Evaluating the Effect of SASP Factors on the Proliferation of Cancer Cells Using a Comparative Analysis of Three Distinct Methodologies

Senescent cancer cells secrete such factors influencing neighboring cells. We present three in vitro models to study SASP-driven paracrine and juxtacrine effects on HeLa cell proliferation, Real-time impedance monitoring and live-cell imaging enabled dynamic, kinetic, and morphological analysis revealing senescent and microenvironmental paracrine effects beyond traditional endpoint assays. To begin, power on the real-time cell analysis system before cell seeding.

Launch RTCA software version 2.0. Click the X notes button and enter the experimental details. Click the Schedule button and add two steps to record background and to monitor cell proliferation every 15 minutes for 72 hours.

Choose the first step and click Play. Manually add 50 microliters of complete medium to each well of the e-plate view. Next, wash both the senescent and non-senescent HeLa cells with five milliliters of sterile, prewarmed PBS.

After aspirating, add one milliliter of prewarmed 0.05%trypsin and incubate. Neutralize the trypsin with five milliliters of antibiotic-supplemented complete DMEM. Then, centrifuge the cells at 500 G at room temperature for 10 minutes.

After discarding the supernatant, re-suspend the cell pellets in five milliliters of antibiotic-supplemented complete DMEM. Now, seed non-senescent HeLa cells into each well of one e-plate view in 100 microliters of antibiotic-supplemented complete DMEM. Place the e-plate view in the cradle of the real-time cell analysis system to allow the cells to attach to the plate surface in the incubator.

Choose the second step and click Play to start impedance measurement. Seed cells in the e-plate insert wells containing 60 microliters of antibiotic-supplemented complete DMEM under three different experimental setups. Maintain the e-plate insert at 37 degrees Celsius in an incubator with 5%carbon dioxide for six hours for attachment.

Click Pause after six hours of cell seeding, then gently aspirate the culture medium from each well of the e-plate view to remove serum and non-adherent cells. Wash wells two times with prewarmed PBS at 37 degrees Celsius. Add 140 microliters of serum-free DMEM to each well of the e-plate view.

Remove the medium. Wash wells of e-plate insert with PBS, then adds 60 microliters of serum-free DMEM. Now, position the e-plate insert into the e-plate view.

Gently press down until the insert is fully seeded in the well. Insert the combined plate into the real-time cell analysis system. Click Play to resume real-time monitoring of cell index values at 15 minute intervals using the software.

Normalize the cell index values at the time point when the e-plate view and e-plate insert are combined and inserted into the real-time cell analysis system. Next, press the Data Analysis button to calculate the proliferation rate and quantity using the maximum cell index and slope data obtained from the real-time cell analysis system. Then, analyze the data using an appropriate statistical program.

Aspirate the culture medium from both six-well plates containing the senescent and non-senescent HeLa cells. Wash the cells two times with prewarmed PBS at 37 degrees Celsius to remove residual serum and cell debris. Add two milliliters of serum-free antibiotic-supplemented DMEM media into each well, then incubate the cells in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for 48 hours.

Collect the conditioned media from each well into sterile tubes in a laminar safety cabinet. Centrifuge the collected conditioned media at 1000 g for 10 minutes at four degrees Celsius to remove cell debris, followed by centrifugation at 10, 000 g for 10 minutes at four degrees Celsius to obtain the clarified supernatant. Then, centrifuge the conditioned media using two-milliliter ultra-centrifugal filter units at four degrees Celsius for 60 minutes at 3000 g to concentrate it to 400 microliters.

Dilute the obtained concentrate with fresh serum-free medium to prepare 1x, 2x, and 3x concentrated conditioned media in sterile tubes in a laminar safety cabin. When the cells have attached, gently aspirate the culture medium from each well of e-plate view to remove serum and non-adherent cells and wash with PBS. Next, pipette 200 microliters prewarmed collected conditioned media at 37 degrees Celsius into each group.

Insert the e-plate view into the real-time cell analysis system and click Play to resume real-time monitoring of cell index values at 15 minute intervals for 48 hours. Seed cells onto a micro slide under three experimental setups and incubate at 37 degrees Celsius in a humidified atmosphere with 5%carbon dioxide. After eight hours of attachment, wash the cells twice with prewarmed PBS at 37 degrees Celsius.

Add 200 microliters of serum-free Dulbecco's Modified Eagle's Medium to each well. Turn on the Temp-controller 2002-2 and set it to 37 degrees Celsius. Turn on the CO2-Controller 2000 and set it to 5%carbon dioxide.

Switch on the fluorescence light source. Place the slide on the stage-top incubation chamber. Launch the software on the computer.

Select the 20x objective. Add the phase contrast channel and the fluorescence FITC channel. Using the stage controller, mark the positions for each location.

Set the time interval to 30 minutes and the acquisition duration to 48 hours, then start the acquisition. Count green fluorescent protein positive cells at both time points and calculate the percentage increase for analysis. Doxorubicin treatment significantly induced cellular senescence in HeLa cells.

Quantification of SA-beta-gal-positive cells showed a significantly higher percentage in the doxorubicin-treated group compared to control. In the first approach, non-senescent HeLa cells co-cultured with senescent HeLa cells exhibited a significantly increased proliferation rate and capacity compared to cells co-cultured with non-senescent HeLa cells. Conditioned media derived from senescent HeLa cells at 2x and 3x concentrations significantly increased the proliferation rate and capacity of non-senescent HeLa cells compared to conditioned media from non-senescent cells.

In the third approach, after 48 hours of co-culture, GFP positive HeLa cells showed a significantly greater increase in proliferation when co-cultured with senescent HeLa cells compared to non-senescent co-culture or GFP-only controls.