poster_final version

Differential response to matrix rigidity correlates with aggressive phenotype of breast cancer cells Ji Li, Yang Wu, Mohammad Ali Al-Ameen, and Gargi Ghosh

Department of Mechanical Engineering Rackham Graduate School

University of Michigan - Dearborn  Introduc*on  As a major component of tumor microenvironment, extracellular matrix (ECM) is believed to play an important role in cancer metastasis. Breast cancer is the second leading cause of cancer mortality in women, with the vast majority of the deaths resulting from metastatic tumors. As the compliance of the stroma within breast carcinomas is approximately 5-20 times more rigid than normal breast tissue, it has recently been postulated that local changes in matrix elasticity contribute significantly to the progression of the disease. However, it is unclear whether restoration of ECM elasticity to normal levels may benefit treatment prognosis. Such an understanding would require a systematic characterization of how cells sense and integrate abnormal ECM dynamics. Here, we report the fabrication and characterization of poly (ethylene glycol) (PEG) based hydrogel matrices of varying stiffness. These matrices were then utilized to investigate cell-material interactions in the context of matrix stiffening.

Materials  and  Methods  

Fabricate scaffold §  The pre-polymer solution, consisting of 10% PEG6kDA, 1% photo-initiator, and

5% gelatin methacrylate. §  Polymer matrix is made by exposure under UV light for multiple time, 2.5 min, 4

min and 6 min. §  Seeding non-treated MDA-MB-231 and treated cells on the matrixes and

incubated in 37℃ for 3 days. Compression test •  Incubate 3 mm thickness scaffolds in 1XPBS for 72 hrs after fabricated •  Carry out the compressive test data by using uniaxial testing machine at a loading

rate of 1.2 mm/min with a precision load up to 9 N •  Record the maximum strain and stress and calculate Young’s modulus from the

initial 10% compression

Cell Treatment •  Aspirate old media form flask •  Incubate cells with 5ml 10µM Y-27832(ROCK) of fresh media in 37℃ for 3hrs Morphology §  Cell area and aspect ratio (Width of cell/length of cell) is measured by AxioVision

Rel. 4.8 software Proliferation §  Aspirate old medium form each well and inject with 100µm fresh medium and

50µm activated-XTT solution which is made with 0.1ml activation reagent and 5ml XTT reagent.

§  Return the plate to the cell culture CO2 incubator for 5 hrs. §  Measure the absorbance of the wells containing the cells and the blank

background control wells at a wavelength between 475 nm using a microtiter plate reader.

Adhesion •  Seed 10K per well of cells on each matrix and incubate for 17hrs •  Wash the matrix by fresh medium three times •  Count the number of cell remaining on the scaffolds.

Protein Assay •  Add 5% Phalloidin solution on each matrix with fixed cells and put plate on shaker

with 37℃ for 2 hrs •  Wash with PBS 3 times •  Using fluorescent analogs the distribution of F-actin in cell can be investigated.

Goals   Results  Characteristics of Matrix

Morphology

Proliferation

Adhesion

Migration

Fluorescent  phalloidin  (green)  marking  ac6n  filaments  in  treated  and  non-­‐treated  cells  on  different  s6ffness  matrixes  

• Breast  cancer  cells  with  differen6al  aggressive  phenotype  respond  differently    to  matrix  rigidity  • Incuba6on   in   the   presence   of   ROCK   inhibitor   (Y-­‐27632)     reduces   ac6n   organiza6on,  adhesion,  and  prolifera6on  of  MDA-­‐MB-­‐231  cells  on  s6ffer  matrices  • Effect   of   matrix   rigidity   on   prolifera6on,   migra6on,   adhesion,   and   cytoskeletal  organiza6on  of  SkBr3  cells  will  be    inves6gated    

Conclusion/  Future  Studies  

§ Developing   and   characterizing   PEG   based   hydrogel   matrices   with   different   s6ffness   by  controlling  the  UV  exposure  6me    § Evalua6on  cellular  responses  to  matrix  rigidity  by  measuring  the  morphology,  prolifera6on,  adhesion  and  cytoskeletal  organiza6on  (phaloidin  staining).      

MDA

-­‐MB-­‐231  

SkBR

3      

17  kPa   21  kPa   25  kPa  

Shown  in  the  pictures  of  cell  morphology  ,  SKBR3  cells  do  not  have  significant  different  of  different  s6ffness,  So  we  concentrate  our  work  on  finding  the  rela6onship  between  treated  and  non-­‐treated    MDA-­‐MB-­‐231  cells.        

Structure of F-actin

Non

-­‐Treated

Treated

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ct  Ra*

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rowth  

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ROCK Inhibition of MDA-MB-231

17  kPa   21  kPa   25  kPa  

Acknowledgement  

Authors  would  like  to  thank  University  of  Michigan,  Dearborn  and  Office  of  Vice  President  of  Research,  University  of  Michigan,  Ann  Arbor  for  their  financial  support  

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Weight(g)

Days  of  incuba*on  in  PBS

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UV  exposure  *me  (min)