Transcript A Biomechanical Comparison of Cancerous and Normal Cell

A Biomechanical Comparison of
Cancerous and Normal Cell
Plasma Membranes
Olivia Beane
Syracuse University
BRITE 2009
Introduction
• Plasma Membranes consist of neutral
phospholipids, creating a bilayer.
Introduction
• Normal cells internalize
anionic phospholipids
Cancer Res. 2002;62:6132–6140.
• Cancerous cells externalize
anionic phospholipids
• The externalization of anionic phospholipids in
cancerous cells may cause a variance from the
biomechanical properties of normal cell plasma
membranes.
=\
Objective
To test this theory, compare the biomechanical
properties of normal Human Bronchial Epithelial
cell plasma membranes (HBE4) and cancerous
Human Bronchial Epithelial cell plasma
membranes (H460), using optical tweezers.
Experimental Setup
Beam Expanders
Diode Pump Laser
Piezo Stage
Piezo Controller
Objective
Lens
Mirror
Adapted from Journal of Biomechanics, 40, 2007, 476- 480
Micromanipulator
Stage
Mirror
Experimental Setup
• Diode Pump Laser
– λ=1064 nm
• Objective Lens
– 100x resolution and 1.47 NA to cause higher
gradient for electrical field to form optical trap.
• Piezo Stage
– Controls fine movement.
Bead
Dish
Cell
Piezo Stage
Laser
Objective
The laser provides enough force to create an
optical trap where the sulfate-modified bead is
unable to escape.
Cell
+
-+ -
Bead
The negatively charged bead attaches to the
positively charged exterior of the cell when they
have been moved into contact with each other.
Bead
Tether
Cell
Piezo Stage
After 5-10 seconds, the Piezo stage moves the cell a controlled
distance away (10 μm, 15 μm, and 20 μm) from the bead and a
tether, nanotubule, is formed.
Cell
Piezo Stage
• The tether has elastic properties and relaxation tendencies.
Tested tether forces after no-delay, 1 minute delay, and 2
minute delay.
• The current at which the force of the tether exceeds that of the
optical trap, portrays the force of the tether.
Static Calibration and Force Measurements
Bead
Piezo Stage
A static calibration is done with each individual
media, due to viscosity variances.
Static Calibration and Force Measurements
Bead
Piezo Stage
The piezo staged is moved a set distance at chosen
velocities until it escapes the optical trap.
Static Calibration and Force Measurements
Bead
Piezo Stage
• This is known as the “escaping velocity”.
• The calibration continues at different currents to
determine their respective escaping velocities.
Stoke’s Law
F= 6πηνr
η= Dynamic Viscosity (known)
ν = Escaping Velocity
r = Bead Radius (known)
F = Viscous Drag Force
• Implement Stoke’s Law to convert the escaping
velocities to Viscous Drag Force.
• This calculates the force of the optical trap at each
current value.
Power Measurements
Power
vsPower
Current
August
10 RPMI
vs Current
0.25
2
Y =0.43118-0.09915 X+0.00493 X
B
Power
Meter Measurement
Polynomial Fit of Data1_B
Power (W)
0.20
0.15
0.10
0.05
0.00
13
14
15
16
17
18
Diode Current (A)
• To calibrate power values from specific currents,
a power meter was used.
Static Calibration and Force Measurements
Cancer Cell Media
Normal Cell Media
LHC-9
RPMI
Output Power (W)
Output Power (W)
• Each media’s viscosity differed, thus resulting
in varying calibration graphs.
No Delay Force Measurements
• The forces of cancerous cell tethers are larger
than those of normal cell tethers.
1 Minute Delay Force Measurements
Tether Force (pN)
August 11 HBE4/H460 1 Minute Delay Comparison
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18
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15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
HBE4
H460
10
12
14
16
18
20
Tether Length (m)
• The forces of normal cell tethers are larger than those
of the cancerous cell tethers.
2 Minute Delay Force Measurements
August 11 HBE4/H460 2 Minute Delay Comparison
14
HBE4
H460
12
Tether Force (pN)
10
8
6
4
2
0
-2
10
12
14
16
Tether Length (m)
18
20
Conclusions
• The no-delay forces generated by cancerous
cell tethers are larger than those generated by
normal cell tethers suggesting carcinoma cell
plasma membranes have higher elasticity than
normal cells.
• The opposite was true after 1 minute delay. This
suggests that the viscous properties of
carcinoma and normal cell membranes differ.
• No conclusions can yet be determined from the
2 minute delay results.
Future Directions
• Perform same experiment with dynamic
force measurements to obtain timeresolved force of plasma membrane.
• Use standing wave microscopy to
measure diameter of tether.
Acknowledgements
Thank you to…
Professor Anvari
The Anvari Lab
Jun Wang
Dr. Victor Rodgers