We thank Simon Hayward (Vanderbilt University Medical Center) for the BPH-1 cells; and Thomas Cox and Janine Erler (Biotech Research &amp; Innovation Centre, University of Copenhagen) for their assistance with rheological measurements. MR-T was funded by Worldwide Cancer Research, formerly The Association of International Cancer Research (Grant 08-0803 to JS), The British Embassy Montevideo and Agencia Nacional de Investigacion e Innovacion (UK_RH_2015_1_2 to MR-T). MC was supported by Prostate Cancer UK (Grant S14-017 to JS and GS). KW was funded by The China Scholarship Council. MAM was funded by The Saudi Arabian Cultural Bureau.

<ol>
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The authors have nothing to disclose.

A protocol for the generation of 3D glandular acini from MECs in pure rBM gels6 was modified in a previous study by the addition of 4 mg/ml type I collagen to the rBM matrix. The addition of collagen resulted in the elastic modulus of the rBM gel increasing from 175 &#177; 37 to 1589 &#177; 380 Pascals. This 9.1-fold increase in stiffness modulated the growth, survival, migration and differentiation of MECs21. The protocol was modified again by including a treatment step with D-(-)-ribose to promote non-enzymatic crosslinking of the type I collagen that had been added to the rBM gel. The resultant 15-fold increase in stiffness was found to cooperate with oncogenic transformation of MECs to promote their invasive behavior22. The experimental approach of adding type I collagen to rBM gels facilitates the direct interaction of MECs with collagen fibres, which only occurs in human tissue after the physical barrier between the stroma and epithelium provided by the BM undergoes proteolytic degradation. By generating 3D glandular acini from PECs in pure rBM gels pre-treated with GLA, the current protocol opens the way to study how BM stiffness per se can trigger their invasive behaviour (Figure 3). The levels of BM stiffness induced in this protocol have physiological relevance. Incubation with 50 mM GLA for 6 hr and 14 hr respectively increased the elastic moduli of the pure rBM gel to 175 &#177; 90 and 322 &#177; 160 compared to 122 &#177; 55 Pascals in rBM gels treated with PBS (Table 1). This 1.7 to 3.2-fold increase in rBM stiffness recapitulates the 2.5- to 3.4-fold increase in stiffness observed in malignant compared to normal prostate or BPH tissue23-26. As outlined in a recent publication13 the morphological changes induced by the accumulation of AGE and rBM stiffness in PEC acini can be quantified for a statistically significant shift from a rounded to polygonal shape, decreased luminal/total acinar area, and protruding cells migrating from the acina into the AGE-rich rBM (Figure 3). Immunoblotting can also be used to assess markers of EMT (e.g. loss of E-cadherin13) and the contractile behavior (e.g. phosphorylated myosin light chain-2, pMLC213) in PECs grown in normal versus stiff rBM (Figure 3). Further evaluation using immunofluorescent staining and confocal microscopy can be applied to visualize the BM (e.g. laminin, collagen IV and AGE accumulation13), cellular apical-to-basal polarity (e.g. apical localization of EEA1: early endosomal antigen 1; and GM130: 130 kDa cis-Golgi marker13) and cellular patterns of adhesion molecules (e.g. E-cadherin localization to cell-cell junctions13) (Figure 3).
<img alt="Figure 3" src="/files/ftp_upload/54230/54230fig3.jpg" /> 
Figure 3: Overview of the Different Protocols Presented Here. The diagram depicts how to prepare and stiffen the reconstituted basement membrane (rBM) with glycolaldehyde (Maillard reaction), how to seed cells on to the stiff rBM, how to analyze the stiff rBM (extent of Maillard reaction) and procedures that can be used to analyze the cellular and molecular changes induced by AGE-rich rBM. AGE, advanced glycation endproducts; BM, basement membrane; DAPI, 4&#39;,6-diamidino-2-phenylindole; EEA1, early endosomal antigen 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLA, glycolaldehyde; GEE, glycine ethyl ester; GM130, 130 kDa cis-Golgi marker; p-MLC2 (Thr18/Ser19), myosin light chain-2 phosphorylated at sites threonine 18 and serine 19; rBM, reconstituted basement membrane; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. For RWPE1 acini Scale bar = 10 &#181;m; for PC3 tumor cell spheroids Scale bar = 100 &#181;m. This figure has been modified from reference13. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Troubleshooting steps will be necessary if D-(-)-ribose is chosen as the crosslinking agent for rBM. During protocol development it was found that treatment with 1 M D-(-)-ribose for 72 hr, as previously described for rBM/collagen gels22, resulted in the dehydration and shrinkage of rBM gels. The evaluation of lower concentrations of D-(-)-ribose and shorter treatment times may help to overcome this limitation.
A potential limitation in future applications of the protocol could be encountered where higher levels of rBM stiffness are desired. If longer incubation times and higher concentrations of GLA are used to induce higher levels of rBM gel stiffness it will be necessary to assess whether these treatment conditions have an impact on cell survival and proliferation, as previously described13. It should also be noted that incubation of RWPE-1 cells with serum induces a phenotypic EMT-like transition and exposure to serum or serum-containing materials should be avoided. For example, if experiments involve the transfection of short interfering RNA (siRNA) oligonucleotides, the procedure should be optimized using RWPE-1 cells grown in KSFM, without switching the cells to low serum transfection media. This drawback could compromise the level of gene silencing achieved when using transient siRNA approaches in the model. For some protein targets it would be advised to employ inducible shRNA vectors for tunable gene silencing and the desired decrease in protein levels. Adaptations that incorporate enzymatic crosslinking by stromal cell or tumor cell associated lysyl oxidase (LOX)17 could also be incorporated into future models.
This protocol will facilitate the future study of pro-invasive mechanisms triggered by AGE-dependent BM stiffness in PECs (RWPE-1, BPH-1) and evaluation of anti-metastatic targets in invasive PTCs (PC3). Given that BPH is considered to be a metabolic disorder27, this protocol also paves the way towards our improved understanding of the link between metabolic disorders and increased prostate cancer risk. Given that BM stiffness induced by its exposure to AGEs may be a trigger for invasiveness in other cancer types, it will be of interest to use the protocol to set up similar models that incorporate normal epithelial cells and tumor cells from other organs (e.g. breast, colon, ovaries, pancreas).
Critical steps within the protocol, together with their timings, are summarized in Figure 4. During the initial step it is essential to maintain the stock solution of rBM at 4 &#176;C while it thaws to prevent its polymerization. Pipette tips should not be placed into the rM stock solution until they have been chilled to 4 &#176;C. For the next step it is also important to ensure the chamber slides have equilibrated to 4 &#176;C before they are coated with the rBM solution. As soon as the temperature of the rBM solution is increased above 4 &#176;C it will undergo irreversible polymerization to form a gel. It essential that the rBM is not disturbed during the polymerization stage to ensure that it forms an even surface suitable for cell culture and microscopic analysis. The duration of incubation with GLA with or without inhibitors of the Maillard reaction (sodium cyanoborohydride and amingoguanidine) will determine how stiff the rBM gel becomes. It is recommended to use a 6 hr incubation with GLA if semi-stiff conditions are required, and 14 hr incubation if stiff conditions are required (Table 1). Alternate incubation times or concentrations of GLA can be used if different levels of stiffness are desired. In this case rheological analysis of the rBM gels need to be incorporated as an essential step. Following the step of quenching the Maillard reaction by incubation with GEE and the subsequent washing steps with PBS, the rBM gels can be used immediately or stored at 4 &#176;C for up to 48 hr prior to their use for cell culture. Once cell cultures are set up it is important to change the culture medium (including any treatments) every two days. It is recommended to maintain the 3D cell cultures for 3-12 days according to the parameters under investigation. For 3D PEC acini it is recommended to analyse the cultures after 6 days, and for 3D PTC spheroids analysis is recommended after 3 days of culture in the first instance.
<img alt="Figure 4" src="/files/ftp_upload/54230/54230fig4.jpg" /> 
Figure 4: Simple Overview of the Protocol with Critical Steps and Timings Indicated. The flow diagram depicts how to prepare and stiffen the reconstituted basement membrane (rBM) with glycolaldehyde (Maillard reaction) with critical steps and timings indicated. Points where the protocol can be stopped, and rBM gels stored, are also indicated. rBM, reconstituted basement membrane; GLA, glycolaldehyde; GEE, glycine ethyl ester; O.N., overnight; PBS, phosphate buffered saline; R.T., room temperature. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The basement membrane (BM) is a sheet of specialized extracellular matrix (ECM) that maintains stable tissue borders by separating layers of epithelial cells from the stroma1. Covalent crosslinking between adjacent triple helices of collagen IV in the BM stabilizes their lateral association by establishing an irregular network of super-twisted helices2. These collagen IV lattices act as a scaffold for its interaction with laminin and other BM components1. The structural arrangement of the BM provides it with the mechanical strength and rigidity necessary for the normal development of glandular epithelia3. 
During aging and disease the BM progressively thickens and stiffens3,4. For example, a 3-fold increase in the elastic modulus (E) of the ocular BM occurs between the ages of 50 and 80 in the normal population, and this stiffening is further exacerbated in metabolic disorders like diabetes5. The structural and biomechanical changes in the BM that result in its increased stiffness occur when its ECM components, collagen IV and laminin, become non-enzymatically crosslinked following their exposure to advanced glycation endproducts (AGEs).
The purpose of the method described here was to establish a model for the investigation of how BM stiffness, due to AGE exposure, promotes prostate epithelial cell (PEC) and prostate tumour cell (PTC) invasiveness in the context of the switch to metastatic prostate cancer (PCa). To do this a previous method used for generating 3D glandular acini from mammary epithelial cells (MECs) in reconstituted rBM gels6 was adapted to include an additional step where the rBM gels are pre-treated with glycolaldehyde (GLA). Several techniques for assessing GLA induced crosslinking and stiffening of pre-treated rBM gels are described, including photometric analysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), confocal microscopy and rheometric analysis. The prostate cell types selected for culture on the pre-stiffened rBM include: RWPE-1, PECs derived from a normal prostate gland7; BPH-1, PECs derived from a prostate gland affected by BPH8; and PC3, metastatic PTCs derived from a secondary tumor located in the vertebral bone of a prostate cancer (PCa) patient9. 
In addition to advancing the study of prostate gland pathology, the protocol for stiffening of rBM gels by their treatment with GLA can be adapted to investigate how BM stiffness contributes to other age-related pathologies and metabolic disorders. For example, the model can be directly applied to investigate how metastatic cancer is induced by BM stiffness in organs such as the breast, colon, ovary and pancreas by the incorporation of appropriate cell types. Furthermore, the protocol can be adapted to investigate how stiff BM promotes biomechanical mechanisms of disease progression in diabetes-related microvascular disease, retinopathy, nephropathy and neuropathy.

<table><tbody><tr><td>&lt;strong&gt;I - Material for monolayer culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BPH-1</td><td>CaP Cell Line Database</td><td>PCaCL-132</td><td>Contact: simon.hayward@mcmail.vanderbilt.edu</td></tr><tr><td>Complete keratinocyte serum-free media&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>17005-075</td><td>Do not warm at 37 &amp;ordm;C before use</td></tr><tr><td>Fetal calf serum&amp;nbsp;</td><td>First Link UK Ltd</td><td>02-00-850</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>PC3&amp;nbsp;</td><td>American Type Culture Collection</td><td>CRL-1435</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>ThermoFisher Scientific</td><td>15070-063</td><td></td></tr><tr><td>Phosphate Buffer Saline (Dulbecco A) tablets</td><td>Oxoid</td><td>BR0014G</td><td></td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Sigma-Aldrich</td><td>R5886</td><td>warm in 37 &amp;ordm;C water bath before use</td></tr><tr><td>RWPE-1&amp;nbsp;</td><td>American Type Culture Collection</td><td>CRL-11609</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich</td><td>T4049</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;II - Material for 3D culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>271004</td><td></td></tr><tr><td>Aminoguanidine hydrochloride</td><td>Sigma-Aldrich</td><td>396494</td><td>Irritating to eyes, respiratory system and skin</td></tr><tr><td>Chamber slides, 8-well&amp;nbsp;</td><td>Thermo Scientific Nunc Lab-Tek</td><td>TKT-210-816M</td><td></td></tr><tr><td>Culture Matrix reconstituted basement membrane (rBM) reduced growth factor extract&amp;nbsp;</td><td>AMS Biotechnology</td><td>3445-005-01</td><td>Store Basement membrane (BM) at -80 &amp;ordm;C in aliquots</td></tr><tr><td>Cyanogen bromide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C91492</td><td>Toxic by contact skin and inhalation</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>695076</td><td></td></tr><tr><td>Glycine ethyl ester hydrochloride (GEE)</td><td>Sigma-Aldrich</td><td>50060</td><td>Irritating to eyes</td></tr><tr><td>Glycolaldehyde dimer (GLA)</td><td>Sigma-Aldrich</td><td>G6805</td><td></td></tr><tr><td>Sodium cyanoborohydride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>71435</td><td>Highly flammable; Toxic by contact skin and inhalation</td></tr><tr><td>Syringe filter 0.22 microns</td><td>Appleton Woods</td><td>BC680</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;III - Material to quantify Maillard reaction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>ThemoFisher Scientific</td><td>D3571</td><td>light sensitive and store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Cloning cylinders</td><td>Sigma-Aldrich</td><td>C1059</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td><td>ThemoFisher Scientific</td><td>A-11001</td><td>light sensitive</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Alexa Fluor 488 conjugate</td><td>ThemoFisher Scientific</td><td>A-11034</td><td>light sensitive</td></tr><tr><td>Goat serum</td><td>Abcam</td><td>ab7481</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Vectashield mounting media&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>Mouse anti-pentosidine clone PEN-12 mAb</td><td>TransGenic Inc</td><td>KH012</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>F8775</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Rabbit anti-human collagen IV polyclonal antibody&amp;nbsp;&amp;nbsp;</td><td>Acris Antibodies</td><td>R1041</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Rabbit anti-laminin A/C pAb</td><td>Santa Cruz Biotechnology Inc</td><td>sc-7292</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Polyethylene glycol tert-octylphenyl ether (Triton-X100)</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Polyethylene glycol sorbitan monolaurate (Tween-20)</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Dialysis cassette Slide-A-Lyzer</td><td>ThemoFisher Scientific</td><td>66333</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;IV - Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ARG2 controlled strain rotational rheometer</td><td>T.A. Instruments</td><td></td><td></td></tr><tr><td>Axiovert S100 (20X magnification) microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>CO2 controlled humidified incubation chamber for Zeiss Axio S100 microscope</td><td>Solent Scientific</td><td></td><td></td></tr><tr><td>Confocal Axiovert 200M (40X, 63X magnification) microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Olympus LH50A microscope fitted with a digital camera using phase-contrast&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td></td><td></td></tr><tr><td>PHERAstar Plus plate reader spectrophotometer</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;V - Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image J 1.47v</td><td>National Institute of Health, USA</td><td></td><td></td></tr><tr><td>MetaXpress</td><td>Molecular Divices</td><td></td><td></td></tr></tbody></table>

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

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.

3D Prostate Acini Cultured on Stiff rBM
After 6 days in culture, PECs derived from normal prostate tissue (RWPE-1) (Figure 1A) and BPH tissue (BPH-1) (Figure 1B) form acini on native (PBS treated) rBM that are organized into uniform spheroids of epithelial cells. These acini also have the characteristics of highly organized PECs with apical-to-basal polarity and a visible luminal space13,20.
The acini formed by PECs derived from normal prostate tissue (RWPE-1) (Figure 1A) and BPH tissue (BPH-1) (Figure 1B) on stiffened (AGE-rich) rBM (treated with GLA) have a disrupted architecture (shifting from spheroidal to polygonal in shape and cells protruding/migrating from the acini into the AGE-rich rBM) (Figure 1A). These acini are also characterized by highly disorganized PECs that have lost their apical-to-basal polarity with a small or non-existent luminal space13.
<img alt="Figure 1" src="/files/ftp_upload/54230/54230fig1.jpg" /> 
Figure 1: Prostate Epithelial Cells Grown as 3D Glandular Acini on Native and Stiff Reconstituted Basement Membrane (rBM). (A) Brightfield images of RWPE-1 cells grown for 12 hr up to 6 days on rBM gels treated with PBS (native) or 50 mM glycolaldehyde for 14 hr (AGE-rich; stiff); Scale bar = 50 &#181;m. (B) BPH-1 cells, grown as described in panel A; Scale bars = 50 &#181;m; data is representative of 3 independent experiments. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54230/54230table1.jpg" /> 
Table 1: Characteristics of Prostate Epithelial RWPE-1 acini Grown on Native, Semi-Stiff and Stiff Reconstituted Basement Membrane (rBM). RWPE-1 acini were grown on rBM pre-treated with PBS for 14 hr (native), glycolaldehyde (GLA) for 6 hr (semi-stiff) or GLA for 14 hr (stiff). For acinar shape, the percentage (%) &#177; standard deviation (SD) of round, semi-polygonal and polygonal acini were calculated from 5 independent experiments (50 acini quantified per condition). Relative acinar size was calculated (native rBM = 100 %) from 3 independent experiments. For invasiveness, % &#177; SD acini with one or more protruding cells were calculated from 3 independent experiments. Fold change is calculated by dividing the average value obtained under semi-stiff or stiff conditions by the corresponding value for native conditions. P values calculated using Student&#39;s t-test (&#945; = 0.05).
AGE dependent increased rBM stiffness promotes PC3 prostate tumor cell migration
PC3 cells grown on native rBM migrate by maintaining continuous cell-cell contact, whereas PC3 cells grown on AGE-rich (stiff) rBM move independently from each other (Figure 2A). After 72 hr in culture PC3 cells form foci (spheroids) on native (PBS treated) rBM, whereas PC3 cells on stiff (AGE-rich) rBM do not from spheroids and migrate independently (Figure 2B). PC3 cells on stiff (AGE-rich) rBM are more elongated than PC3 cells grown on native rBM (Figure 2C). PC3 cells on stiff rBM migrate faster than PC3 cells grown on native rBM (Figure 2D). PC3 cells on stiff rBM display a decrease in persistence compared to PC3 cells grown on native rBM (Figure 2E).
<img alt="Figure 2" src="/files/ftp_upload/54230/54230fig2.jpg" /> 
Figure 2: Prostate Tumor Cell Migration on Native and Stiff Reconstituted Basement Membrane (rBM). (A) Brightfield images of PC3 cells grown on rBM gels treated with PBS (native) or 50 mM glycolaldehyde for 14 hr (AGE-rich, stiff). Cells were imaged using a brightfield microscope (10X objective) and an acquisition rate of 1 image per hr for 12 hr followed by cell tracking to generate trajectories. Images shown correspond to the time points after 0, 3, 6, 9 and 12 hr. Trajectories of single cells are shown for the 12 hr time point. Scale bar = 100 &#181;m. (B) PC3 cells cultured on native or stiff rBM for 72 hr, and imaged as described in panel (A). Scale bar = 100 &#181;m. Detail shows selected area at 2X magnification. (C) Mean &#177; S.D. cell length (&#181;m); significant difference between native rBM and stiff rBM (p = 1.2 x 10-23). (D) Mean &#177; S.D. velocity (&#181;m/hr) calculated from cell trajectories; significant difference between native rBM and stiff rBM (p = 0.004). (E) Mean &#177; S.D. persistence of cell movement (ratio D/T, where D = distance from start to end of cell trajectory, T = total length of cell trajectory); significant difference between native rBM and stiff rBM (p = 0.0007). For panels C-E &#62;10 cells were analyzed, data is representative of 3 independent experiments. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases at JoVE.com

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

how-to-study-basement-membrane-stiffness-as-biophysical-trigger

Research

JoVE Journal

Cancer Research

9.6K Views.  Universidad de la República (UDELAR). Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

Video: How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Bioengineering

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

Engineering

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Biology

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Immunology and Infection

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Extracting the Young's Modulus of Native Murine Pulmonary Basement Membranes from Atomic Force Microscopy Derived Force Maps

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of nanometers. Because mammalian cells sense and react to the mechanics of their immediate microenvironment, the characterization of biomechanical properties of tissues with high spatial resolution is crucial for understanding various developmental, homeostatic, and pathological processes. The basement membrane (BM), a roughly 100 - 400 nm thin extracellular matrix (ECM) substructure, plays a significant role in tumor progression and metastasis formation. Although determining Young&#39;s modulus of such a thin ECM substructure is challenging, biomechanical data of the BM provides fundamental new insights into how the BM affects cell behavior and, in addition, offers valuable diagnostic potential. Here, we present a visualized protocol for assessing BM mechanics in murine lung tissue, which is one of the major organs prone to metastasis. We describe an efficient workflow for determining the Young&#39;s modulus of the BM, which is located between the endothelial and epithelial cell layers in lung tissue. The step-by-step instructions comprise murine lung tissue freezing, cryosectioning, and AFM force-map recording on tissue sections. Additionally, we provide a semi-automatic data analysis procedure using the CANTER Processing Toolbox, an in-house developed user-friendly AFM data analysis software. This tool enables automatic loading of recorded force maps, conversion of force versus piezo-extension curves to force versus indentation curves, computation of Young&#39;s moduli, and generation of Young&#39;s modulus maps. Finally, it shows how to determine and isolate Young&#39;s modulus values derived from the pulmonary BM through the use of a spatial filtering tool.

This protocol visualizes how to prepare cryosections of murine lung tissue, perform atomic force microscopy force map experiments, and analyze the data to determine Young's modulus of the native murine pulmonary basement membrane.

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The cellular mechano-sensation of extracellular mechanical stimuli is believed to be through the membrane receptors, the associated protein complex, and the cytoskeleton. Recent advances in mechanobiology demonstrate that the cell nucleus in cytoplasm itself can independently sense mechanical stimuli simultaneously. However, a mechanistic understanding of how the cell nucleus senses, transduces, and responds to mechanical stimuli is lacking, mainly because of the technical challenges in accessing and quantifying the nucleus mechanics by conventional tools. This paper describes the design, fabrication, and implementation of a new magnetic force actuator that applies precise and non-invasive 3D mechanical stimuli to directly deform the cell nucleus. Using CRISPR/Cas9-engineered cells, this study demonstrates that this tool, combined with high-resolution confocal fluorescent imaging, enables the revelation of the real-time dynamics of a mechano-sensitive yes-associated protein (YAP) in single cells as a function of nucleus deformation. This simple method has the potential to bridge the current technology gap in the mechanobiology community and provide answers to the knowledge gap that exists in the relation between nucleus mechanotransduction and cell function.

This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging.

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The ...

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

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