Name: Evaluating the Effect of SASP Factors on the Proliferation of Cancer Cells Using a Comparative Analysis of Three Distinct Methodologies (Video) | JoVE Journal
Uploaded: 2025-09-19T14:20:00.000+00:00
Duration: 8 min 57 s
Description: Learn three in vitro models to evaluate SASP effects on HeLa cell proliferation using real-time impedance monitoring and live-cell imaging. This method reveals dynamic paracrine interactions in cancer microenvironments.

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This study was supported by the Scientific Project Unit of Gazi University (Grant code: TOA-2021-7321) and TUBITAK (Grant code: 122S564 and 223S969).

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<li>Kuilman, T., Peeper, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Senescence-messaging+secretome%3A+SMS-ing+cellular+stress%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Senescence-messaging secretome: SMS-ing cellular stress.</a> Nat Rev Cancer. 9 (2), 81-94 (2009).</li>
<li>Simay Demir, Y. D., Ozdemir, A., Sucularli, C., Benhur, E., Ark, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+implication+of+ROCK+2+as+a+potential+senotherapeutic+target+via+the+suppression+of+the+harmful+effects+of+the+SASP%3A+Do+senescent+cancer+cells+really+engulf+the+other+cells%5BTitle%5D)+AND+%22Cell+Signal%22%5BJournal%5D)">The implication of ROCK 2 as a potential senotherapeutic target via the suppression of the harmful effects of the SASP: Do senescent cancer cells really engulf the other cells.</a> Cell Signal. 84, 110007(2021).</li>
<li>Demaria, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+Senescence+Promotes+Adverse+Effects+of+Chemotherapy+and+Cancer+Relapse%5BTitle%5D)+AND+%22Cancer+Discov%22%5BJournal%5D)">Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse.</a> Cancer Discov. 7 (2), 165-176 (2017).</li>
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<li>zdemir, A., Ibisoglu, B., Simay, Y. D., Polat, B., Ark, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ouabain+induces+Rho-dependent+rock+activation+and+membrane+blebbing+in+cultured+endothelial+cells%5BTitle%5D)+AND+%22Mol+Biol%22%5BJournal%5D)">Ouabain induces Rho-dependent rock activation and membrane blebbing in cultured endothelial cells.</a> Mol Biol. 49 (1), 138-143 (2015).</li>
<li>Lee, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Senescence-associated+beta-galactosidase+is+lysosomal+beta-galactosidase%5BTitle%5D)+AND+%22Aging+Cell%22%5BJournal%5D)">Senescence-associated beta-galactosidase is lysosomal beta-galactosidase.</a> Aging Cell. 5 (2), 187-195 (2006).</li>
<li>Simay Demir, Y. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimigratory+effect+of+pyrazole+derivatives+through+the+induction+of+STAT1+phosphorylation+in+A549+cancer+cells%5BTitle%5D)+AND+%22J+Pharm+Pharmacol%22%5BJournal%5D)">Antimigratory effect of pyrazole derivatives through the induction of STAT1 phosphorylation in A549 cancer cells.</a> J Pharm Pharmacol. 73 (6), 808-815 (2021).</li>
<li>Ariffin, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+CellProfiler+pipeline+analysis+of+cell+migration%5BTitle%5D)+AND+%22Acta+Histochem%22%5BJournal%5D)">The CellProfiler pipeline analysis of cell migration.</a> Acta Histochem. 125 (7), 152074(2023).</li>
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The authors have nothing to disclose.

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 resistance4,13...

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 senescence3,4. 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 &#946;-galactosidase (SA-&#946;-gal) activity, a lysosomal enzyme that remains active even at acidic pH 6.0. SA-&#946;-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 arrest8,9. 
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 &#181;m 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.

<table><tbody><tr><td>&amp;micro;-Slide 8 Well high</td><td>&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/68883/68883eq1.jpg&quot; style=&quot;margin: auto;&quot;/&gt;bidi</td><td>80806</td><td></td></tr><tr><td>0.05 % Trypsin-EDTA (1X)</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Amicon Ultra centrifugal filter units (Ultracel-3K)</td><td>Millipore</td><td>UFC200324</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator (37&amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sanyo</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-Controller 2000&amp;nbsp;</td><td>Pecon&amp;nbsp;</td><td></td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>41966-029</td><td></td></tr><tr><td>Doxorubicin</td><td>Tocris</td><td>2252</td><td></td></tr><tr><td>E-plate INSERT 16&amp;nbsp;</td><td>ACEA Biosciences/Agilent</td><td>6 465 382 001</td><td></td></tr><tr><td>E-plate VIEW 16&amp;nbsp;</td><td>ACEA Biosciences/Agilent</td><td>300 601 140</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>A5256701</td><td></td></tr><tr><td>HeLa Cells</td><td>&lt;img alt=&quot;Equation 2&quot; src=&quot;/files/ftp_upload/68883/68883eq2.jpg&quot; style=&quot;margin: auto;&quot;/&gt;AP Institute, Ankara, Turkey</td><td>H&amp;Uuml;K&amp;Uuml;K No: 90061901&amp;nbsp;</td><td></td></tr><tr><td>Leica DMi8 inverted fluorescence microscope&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Gibco</td><td>70011-044</td><td></td></tr><tr><td>Senescence-associated &amp;beta;-galactosidase (SA-&amp;beta;-gal) staining kit&amp;nbsp;</td><td>Cell Signalling</td><td>9860S</td><td></td></tr><tr><td>Stage-top incubation system</td><td>Pecon&amp;nbsp;</td><td></td><td></td></tr><tr><td>Temp-controller 2002-2&amp;nbsp;</td><td>Pecon&amp;nbsp;</td><td></td><td></td></tr><tr><td>Trypan Blue Dye</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>xCELLigence RTCA DP system&amp;nbsp;</td><td>ACEA Biosciences/Agilent</td><td></td><td></td></tr></tbody></table>

evaluating the effect of sasp factors on the proliferation of cancer cells using a comparative analysis of three distinct methodologies

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

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.

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...

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.

Chemotherapy-induced senescent cancer cells also secrete various factors, called senescence-associated secretory phenotype, to regulate their ...

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.

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Learn three in vitro models to evaluate SASP effects on HeLa cell proliferation using real-time impedance monitoring and live-cell imaging. This method reveals dynamic paracrine interactions in cancer microenvironments.

Evaluating the Effect of SASP Factors on the Proliferation of Cancer Cells Using a Comparative Analysis of Three Distinct Methodologies (Video) | JoVE Journal

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

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