cm )
T98G TD= 24+/-2 h
4
10
4
10
U373MG TD=23+/-3 h
3
10
0
20
40
60
80
100
0
120
20
40
80
100
120
100
120
2
Cell density (cell/cm )
2
Cell density (cell/cm )
60
Time (h)
Time (h)
4
10
4
10
LN229 TD= 25+/-2,5h
U87MG TD = 28+/-2 h
3
3
10
10
0
20
40
60
Time (h)
80
100
120
0
20
40
60
Time (h)
80
Growth parameters (measured in Naples)
Cell
line
Lag time
(h)
Td
(h)
High (1)
density
Low (2)
density
High
density
Low
density
T98G
6
6
22 ± 2
27 ± 1
LN229
2
18
33 ± 1
33 ± 2
U87MG
15
20
25 ± 4
29 ± 1
1:
~1.2 103 cells/cm2
2: ~ 6 102 cells/cm2
 Td
 Good agreement with literature for T98G
 In a paper a Td= 24 was reported for LN229 (which agrees best with Milan)
 For U87MG values greater than 40h have been recorded by others
Cellular sensitivity to DMSO and TMZ (Naples)
U87 MG
LN229
0,8
0,8
0,7
0,7
0,7
0,6
0,6
0,6
0,5
0,5
0,5
0,4
P.E.
0,8
P.E.
P.E.
T98G
0,4
0,4
0,3
0,3
0,3
0,2
0,2
0,2
0,1
0,1
0,1
0,0
0,0
0
1
2
3
4
0,0
0
1
Time (h)
Controllo
TMZ 50 M
DMSO
2
3
4
0
Time (h)
Controllo
TMZ 50 M
DMSO
1
2
Time (h)
Control
TMZ 50M
DMSO
3
4
Plating efficiency of LN229 (a) and U373 (b) cells
exposed to DMSO (0.02 -1 %) for 1 and 3 hours .
b)
a)
LN 229
1h
3h
1,2
1,2
1,1
1,1
1,0
1,0
0,9
Plating efficiency
0,9
Plating Efficiency
1h
3h
U 373
0,8
0,7
0,6
0,5
0,4
0,3
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,2
0,1
0,1
0,0
0,0
0
1
2
3
4
5
6
7
8
DMSO concentration ( microl/ml)
9
10
11
0
1
2
3
4
5
6
7
8
DMSO concentration (microl/ml)
9
10
11
Plating efficiency of LN229 cells exposed to a) 0.05% DMSO, 50 M
TMZ + 0.05% DMSO, 50 M TMZ; b)0.02% DMSO, 20 M TMZ +
0.02% DMSO, 20 M TMZ, vs exposure time interval.
b)
a)
1
1
LN229
50M TMZ+0.05% DMSO
0.05% DMSO
50M TMZ
0,1
Plating efficiency
Plating efficiency
LN229
20M TMZ+0.02% DMSO
0.02% DMSO
20M TMZ
0,1
0
1
2
3
exposure time interval (h)
4
0
1
2
3
exposure time interval (h)
4
Plating efficiency of U373 cells exposed to a) 0.05% DMSO, 50 M
TMZ + 0.05% DMSO, 50 M TMZ; b)0.02% DMSO, 20 M TMZ +
0.02% DMSO, 20 M TMZ, vs exposure time interval.
a)
b)
1
1
U373
Plating efficiency
Plating efficiency
U373
0,1
0,1
50M TMZ+0.05% DMSO
0.05% DMSO
50M TMZ
0
1
2
3
exposure time interval (h)
20M TMZ+0.05% DMSO
0.02% DMSO
20M TMZ
4
0
1
2
3
exposure time interval (h)
4
Plating efficiency of T98 cells exposed to a) 0.05% DMSO, 50 M TMZ
+ 0.05% DMSO, 50 M TMZ; b)0.02% DMSO, 20 M TMZ + 0.02%
DMSO, 20 M TMZ, vs exposure time interval.
b)
a)
1
Plating efficiency
Plating efficiency
1
T 98
T 98
0,1
0,1
20M TMZ+0.02% DMSO
0.02% DMSO
20M TMZ
50M TMZ+0.05% DMSO
0.05% DMSO
50M TMZ
0
1
2
3
exposure time interval (h)
4
0
1
2
3
exposure time interval (h)
4
Survival for T98G (Naples)
T98G dose-response to x-rays in the presence or absence of TMZ
Surviving fraction
1
α
(Gy-1) ± SE
β
(Gy-2) ± SE
X-rays
0.20 ± 0.07
0.11 ± 0.02
TMZ
(50 M)
0.05 ± 0.09
0.16 ± 0.05
DMSO
0.01 ± 0.07
0.13 ± 0.02
0,1
0,01
TMZ
DMSO
X-rays only
0,001
0,0001
0
1
2
3
4
5
Dose (Gy)
6
7
8
9
Survival for LN229 (Naples)
Dose response of LN229 to x-rays: with or without TMZ (plus DMSO)
1
α
(Gy-1) ± SE
β
(Gy-2) ± SE
X-rays
0,19 ± 0,02
0,065
±0,003
TMZ
(50 M)
0,64 ± 0,07
0,00 ±0,01
DMSO
0,37 ± 0,04
0,040 ±
0.007
Surviving fraction
0,1
0,01
TMZ
DMSO
x-rays only
0,001
0,0001
0
1
2
3
4
5
Dose (Gy)
6
7
8
9
10
Survival of LN229 cells vs X rays dose with or without
TMZ ( 50 microM )-DMSO concentration 0.05 % (Milan)
Surviving fraction
1
0,1
LN229 cell line
X
X+DMSO
X+DMSO+TMZ
0,01
0
2
4
Dose ( Gy )
6
Survival for U87MG
U87-MG dose response to x-rays in the presence or absence of TMZ
100
α
(Gy-1) ± SE
β
(Gy-2) ± SE
X-rays
0,17 ± 0,14
0,13 ± 0,04
TMZ
(50 M)
0,5 ± 0,2
0,03 ± 0,04
DMSO
0,24 ± 0,08
0,06 ± 0,02
Surviving fraction
10-1
10-2
10-3
x-rays only
TMZ
DMSO
10-4
10-5
0
1
2
3
4
Dose (Gy)
5
6
7
8
Pooled dose-responses to x-rays of human gliomas
100
10-1
Surviving fraction
10-2
10-3
T98G
LN 229
U87MG
10-4
MCF-7 breast cancer
10-5
10-6
10-7
0
1
2
3
4
5
Dose (Gy)
6
7
8
9
10
Cumulative response to TMZ for x ray-irradiated gliomas
100
10-1
SF
10-2
10-3
T98G
LN229
U87-MG
10-4
10-5
10-6
0
1
2
3
4
5
Dose (Gy)
6
7
8
9
10
Future work
-
12C
ion irradiation at LNL
- 12C ion beam well characterised
(Belli et al. - Effectiveness of Monoenergetic and Spread-Out Bragg Peak Carbon-Ions for Inactivation of
Various Normal and Tumour Human Cell Lines. J. Rad. Res. 2008)
Incident
energy*
(MeV/amu)
Incident
LET§
(keV/m)
Range§
(mm)
ALPI
19
94
1.2
Tandem
6.7
222
0.2
Tandem
4.5
303
0.1
Facility
* Peak value of the measured ion beam energy incident on the cell layer.
§ Evaluated from the ICRU tables for MS20 tissue (ICRU 1993).
High-LET exposure set-up
Cells
Mylar 6 m
Radiation
Plating efficiency of LN229 and U373 cells for conventional
flasks and vessels with a mylar bottom
LN229 cells
Mylar
polystyrene flasks
1,0
1,0
0,9
0,8
0,8
0,7
0,7
0,6
0,6
Plating Efficiency
Plating Efficiency
0,9
0,5
0,4
0,5
0,4
0,3
0,3
0,2
0,2
0,1
0,1
0,0
0,0
Control
DMSO
0.05 %
TMZ
50 
U373 cells
Mylar
polystyrene flasks
Control
DMSO
0.05%
TMZ
50
Schedule for 2010
irradiations with 12C (LNL + LNS)
-Clonogenic survival, apoptosis induction, cellcycle analysis
- Data analysis and RBE calculation
- Further