The Effects of Ionising Radiation on Cell Mechanics Greg Madden 13 th February

1. The Effects of Ionising Radiation on Cell Mechanics Greg Madden 13th February Supervisory Team: Dr. Mark Murphy; Dr. Francis Lilley Prof. Dave Burton

2. Contents • Who are you? • Project outline and Aims • Cell mechanics • Radiation experimental procedure • Baseline data • Radiation Results • Further experiments • Summary The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

3. Who am I? • BSc in Medical Biochemistry • Cell biology in normal and diseased states • Molecular and cellular pathways • Molecular studies of bacteria with UniLever • Gene expression evaluation of microorganisms inhabiting washing machines • Biofilm formation The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

4. TeRaFS Project • Feasibility study – is it possible? • Funding body - EPSRC The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

5. Project Outline • Aims • Investigate mechanical changes as cancer progresses • Determine the effects of ionising radiation on major mechanical structures • Exploit apparent mechanical weakness • Atomic Force Microscopy (AFM) • Measure force response of cells pre- and post-irradiation • Immunofluorescence staining & Laser Scanning Confocal Microscopy (LSCM) • Monitor changes in actin cytoskeleton organisation • DNA damage and biochemical assays • Determine underlying mechanism for loss of mechanical integrity The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

6. What is Cell Mechanics? • Cell Mechanics • Cytoskeleton – Actin filaments, Microtubules and intermediate filaments • Provides shape and structure to the cell • Vital role in many cellular processes • Influence of Cell Mechanics • Physical interaction with environment • Mechanotransduction – increasing importance on cell behaviour • Cell Mechanics and disease • Cancer cells exhibit reduced stiffness compared to normal cells The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

7. Radiation Source Sealed Strontium-90 β emitter Mimic therapeutic regimens i.e. Samples will receive ‘fractions’ daily until a total dosage is met The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

8. Experimental procedure -ve Control Repeat Results The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

9. 3 Day Experiment • samples received 1 gray (Gy) fractions – total dose 3Gy • AFM, Confocal analysis • 2 irradiated and 2 non-irradiated samples analysed per day • 1 irradiated undergoes AFM analysis plus –ve control • 1 irradiated undergoes staining procedure plus –ve control • Irradiation Chamber • 2cm thick Perspex box • Moveable stage for Strontium-90 • Drawer for sample presentation The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

10. Model cell lines • Prostate epithelia • Allow a comparison from normal to a range of cancer severity • Morphology between cell lines is markedly different • Epithelial cells are generally the first contact point of radiotherapy • Prostate cancers are regularly treated with radiotherapy • Morphology of cells differ despite originating from same tissue • Morphology effects cell mechanics • Cancer progression results in loss of control of morphology PC-3 DU145 PNT2 The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

11. Force Curve Generation • Deflection used to calculate indentation • Produces a force vs indentation plot • Standard Hertz model assumes linear response to force • Non-linear Hertz Hybrid • Three parameters – Eb, K1, K2 Laser PhotoDetector Z-piezo X-Y Stage Objective Lens The Effect of Ionising Radiation on Cell Mechanics 18th February 2011 Force vs Indentation curve fit. Force/N Indentation/m

12. Baseline Data • Blue – PNT2 • Green – DU145 • Red – PC-3 The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

13. Results 1 Gy Fraction – PNT2 vs Control – Zero delay Control = Red X Exposed = Green O The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

14. Results 2 Gy Fraction - PNT2 vs Control - Zero delay Control = Blue X Exposed = Red O The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

15. Results • 3 Gy Fraction - PNT2 vs Control - Zero delay Control = Red X Exposed = Green O The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

16. Results 10 Gy singular dose PNT2 vs Control (no dose) Zero delay Control = Red O Exposed = Blue X The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

17. Results 10 Gy singular dose PNT2 vs Control (no dose) 24hr delay Control = Red O Exposed = Blue X The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

18. Results 10 Gy singular dose PNT2 vs Control (no dose) 2 - 24hr delay Control = Green X Exposed = Red X The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

19. Summary of Results • 1 Gy fractions • associated increased stiffness with irradiation • most significant at 3 Gy • 10Gy singular dose • immediate analysis showed no significant difference • 24 hr delay • 2-24hr delay following 10Gy exposure • suggestion of effect up to 4 hrs • recovery The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

20. Further work • Reproducible • is the pattern seen with repeated experiments? • Compare and contrast with other cell lines • do we see similar pattern • are cancer cell lines effected differently • Confocal analysis • cytoskeleton organisation • Analyse at different indentations • compare force response • Understand underlying mechanism • DNA damage assay The Effect of Ionising Radiation on Cell Mechanics 18th February 2011

21. Fin Questions, Questions, Questions. The Effect of Ionising Radiation on Cell Mechanics 18th February 2011