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 GERI Bimolecular Sciences and Pharmacy
Feasibility study – is it possible?
Funding body - EPSRC
GERI
Bimolecular Sciences and Pharmacy
University of Central Lancashire
Christie Hospital
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
Prepare samples for exposure
Expose to Radiation
Prepare samples for analysis
Analyse effects of treatment against no treatment
Results
-ve Control
Repeat
Results
The Effect of Ionising Radiation on Cell Mechanics
18th February 2011

9. Irradiation Chamber 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
Epithelial Cell lines
Designation
Source
Disease state
Characteristics
PC-3
Prostate
Carcinoma
High metastatic potential
DU145
Adenocarcinoma
Low metastatic potential
PNT2
Normal
Normal 
PC-3
DU145
Does loss of morphology control effect cytoskeleton organisation?
Do mechanical properties differ between cell lines?
Morphological differences between single and monolayer cells.
Growing cells in a monolayer may provide more uniform morphology for force measurements & cytoskeleton organisation
In vivo epithelial cells naturally form a monolayer – better representation of in vivo effects
Is a difference in the mechanical properties between single cells and cell growing in a monolayer?
The cytoskeleton is important for mechanical structure it connects at cell boundaries to help tissue withstand stress
PNT2
The Effect of Ionising Radiation on Cell Mechanics
18th February 2011

11. Force vs Indentation curve fit.
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
Z-piezo
Laser
Photo Detector
Objective Lens
X-Y Stage
Force/N
Indentation/m
Force vs Indentation curve fit.
Extended (Quadratic) Hertzian model, results in closer curve fit than the smaller image (standard Hertzian mdoel fit), assumes constant response to stress.
This indicates that stress response in cells is not linear.
The Extended Hertzian model provided a good fit over the cells under investigation.
The Effect of Ionising Radiation on Cell Mechanics
18th February 2011

12. Baseline Data The Effect of Ionising Radiation on Cell Mechanics
Blue – PNT2
Green – DU145
Red – PC-3
Cell line
Mean Force(N)
PNT2
1.1 x 10-8
DU145
6.7 x 10-9
PC3
4.2 x 10-9
The Effect of Ionising Radiation on Cell Mechanics
18th February 2011

13. 1 Gy Fraction – PNT2 vs Control – Zero delay
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. 2 Gy Fraction - PNT2 vs Control - Zero delay
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. 3 Gy Fraction - PNT2 vs Control - Zero delay
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. 10 Gy singular dose PNT2 vs Control (no dose) Zero delay
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. 10 Gy singular dose PNT2 vs Control (no dose) 24hr delay
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. 10 Gy singular dose PNT2 vs Control (no dose) 2 - 24hr delay
Results
10 Gy singular dose PNT2 vs Control (no dose) hr 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. Questions, Questions, Questions.
Fin
Questions, Questions, Questions.
The Effect of Ionising Radiation on Cell Mechanics
18th February 2011