Development of 3D Breast Cancer Models in PEG-Fibrinogen Hydrogels

Kelsey L. Henderson, Shantanu Pradhan, Dr. Elizabeth A. LipkeDepartment 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

Acknowledgements1. Auburn University Cellular and Molecular Biosciences2. Auburn University Research In Cancer

HER2+Luminal B HER2+

Luminal A Basal-like“TNBC”

ER+, PR+ ER-, PR-

Types of Breast CancerRatio of Deaths to New Cases of

Breast Cancer in the United States

Biomimetic Material:PEG-Fibrinogen

Fibrinogen promotes tumorogenesis and angiogenic growthPEGDA 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)