TPS Status report– November 2009 Turin 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 a e b) 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 a/b) 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 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)

Growth curves (Milan)

Growth parameters (measured in Naples) Cell line Lag time (h) Td High (1) density Low (2) High density Low T98G 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

Sensibilità cellulare al DMSO e al TMZ c/o Milano La sensibilità cellulare al solo DMSO e al TMZ+DMSO è misurata tramite saggio clonogenico che fornisce il parametro efficienza di piastramento (E.P.). Le cellule sono esposte a DMSO e TMZ+DMSO per tempi diversi: 0, 1, 2 e 3 h Concentrazioni DMSO: 2/1000 e 10/1000 Concentrazione TMZ: 50 μM (DMSO 2/1000)

Cellular sensitivity to DMSO and TMZ (Naples)

Plating efficiency of LN229 (a) and U373 (b) cells exposed to DMSO (0 Plating efficiency of LN229 (a) and U373 (b) cells exposed to DMSO (0.02 -1 %) for 1 and 3 hours . b) a)

Plating efficiency of LN229 cells exposed to a) 0 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)

Plating efficiency of U373 cells exposed to a) 0 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)

Plating efficiency of T98 cells exposed to a) 0 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)

Survival for T98G (Naples) α (Gy-1) ± SE β (Gy-2) ± SE X-rays 0.20 ± 0.07 0.11 ± 0.02 TMZ (50 mM) 0.05 ± 0.09 0.16 ± 0.05 DMSO 0.01 ± 0.07 0.13 ± 0.02

Survival for LN229 (Naples) α (Gy-1) ± SE β (Gy-2) ± SE X-rays 0,19 ± 0,02 0,065 ±0,003 TMZ (50 mM) 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)

Survival for U87MG α (Gy-1) ± SE β (Gy-2) ± SE X-rays 0,17 ± 0,14 0,13 ± 0,04 TMZ (50 mM) 0,5 ± 0,2 0,03 ± 0,04 DMSO 0,24 ± 0,08 0,06 ± 0,02

MCF-7 breast cancer

Attività previste nel secondo semestre 2009 Ulteriori curve clonogeniche per raggi X con e senza TMZ+DMSO (e DMSO) Esposizione a fasci di particelle (12C) ai LNL Risposta a raggi X con e senza TMZ+DMSO (e DMSO) Vitalità (apoptosi e MTT assay) Analisi del ciclo cellulare

Attività prevista per il 2° semestre 2009 - 1 - Ulteriori irraggiamenti con raggi X a Napoli e a Milano per morte clonogenica, apoptosi e ritardo della crescita con e senza TMZ+DMSO (e DMSO) Morte clonogenica radioindotta (NA + MI) misurata tramite saggio clonogenico. - Apoptosi radioindotta (NA) misurata tramite: - analisi al citofluorimetro, - misura dell’attività delle caspasi tramite Western blotting. - saggio dell’Annexina V, 

Attività prevista per il 2° semestre 2009 - 2 - Ritardo della crescita (MI) misurata tramite: - analisi citofluorimetrica del ciclo cellulare, - incorporazione della bromodeossiuridina.  - Analisi dati irraggiamenti con raggi X Curve di inattivazione analizzate con il modello lineare-quadratico SF = exp (- aD - bD2) per ricavare i parametri caratterizzanti la radiosensibilità cellulare a/b SF2 D10 SF2 2 D10

Incident energy* (MeV/amu) Incident LET§ (keV/m) 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 4.5 303 0.1

High-LET exposure set-up Cells 6 mm Mylar Radiation

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

Clonogenic survival, apoptosis induction, cell-cycle analysis Schedule for 2010 Further irradiations with 12C (LNL + LNS) Clonogenic survival, apoptosis induction, cell-cycle analysis - Data analysis and RBE calculation

Preventivo di spesa 2010 - Milano Consumo 15.0 k€ - Materiale monouso  per colture cellulari ( bottiglie, capsule di Petri, puntali sterili, provette , pipette , filtri per produzione acqua ultrapura, filtri per sterilizzazione terreni di coltura )  4.0 k€ - Terreni di coltura, antibiotici , soluzioni saline, siero fetale  4.0 k€ - Reagenti e materiali per irraggiamento ( alcool  etilico, colorante Giemsa, tripsina, EDTA, TMZ, Ioduro di Propidio e BrdU (per analisi al citofluorimetro del ciclo cellulare e del ritardo di crescita ); fogli di mylar  (da usare come substrato per contenitori per irraggiamento cellule), rivelatori plastici CR39. 7.0 k€ Missioni interne 9.0 k€ - Due persone per due turni (~7 giorni/turno) per anno (c/o LNL) 3.0 k€ - Due persone per tre turni (~7 giorni/turno) per anno (c/o LNS) 6.0 k€ - Missioni estere Un congresso internazionale (per 2 persone) 4.0 k€ Totale 28.0 k€

Preventivo di spesa 2010 - Napoli Consumo 20.00  16.00 k€ Materiale monouso (fiasche, capsule, provette, pipette, puntali, sistemi filtrazione membrana 0,2 μm), terreni per colture cellulari, soluzioni saline, reagenti chimici, kit filtri per sistema purificazione acqua, bombole CO2 10.00  8.00 k€ - Prodotti chimici per saggi funzionali al citofluorimetro 10.00  8.00 k€ Missioni interne 13.50 k€ - Due persone per due turni (~7 giorni/turno) c/o LNL 4.00 k€ - Due persone per tre turni (~7 giorni/turno) c/o LNS 6.00 k€ - Riunioni della collaborazione (4 riunione per anno per 2 persone) 3.00 k€ Missioni estere 5.50 k€ - Un congresso internazionale (per 2 persone) 4.00 k€ Un corso IBA per una persona 1.50 k€ Totale 39.00  35.00 k€

Team for 2010 Milano Napoli