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

• T98G: GBM, fibroblast morphology – In vitro growth parameters characterised– Genotype: mut. PTEN, p53, p16, p14ARF– Data on x-rays and TMZ (fewer on e )– No data on ions

• LN229: GBM, epithelial morphology– Unsatisfactory in vitro characterisation– Genotype: PTEN wt; mut. p53, p16, p14ARF– Known response to x-rays and TMZ (scant on /– Preliminary studies on combined TMZ and 12C

• 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

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)LN229 growth curve

Time (h)

0 50 100 150 200 250

Cel

l den

sity

(ce

lls/c

m2 )

103

104

105

106

U87 MG growth curve

Time (h)

0 50 100 150 200 250 300

Cel

l den

sity

(ce

lls/c

m2 )

103

104

105

106

T98G growth curve

Time (h)

0 20 40 60 80 100 120 140 160 180

Ce

ll d

en

sity

(ce

lls/c

m2)

103

104

105

Growth curves (Milan)

0 20 40 60 80 100 120103

104C

ell

de

nsi

ty (

cell/

cm2)

Time (h)

LN229 TD= 25+/-2,5h

0 20 40 60 80 100 120

104

105

Ce

ll d

en

sity

(ce

lls/c

m2)

Time (h)

T98G TD= 24+/-2 h

0 20 40 60 80 100 120103

104

Cel

l den

sity

(ce

ll/cm

2)

Time (h)

U373MG TD=23+/-3 h

0 20 40 60 80 100 120103

104

U87MG TD = 28+/-2 h

Ce

ll d

en

sity

(ce

ll/cm

2)

Time (h)

Growth parameters (measured in Naples)

Cell line

Lag time(h)

Td (h)

High (1)

densityLow (2)

densityHigh

densityLow

density

T98G 6 6 22 ± 2 27 ± 1

LN229 2 18 33 ± 1 33 ± 2

U87MG 15 20 25 ± 4 29 ± 1

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

1: ~1.2 103 cells/cm2 2: ~ 6 102 cells/cm2

Cellular sensitivity to DMSO and TMZ (Naples)

T98G

Time (h)

0 1 2 3 4

P.E

.

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

ControlloTMZ 50MDMSO

U87 MG

Time (h)

0 1 2 3 4

P.E

.

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

ControlloTMZ 50 MDMSO

LN229

Time (h)

0 1 2 3 4

P.E

.

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

ControlTMZ 50MDMSO

Plating efficiency of LN229 (a) and U373 (b) cells exposed to DMSO (0.02 -1 %) for 1 and 3 hours .

a) b)

0 1 2 3 4 5 6 7 8 9 10 110,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

1,2LN 229 1 h

3 h

Pla

ting

Effi

cien

cy

DMSO concentration ( microl/ml)0 1 2 3 4 5 6 7 8 9 10 11

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

1,2

U 373 1 h 3 h

Pla

ting

effic

ienc

y

DMSO concentration (microl/ml)

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.

0 1 2 3 4

0,1

1

50M TMZ+0.05% DMSO 0.05% DMSO 50M TMZ

Pla

ting

eff

icie

ncy

exposure time interval (h)

LN229

0 1 2 3 4

0,1

1

20M TMZ+0.02% DMSO 0.02% DMSO 20M TMZ

Pla

ting e

ffic

iency

exposure time interval (h)

LN229

a) b)

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.

0 1 2 3 4

0,1

1

50M TMZ+0.05% DMSO 0.05% DMSO 50M TMZ

Pla

tin

g e

ffic

ien

cy

exposure time interval (h)

U373

0 1 2 3 4

0,1

1

20M TMZ+0.05% DMSO 0.02% DMSO 20M TMZ

Pla

ting

eff

icie

ncy

exposure time interval (h)

U373

a) b)

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.

0 1 2 3 4

0,1

1

50M TMZ+0.05% DMSO 0.05% DMSO 50M TMZ

Pla

ting

effic

ienc

y

exposure time interval (h)

T 98

0 1 2 3 4

0,1

1

20M TMZ+0.02% DMSO 0.02% DMSO 20M TMZ

Pla

ting

eff

icie

ncy

exposure time interval (h)

T 98

a) b)

Survival for T98G (Naples)

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

T98G dose-response to x-rays in the presence or absence of TMZ

Dose (Gy)

0 1 2 3 4 5 6 7 8 9

Surv

ivin

g fr

acti

on

0,0001

0,001

0,01

0,1

1

TMZ DMSO X-rays only

Survival for LN229 (Naples)

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

Survival of LN229 cells vs X rays dose with or without TMZ ( 50 microM )-DMSO concentration 0.05 % (Milan)

0 2 4 60,01

0,1

1

LN229 cell line

X X+DMSO X+DMSO+TMZ

Sur

vivi

ng fr

actio

n

Dose ( Gy )

Survival for U87MG

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

Pooled dose-responses to x-rays of human gliomas

Dose (Gy)

0 1 2 3 4 5 6 7 8 9 10

Sur

vivi

ng f

ract

ion

10-7

10-6

10-5

10-4

10-3

10-2

10-1

100

T98G LN 229 U87MG

MCF-7 breast cancer

Cumulative response to TMZ for x ray-irradiated gliomas

Dose (Gy)

1 3 5 7 90 2 4 6 8 10

SF

10-6

10-5

10-4

10-3

10-2

10-1

100

T98GLN229U87-MG

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)

* Peak value of the measured ion beam energy incident on the cell layer.

§ Evaluated from the ICRU tables for MS20 tissue (ICRU 1993).

Facility 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

High-LET exposure set-up

Cells

Mylar

Radiation

6 m

Plating efficiency of LN229 and U373 cells for conventional flasks and vessels with a mylar bottom

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

Pla

ting E

ffic

iency

LN229 cells Mylar polystyrene flasks

Control DMSO 0.05 %

TMZ50

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

Pla

tin

g E

ffic

ien

cy

U373 cells Mylar polystyrene flasks

Control DMSO0.05%

TMZ50

Schedule for 2010

- Further irradiations with 12C (LNL + LNS) -Clonogenic survival, apoptosis induction, cell-cycle analysis

- Data analysis and RBE calculation