Cancer remains a major health problem, encompassing diseases that cannot yet always be treated successfully. Tumor tissue is a complex cellular network containing not only tumor cells but also infiltrating immune cells, including tumor-associated macrophages. The tumor microenvironment is thought to actively direct macrophages toward the development of the M2 phenotype, i.e., the immunosuppressive phenotype, which promotes tumor cell proliferation, angiogenesis, invasion, metastasis, tumor microenvironment remodeling, and therapy resistance. In some tumors, increased activity of the cysteine peptidase cathepsin X, which cleaves the C-terminal region of enolase, has been detected in M2 macrophages. Enolase is primarily a glycolytic enzyme with neurotrophic and neuroprotective functions in the central nervous system. Changes in its function have been linked to both neurodegenerative diseases and cancer, while the role of cathepsin X in regulating enolase, particularly in tumor-altered macrophages, remains unclear. The aim of this master’s thesis was to investigate the activity and role of the cysteine peptidase cathepsin X in relation to the expression levels of enolase isoforms in macrophages obtained by differentiation of the THP-1 monocyte cell line and exposed to tumor cell conditioned medium. We used conditioned medium from cell lines derived from glioblastoma (U87), breast cancer (MDA-MB-231), pancreatic adenocarcinoma (Colo-357), and hepatocellular carcinoma (HepG2). Prior to exposure, we differentiated THP-1 cells for 72 hours into a macrophage-like adherent phenotype, confirmed by the expression levels of surface markers. We observed increased cathepsin X activity in differentiated cells exposed to conditioned U87 and MDA-MB-231 medium and simultaneously reduced expression of both γ-enolase forms after 48 hours, followed by increased expression after 72 hours. Conditioned Colo-357 medium further enhanced the reduction in cathepsin X activity, while conditioned HepG2 medium attenuated this reduction. Both conditioned media also reduced expression of the active γ-enolase form while expression of the total γ-enolase form remained relatively unchanged. Regardless of the conditioned medium, the differentiated cells adopted a less glycolytic phenotype. Cathepsin X inhibition suppressed cell differentiation and increased CD11b expression in differentiated cells exposed to conditioned MDA-MB-231 medium. A better understanding of the role of cathepsin X in the development of the M2 phenotype of tumor-associated macrophages and their repolarization to the M1 phenotype using cathepsin X inhibitors could contribute to the development of more effective cancer treatments.
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