1. Development of 3D Breast Cancer Models in
PEG-Fibrinogen Hydrogels
Kelsey L. Henderson, Shantanu Pradhan, Dr. Elizabeth A. Lipke
Department of Chemical Engineering, Auburn University, Auburn, AL
Background & Significance
2D Cell Culture vs 3D Cell Culture
• PEG-Fibrinogen was established as a novel biomaterial for 3D
breast cancer cell culture.
• EMT markers and integrins were differentially expressed in 3
breast cancer cell lines.
• Morphological characteristics of breast cancer cells were
quantified.
• By creating an improved in vitro model, tumorigenic phenomena
can be investigated and anti-cancer drug efficacy can be tested.
Hoechst &
Phalloidin:
Indicators of cell
morphology and structure.
Shows nuclei and actin
filaments.
Ki67:
Prognostic marker of
breast cancer
progression. Shows
cell proliferation.
Vimentin:
Shows progression
towards invasive
morphology. MDA-MB-231
exhibits this marker as a
result of its mesenchymal
morphology.
E-Cadherin:
Native luminal mammary
tissue marker. MCF-7
exhibits this marker as a
result of its epithelial
morphology.
αvβ3 integrin:
Expressed when
cancer cells bind to
fibrinogen.
Overexpressed in
MDA-MB-231 cells.
β1 integrin:
Signal of malignant cancer
progression. Nuclear
expression in MCF-7 &
SK-BR-3 vs. cytoplasmic
expression in
MDA-MB-231.
MCF-7
(ER+, PR+)
SK-BR-3
(HER2+)
MDA-MB-231
(TNBC)
2D Culture
• Cells grow in flat
monolayer
• Easy to culture,
observe, and
analyze
• Does not
accurately
represent
physiological
environment
3D Culture
• Cells grown in
aggregate colonies
• Difficult to study
and analyze
• More closely
represents native
environment
EMT & Integrin Expression in Breast Cancer Cells Morphological Quantification
Conclusions
Breast cancer is the most common cancer in women. Over 200,000 women are diagnosed and over 40,000
killed per year in the United States. In the recent years, the ratio of deaths to new cases of breast cancer
has steadily declined. This is due in part to early detection, but is also a product of increased breast cancer
research that has lead to more effective treatment. In order to efficiently study cancer behavior and to test
newly developed drugs, a system that imitates natural environment and tissues in the body must be
developed. Here, cancer cells were encapsulated in 3-dimensional hydrogels that mimic the body’s natural
environment. The development of novel methods of in vitro models helps to identify invasive cancer
behavior as well as test the efficacy of new anti-cancer drugs. When studying behavior of cancer, one
aspect of particular interest is the epithelial and mesenchymal morphologies.
Epithelial cells exhibit normal morphology of healthy cells. The cells have tight cell-cell junctions and are
mostly stationary. Mesenchymal cells are elongated in structure and do not adhere to each other, allowing
them to move about much more freely. It is this morphology that characterizes very aggressive cancer cells.
The transition of cells from epithelial morphology to mesenchymal morphology is called the EMT: epithelial-
mesenchymal-transition, which is also widely studied.
http://www.keep-a-breast.org/get-
educated/cancer-facts/
http://keionline.org/node/1042
Acknowledgements
1. Auburn University Cellular and Molecular Biosciences
2. Auburn University Research In Cancer
HER2+
Luminal B HER2+
Luminal A Basal-like
“TNBC”
ER+, PR+ ER-, PR-
Types of Breast Cancer
Ratio of Deaths to New Cases of
Breast Cancer in the United States
Biomimetic Material:
PEG-Fibrinogen
Fibrinogen promotes tumorogenesis
and angiogenic growth
PEGDA provides stiffness and
architectural support
Begin with
isolated
cancer cells
Hydrogel
precursor is
added
Precursor and
cells added to
PDMS mold
and cross-
linked with
visible light
Hydrogel
encapsulated cancer
cells maintained in
culture for 15 days
Hydrogel Formation Procedure:
Experimental Plan:
• Encapsulate 3 different breast cancer cell lines in hydrogels
• Observe and record morphological quantification data over a period of 15 days.
• Perform immunostaining to observe cell structure, epithelial-mesenchymal transition,
and various integrins.
A B B C C C
MCF-7 (ER+, PR+)
A B B B B B
MDA-MB-231
(TNBC)
A B C C C C
SK-BR-3 (HER2+)
A B C D D D
MCF-7 (ER+, PR+)
A B B B B B
MDA-MB-231 (TNBC)
A B C C C C
SK-BR-3 (HER2+)
Seliktar et al. Biomaterials 2005. May 26 (15). Volume 26. Issue 15
Colony area increases with time
Colony diameter increases with time
Colony circularity decreases with time
Images of MCF-7, MDA-MB-231, and SK-BR-3 taken every three days for fifteen days after encapsulation
showed an increase in colony area with time followed by a period of constant area. Comparison of the three cell
lines showed a more rapid increase in the area of MCF-7.
Images of the same cell lines for the same amount of time also showed an increase in colony diameter over
time followed by a period of constant diameter. MCF-7 still shows the fastest increase.
Measurement of circularity showed a steady decrease in circularity that is relatively similar among the three test
cell lines.
Colony area, diameter, and circularity are indicators of aggressiveness of cancer cells. As colony area and
diameter increase, and circularity decreases, the cells become larger, with a more elongated shape, thus
invading into surrounding tissues. Images were taken and these morphological characteristics were quantified.
The letters above the dot plots show the statistical analysis.
A B BC BC CD D
MCF-7 (ER/PR+)
A B B B C C
SK-BR-3 (HER2+)
A B BC BC BC C
MDA-MB-231 (TNBC)