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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.

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TPS Status report– November 2009 Turin

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  1. Radiation Biology TaskRadiosensibilization of gliomas for hadron therapy INFN Sections of Milan and Naples TPS Status report– November 2009 Turin

  2. 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

  3. Working hypothesis Possible enhancement of cell killing by TMZ as a result of high-LET irradiation (12C e 1H)

  4. 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 • Genotype: p53 wt; mut. PTEN, p53, p16, p14ARF • Data on low-LET radioresponse

  5. 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)

  6. Cellular growth parameters in vitro • Initial lag phase • Exponential growth phase • Plateau

  7. Growth curves (Naples)

  8. Growth curves (Milan)

  9. Growth parameters (measured in Naples) 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

  10. Cellular sensitivity to DMSO and TMZ (Naples)

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

  12. 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)

  13. 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)

  14. 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)

  15. Survival for T98G (Naples)

  16. Survival for LN229 (Naples)

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

  18. Survival for U87MG

  19. MCF-7 breast cancer

  20. 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).

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

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

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

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