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

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

doi:10.3791/54230

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

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

^*Corresponding author for this work

Imaging Chemistry & Biology

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

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.

Original language	English
Article number	e54230
Journal	Journal of Visualized Experiments
Volume	2016
Issue number	115
DOIs	https://doi.org/10.3791/54230
Publication status	Published - 20 Sept 2016

Keywords

Advanced glycation endproducts
Basement membrane
Biophysical strain
Cancer research
Cell migration
Collagen IV
Epithelial cells
Extracellular matrix
Issue 115
Laminin
Medicine
Non-enzymatic crosslinking
Prostate cancer
Stiffness

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.3791/54230

Cite this

Rodriguez-Teja, M., Breit, C., Clarke, M., Talar, K., Wang, K., Mohammad, M. A., Pickwell, S., Etchandy, G., Stasiuk, G. J., & 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, 2016(115), Article e54230. https://doi.org/10.3791/54230

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

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.",

keywords = "Advanced glycation endproducts, Basement membrane, Biophysical strain, Cancer research, Cell migration, Collagen IV, Epithelial cells, Extracellular matrix, Issue 115, Laminin, Medicine, Non-enzymatic crosslinking, Prostate cancer, Stiffness",

author = "Mercedes Rodriguez-Teja and Claudia Breit and Mitchell Clarke and Kamil Talar and Kai Wang and Mohammad, {Mohammad A.} and Sage Pickwell and Guillermina Etchandy and Stasiuk, {Graeme J.} and Justin Sturge",

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T1 - How to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases

AU - Rodriguez-Teja, Mercedes

AU - Breit, Claudia

AU - Clarke, Mitchell

AU - Talar, Kamil

AU - Wang, Kai

AU - Mohammad, Mohammad A.

AU - Pickwell, Sage

AU - Etchandy, Guillermina

AU - Stasiuk, Graeme J.

AU - Sturge, Justin

PY - 2016/9/20

Y1 - 2016/9/20

N2 - 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.

AB - 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.

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KW - Cell migration

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KW - Epithelial cells

KW - Extracellular matrix

KW - Issue 115

KW - Laminin

KW - Medicine

KW - Non-enzymatic crosslinking

KW - Prostate cancer

KW - Stiffness

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

Abstract

Keywords

UN SDGs

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