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