Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

This protocol highlights the importance of the tumor microenvironment in activating the cancer-associated fibroblasts or CAF. It could be excellent in vitro model to study phenotypic characteristics. The major constraints in CAF biology lies in the limited availability of CAF from primary tumor biopsy samples.

So this technique can be used to study various aspects of CAF biology. This study depicts aberrant cross production and mitochondrial dysfunction associated with CAF activation leading to a poor tumor prognosis. The application of this method would be useful for assessing the tumor's trauma interactions particularly CAF activation and mitochondrial health.

In order to explore the novel drug targets. Individuals must have experience in cell procedure before performing this technique. The visual demonstration of this technique will allow understanding the critical steps in isolating the CAF population from the tumor spheroids and downstream applications to study CAF biology.

Miss Leena Arora, Miss Moyna Kalia, and Mrs. Soumyajit Roy, assistants of my lab perform the experiments. To begin grow human lung adenocarcinoma A549 and human lung fibroblast MRC-5 adherent cells in DMEM and human monocytes THP-1 cells in RPMI complete growth medium in T25 flasks at 37 degrees Celsius in a humidified chamber with 5%carbon dioxide.

After three days, at 80 to 85%cell confluency, wash the A549 and MRC-5 cells with one milliliter of PBS for one minute. Next, harvest the cells by incubating them with 500 microliters of 0.25%trypsin EDTA solution for five minutes at 37 degrees Celsius. Then, immediately add four milliliters of complete growth medium to inactivate the trypsin.

Collect the cell suspension into a 15 milliliter tube and pellet it down at 125 x g for five minutes. Remove the supernatant and resuspend the cells in a five milliliter complete growth medium. To establish multicellular tumor spheroids, count A549, MRC-5, and THP-1 cell numbers using a cell counter for one milliliter volume.

After counting, prepare the cell suspension at a ratio of 5:4:1 and make up the volume to one milliliter with complete DMEM. Next, place a drop of 25 microliters of cell suspension mixture onto the cover of a 90 millimeter culture dish. Fill the bottom of the dish with 10 milliliters of sterile water.

Carefully invert the lid over the water-filled hydration chamber and place the dish in a cell culture incubator for three days. On day four, monitor the spheroids under the microscope. To acquire images, switch on the power switch.

Place the 90 millimeter culture dish on the stage and select the 10 x magnification. Adjust the lenses and examine the cells to analyze the cell aggregation and proliferation. Press the freeze and save buttons provided to capture the image.

After imaging, replace the growth medium by carefully aspirating 20 microliters of medium from each droplet with a fresh medium. For live-dead imaging, switch on Ctr advanced, which is switch one, then the CPU and wait for the software system to boot. When booted, place the 90 millimeter dish containing spheroids onto the stage of the inverted fluorescent microscope.

Then observe and capture images at 10 x magnification by selecting the fluorescence option in the software and selecting the FITC for the calcein and Texas Red channel. Next, select the option, live and view the image. Adjust the fluorescence intensity for appropriate optimization and click the safe button.

Collect 200 tumors, spheroids each on day seven and 10 using a one milliliter pipette and a 15 milliliter tube. Then pellet the spheroids by centrifugation at 125 x g for five minutes and carefully aspirate the supernatant. Wash the spheroids with 200 microliters of PBS, centrifuge again and discard the supernatant.

Add 400 microliters of 0.25%trypsin EDTA solution for spheroid disintegration and incubate at 37 degrees Celsius for 10 minutes. Perform vigorous pipetting for complete disintegration of the spheroids. Neutralize the trypsin by adding one milliliter of complete DMEM growth medium.

Then centrifuge the spheroid suspension and discard the supernatant before resuspending the pellet in one milliliter of complete DMEM medium. Count the total number of cells as demonstrated. For the isolation of Cancer-Associated Fibroblasts or CAFs from the tumor spheroids, resuspend 1 x 10 to the seventh cells in 80 microliters of cold magnetic activated cell sorting or MACS buffer.

Add 20 microliters of anti-fibroblast microbeads into the cell suspension, mix well by gently tapping the tube and incubating it at room temperature for 30 minutes. After incubation, wash the cells with one milliliter of cold MACS buffer. Then centrifuge and aspirate the supernatant before resuspending the pellet in 500 microliters of MACS buffer.

For magnetic bead based cell separation, prepare the MACS column by rinsing it with three milliliters of MACS buffer. Place the cell suspension into the column and collect the flow through containing the unlabeled cell population. Next, wash the column thrice with three milliliters of MACS buffer before removing it from the separator.

Then place the column on the collection tube. Collect the anti-fibroblast microbead labeled cells by adding five milliliters of MACS buffer and firmly pushing the plunger into the column. Centrifuge the labeled cells before proceeding for downstream applications.

Count the number of isolated CAFs and use approximately 6 x 10 to the fourth cells for flow cytometry analysis. Wash the cells once with PBS, then centrifuge and aspirate the supernatant. Next, add 100 microliters of cell permeabilization buffer and incubate the cells at four degree Celsius for 30 minutes with intermittent vortexing to maintain a single cell suspension.

Again, after centrifugation and resuspending the cells in permeabilization buffer, add two microliters of APC conjugated anti-human alpha SMA antibody and incubate at four degree Celsius for 45 minutes. After incubation, add one milliliter of permeabilization buffer and centrifuge to remove excess antibody. Resuspend the pellet in 400 microliters of permeabilization buffer for flow cytometry.

Select ACTA2 positive cells after distinguishing the populations of cells based on their forward and side scatter. Selecting the singlet population followed by selecting the ACTA2 positive cells which appear as a single peak on the single parameter histogram. The development of multicellular tumor spheroids and live-dead analysis are shown here.

On day seven, spheroids were compact and rigid with approximately 260 microns in diameter. On day 10, spheroids were 480 microns in diameter. In cell viability assay, a decrease in propidium iodide fluorescence and an increase in calcein-AM fluorescence from day seven to day 10 was observed.

Hypoxic staining revealed a hypoxic core at the center of the day 10 spheroids. Furthermore, upregulation of E-cadherin and Mki-67 gene expression with the increase in days of tumor spheroid formation was observed. CAFs isolated from day 10 tumor spheroids were approximately 69%ACTA2 positive.

The CAFs showed threefold upregulation in ACTA2 and tenfold in collagen type 1 alpha 2 chain compared to the MRC-5 fibroblasts. A significant increase in ROS levels on day 10 tumor spheroids was observed compared to day seven, suggesting the possible involvement of ROS generation on CAF activation. A significant increase was observed in cellular ROS levels in CAFs, isolated from day seven and day 10 tumor spheroids.

Moreover, an alteration of mitochondrial membrane potential was seen by JC-1 staining. An induction of GLUT1 and MCT4 gene expression was observed in CAFs compared to normal fibroblasts. Significant induction of lactate dehydrogenase, Cytochrome C oxidase and succinate dehydrogenase enzyme activity were observed in CAFs compared to normal fibroblasts.

A ratio of 5:4:1 of A549, MRC-5, and THP-1 cells must be used for the successful development of 3D multicellular tumor spheroid. Fibroblast population is critical for the rigidity of the tumor spheroids. Similar to cation solution and characterization, one can also use this tumor spheroid to obtain tumor associated macrophage cell population, which is an accomplice in tumor progression.

This spheroid analysis would help us to understand how cancerous cells crossed fibroblast are involved in the CAFs activation, and also can be used to check the mitochondrial specific drug candidates.