Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

Kathryn Woods,Abigail Hielscher,Catlyn Thigpen,Jennifer Peyling Wang,Hana Park

doi:10.1186/s12860-017-0151-y

Abstract

BackgroundCarcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance. As such, recent efforts have been directed toward understanding the factors responsible for activation of the phenotype. In this study, we have investigated how changes in the mechanical stiffness of a 3D hydrogel alter the behavior and myofibroblast-like properties of human mammary fibroblasts (HMFs).ResultsHere, we utilized microbial transglutaminase (mTG) to mechanically tune the stiffness of gelatin hydrogels and used rheology to show that increasing concentrations mTG resulted in hydrogels with greater elastic moduli (G’). Upon encapsulation of HMFs in 200 (compliant), 300 (moderate) and 1100 Pa (stiff) mTG hydrogels, it was found that the HMFs remained viable and proliferated over the 7 day culture period. Specifically, rates of proliferation were greatest for HMFs in moderate hydrogels. Regarding morphology, HMFs in compliant and moderate hydrogels exhibited a spindle-like morphology while HMFs in stiff hydrogels exhibited a rounded morphology with several large cellular protrusions. Quantification of cell morphology revealed that HMFs cultured in all mTG hydrogels overall assumed a more elongated phenotype over time in culture; however, few significant differences in morphology were observed between HMFs in each of the hydrogel conditions. To determine whether matrix stiffness upregulated expression of ECM and myofibroblast markers, western blot was performed on HMFs in compliant, moderate and stiff hydrogels. It was found that ECM and myofibroblast proteins varied in expression during both the culture period and according to matrix stiffness with no clear correlation between matrix stiffness and a myofibroblast phenotype. Finally, TGF-β levels were quantified in the conditioned media from HMFs in compliant, moderate and stiff hydrogels. TGF-β was significantly greater for HMFs encapsulated in stiff hydrogels.ConclusionsOverall, these results show that while HMFs are viable and proliferate in mTG hydrogels, increasing matrix stiffness of mTG gelatin hydrogels doesn’t support a robust myofibroblast phenotype from HMFs. These results have important implications for further understanding how modulating 3D matrix stiffness affects fibroblast morphology and activation into a myofibroblast phenotype.

Highlights

Carcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance
It was determined that human mammary fibroblasts (HMFs) were viable at most microbial transglutaminase (mTG) concentrations tested, suggesting that overall the enzyme is non-toxic to HMFs (Fig. 1)
During the 7 day culture period no observable changes in hydrogel contraction were found, suggesting that HMFs may not have fully differentiated into a myofibroblast phenotype following encapsulation in the tested hydrogel conditions. It appeared that following release of the hydrogels from the dish, the HMFs became gradually more rounded, suggesting that the tension exerted by the attachment of the hydrogel to the sides of the dish is an important mediator for supporting the spreading and growth of the fibroblasts. These results indicate that extracellular matrix (ECM) and myofibroblast proteins in HMFs vary in expression during both the culture period and according to the mechanical stiffness of the hydrogel and further suggest that HMFs may not be fully differentiated into a myofibroblast phenotype as they didn’t contract the mTG hydrogels

Summary

Introduction

Carcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance. The mortality rate of breast cancer has diminished due to the availability of treatments including total or partial removal of the breast, chemotherapy and radiation therapy, as well as endocrine and targeted therapies. While the growth and metastatic potential of breast cancer is dependent on a number of factors, the tumor microenvironment which includes fibroblasts, immune cells, endothelial cells and the extracellular matrix (ECM), have been reported to play a role in these tumor cell behaviors [2]. CAFs have been documented to secrete several growth factors including TGF-β1 [10, 11], and stromal derived factor 1 (SDF1) [12] amongst others and have further been reported to remodel the ECM as a result of their deposition of new ECM proteins and expression of matrix degrading enzymes such as matrix metalloproteinases (MMPs). Given the prevalence of these cells in breast tumors, there is much interest devoted to better understanding the factors which drive the acquisition of the CAF phenotype but how these CAFs contribute to aberrant epithelial cell behavior and breast tumor progression

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