Advancements in the Metabolic Profiling of Three-Dimensional Brain Tumor Spheroids for Drug Screening

The research aims to optimize 3D cell cell root formation for studying energy metabolism using Seahorse technology, focusing on cell behavioral metabolic activity and drug responses in our clinically-relevant model. This findings will enhance drug casting models by providing a more accurate representation of brain tumors, improving therapy evaluation, and metabolic analysis for better treatment strategies. The results paved the way for investigating how energy metabolism alters in 3D spheroid models, how drugs exit effects on metabolic pathways, and the mechanisms by which metabolic changes contribute to the development of tumor resistance to therapy.

After resuspending the collected U87 cells in DMEM, preheat the unused medium to 37 degrees Celsius. Maintain the cell suspensions at room temperature for 30 minutes. Based on the initial cell count, dilute the cell suspension with DMEM containing 10%FBS to a final volume of 20 milliliters, ensuring approximately 200, 000 cells.

Mix the final cell suspension thoroughly by repeated inversion. Slowly add 200 microliters of the suspension along the edge of each well in a transparent round bottom ultra-low attachment 96-well plate, then seal the plate tightly with Parafilm to prevent leakage during centrifugation. Place the sealed plate in the centrifuge and run at 200 G for two minutes to pellet the cells at the bottom of each well.

Remove the plate from the centrifuge. Carefully remove the Parafilm, and place the plate in a 37 degrees Celsius incubator with 5%carbon dioxide. To prepare a coating solution, dissolve five milligrams of Poly-D-Lysine in 50 milliliters of sterile water, and mix the solution thoroughly, until completely dissolved.

Add 30 microliters of the coating solution to each well of the metabolic assay plate. Ensure the absence of bubbles by gently shaking the plate or aspirating the excess solution. Incubate the plate with the cover on for 20 minutes at room temperature, then aspirate the coating solution, and wash the wells twice with 200 microliters of sterile water.

Air dry the plate for at least 30 minutes, then heat the plate at 37 degrees Celsius without carbon dioxide for 30 minutes to stabilize the coating. Afterward, add 175 microliters of preheated medium to each well. Keep the plate at 37 degrees Celsius in a carbon dioxide-free incubator, until it is ready for cell spheroid transfer.

Transfer the spheroid plates to a high-content imaging system after five days of culture. To configure the imaging parameters, set the temperature to 37 degrees Celsius and the carbon dioxide level to 5%Adjust the plate type, channels, and focus range on the system settings. Next, acquire spheroid images using a confocal microscope with 10 times objectives and the imaging software, then identify wells containing intact spheroids, and record their positions for transfer.

Assign the intact spheroids to two groups, a control group with six replicate wells and a treatment group with six replicate wells for fenofibrate treatment. Prepare the fenofibrate working solution in fresh prewarm medium at the optimal concentration. Carefully remove the spheroid containing low-attachment plate from the imaging system and place it on a sterile surface inside a biosafety cabinet.

Position the Poly-D-Lysine-coated assay plate adjacent to it for transfer, then trim the tip of a 20 microliter pipette to allow gentle aspiration of the cell spheroids without causing damage. Using the trimmed pipette tip, gently aspirate each intact spheroid from the bottom of its well. Transfer each spheroid to the corresponding well of the coated plate, and allow it to settle for 20 seconds.

Transfer the 3D spheroid assay plates to a high-content imaging system. To analyze the morphological data, apply the texture region module to coarsely separate 3D spheroids from the background by dividing the image content into three clusters based on texture features. Apply the find image region module to refine the separation of preisolated 3D spheroids by using volume-based size characteristics, ensuring impurities are removed and intact spheroids are retained.

Measure the morphological parameters of the 3D spheroids using the morphology properties module. Apply the select population module to filter and retain only genuine 3D cell spheroids, then export the morphological data results. Spheroids were visibly formed by the fifth day of culture, maintaining a uniform spherical morphology across all observed samples.

A disintegrated spheroid with visibly disrupted structural integrity was also observed on day five. Post-transfer analysis revealed that both control and fenofibrate-treated 3D spheroids remained intact in most cases, but some fenofibrate-treated spheroids were disintegrated. Oxygen consumption rate was significantly reduced in fenofibrate-treated spheroids across all time points compared to the control group.