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.