Benjamin Yanga, Sung Sik Hura,b, Yingxiao Wanga,b, and Shu Chiena,b
aDepartment of Bioengineering, bThe Institute of Engineering in Medicine, University of California at San Diego, La Jolla, CA 92093
1. 3D Traction Force Microscopy (3D-TFM): To determine cell-ECM forces
INTRODUCTION
Mechanical force can modulate cell functions
FAs mediate cell-ECM force (traction force)
AJs mediate cell-cell force (intercellular force)
2. Glioblastoma (GBM), Doxorubicin
1. Cells and their environment can communicate through mechanical interactions.
Force
AdherensJunction
(AJ)
Mechanosensing
Extracellular Matrix
Force
Mechanosensing
Focal Adhesion (FA)
Gene Expression Cell Responses
Adherent Cells
SPECIFIC AIMS
1. To develop a method to simultaneously monitor epigenetic modifications and intracellular stress in real-time
2. To quantify cell-ECM and cell-cell stresses of cancer cells in differentmechanical microenvironments
3. To investigate the molecular mechanisms by which cancer cells react tochemotherapeutics
METHODS
Image
Displacement
Traction Force
Cells on PAA
Image Processing
Confocal Microscopy
Finite Element Analysis
TFM Sequence Confocal Microscopy
Hur SS et al. Cell Mol Bioeng. 2009
TS: Traction StressJT: Junctional TensionIT: Intracellular Tension
Single Cell
Multiple Cells
TS
JT
IT
TS
TS
TS
TS
TS
∫∫ TS = 0
∫∫ TS + ∫ JT = 0
∫∫ TS + ∫ IT = 0
When a cell is isolated as a single, total sum of cell-ECM force should be balanced to be zero. TS can be determined from TFM.
When a cell is in contact with other cells, total sum of cell-ECM force and cell-cell force from neighboring cell should be balanced to be zero. Junctional tension (JT) can be determined from TS.
Intracellular tension (IT) can be determined similarly from TS by splitting the domain along an intracellular section line.
Hur SS et al. PNAS. 2012
2. 3D Intracellular Force Microscopy (3D-IFM) : To determine cell-cell and intracellular forces
CONCLUSIONSRESULTS
Finite Element Analysis
Computing force from deformation
Image ProcessingTracking Substrate Deformation
Progression of glioblastoma cells and mechanical-epigenetic behaviors
3. FRET Biosensors: To monitor tri-methylation of H3K9
u87 u87vIIISoft
u87 u87vIIIHard
CT DOX CT DOX CT DOX CT DOX
Ce
ll-EC
MIn
trac
ellu
lar
Doxorubicin is a common chemotherapeutic anti-mitotic drug
GBM are usually highly malignant cancerous astrocytes
u87 and u87vIII are GBM cell lines; u87vIII is a “sister” maligned line that overexpresses the EGFRvIII gene
Figure 1. Intracellular and cell-ECM forces for u87 and u87vIII cells on soft (1.7 kPa) and hard (21 kPa) gels. CT = control, D=doxorubicin for 30 min* p < 0.05, ** p<0.01, *** p < 0.001
u87 u87vIIISoft
u87 u87vIII
Hard
CT DOX CT DOX CT DOX CT DOX
Figure 2: H3K9me FRET biosensor signal intensity for u87 and u87vIII cells on soft (1.7 kPa) and hard (21 kPa) gels
a.u
PaCT DOX (30m)
u87
CT DOX (30m)
u87vIII
Figure 3: FRET imaging of u87 and u87vIII cells
REFERENCES
u87 and u87vIII cells respond differently to treatment with DOX
The more malignant u87vIII cells exhibit lower intracellular and cell-ECM stresses than u87 cells for soft and hard microenvironments with DOX
H3K9me trimethylation increases with DOX treatment on stiff and soft gels for both u87 and u87vIII cells except for u87 cells on soft gels
There is a correlation between H3K9 trimethylation and intraceullarstress in nuclear regions
Parekh A & Weaver AM, 2009, Cell Adhesion & MigrationJiang P, Mukthavavam R et al., 2014, Journal of Translational MedicineHur SS et al., 2009, Cellular and Molecular Bioengineering.Hur SS et al., 2012, PNASSeong J et al. 2009, Chemistry & biology
Doxorubicin intercalating DNA
FUTURE WORK
Develop method to directly quantify 3D intracellular forces
Explore the effect of different drugs on the epigenetic and mechanical responses of glioblastoma cells
**
*****
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20µm
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