Modulatory effects of Pioglitazone on the CAF-driven malignant phenotype in cervical cancer: an in vitro bidirectional study

Cervical cancer (CC) remains one of the leading causes of morbidity and mortality in middle- and low-income countries. The tumor microenvironment (TME), especially cancer-associated fibroblasts (CAF), plays a key role in tumor development and progression through diverse mechanisms, including cytokine secretion, angiogenesis, metabolism, and immune modulation. Pioglitazone (Pgz) has shown anti-fibrotic effects, preventing fibroblast activation and differentiation into myofibroblasts; anti-inflammatory effects, reducing the expression of IL-1, IL-6, and TNFα; and even anti-tumor properties, decreasing tumor cell migration and proliferation. In our study, we investigated the impact of Pgz on CAF-mediated effects on HeLa cervical cancer cells, focusing on metabolism, migration, invasion, and proliferation, and on how Pgz affects HeLa cells’ ability to activate and induce a CAF phenotype. After CAF obtention from cervical cancer biopsies, conditioned medium was collected from CAF or HeLa cell cultures after 24 h in free-serum media. Cells were treated with the conditioned media with or without pioglitazone for 24 h. To evaluate cell activity, we performed an MTT assay, measured ROS production, and glucose present in the medium. We also evaluate cell proliferation by bioimpedance, migration by a wound-healing assay, and invasion using the Matrigel and transwell systems. The cytokines produced by CAFs were evaluated by multiplex ELISA, and Mesenchymal Stem Cell differentiation into a CAF phenotype was evaluated by immunofluorescence for the markers αSMA and FAP. Statistical analyses were performed using GraphPad Prism software, using One-way analysis of variance with Tukey post hoc tests or the Kruskal-Wallis test for non-normal distributions. Our findings reveal that Pgz reduces CAF-induced metabolic activity, ROS production, glucose uptake, migration, invasion, and proliferation in HeLa cells. Additionally, Pgz suppresses HeLa-induced expression of αSMA and FAP in mesenchymal stem cells, suggesting it may help prevent CAF differentiation. CAFs appear to be a promising target for future cancer therapies; disrupting their tumor-supportive role through Pgz might help slow cancer progression and improve therapeutic outcomes.