AcknowledgementsA special thanks to:

The Anvari Lab

Jun Wang

Instrumentation and Experimental Methods

• Culturing Methods

• H460: Cells are grown in RPMI medium containing 10% FBS. Cells adhere to flask walls.

•HBE4: Cells are grown in serum-free, LHC-9 medium containing cholera toxin (10 ng/ml ratio).

•Force Calibration• A 2 μm diameter sulfate modified fluorescent polystyrene microsphere was trapped at known currents. The piezo stage on which the plate resides was moved at set velocities, pushing media through the bead. The velocities at which the force of the media exceeded the optical trap were recorded and used in Stock’s law to generate viscous drag forces.

Stock’s Law

• η= Dynamic Viscosity (known)

ν = Solution Velocity

r = Bead Radius (known)

h = Distance between center of bead and coverslip

F = Viscous Drag Force

• Viscosity Measurement• Viscosities of both RPMI and LHC-9 complete growth mediums were measured using a Cannon- Fenske Routine Calibrated Viscometer at 25ºC, room temperature. Their kinematic values were calculated as 1.01325 x 10-6 m2/s for RPMI and 9.855 x 10-7 m2/s for LHC-9. Converting to dynamic required being multiplied by their respective densities.

• Force Measurements• A bead was optically trapped within close proximity of the cell. The cell was then manually maneuvered by a stage until in contact with the bead. After 5-10 seconds, the bead was moved 10, 15, or 20 μm away from the cell at a velocity of 1 μm/s. To test non-delay forces, as soon as the bead had completed its shift, the power was decreased until the force of the tether exceeded that of the optical trap and sprung back to the cell. To test the delay forces, once the bead had completed its shift from the cell, it was kept in place for times of 1 and 2 minutes. After these specific times, the power was then decreased until the bead sprung back to the cell.

• System Setup

A Biomechanical Comparison of Cancerous and Normal Cell Plasma Membranes

Olivia Beane1, Nima Khatibzadeh2, Sharad Gupta3, and Bahman Anvari31Department of Bioengineering, Syracuse University, Syracuse, NY 13210

2Department of Mechanical Engineering, UC Riverside, Riverside, CA 925063Department of Bioengineering, UC Riverside, Riverside, CA 92506

Abstract Cancer research can cover a wide spectrum of areas. One of which is focusing on the cellular mechanics of cancer cells to determine its variances from the mechanics of healthy cells, a useful tool for areas focusing on diagnostics. Because the plasma membrane is known to provide mechanical strength for the cell and maintain the internal structure of organelles, it is of importance to study. While healthy cells maintain a plasma membrane consisting of neutral phospholipids, they also internalize anionic phospholipids. It has recently been discovered that cancer cells differ in that they externalize these anionic phospholipids. This has been hypothesized to occur due to the environments in which cancer cells metastasize. This surplus of phospholipids in the plasma membrane may thus cause a variation in the mechanical properties of cancerous plasma membranes from those of healthy plasma membranes. Thus, this project was designed to:• Compare the biomechanics of cancerous and non-cancerous plasma membranes.• Determine what affects relaxation periods have on tether forces of both cell types. Optical tweezers were employed to compare the forces generated by tethers, nanotubules comprising the plasma membranes, of both healthy bronchial epithelial cells (HBE4) and bronchial epithelial cells plagued with carcinoma (H460). These cells were chosen for this particular study based on previous results proving that the externalization of anionic phosopholipids occurs in human non-small cell lung carcinoma.

ResultsConclusions

• After no delay, the forces generated by the canerous cell tethers (H460) were larger than those generated by the normal cell tethers (HBE4). This indicates that the bronchial carcinoma cell plasma membranes have higher elasticity than normal cell plasma membranes.

• After allowing for one minute relaxation, tether forces for cancerous cells were less than those tether forces of normal cells. This indicates that the viscous component of cancer cell plasma membranes and normal cell plasma membranes also differ.

Future Directions• Perform same experiment with dynamic force measurements to obtain time-resolved force of plasma membrane.

• Standing wave microscopy to measure diameter of tether.

Literature Cited1. Ran S, Downes A, Thorpe PE. Increased exposure of anionic phospholipids on the surface of tumor blood vessels. Cancer Res. 2002;62:6132–6140.2. Zhiwei Li et al, Membrane Tether Formation from Outer Hair Cells with

Optical Tweezers. Biophysical Journal- 1 March 2002 (Vol. 82, Issue 3, pp. 1386- 1395)

3. Ermilov et al, Journal of Biomechanics, 40, 2007, 476- 480

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