Title

The Influence of Matrix Stiffness on Acquisition of a Myofibroblast Phenotype in 3D Encapsulated Human Mammary Fibroblasts

Date of Award

2015

Degree Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Abigail Hielscher, PhD

Second Advisor

Huo Lu, PhD

Third Advisor

Francis Jenney, PhD

Fourth Advisor

Brian Matayoshi, PhD

Abstract

Much of the tumor microenvironment is composed of fibroblasts, which contribute to the spread and metastasis of breast cancer. In the presence of cancer cells or when cultured atop mechanically stiff substrates, fibroblasts undergo a phenotypic transition into activated myofibroblasts that continuously secrete extracellular matrix proteins and growth factors that aid in tumor growth. Previous studies have examined the effect of growing fibroblasts on stiff two-dimensional substrates, but the correlation between their transition into myofibroblasts and the surrounding matrix's stiffness has not been fully explored in three-dimensions. This study's focus was examining the effect of increased matrix stiffness on human mammary fibroblasts (HMF) and their acquisition of the myofibroblast phenotype. HMFs were encapsulated within 7.5% gelatin mechanically tuned using microbial transglutaminase, which yielded compliant, moderately stiff, and stiff hydrogels. The encapsulated cells were cultured and monitored over a 7 day culture period for changes in proliferation, morphology, and myofibroblast markers, Vimentin and a-SMA expression were examined using western blot analyses. It was noted that a-SMA expression was upregulated at different time points over the culture period. However, vimentin expression in moderately stiff gels remained constant, while slightly increasing in compliant gels. Immunofluorescence demonstrated the presence of actin stress fibers in the HMFs encapsulated within the compliant and moderately stiffhydrogels. Proliferation was evaluated and indicated a direct correlation between increased matrix stiffness and proliferation. However, this appears to apply up to a certain degree of stiffness, and when stiffness becomes too high proliferation decreases. These findings suggest that matrix stiffness alters HMF behavior and affects the acquisition of some aspects of the myofibroblast phenotype.

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