The overall goal of this model is to investigate how basement membrane stiffness and thickness, due to advanced glycation and product exposure can modulate invasive cell behavior corresponding to the early and advanced stages of metastatic prostate cancer. This method can help answer key questions in the age related pathologies field. Such as how increased basement membrane stiffness and thickness contributes to cancer progression, and also metabolic disorders like diabetes.
The main advantage of this technique is that it provides a simple method for mimicking the level of stiffness measured in human prostate tumors in the clinical setting. The implication of this technique extends to our therapy and diagnosis of prostate cancer by improving our understanding of how basement membrane stiffness promotes molecular and cellular changes. Individuals new to this method may struggle, because the model is multi-disciplinary.
Combining techniques from the fields of chemistry, physics, and biology. We first had the idea for this method when we hypothesized basement membrane stiffness in the prostate gland can promote de novo invasive behavior in known transformed epithelial cells. Video demonstration of this method is critical, because inter-disciplinary steps need to be described.
Including cross-linking of basement membrane, followed by 3D cell culture. Start by preparing an even surface of ice, and using it to chill an eight well chamber glass slide to four degrees Celsius. Then, cut off the end of a 200 microliter pipette tip, and cool the tip to four degrees Celsius as well.
Once cooled, place the tip onto a 200 microliter capacity pipetting aid. And use it to transfer 40 microliters of ice-cold BM matrix solution into each well of the chilled eight well chamber glass slide. Then place the chamber slide at 37 degrees Celsius for 30 minutes to promote polymerization of the basement membrane.
Close the incubator door very carefully to avoid disturbing the liquid reconstituted basement membrane. After exactly 30 minutes, add 250 microliters of a prepared glycolaldehyde solution to cover the polymerized, reconstituted basement membrane. Next, prepare a negative control by adding 250 mircoliters of sterile PDS to a native, reconstituted basement membrane gel.
Also, prepare two additional controls by adding either 50 millimolar sodium cyanoborohydride, or 250 millimolar aminoguanidine to glycolaldehyde solution before adding it to the basement membrane gel. Then, incubate the gels at 37 degrees Celsius for six hours to produce a semi-stiff gel. Or for 14 hours to produce a stiff gel.
Following incubation, carefully remove the solutions from the gels and add 250 microliters of one molar glycine ethyl ester to each gel. And incubate them at 37 degrees celsius for one hour. Wash each gel 10 times in 500 mircoliters of PBS to remove all traces of the glycolaldehyde solution, and the glycine ethyl ester.
Then add 400 microliters of PBS to prevent dehydration and incubate the reconstituted basement membrane gels overnight at 37 degrees Celsius. When ready to continue, remove the PBS from the reconstituted basement membrane gels, and add 250 microliters of ice-cold, double-distilled water to each well. Incubate the gels at four degrees Celsius for 16 to 24 hours to ensure that the matrix is completely liquefied.
Then transfer the liquefied basement membrane into a 1.5 milliliter tube, and measure the fluorescent emission of the solution using a spectrophotometer. Then centrifuge the solution and remove the resulting supernatant. Re-suspend the pellet in 500 microliters of a solution containing 20 milligrams per milliliter cyanogen bromide and 70%formic acid.
Incubate the samples overnight at room temperature. Next, use a one milliliter disposable syringe to transfer the re-suspended basement membrane gel into a dialysis cassette with a molecular weight cutoff of 3.5 kilodaltons. Submerge the cassette into a 500 milliliter glass beaker containing 500 milliliters of double-distilled water.
And stir the water with a magnetic stir bar. Place this setup overnight at four degrees Celsius to remove all traces of cyanogen bromide and formic acid. Use a one milliliter disposable syringe to transfer the dialized solution from the cassette into a 1.5 milliliter tube.
Then analyze the solution by running it on a 12%polyacrylamide gel. Stain the gel with silver stain to visualize the electrophoretic pattern of the cyanogen bromide matrix peptides. Starting with the basement membrane gels covered with PBS, rinse the gels two additional times, with 300 microliters of PBS containing 0.1 millimoles of calcium chloride.
And 0.5 millimoles of magnesium chloride. For five minutes at room temperature. Then, remove the PBS and add 300 microliters of 4%paraformaldehyde to cover each reconstituted basement membrane gel.
Incubate the gels for 30 minutes at room temperature to fix the reconstituted basement membrane components. Next, remove the fixative and add 300 microliters of a quenching solution containing 75 millimoles of ammonium chloride and 0.5 millimoles of magnesium chloride. Incubate the solution for five minutes at room temperature, and then remove the solution and repeat the rinse four more times.
Following the final rinse, add 300 microliters of immunofluorescence buffer to the reconstituted basement membrane gels to prevent non-specific reactions. Incubate the gels in the solution for two hours at room temperature on a shaking platform. Then remove the immunofluorescence blocking buffer.
And add 300 microliters of primary antibody that is diluted in the blocking buffer. Incubate the reconstituted basement membrane gels at four degrees Celsius for 16 hours. The next day, remove the primary antibody and wash the gels three times with 300 microliters of immunofluorescence buffer.
Shake the gels at room temperature on a shaking platform for 10 minutes after each buffer addition. Then remove the buffer and add 300 microliters of the florescently labeled secondary antibody, diluted in the blocking buffer. Place the samples on a shaking platform, and incubate the gels for two hours at room temperature.
Remove the secondary antibody. Rinse the sample in a immunofluorescence buffer, followed by PBS, and then fix and quench the samples a second time. Finally, mount the gels and use a microscope to analyze the formation of dense bundles.
To form prostate gland acini, dilute 5, 000 RWPE-1 cells in 300 microliters of complete KSFM, supplemented with 2%of the basement membrane solution. For prostate tumor cell aggregates, dilute 2, 500 PC3 cells in 300 microliters of RPMI-1640 culture medium, supplemented with 2%of the basement membrane solution. Following overnight incubation of the reconstituted basement membrane gels in PBS, rinse them twice with 500 microliters of culture medium.
Before seeding the cells. Gently seed the cells onto the native and advanced glycation end product-stiffened reconstituted basement membranes. And carefully place the cultures in an incubator to ensure an even distribution of growing acini and spheroids.
For the acini forming cells, change the medium every two days with fresh culture medium, containing 2%of the basement membrane solution. To ensure that cells have the growth factors required for normal acini homeostasis. After six days in culture, prostate epithelial cells form acini on native reconstituted basement membrane.
And are organized into uniformed spheroids of epithelial cells. These acini also have the characteristics of highly organized PECs with apical to basal polarity, and a visible luminal space. When cultured for six days on the stiffened membrane, the cells have a disrupted architecture.
And change from spheroidal to polygonal in shape. In addition these acini are highly disorganized. And have lost their apical to basal polarity, with a small or non-existent luminal space.
This is also true for the cells derived from a prostate gland affected by benign prostatic hyperplasia. When the prostate tumor cells were measured, cells were found to be longer on the stiff, reconstituted basement membrane, and also migrated faster and had increased cell persistence. After watching this video, you should have a good understanding of how to model the effects of basement membrane stiffness on epithelial and tumor cell behavior.
While attempting this procedure, it is important to remember that the desired level of basement membrane stiffness is induced, and individual acini or s-spheroids are properly formed. After its development, this technique paved the way for researchers in the field of oncology to explore the induction of cancer cell invasion by basement membrane stiffness in the prostate gland. Following this protocol, other method, like immunoblotting of cell lysate can be performed to answer additional questions relating to signaling pathway, modulated by basement membrane stiffness.
Don't forget that working with sodium cyanoborohydride, cyanogen bromide and formic acid can be extremely hazardous, and precautions such as wearing a lab coat, gloves, face shield and a respirator while working in the fume hood should always be taken while performing this procedure.
