University of Hull logo

How to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases

Rodriguez-Teja, Mercedes; Breit, Claudia; Clarke, Mitchell; Talar, Kamil; Wang, Kai; Mohammad, Mohammad A.; Pickwell, Sage; Etchandy, Guillermina; Stasiuk, Graeme J.; Sturge, Justin

Authors

Mercedes Rodriguez-Teja

Claudia Breit

Mitchell Clarke

Kamil Talar

Kai Wang

Mohammad A. Mohammad

Sage Pickwell

Guillermina Etchandy

Abstract

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 Article Type Article
Publication Date Sep 20, 2016
Journal Journal of visualized experiments
Print ISSN 1940-087X
Electronic ISSN 1940-087X
Publisher Journal of Visualized Experiments
Peer Reviewed Peer Reviewed
Volume 2016
Issue 115
Institution Citation Rodriguez-Teja, M., Breit, C., Clarke, M., Talar, K., Wang, K., Mohammad, M. A., …Sturge, J. (2016). How to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases. Journal of visualized experiments : JoVE, 2016(115), https://doi.org/10.3791/54230
DOI https://doi.org/10.3791/54230
Keywords Advanced glycation endproducts
Publisher URL https://www.jove.com/video/54230/how-to-study-basement-membrane-stiffness-as-biophysical-trigger
Copyright Statement © JoVE 2016
Additional Information This is a description of an article published in Journal of visualized experiments, 2016, v.115.

Files

Figure_1 (17.4 Mb)
Other

Copyright Statement
© JoVE 2016



Figure_2 (9.8 Mb)
Other

Copyright Statement
© JoVE 2016



Figure_3 (31.7 Mb)
Other

Copyright Statement
© JoVE 2016




You might also like


Downloadable Citations