Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

My lab specializes in three and four dimensional tumor modeling. Tumor spheroid models are widely used throughout biology and drug discovery. However, existing protocols for collecting data from these experiments are expensive, difficult, and limited.

This protocol requires easily accessible chemicals and equipments, and is not very labor intensive compared to current methods that provide similar results. This protocol was developed to be relatively easy to implement for beginners and robust to small mistakes, as many of the steps are reversible. Begin by weighing 0.5 grams of low-melting agarose in a glass bottle.

And add 25 milliliters of PBS. Melt the agarose by boiling the solution in a microwave for 30 to 60 seconds with the lid closed, and constant swirling. Weigh nine grams of N, N, N'N'Tetrakis(2-hydroxypropyl)ethylenediamine, 22 grams of urea, 44 grams of sucrose, 0.1 grams of Triton X-100, and 24.9 grams of deionized water.

Heat the solution in a 56 degrees Celsius water bath with constant mixing. After the crystals are dissolved, rest the solution at room temperature to allow the bubbles formed during mixing, to rise to the surface. Generate spheroids using the agarose overlay method described in the text manuscript.

Cut the tip of a one milliliter pipette tip using a scalpel to enlarge the orifice. Using the pipette, aspirate the spheroids from the wells and transfer them to a clear 1.5 milliliter conical tube. Multiple spheroids from the same conditions can be combined in the same tube.

Wash the spheroids twice in one milliliter of PBS, then add neutral 4%pre-warmed paraformaldehyde solution and incubate the spheroids at 37 degrees Celsius. After 20 minutes, remove the paraformaldehyde solution and wash the spheroids twice with one milliliter of PBS. Then for staining, transfer up to 15 spheroids into a 200 microliter PCR tube, using a pipette.

Permeabilize the cells using 200 microliters of 0.5%Triton X-100 in PBS. Place the PCR tubes inside 50 milliliter, screw cap tubes and cover the tube with aluminum foil and incubate the spheroids at room temperature with mild agitation. After two hours, replace the solution with 200 microliters of antibody dilution buffer and incubate overnight at room temperature.

The next day, remove antibody dilution buffer before adding 75 microliters of primary antibody and incubate at four degrees Celsius for two and a half days. After incubation, remove the supernatant and wash the spheroids twice with 200 microliters of PBS, containing 0.1%Tween and 0.1%Triton X-100 or PBS-TT. Then incubate with 200 microliters of PBS-TT for four hours at room temperature.

Next, remove PBS-TT before adding 100 microliters of the secondary antibody and incubate at four degrees Celsius for two and a half days. After incubation, remove the supernatant and wash twice with PBS-TT, before incubating with 200 microliters of PBS-TT for four hours. For mounting, use a 200 microliter pipette, transfer fixed and stained spheroids to 500 microliter PCR tubes, at one tube per condition.

Replace the solution with 200 microliters of 2%liquid agarose gel, and centrifuge on the quick spin, at the fixed maximum speed, at room temperature, for 30 seconds. Aspirate the spheroids in 50 microliters of liquid agarose gel and dispense in the well of a 24-well glass bottom plate. Before the gel hardens, separate the spheroids using a pipette tip in the surrounding gel, and ensure the spheroids are covered with gel.

Next, submerge the gel by adding 500 microliters of clearing solution per well, and incubate at room temperature, in the dark, for 24 hours before imaging. For imaging, choose an objective with a working distance long enough to encompass the entire spheroid, including the mounting height of the spheroid in the vessel. To identify the equatorial plane, adjust the focus until the largest surface area is reached in the XY plane, and image with the required laser power percentage, detector voltage, gain, and offset settings.

For three dimensional images, set the start and end of the spheroids and choose the appropriate signal intensity at various Z-depths, using Z intensity correction settings. This study shows a comparison of cleared and uncleared FUCCI human melanoma spheroids, compared with PBS mounted spheroids. The clearing solution provides high clarity images with minimal size distortion.

Clearing solution further allows for high resolution imaging of cellular level details deeper into the spheroids without histological sectioning. With three-dimensional confocal images, cellular level details at a depth of 200 microns can be obtained. Further, a comparison between cryosectioned and cleared hole spheroid, stained for pimonidazole and p27kip1, is shown.

Pimonidazole staining shows the hypoxic region of the spheroid, and p27kip1 staining, marks cell cycle arrest and DAPI nuclear stain. Deeper penetration and less scattering, allow improved three-dimensional spheroid structure representation with minimal light loss. The three-dimensional rendering of the FUCCI spheroids, stained with DRAQ7, is shown.

Clearing solution has minimal impact on spheroid size. The diameter of the spheroid was defined based on a sphere with the same cross-sectional area as the spheroid. The spheroids were observed to slightly increase in size over the first six hours, as indicated by a diameter fold-change of between 2%and 6%In contrast, after 24 to 72 hours, the spheroids returned to a size approximately equal to the corresponding size in PBS post-paraformaldehyde fixation.

It is important to wait at least 24 hours before imaging, to let the clearing solution equilibrate with the spheroids and the gel. This protocol allows practitioners to rapidly collect data from large numbers of spheroids, allowing for detailed quantitative mathematical and statistical analysis that provides detailed insights into the biology.