The Combined Effects of Matrix Stiffness and TGF-β on the Acquisition of a Myofibroblast Phenotype in 3D Encapsulated Human Mammary Fibroblasts
Date of Award
Master of Science in Biomedical Sciences
Abigail Hielscher, PhD
Eric Wang, PhD
Harold Komiskey, PhD
Richard White, PhD, FAHA
The tumor microenvironment has been reported to play an active role in breast cancer progression. One component of the breast cancer environment which facilitates tumor growth and metastasis is the activation of tissue resident fibroblasts to a myofibroblast or carcinomaassociated fibroblast (CAFs) phenotype. Current research shows that matrix stiffness and the multi-functional cytokine TGF-β independently alter fibroblast behavior and activate the myofibroblast/CAF phenotype in 2D environments. It is currently unknown whether matrix stiffness and TGF-β promote the activation of fibroblasts to myofibroblasts in 3D encapsulated hydrogels. Thus, the goal of this study is to determine whether the introduction of bioactive factors such as TGF-β and mechanical stiffness are necessary for the transition of human mammary fibroblasts (HMFs) to myofibroblasts in 3D gelatin hydrogels. To tune the mechanical stiffness, hydrogels were crosslinked with microbial transglutaminase (mTGase) to generate compliant (20 µg/mL mTG, 200 Pa G’) and stiff (60 µg/mL mTG, 1100 Pa G’) gels. To address the combined role of matrix stiffness and TGF-β on a myofibroblast phenotype, compliant and stiff hydrogels were mixed with 0, 1, 5, and 10 ng/mL TGF-β prior to HMF encapsulation. Changes in HMF proliferation, morphology, and myofibroblast marker expression were measured via imaging, WST, and western blot analyses at day 1, 3, 5, and 7 along the culture period. Results from these studies indicated that during the 7-day culture period, HMF proliferation was not significantly altered at different concentrations of TGF-β or varying gel stiffness, however there was a significant difference in proliferation among different culture time points. To determine the influence of TGF-β and matrix stiffness on activation of a myofibroblast phenotype, HMFs were isolated at days 3, 5 and 7 from TGF-β treated compliant and stiff hydrogels and expression of myofibroblast markers (α-SMA, fibronectin, collagen I and vimentin) were measured via western blot. It was shown that increasing concentrations of TGF-β resulted in higher protein expression for fibronectin at all time points tested with maximal expression on Day 7 at 10 ng/mL TGF-β for stiff gels. Furthermore, the results showed that α- SMA expression, a well-characterized marker of the myofibroblast phenotype, was more highly expressed for stiff gels than compliant gels with maximal expression at Day 5 at 5 ng/mL TGF-β. Vimentin, an intermediate filament involved in mesenchymal cell processes was most highly expressed in stiff gels at lower concentrations of TGF-β with maximal expression on Day 5 at 0 ng/mL TGF-β. Lastly, expression of collagen I, a protein in the breast microenvironment, had higher levels of expression in compliant gels at higher concentrations of TGF-β indicating that matrix stiffness may not affect collagen I expression as greatly as TGF-β concentration compared to other myofibroblast markers. To assess whether TGF-β could be inhibited in a 2D culture, HMFs were treated with anti-TGF-β and HMF morphology and protein expression of SMAD, pSMAD and TGF-βR1 were measured. Phase contrast imaging demonstrated more cell clustering, elongation, and spindle-formation of HMF cells, indicating activation to a myofibroblast-like phenotype, after exposure to TGF-β and a less activated phenotype with higher concentrations of TGF-β inhibitor. According to western blot analysis, pSMAD was lowest for the control and the highest concentration of TGF-β inhibitor, indicating the TGF-β in the HMFs were successfully inhibited in 2D. Based on these analyses, it is apparent that during the culture period of 3D encapsulated gels, matrix stiffness in concert with TGF-β does not significantly affect the proliferation of HMFs. However, greater concentrations of TGF-β and increased matrix stiffness may lead to a more activated phenotype due to higher expression of fibronectin and α-SMA. Analysis of TGF-β and matrix stiffness on the activation of the myofibroblast phenotype in a biologically relevant 3D culture system will elucidate how a more complex in-vivo-like environment regulates this phenotype.
Parham, Mahtab, "The Combined Effects of Matrix Stiffness and TGF-β on the Acquisition of a Myofibroblast Phenotype in 3D Encapsulated Human Mammary Fibroblasts" (2018). PCOM Biomedical Studies Student Scholarship. 140.