Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e. g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.
Journal of visualized experiments : JoVE. 2016 Sep 20*** epublish ***
Mercedes Rodriguez-Teja, Claudia Breit, Mitchell Clarke, Kamil Talar, Kai Wang, Mohammad A Mohammad, Sage Pickwell, Guillermina Etchandy, Graeme J Stasiuk, Justin Sturge
Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR)., Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology., School of Biological, Biomedical & Environmental Sciences, University of Hull., School of Biological, Biomedical & Environmental Sciences, University of Hull; .