Event Title
Mechanically tuned hydrogels for investigation of the myofibroblast phenotype
Location
Georgia
Start Date
12-5-2015 1:00 PM
Description
Much of the tumor microenvironment is composed of fibroblasts, which contribute to the spread and metastasis of breast cancer. While in the presence of cancer cells or when cultured a top mechanically stiff substrates, fibroblasts undergo a phenotypic transition into activated myofibroblasts that continuously secrete extracellular matrix (ECM) proteins and growth factors that aid in tumor growth. Several studies have previously examined the effect of growing fibroblasts in mechanically stiff 2D environments, but the possible correlation between the transition of fibroblasts into myofibroblasts and the stiffness of the surrounding matrix has yet to be fully explored in 3D. The focus of this study was to examine the effect of increased matrix stiffness on human mammary fibroblast (HMF) growth and acquisition of the myofibroblast phenotype. HMFs were encapsulated within 7.5% gelatin hydrogels that were mechanically tuned using different concentrations of microbial transglutaminase (mTg), which yielded compliant (300 Pascal) and stiff (1200 Pascal) hydrogels. The encapsulated cells were cultured and monitored over a 7 day culture period for changes in proliferation in addition to differences in morphology and phenotypic markers indicative of a myofibroblast transition. Vimentin and α-SMA expression were examined using western blot analysis. It was noted that when comparing the compliant and stiff hydrogels, α-SMA expression was notably upregulated at different time points over the 7 day culture period. Vimentin expression, however, remained constant within the stiff gels while gradually increasing within the compliant gels. Immunofluorescence demonstrated the presence of actin stress fibers within the HMFs encapsulated within both the compliant and stiff hydrogels. HMF proliferation was evaluated using the WST-1 assay and results indicated a direct correlation between increased matrix stiffness and cell proliferation. These findings suggest that matrix stiffness does alter the behavior of HMFs and affects the acquisition of some aspects of the myofibroblast phenotype.
Mechanically tuned hydrogels for investigation of the myofibroblast phenotype
Georgia
Much of the tumor microenvironment is composed of fibroblasts, which contribute to the spread and metastasis of breast cancer. While in the presence of cancer cells or when cultured a top mechanically stiff substrates, fibroblasts undergo a phenotypic transition into activated myofibroblasts that continuously secrete extracellular matrix (ECM) proteins and growth factors that aid in tumor growth. Several studies have previously examined the effect of growing fibroblasts in mechanically stiff 2D environments, but the possible correlation between the transition of fibroblasts into myofibroblasts and the stiffness of the surrounding matrix has yet to be fully explored in 3D. The focus of this study was to examine the effect of increased matrix stiffness on human mammary fibroblast (HMF) growth and acquisition of the myofibroblast phenotype. HMFs were encapsulated within 7.5% gelatin hydrogels that were mechanically tuned using different concentrations of microbial transglutaminase (mTg), which yielded compliant (300 Pascal) and stiff (1200 Pascal) hydrogels. The encapsulated cells were cultured and monitored over a 7 day culture period for changes in proliferation in addition to differences in morphology and phenotypic markers indicative of a myofibroblast transition. Vimentin and α-SMA expression were examined using western blot analysis. It was noted that when comparing the compliant and stiff hydrogels, α-SMA expression was notably upregulated at different time points over the 7 day culture period. Vimentin expression, however, remained constant within the stiff gels while gradually increasing within the compliant gels. Immunofluorescence demonstrated the presence of actin stress fibers within the HMFs encapsulated within both the compliant and stiff hydrogels. HMF proliferation was evaluated using the WST-1 assay and results indicated a direct correlation between increased matrix stiffness and cell proliferation. These findings suggest that matrix stiffness does alter the behavior of HMFs and affects the acquisition of some aspects of the myofibroblast phenotype.