Extracellular Flux Assay in 3D Spheroids: A Method for Measuring the Metabolic Activity of Pancreatic Tumor Spheroids

- Pancreatic cancer cells interact with cancer-associated fibroblasts in the tumor microenvironment or TME. These interactions modulate the metabolic activity of the cancer cells and affect cancer progression.

Spheroids are the cellular aggregates that recapitulate the TME in vitro. Therefore, we can use these spheroids to test the changes in tumor's metabolic functions by extracellular flux assay. This assay measures the extracellular concentration of oxygen and protons to determine aerobic and glycolytic metabolism, respectively.

Begin by taking appropriately sized 3D spheroids made by co-culturing pancreatic cancer cells with pancreatic fibroblasts. Now, transfer the spheroids into a flux analysis plate and cover it with a sensor cartridge containing test drugs. Run the assay in an extracellular flux analyzer.

Flux analyzer pneumatically injects test drugs from the sensor cartridge to the analysis plate at specific time points. In response to the test drugs, cells release protons and oxygen into the plate micro-chamber and the fluorophores present in the sensor probe measure these levels. Finally, using appropriate software, analyze the functionality of the spheroids.

In the following protocol, we will demonstrate a method for measuring the metabolic function of the pancreatic tumor cells present in 3D spheroids using the extracellular flux assay.

The day before the metabolic assay, hydrate the probe cartridge with 200 microliters of assay calibrant per well in the provided utility plate and incubate the utility plate overnight in a humidified 37 degrees Celsius non-CO2 incubator.

The next morning, observe the spheroids in the growth plate under a light microscope to check their morphology and overall uniformity. Transfer the growth plate onto a magnetic holding drive and carefully aspirate approximately 120 microliters of growth medium per well.

Gently wash the spheroids with 120 microliters of warmed assay medium three times. After the last wash, inspect the spheroids under the microscope again to confirm that the spheroids were not washed away and add 180 microliters of warm assay medium to each well.

Use a wide bored tip to carefully aspirate a single pre-washed spheroid from the cell repellent growth plate and gently transfer the spheroid directly into the center of one well of a spheroid assay plate, allowing the spheroid to fall into the central micro-chamber of the well by gravity.

When all of the spheroids have been transferred, place the assay plate in the non-CO2, 37 degrees Celsius humidified air incubator for one hour. During the incubation, orient the sensor cartridge so that rows A through H are on the left side and place a loading guide on top of the cartridge such that the letter corresponding to the port to be loaded is in the upper left-hand corner.

If the plate has been evenly loaded, open the analysis software and click Templates. Double click on Blank Template and click Generate Groups. Under the Plate Map tab, assign groups to the assay plate corresponding to the experimental design and select the four corner wells as the background wells.

Under the Instrument Protocol tab, check Calibrate, Equilibrate, and baseline Measurements Cycle. Click Injections and define the compound for each port. Change the measurement cycles to 6. Review the Protocol and Group summary, then, save the assay design template.

Based on the assay design as a guide, use a multichannel pipette to dispense each reagent directly into the injection ports, holding the loading guides in place with fingertips. When all the reagents have been loaded, remove the loading guide and hold the cartridge at eye level to visually inspect the injection ports for even loading.

Transfer the cartridge and the utility plate to the extracellular flux analyzer and initiate the pre-assay calibration. Once calibrated, replace the utility plate with the pre-warmed assay plate containing the 3D spheroids and begin the assay. When the assay is finished, export the data into the appropriate data analysis software.