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ECM Stiffness and Modulus as Independent Controllers of
Cancer Metastasis
Stiffness: Defined as F/ΔL where F is the force
applied to a material in a single direction and ΔL is
the change in length in the same direction. Stiffness
is an extrinsic property and is geometrically
dependent.
Modulus: Defined as (F/A)/(ΔL/L), where A is the
cross sectional area through which the force is
applied and L is the original length of the object.
Modulus is a normalized form of stiffness thus
making it an intrinsic property (independent of
geometry).
Ligand Availability: The concentration of protein
in the microenvironment that create potential
binding sites for transmembrane proteins and other
molecules located at the cell’s outer surface.
Materials and Methods
Conclusions
Acknowledgments
References
Exploring
Engineered
Cells Summer
REU
NSF EEC-1005083
Objective: To observe the changes in cell
migration and adhesion under the influence of
varying mechanical and biochemical cues during
breast cancer metastasis. We have created novel
biomaterials which mimic the extracellular matrix
(ECM) of the “primary tumor” site.
Adhesion Experiment
Polarization is indicative of directionality
and the cell’s “readiness” to migrate.
Rate of adhesion is quantified by measuring
the two-dimensional cell area in contact with
the gel surface as a function of time.
Time 0 Cell Spreading Cell Polarized
Migration Experiment
Cells are allowed to become fully
adhered and polarized overnight (12hrs).
Cells are tracked at 15 minute intervals
for a total of 12 hours. The cells’
trajectories are tracked, which allows for
the calculation of their velocity using
Imaris software and a Brownian Motion
based algorithm.
Results and Discussion
Metastasis: is the translocation of a
cancer cell from the primary tumor
site to a non-adjacent tissue by
means of the circulatory or
lymphatic systems.
•Over 90% of deaths result from
cancer metastasis
Figure 3 [3] Illustrates the many ways in which the
extracellular matrix (ECM) can influence the behavior of a
cell.
Figure 2 [2] Illustrates the pathways which may be taken by a
departed cell
Figure 1 [1]
Cells are motivated to metastasize by three
major types of stimuli…
• biochemical (protein concentration)
• mechanical (resting to active state)
• topography (material surface cues)
h
Modulus is
dependent on
PEG
crosslinking
ratio.
Stiffness is
determined
by the height
of the PEG-
MPC gel.
Spacers: 7,
14, 24um
height
1. Place spacers onto Methacrylate treated
coverslip. Add Sigmacote coverslip on top,
clamp down with small binder clips.
2. Flow in PEGMPC solution in-between
coverslips.
3. Photo crosslink with UV for 7mins (3
samples per time interval)
4. Store coverslip-gel assembly in 1x PBS
until Sigmacote coverslip floats off wafer.
PEGMPC Gel Preparation
1.
2.
3.
4.
Tested Sample Testing Method Measured Parameter Measured value Stiffness *
Mammary Gland1
Unconfined
Compression Test
Elastic Modulus of Normal
to Premalignant to Invasive
Cancer
200~2000 Pa 0.004-0.04
N/m
Human Breast
Carcinoma2
MR elastography
(MRE)
Elastic Modulus
60-100 Pa 1.2-2 N/m
Normal Breast Tissue
Active State 3
Indentation, MRI Elastic Modulus 0.4-2 kPa
4-12 kPa
0.008-0.04
N/m
0.08-0.24
N/m
PEGDMA gels4
Tensile
Compression Test
Tensile Modulus 13.7-423.9 kPa 0.06-2 N/m
Polyacrylamide gels5
Compression Test Young’s Modulus 1.0-308 kPa 0.06-0.62
N/m
Relative Modulus and Stiffness of Biological and Material Samples
Figure 4 [4]
Figure 6. [6] The modulus value of our gels fell within the
same range as mammary gland tissue.
Mechanical Testing of PEGMPC hydrogels
Figure 5. [5] Modulus testing was done with a constant 20% MPC
concentration and varying PEG. Our gels consisted of 10% PEG.
Background Ligand Availability was controlled by the concentration of monomeric collagen I covalently bound
to the surface of the PEG-MPC gels. The gels were Sulfo-SANPAH treated to aid in covalent
bonding of Collagen I to the PEG-MPC hydrogel.
[1] 2011, American Cancer Society, Inc, Surveilliance Research
[2] http://geneticaysexologiaintegral.blogspot.com/2012/06/metastasis-en-cancer-de-mama.html
[3] Pengfei Lu, Valerie M. Weaver, and Zena Werb. 2012. The extracellular matrix: A dynamic niche in cancer
progression. JCB. 196. 395-406
[4] Dannielle Ryman. 2012. University of Massachusetts Amherst.
[5] Will Herrick. 2012. University of Massachusetts Amherst.
[6] Dannielle Ryman. 2012. University of Massachusetts Amherst.
Levental, K. R., Yu, H., Kass, L., Lakins, J. N., Egeblad, M., Erler, J. T., Fong, S. F. T., et al. (2009). Matrix
crosslinking forces tumor progression by enhancing integrin signaling. Cell, 139(5), 891-906. Elsevier Ltd.
doi:10.1016/j.cell.2009.10.027
Chaz Cuckler1
Dannielle Ryman2
Shelly Peyton2
Ohio University Athens1
, University of Massachusetts Amherst 2
A special thanks to Dannielle Ryman, Shelly Peyton,
Aidan Gilchrist, and the ICE REU program
This material is based upon work supported by the National
Science Foundation under Grant No. EEC-1005083
Cells exposed to the higher collagen I concentration adhere to the gel surface at a slower rate, but are
more readily polarized and thus become capable of migrating more quickly. These cells also migrate
at a quicker pace, particularly at the 14um gel height. This suggests that a higher collagen
concentration and a stiffness corresponding to a 14um gel correlates to cells that are more likely to
metastasize in an in vivo environment. Cell adhesion and polarization appears to be independent of
stiffness (gel height), but further testing may unveil a relationship at greater gel heights (less stiff)
that cannot be appreciated at these levels. A significant difference in migration velocity is observed
only at a 14um gel height, but further testing may show this to occur at a different range of
stiffnesses.
Cell adhesion is independent of
gel height and therefore stiffness,
but is dependent on collagen I
concentration. The lower collagen
I concentration result a faster rate
of cell spreading.
The percentage of cells polarized at
each condition by the end of the 160
min time interval is greatest for the
cells exposed to the higher collagen I
concentration.
At lower collagen concentrations
velocity decreases as stiffness
increases. At higher collagen
concentrations stiffness demonstrates
a biphasic response.
MDA MB-231 Cell Migration
1 and 10 µg/cm2
Collagen I
Gel Height
Velocity(µm/hr)
24µm 14µm 7µm
0
10
20
30
40
50
1ug/cm2
10ug/cm2