Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

This protocol is demonstrating that we can now determine the effect of migrastatic inhibitors in detail in 3D cancer models. The main advantage is the ease and all-in-one approach with which this technique can be carried out. This technique allows the assessment of migrastatic drugs in cancer research in a 3D setting, allowing the testing of drugs in a more tumor environment-relevant setting.

This method may be applied to testing of cytotoxic drugs in cancer or, for example, to assess the effect of radiation on cancer proliferation and cell migration. It is useful to practice the medium removal step and collagen replacement with ordinary 96-well plates to become confident. Also, training on a confocal microscope is essential.

Visual demonstration of this technique is critical for researchers to understand the required delicate handling of the spheroids and their use in confocal microscopy. On the first day of experimentation, have the standard culture medium, in this case, U251, ready for the appropriate cell line. In a tissue culture hood, add 0.5 milliliters of trypsin into the culture to trypsinize the cancer cells.

Count the cancer cells on a hemocytometer, and dilute to a concentration of five times 10 to the third cells per milliliter. Keep the cell suspensions in clearly labeled, sterile universal tubes. Resuspend the cells by gentle inversion to avoid cell clumping.

Use a pipette to add 200 microliters of the cell culture to each well in a 96-well plate. Add 200 microliters of 1X PBS to each empty well to avoid evaporation. Incubate the cells in an incubator at 37 degrees Celsius.

After 24 hours, check the cells by bright-field microscopy. Cell lines such as glioma cancer cell lines form spheroids in the bottom of the well within 24 hours. Incubate the spheroids for another 48 hours to allow a 3D cellular architecture to form.

On the third day, place collagen, 5X culture medium, one-molar sodium hydroxide, and one 20-milliliter tube on ice. Carefully and slowly add 10.4 milliliters of cold collagen into the chilled culture tube. Avoid bubbles.

Then, gently add 1.52 milliliters of cold sterile 5X culture medium. Avoid bubbles. Just before use, gently add 72 microliters of cold sterile one-molar sodium hydroxide, and keep the solution on ice.

Mix gently by pipetting and avoiding bubbles. Efficient mixing leads to a color change from red to orange-red in the medium. Leave the mixture on ice until use.

Next, to remove 190 microliters of supernatant from the 96-well plate prepared on day one, use a pipette at an angle towards the side of the well. Be very careful not to disturb the spheroids that formed in the bottom of the well. Gently add 100 microliters of the collagen mix to each well, pipetting down the side of the well.

Avoid bubbles and any spheroid disturbance. Keep any remaining collagen mix in the 20-milliliter tube at room temperature to assess polymerization. Incubate the plate in the incubator for at least 10 minutes to allow the collagen to polymerize.

When the leftover collagen becomes semisolid and sponge-like, the spheroids are ready to be treated with inhibitor. Add the inhibitors at 2X concentration to five milliliters of culture medium. Pipette 100 microliters of the medium gently down the side of each well to avoid spheroid disturbance.

Observe and image each spheroid by bright-field microscopy at times of zero hour, 24 hours, 48 hours, and 72 hours to assess drug activity. Then, return the plate to the incubator. Now, place the plate in a tissue culture hood, and gently replace the supernatant with 100 microliters of 1X PBS.

Take care not to disturb the spheroid, and avoid touching the collagen. Repeat this wash step three more times. Remove the final wash in each well, and replace with 100 microliters of 4%formaldehyde in 1X PBS.

Place the 96-well plate on a lab bench, cover with foil, and leave for 24 hours at room temperature. The next day, carefully remove the formaldehyde, and replace with 1X PBS. Repeat this wash three more times.

Then, replace the 1X PBS wash with 100 microliters of freshly prepared Triton X-100 solution. Incubate for 30 minutes at room temperature. In the meantime, prepare the blocking solution with 50 milliliters of 1X PBS and 05%skimmed milk powder in a 50-milliliter tube, and mix thoroughly.

After 30 minutes, remove the Triton X-100, and wash three times with 1X PBS. Add 100 microliters of the blocking solution to each well, and incubate for 15 minutes. Next, dilute anti-mouse IgG acetylated tubulin antibody in the blocking buffer at a ratio of one to 100.

Centrifuge the primary antibody-blocking buffer mix for five minutes at 15, 682 times g. Carefully remove the blocking solution in each well, and add 25 to 50 microliters of the antibody blocking buffer supernatant from the tube. Incubate in the dark at room temperature for one hour.

Then, remove the antibody solution from each well, and wash three times with 100 microliters of 1X PBS. Dilute secondary antibody in the blocking buffer at the recommended concentration in addition to any additional fluorescent stains. Again, centrifuge the secondary antibody solution for five minutes at 15, 682 times g.

Remove the blocking solution from each well, and add 25 to 50 microliters of the secondary antibody supernatant. Incubate in the dark for 1 1/2 hours at room temperature. Remove the secondary antibody dye solution, and wash three times with 1X PBS, 100 microliters per well.

Carefully lift individual collagen plugs by suction with a plastic 200-microliter pipette onto the center of a glass slide. Add one drop of a suitable mountant to cover the collagen plug. Position a coverslip on top of the sample, and store the slide at room temperature in the dark.

Three-dimensional spheroid technology is advancing the understanding of drug-tumor interactions because it is more representative of the cancer-specific environment. In this study, potential anti-or pro-migratory effects were detected. This is especially noticeable in KNS42 with seemingly no migration in either the control or treated spheroids.

Cell death was observed at the highest inhibitor concentration of 10 micromolar. Nuclear fragmentation and migrating cells were evident in U251 cells. U251 cell spheroids had spikes radiating away from the original spheroid, with increasing cell rounding in cells apparent with increasing inhibitor concentration.

Collapsed microtubules and nuclear fragmentation were observed in KNS42. Sheetlike protrusions with single cell spikes indicate KNS42 migration. At the lowest inhibitor concentration, this phenotype was pronounced but was lost with increasing inhibitor concentration.

It is crucial to leave all the reagents on ice until ready. Otherwise, the collagen starts polymerizing before it has all been added to the spheroids. There is scope to quantify the images generated by confocal microscopy to assess the effects of, for example, migrastatic drugs on cell morphology.

It allowed us, for the first time, to confirm the effect of the migrastatic inhibitor MI-192 on microtubule acetylation levels and to explore this further in terms of cell migration. Extra care should be taken when handling formaldehyde for the fixation step. The usual health and safety guidelines apply.