This protocol exposes cancer cells in a suspension to brief pulses of fluid shear stress to mimic certain aspects of how metastatic cancer cells are exposed to hemodynamic forces while they're in the circulatory system. It is a relatively simple technique to apply brief pulses of high level fluid shear stress. When attempting this protocol, be careful on uncapped needles and do not bend them as this will change applied force shear stress.
This procedure may produce aerosols, so use appropriate safety measures. To begin, release 70 to 90%confluent PC3 cells from the tissue culture dish by aspirating the growth medium and washing the 10 centimeter dish with five milliliters of calcium and magnesium-free PBS. Then, aspirate the PBS and add one milliliter of 0.25%trypsin.
Post-trypsinization, observe the detachment of the cells under an inverted microscope. To inhibit the trypsin, add five milliliters of DMEM/F12 medium containing 10%FBS. Next, place the cell suspension into a conical tube and determine the cell concentration and total cell number.
Centrifuge the cell suspension at 300 x g for three minutes, then aspirate the supernatant and resuspend the pellet in a serum-free tissue culture medium. Cut around bottom of 14 milliliter polystyrene tube at the seven milliliter line and place the mixed cell suspension into the cut tube. Separately collect static control samples of cells before fluid shear stress exposure to use for performing assays.
To expose the remaining cell suspension sample to fluid shear stress, draw the cell suspension into a five milliliter syringe and attach a 30 gauge half inch needle. Place and secure the syringe with an uncapped needle onto a syringe pump and set the flow rate to achieve the desired level of fluid shear stress. Run the syringe pump and collect the sheared sample in the cut tube at an approximately 45 degree angle to reduce foaming.
Carefully remove the syringe from the syringe pump and use pliers to remove the needle, taking care not to touch it. Repeat the procedure until the cell suspension has been exposed to the desired number of pulses of fluid shear stress. Perform viability assays with the static samples before exposing the cells to fluid shear stress.
For enzymatic assays, transfer 100 microliter aliquots from the static samples in duplicate into a 96-well plate. Then, collect 100 microliters of samples after fluid shear stress exposure and place them in a 96-well plate. Add 20 microliters of a 0.15 milligram per milliliter resazurin solution to each sample well and to wells containing 100 microliters of medium alone.
Incubate the well plates in the 37 degrees Celsius tissue culture incubator for two hours, then measure the fluorescence and absorbance using a plate reader and obtain the percentage of viable cells by comparing the average signal from each of the fluid shear stress exposed samples to the average to static control sample. Similarly, collect aliquots from static samples to perform flow cytometry and clonogenic assays as described in the text manuscript. In the representative analysis, the viability of syngeneic biopsy mammary epithelial cancer cells was assessed using resazurin conversion after exposing cells to a number of fluid shear stress pulses.
Even though each cell line displayed different resistance profiles, there was no significant difference in viability after 10 pulses of fluid shear stress exposure. Additional cancer cell lines from a variety of tissue origins demonstrated the viability of more than 20%after 10 pulses of fluid shear stress, except for MIA PaCa-2 cells, which showed a viability of less than 10%due to sensitivity towards mechanical destruction from fluid shear stress. When collecting the static sample prior to applying fluids shear stress, ensure that the cell suspension is a homogeneously mixed.
Carefully remove the needle with pliers and do not bend or touch the needle. In addition to measuring cell viability, one can evaluate changes in gene expression, cell signaling, proliferation, and migration. Using this as a model of exposure to fluid shear stress, one can study both how cancer cells resist destruction by fluid shear stress and evaluate the effects of fluid shear stress on the biology of cancer cells.
This technique has been used by our laboratory and others to explore the effects of fluid shear stress on circulating tumor cells. These studies have shown that fluid shear stress rapidly alters the mechanical properties of cancer cells and that this contributes to the ability of cancer cells to resist destruction by fluid shear stress.
