1. Force Microscopy of Nonadherent Cells: A Comparison of Leukemia Cell Deformability Rosenbluth, Lam, and Fletcher (2006) Presented by: Aisha Bobb-Semple 20.309 November 20, 2008

2. Background  Leukemia causes decreased deformability and increased adhesion in myeloblasts and lymphoblasts.  This can lead to leukostasis – cell aggregation in vasculature.  Leukostasis can cause intracranial hemorrhages, respiratory failure, and ultimately death.  More knowledge about deformability is necessary for a better understanding of the disease.

3. Examples of Leukostasis http://tbn0.google.com/images?q=tbn:QtokgQOY0qW2ZM:http://content.nej m.org/content/vol357/issue16/images/medium/10f1.gif http://tbn0.google.com/images?q=tbn:hazJjIDkN2f6oM:http://www.med- ed.virginia.edu/courses/path/innes/images/wcdjpeg/wcd%2520leukostasis %2520x40a.jpeg

4. Goals  Develop a technique for probing non- adherent cells.  Develop a model for characterization mechanical properties.  Compare the deformability of: Acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) cell lines, and human neutrophils

5. Use of AFM for measuring stiffness  Cells are usually firmly adhered to a substrate  Cell is indented by a cantilever at a constant rate  Deflection of the cantilever is measured and related to force.  Linear elastic model of cell based on Hertzian mechanics is commonly used to determine elasticity. http://tbn0.google.com/images?q=tbn:JVssYYJ1qxghVM:http://www.biologicalprocedures.com/bpo/arts/1/67/m67f5sm.jpg

6. Use of AFM for measuring stiffness  Leukocytes are non- adhesive.  One solution is to coat the surface of the substrate with polypeptides  Microfabricated wells were developed to immobilize the cells

7. Model for Characterization Which of two models best describes deformability of the cells? At what deformation rates are viscous contributions from the cytoplasm of the cell minimized?

8. Model Comparison Hertzian Mechanics Model  Indentation is related to the load  Piezo position related to cantilever deflection Liquid Droplet Model  Used predominantly in micropipette experiments  Difference in pressure related to cortical tension which is then related to deflection of the cantilever

9. Model Comparison  Both models were fit to experimental data  Mean squared error for the Hertz model was lower than the liquid drop model  Hertzian model used for cell stiffness comparison

10. Optimal Deformation Rate? Stiffness remained constant at and below 415nm/s for AML cells. (948 nm/s for ALL cells and 501nm/s for neurtrophils)

11. Comparison of Deformability  AML cells found to be significantly stiffer than others.  This is not surprising since leukostasis occurs in much higher frequency in AML.  Common for clinicians to accept that AML cells are more rigid than ALL and neutrophils.

12. Summary  Developed microwells to immobilize non-adherent cells for atomic force microscopy.  Determined that the Hertz model best fits the data.  Determined that AML cells were significantly stiffer than ALL cells and neutrophils  Consistent with finding that myeloid leukemias result in leukostasis more frequently than lymphoid leukemias do.

13. QUESTIONS?? ~Thank you for your attention~