Mechanically tuned sandwich based 3D hydrogels for investigating the fibroblast to myofibroblast transition
Location
Suwanee, GA
Start Date
14-5-2019 1:00 PM
End Date
14-5-2019 4:00 PM
Description
The breast cancer microenvironment is characterized by having an abnormal extracellular matrix (ECM), a protein-rich, non-cellular entity that provides structural support to the tissue. The abnormal ECM in the breast cancer microenvironment may be a result of fibroblast to myofibroblast transition. These myofibroblasts can promote growth and metastasis of the breast tumor by depositing additional ECM and making a stiffer stroma. The purpose of this study is to establish whether human mammary fibroblasts (HMFs) transition to activated myofibroblasts following culture in a mechanically tuned 3D gelatin based sandwich hydrogel. To tune the mechanical stiffness, microbial transglutaminase (mTG), an enzyme that crosslinks lysine and glutamine residues, was used in concentrations of 30, 100, and 200 μg/ml to generate a compliant, moderate, and stiff hydrogel, respectively. The HMFs were added to the compliant, moderate and stiff hydrogels and allowed 2 hours to adhere. To create the 3D hydrogel sandwich, a top compliant gel (30 μg/ml) was added. A western blot was preformed on conditioned media of 3D hydrogels at day 2 and 4 to look at ECM protein expression for collagen I, collagen IV, Fibronectin, and Fibronectin ED-a results from this were inconclusive. Next, we examined the mechanical stiffness of the 3D hydrogels to determine if an increase in stiffness promotes TGF-β signaling. An ELISA was performed to quantify secreted TGF-β and results show that over the culture periods of day 2 and 4 there is an increase in TGF-β expression, regardless of mechanical stiffness. Results from ELISA also showed as mechanical stiffness increased the expression of TGF-β decreased. Confocal microscopy was used to show an increased mechanical stiffness promotes α-SMA co-localization with actin stress fibers. Findings from confocal microscopy show an increase in actin stress fibers with an increased mechanical stiffness, Overall these results show an increased mechanical stiffness promotes fibroblast transition into myofibroblasts. These findings will help further understanding of the tumor microenvironment as a possible target for therapeutic agents and predict prognosis of breast tumors.
Embargo Period
1-28-2020
Mechanically tuned sandwich based 3D hydrogels for investigating the fibroblast to myofibroblast transition
Suwanee, GA
The breast cancer microenvironment is characterized by having an abnormal extracellular matrix (ECM), a protein-rich, non-cellular entity that provides structural support to the tissue. The abnormal ECM in the breast cancer microenvironment may be a result of fibroblast to myofibroblast transition. These myofibroblasts can promote growth and metastasis of the breast tumor by depositing additional ECM and making a stiffer stroma. The purpose of this study is to establish whether human mammary fibroblasts (HMFs) transition to activated myofibroblasts following culture in a mechanically tuned 3D gelatin based sandwich hydrogel. To tune the mechanical stiffness, microbial transglutaminase (mTG), an enzyme that crosslinks lysine and glutamine residues, was used in concentrations of 30, 100, and 200 μg/ml to generate a compliant, moderate, and stiff hydrogel, respectively. The HMFs were added to the compliant, moderate and stiff hydrogels and allowed 2 hours to adhere. To create the 3D hydrogel sandwich, a top compliant gel (30 μg/ml) was added. A western blot was preformed on conditioned media of 3D hydrogels at day 2 and 4 to look at ECM protein expression for collagen I, collagen IV, Fibronectin, and Fibronectin ED-a results from this were inconclusive. Next, we examined the mechanical stiffness of the 3D hydrogels to determine if an increase in stiffness promotes TGF-β signaling. An ELISA was performed to quantify secreted TGF-β and results show that over the culture periods of day 2 and 4 there is an increase in TGF-β expression, regardless of mechanical stiffness. Results from ELISA also showed as mechanical stiffness increased the expression of TGF-β decreased. Confocal microscopy was used to show an increased mechanical stiffness promotes α-SMA co-localization with actin stress fibers. Findings from confocal microscopy show an increase in actin stress fibers with an increased mechanical stiffness, Overall these results show an increased mechanical stiffness promotes fibroblast transition into myofibroblasts. These findings will help further understanding of the tumor microenvironment as a possible target for therapeutic agents and predict prognosis of breast tumors.