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

Summary

This study evaluates the proliferative effects of senescence-associated secretory phenotype (SASP) factors from senescent HeLa cells on non-senescent HeLa cells using three in vitro models with real-time monitoring. The advantages and limitations of each method were systematically compared to better understand cell-cell interactions in cancer.

Abstract

Chemotherapy-induced senescent cancer cells also secrete various factors, called senescence-associated secretory phenotype, to regulate their extracellular microenvironment. Previous studies have reported that these SASP factors exert detrimental paracrine effects on surrounding cells, including promoting proliferation, epithelial-mesenchymal transition (EMT), angiogenesis, and migration. Several in vitro co-culture techniques are widely used to understand the cellular processes resulting from interaction by culturing the same and different types of cells together. Here, the potential proliferative effects of SASP factors secreted by senescent HeLa cells on non-senescent HeLa cells were investigated using three complementary in vitro approaches. The first approach involved co-culturing senescent and non-senescent cells to investigate the paracrine signaling mediated by the SASP. In the second approach, conditioned media collected from senescent cancer cells were concentrated and subsequently used to examine the impact of the conditioned media with real-time monitoring of proliferation. In the third method, senescent and non-senescent cells were cultured side by side to assess the juxtacrine effects of SASP through direct cell-to-cell contact. All three experimental models consistently demonstrated that SASP factors significantly enhanced the proliferation of non-senescent cancer cells. Notably, senescent cell co-culture increased the proliferation rate by 64.6%, and 3x concentrated SASP-conditioned media increased proliferation by over 50% compared to controls. Fluorescence-based imaging showed a 49.3% increase in GFP-positive cell numbers under juxtacrine conditions. These methods collectively enabled quantitative and qualitative evaluation of SASP-induced proliferative changes. The comparative analysis of these approaches highlights their respective strengths and limitations, providing valuable insights into the paracrine effects of senescent cells.

Introduction

In multicellular organisms, cells communicate through paracrine and juxtacrine signaling by exchanging messages with their microenvironment, thereby influencing each other's behavior. This communication can occur either directly, through tunneling nanotubes and gap junctions, or indirectly through the secretion of soluble chemical molecules such as cytokines, growth factors, and chemokines into the extracellular matrix. These signaling mechanisms play essential roles in regulating various physiological processes, including tissue development and immune cell responses, and are also implicated in the pathogenesis of numerous diseases, such as cancer, HIV, hypertension, and Alzheimer's¹.

In the 1960s, Leonard Hayflick discovered that normal human fibroblasts have a limited ability to replicate in vitro². This limited cell proliferation state that develops due to telomerase shortening is called replicative senescence. Subsequent studies revealed that various forms of cellular stress can also trigger cellular senescence. It has been determined that common cancer treatments such as chemotherapy and radiotherapy also induce cellular senescence in both cancer and stromal cells, called therapy-induced senescence^3,4. Cellular senescence and aging are hierarchically related processes; they represent distinct biological processes. The accumulation of senescent cells in the organism contributes to aging by promoting inflammation, tissue dysfunction, and the development of age-related diseases. However, aging encompasses a much broader range of systemic events and mechanisms compared to senescence⁵.

Senescent cells are characterized by their expanded cytoplasm being significantly larger and having a flattened morphology compared to normal cells⁶. These senescent cells have increased lysosomal activity and are positive for senescence-associated β-galactosidase (SA-β-gal) activity, a lysosomal enzyme that remains active even at acidic pH 6.0. SA-β-gal staining is the most widely used biomarker for detecting senescent cells⁷. Enlargement and irregular morphology of nuclei are commonly observed in cells undergoing senescence. In addition, these cells are characterized by unique chromatin rearrangements called senescence-associated heterochromatic foci (SAHF) that mediate irreversible cell cycle arrest^8,9.

Although senescent cancer cells ultimately lost their ability to proliferate, they remained viable and metabolically active. It is known that senescent cells also secrete various factors to regulate their extracellular tumor microenvironment, called senescence-associated secretory phenotype (SASP)^10,11. These SASP factors include soluble signaling factors (interleukins, chemokines, and growth factors), secreted proteases, and secreted insoluble proteins/ extracellular matrix (ECM) components. Collectively, these factors exert detrimental paracrine effects, including promoting cancer cell proliferation, migration, invasion, and the induction of epithelial-to-mesenchymal transition (EMT)^4,12. In addition, chemotherapy-induced adverse effects have been partially attributed to the accumulation of senescent cells¹³. Therefore, investigating the interaction between senescent and non-senescent cells has become an important area of research in the context of cancer therapy. Especially the analysis of SASP factors, particularly their compositions and the effects on the cancer microenvironment, is considered a promising strategy for identifying new target molecules for cancer therapy and discovering new drugs for these molecules.

In vitro co-culture techniques have long been used to study the understanding of cell-to-cell interactions¹⁴. In this methodological article, the aim was to evaluate the possible proliferative activity of factors secreted from senescent cells on non-senescent cancer cells using three different methods. Additionally, the results obtained from the three different methods were compared, and each approach's respective advantages and disadvantages were critically evaluated.

First, a co-culture model was set up between senescent HeLa and non-senescent HeLa cells to monitor the possible changes in cancer cell proliferation in real-time using the real-time cell analysis system¹². This system consists of two plates to evaluate cell-cell interactions. The e-plate view, which forms the lower layer, has a surface where target cells are seeded, with microelectrodes positioned at the bottom of the plate. The electrical resistance created by the adhesion of target cells to this surface is expressed by the measurable parameter Cell Index (CI). The CI value is proportional to the number of cells and the degree of adhesion to the surface¹⁵. In addition, the e-plate view allows morphological examination of cells under a phase contrast microscope. The e-plate insert, which forms the upper layer, is the area where effector cells are seeded. The bottom of this plate is covered with a 0.4 µm pore size semi-permeable membrane. Factors secreted from effector cells can pass through the membrane and affect the target cells in the e-plate view. If this interaction stimulates cell proliferation, this is observed as an increase in the CI value. Thus, interactions between cells can be evaluated quantitatively.

In the second method, conditioned media collected from senescent HeLa cells were concentrated and added to non-senescent HeLa cells. The potential proliferative effect on these cells was monitored using the real-time cell analysis system.

In the third method, it was planned to co-culture the senescent HeLa cells and the non-senescent GFP-HeLa cells directly in 2D and monitor the possible proliferative alterations in cancer cells in real time under a microscope.

The goal of this experiment is to set up three models to study the interaction between senescent HeLa and non-senescent HeLa cells and monitor possible changes in cancer cell proliferation in real time. Thus, the effect of SASP factors secreted by senescent cancer cells on the other cancer cells can be easily investigated. Accordingly, the advantages and limitations of the three approaches were systematically evaluated.

Protocol

NOTE: All cell culture procedures were conducted under biosafety level II+ (BSL2+) conditions within a certified safety cabinet. Doxorubicin is a hazardous chemotherapeutic agent. All procedures must be performed in a certified biosafety level II+ (BSL2+) cabinet using appropriate personal protective equipment (PPE), including laboratory coats and gloves. All wastes containing doxorubicin, including media and fixatives, should be collected in designated and clearly labeled hazardous waste containers and disposed of through the institutional hazardous waste management system.

1. Model 1: Co-culturing using the real-time cell analysis system

1. Culturing cells and induction of senescence (Day 1-2)
   1. Seed 8 x 10⁵ HeLa cells into a T25 culture flask containing 5 mL of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (0.1 µg/mL) and incubate cells at 37 °C in a humidified atmosphere with 5% CO₂.
   2. Next day, replace the medium of the cells with 5 mL of fresh complete medium. Treat the cells with 300 nM doxorubicin directly by adding into the medium to induce cellular senescence. Maintain the T25 flask in an incubator with 5% CO₂ at 37 °C for 72 h.
2. Non-senescent cell seeding (Day 4)
   1. Seed 8 x 10⁵ untreated HeLa cells (non-senescent control) in a new T25 flask in 5 mL of DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL) and maintain in a humidified incubator at 37 °C with 5% CO₂.
3. Confirming the induction of senescence (Day 5)
   1. To determine whether 72 h of doxorubicin treatment induced senescence, perform SA-β-gal staining⁶. Perform this assay on a separate set of cells not used in the main experiments. For the results obtained, see Figure 1A,B.
   2. Measure IL-6 in the conditioned media of senescent cancer HeLa cells to evaluate their secretory activity. Treat cells with doxorubicin for 72 h, followed by incubation in serum-free and phenol red-free medium for an additional 2 days. Normalize the collected conditioned media to cell number and measure IL-6 levels.
      NOTE: IL-6 is a hallmark cytokine of SASP¹². The results of IL-6 measurement are presented in Figure 1C.
4. Real-time cell analysis system setup (Day 5)
   1. Power on the real-time cell analysis system before cell seeding. Launch RTCA software 2.0. Click the Exp Notes button (Figure 2) and enter experimental details.
   2. Click the Schedule button and add two steps. The first step is for the background. The second step is to monitor cell proliferation every 15 min for 72 h.
   3. Choose the First Step and click Play to record the background impedance by adding 50 µL of complete medium to each well of the e-plate view.
5. Seeding cells into the e-plate view (Day 5)
   1. Check both doxorubicin-treated (senescent) and untreated (non-senescent) HeLa cells under a phase contrast microscope using 10x magnification before seeding cells. Under the microscope, senescent cells have a much more enlarged morphology than normal cells.
   2. Wash both the senescent and non-senescent HeLa cells with 5 mL of sterile pre-warmed PBS, then aspirate.
   3. Add 1 mL of pre-warmed 0.05% trypsin and maintain the flasks at 37 °C for 2 min to detach cells. Neutralize the trypsin with 5 mL of complete DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL).
   4. Centrifuge the cells at 500 x g at room temperature for 10 min, discard the supernatant, and resuspend the cell pellets in 5 mL of complete DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL).
   5. Count the viable cells using the trypan blue dye exclusion method using a hemocytometer.
   6. Seed non-senescent HeLa cells at a density of 5 x 10³ cells/well in each well of one e-plate view in complete 100 µL of DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL).
   7. 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.
   8. Choose the Second Step on the schedule button and click the Play button to start impedance measurement using RTCA software 2.0 for 72 h (Figure 2).
6. Seeding cells into the e-plate insert
   1. Seed cells in one e-plate insert wells in 60 µL of complete DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL).
   2. Seed cells onto the e-plate insert under three different experimental setups: Group 1 (Negative Control): No cells seeded (media only)
      Group 2: 2 × 10⁴ non-senescent HeLa cells
      Group 3: 1 × 10⁵ senescent HeLa cells
   3. Maintain e-plate insert at 37 °C in an incubator with 5% CO₂ for 6 h for attachment.
7. Co-culturing cells
   1. Click Pause after 6 h of cell seeding. Gently aspirate the culture medium from each well of the e-plate view to remove serum and non-adherent cells.
   2. Wash wells 2x with pre-warmed PBS at 37 °C.Add 140 µL of serum-free DMEM to each well of the e-plate view.
   3. Take the e-plate insert and aspirate the culture medium. Wash wells gently with PBS at 37 °C 2x and add 60 µL of serum-free DMEM.
   4. Position the e-plate insert into the e-plate view and gently press down until the insert is fully seated in the well.
   5. Insert the combined plate into the real-time cell analysis system and click Play to resume real-time monitoring of cell index values at 15 min intervals using the software (Figure 2).
   6. Turn off the real-time cell analysis system when the experiment time is over (Figure 3A).
8. Imaging cells on e-plate insert
   NOTE: Since there is a membrane in the e-plate insert well, the cells seeded here cannot be visualized on the membrane using phase-contrast microscopy. Therefore, to test whether the cells maintained viability until the end of the experiment and whether the senescent cells preserved their structural integrity, they were stained with DAPI (Figure 3B).
   1. Remove the e-plate insert from the e-plate view. Stain the cells in the e-plate insert with DAPI and check the presence of cells under a fluorescent microscope¹⁶. The nuclei of senescent cells are known to be larger than those of normal cells. Therefore, based on the nuclear sizes of the cells stained with DAPI, determine which well was seeded with non-senescent and which with senescent cells.
9. Imaging cells on the e-plate view
   1. Directly examine the e-plate view under a phase-contrast microscope using 10x magnification (Figure 3B). Since four rows of microelectrode sensors were removed from the center of each well in the e-plate view, space 6 was created to enable cell monitoring by microscopy.
10. Data analysis
    1. Normalize the CI 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.
    2. Click 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 (Figure 2). Then, analyze the data using an appropriate statistical program (Figure 3C,D).

2. Model 2: Conditioned media treatment assay using the real-time cell analysis system

1. Culturing cells and induction of senescence (Day 1-2)
   1. Seed 35 x 10⁴ HeLa cells into each well of 6 well-plate containing 2.5 mL of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (0.1 µg/mL) and incubate cells at 37 °C in a humidified atmosphere with 5% CO₂.
   2. Next day, replace the medium of cells with 2.5 mL of fresh complete medium. Treat the cells with 300 nM doxorubicin directly by adding it to the medium to induce cellular senescence. Maintain the 6-well plate in an incubator with 5% CO₂ at 37 °C for 72 h.
2. Non-senescent cell seeding (Day 4)
   1. Seed 35 x 10⁴ untreated HeLa cells (non-senescent control) to each well of a new 6-well plate in 2.5 mL of DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL) and maintain in a humidified incubator at 37 °C with 5% CO₂.
3. Carry out confirmation of induction of senescence as described in step 1.3.
4. Preparation of conditioned medium from senescent and non-senescent HeLa cells
   1. Check both doxorubicin-treated cells (senescent cells) and untreated HeLa cells (non-senescent cells) under a phase contrast microscope. Under the microscope, senescent cells have a much more enlarged morphology than normal cells.
   2. Aspirate the culture medium from both two 6 well-plates (senescent and non-senescent HeLa cells). Wash the cells 2x with pre-warmed PBS at 37 °C to remove residual serum and cell debris.
   3. Add 2 mL of serum-free DMEM supplemented with penicillin (100 U/mL) and streptomycin (0.1 µg/mL) to each well. Incubate the cells in a humidified incubator at 37 °C with 5% CO₂ for 48 h.
5. Collecting and concentrating conditioned media (Day 7)
   1. Collect the conditioned media from each well into sterile tubes in a laminar safety cabin.
   2. Trypsinize the cells and count the cells using the trypan blue dye exclusion method using a hemocytometer to normalize conditioned media to cell numbers.
   3. Adjust the conditioned media, normalized according to cell number, to a final volume of 2 mL with serum-free DMEM supplemented with penicillin (100 U/mL) and streptomycin (0.1 µg/mL) in sterile 2 mL tubes.
   4. Centrifuge the collected conditioned media at 1,000 x g for 10 min at 4 °C to remove cell debris, followed by centrifugation at 10,000 x g for 10 min at 4 °C to obtain the clarified supernatant.
   5. Centrifuge the conditioned media using 2 mL ultra-centrifugal filter units at  4 °C for 60 min at 3,000 x g to concentrate it. This procedure concentrates the 2 mL conditioned media to 5x the concentrated 400 µL of conditioned media.
   6. Dilute the obtained 40 µL of conditioned media with fresh serum-free medium to prepare 1x, 2x, and 3x concentrated conditioned media in sterile tubes in a laminar safety cabin.
      NOTE: Since the medium remaining on the cells for 48 h is depleted due to cellular consumption, the collected and concentrated conditioned media are subsequently diluted with new medium.
6. Seeding cells to e-plate view
   1. Set up the real-time cell analysis system as described in steps 1.1-1.4.Wash non-senescent HeLa cells with pre-warmed PBS, then aspirate.
   2. Add 1 mL of pre-warmed 0.05% trypsin and maintain the flasks at 37 °C for 2 min to detach cells. Neutralize the trypsin with 5 mL of complete DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL).
   3. Centrifuge the cells at 500 x g at room temperature for 10 min, discard the supernatant, and resuspend the cell pellets in 5 mL of complete DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL).
   4. Count the viable cells using the trypan blue dye exclusion method using a hemocytometer.
   5. Seed non-senescent HeLa cells at a density of 5 x 10³ cells/well in each well of one e-plate view in 200 µL of complete DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL).
   6. 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.
   7. Choose the second step on the schedule button and click the Play button to start impedance measurement using RTCA software 2.0 (Figure 2). Click the Pause button on the software after 6 h of plating non-senescent HeLa cells (Figure 2).
   8. Gently aspirate the culture medium from each well of the e-plate view to remove serum and non-adherent cells. Wash wells 2x with pre-warmed PBS at 37 °C.
   9. Add the pre-warmed conditioned media to 37 °C into the appropriate groups:
      Group 1: Fresh media
      Group 2: 1x non-senescent HeLa cell conditioned media
      Group 3: 2x non-senescent HeLa cell conditioned media
      Group 4: 3x non-senescent HeLa cell conditioned media
      Group 5: 1x senescent HeLa cell conditioned media
      Group 6: 2x senescent HeLa cell conditioned media
      ​Group 7: 3x senescent HeLa cell conditioned media
   10. 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 min intervals for 48 h (Figure 2).
   11. Turn off the real-time cell analysis system when the experiment time is over (Figure 4A).
7. Imaging cells on the e-plate view
   1. Directly examine the e-plate view under a phase-contrast microscope at 10x magnification (Figure 4B). Since four rows of microelectrode sensors were removed from the center of each well in the e-plate view, space was created to enable cell monitoring by microsopy.
8. Data analysis
   1. Normalize the CI values at the time point when the concentrated conditioned media were administered to the non-senescent HeLa cells.
   2. Click 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 (Figure 2). Then, analyze the data using an appropriate statistical program (Figure 4C,D).

3. Model 3: Real-time monitoring of cell-cell interaction

1. Culturing cells and induction of senescence (Day 1-2)
   1. Seed 8 x 10⁵ HeLa cells into a T25 culture flask containing 5 mL of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (0.1 µg/mL) and incubate cells at 37 °C in a humidified atmosphere with 5% CO₂.
   2. Next day, replace the medium of the cells with 5 mL of fresh complete medium. Treat the cells with 300 nM doxorubicin directly by adding into the medium to induce cellular senescence. Maintain the T25 flask in an incubator with 5% CO₂ at 37 °C for 72 h.
2. Non-senescent cell seeding (Day 4)
   1. Seed 8 x 10⁵ GFP-HeLa cells (non-senescent control) in a new T25 flask in 5 mL of DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL) and maintain in a humidified incubator at 37 °C with 5% CO₂.
   2. Seed 8 x 10⁵ HeLa cells (non-senescent control) in a new T25 flask in 5 mL of DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL) and maintain in a humidified incubator at 37 °C with 5% CO₂.
3. Carry out confirmation of induction of senescence as described in step 1.3 (day 5).
4. Co-culturing cells
   1. Check doxorubicin-treated cells, untreated GFP-HeLa, and untreated HeLa cells under a phase contrast microscope before seeding cells. Under the microscope, senescent cells have a much more enlarged morphology than normal cells.
   2. Wash all three T25 flasks with 5 mL of sterile pre-warmed PBS, then aspirate. Add 1 mL of pre-warmed 0.05% trypsin and maintain the flasks at 37 °C for 2 min to detach cells.
   3. Neutralize the trypsin with 5 mL of complete DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL).
   4. Centrifuge the cells at 500 x g at room temperature for 10 min, discard the supernatant, and resuspend the cell pellets in 5 mL of complete DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 µg/mL).
   5. Count the viable cells using the trypan blue dye exclusion method using a hemocytometer. Seed cells in 200 µL of complete DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (0.1 µg/mL).
   6. Seed cells onto µ-slide 8-well high under three different experimental setups:
      Group 1: 7 x 10³ GFP-HeLa cells
      Group 2: 18 x 10³ non-senescent HeLa cells + 7 x 10³ GFP-HeLa cells
      ​Group 3: 18 x 10³ senescent HeLa cells + 7 x 10³ GFP-HeLa cells
   7. Maintain the µ-slide for 8 h for attachment at 37 °C in an incubator with 5% CO₂. After 8 h, wash the cells with pre-warmed PBS (37 °C) 2x to eliminate the effect of serum on the cells.
   8. Add 200 µL of serum-free DMEM containing penicillin (100 U/mL) and streptomycin (0.1 µg/mL) to each well.
5. Microscope set up
   1. Turn on the temperature controller and CO₂-Controller and allow the stage-top incubation system to stabilize for 30 min.
   2. Check the CO₂ concentration and temperature settings manually and ensure that physiological conditions (5% CO₂, 37 °C) are reached.
   3. Turn on the inverted fluorescence microscope and allow the system to complete self-calibration. Switch on the fluorescence light source. Launch the imaging and analysis software to control imaging parameters and image acquisition.
6. Time-lapse imaging
   1. Place the slide on the stage-top incubation chamber of the inverted fluorescence microscopy with a high-resolution digital camera. Open the software and select Part X, Y, Z, and T Modes, and stage experiments in acquisition settings (Figure 5A).
   2. Select a 20x objective with high contrast for fluorescence and phase contrast imaging (Figure 5A). Add the phase contrast and fluorescence (FITC filter) channels to enable imaging in both modes (Figure 5A).
   3. Click the Live button to initiate live-cell imaging, then bring the cells into focus under phase contrast (Figure 5A). Open the z-stack window and mark the position of the cells of interest (containing both senescent/non-senescent HeLa cells and GFP HeLa cells). Navigate the stage between wells using the automatic stage controller, allowing precise location of the selected areas for position marking (Figure 5A).
      NOTE: In this way, more than one area can be selected in the same well of a slide.
   4. Switch the channel to the fluorescence channel to begin fluorescence imaging. Set the appropriate exposure (89.506 ms), gain (10), and intensity (136) settings (Figure 5A).
      NOTE: These parameters vary depending on the fluorescence intensity of the cells expressing the fluorescent protein.
   5. Select a  µm z-stack interval, set the top and bottom positions, and configure the z-stack under phase contrast (Figure 5A). Click Stop to close the Live option.
   6. Set the duration (48 h) and the time interval (30 min) using the Duration and Time Interval fields for time-lapse imaging.
   7. Click Start to begin imaging for 48 h (Figure 5A). Save the project after 48 h (Figure 5A).
   8. At each time point (0 h and 48 h), select the best frame from the z-stack, and export the image (Figure 5A).
   9. Count the GFP-positive cells at 0 h and 48 h, calculate the percentage increase in GFP-positive cells, and analyze the data using an appropriate software program (Figure 1D,E).
7. Generating movie
   1. Click Show Gallery to display all captured images organized by time points and z-stack layers (Figure 5B).
   2. For each time point, right-click on the Best-focused Image from the corresponding z-stack and select Select Thumbs in a Row. Save the selected frames (Figure 5B).
   3. Repeat this process individually for each position. To assist in selecting the most in-focus images, use the Find Best Focused Frames function. Save selected images through the Save Selected Frames option. The saved images are automatically uploaded into the Open Project section.
   4. Right-click on the Movie and select Export Image once the image sequences are fully loaded.
   5. Navigate to the Movie tab in the export window, set the desired frame rate (frames per second), select additional options such as scale bar, timestamp, etc., if needed, and finalize the export by choosing a destination using the Browse option (Figure 5B, Video 1, Video 2, and Video 3).

Results

This representative set of experiments investigated the potential effects of the senescence-associated secretory phenotype derived from senescent cancer cells on other cancer cells. Therefore, it was first confirmed whether treatment with doxorubicin for 72 h could induce senescence in HeLa cells using SA-β-Gal staining. In senescent cells, the lysosomal enzyme β-galactosidase remains active even at low pH and cleaves the chromogenic substrate X-gal, forming a blue precipitate¹⁷. Thus, the presenc...

Discussion

Dynamic interactions of cancer cells in their microenvironment are crucial for tumor progression, which influences the induction of metastasis and the development of a secondary tumor. It is also reported that senescent cancer and stromal cells regulate tumor microenvironments through the factors secreted from senescent cells⁴. These factors stimulate cell proliferation, migration, invasion, differentiation, and increase multidrug resistance^4,13...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
µ-Slide 8 Well high	bidi	80806
0.05 % Trypsin-EDTA (1X)	Gibco	25300-054
Amicon Ultra centrifugal filter units (Ultracel-3K)	Millipore	UFC200324
CO2 incubator (37°C, 5% CO2)	Sanyo
CO2-Controller 2000	Pecon
DMEM	Gibco	41966-029
Doxorubicin	Tocris	2252
E-plate INSERT 16	ACEA Biosciences/Agilent	6 465 382 001
E-plate VIEW 16	ACEA Biosciences/Agilent	300 601 140
FBS	Gibco	A5256701
HeLa Cells	AP Institute, Ankara, Turkey	HÜKÜK No: 90061901
Leica DMi8 inverted fluorescence microscope	Leica
Phosphate-buffered saline (PBS)	Gibco	70011-044
Senescence-associated β-galactosidase (SA-β-gal) staining kit	Cell Signalling	9860S
Stage-top incubation system	Pecon
Temp-controller 2002-2	Pecon
Trypan Blue Dye	Sigma	T8154
xCELLigence RTCA DP system	ACEA Biosciences/Agilent