verma, v. k.; Beevi, S. S.; Panchal, N. K.; Reddy, K. S.; Darapuneni, R. C.

Introduction: Cancer is the second leading cause of mortality in India, with conventional chemotherapy often limited by drug resistance, off-target toxicity, and metastasis promotion. These treatments indiscriminately attack rapidly dividing cells, including healthy tissues and immune cells, leading to severe side effects and weakened antitumor immunity. Hence, alternative strategies are needed to selectively target cancer cells while minimizing collateral damage. Methods: Mesenchymal stem cells (MSCs) were isolated, cultured up to passage 4 (P4), and characterized via flow cytometry and immunocytochemistry for stemness markers. Their differentiation potential was validated using adipogenic and chondrogenic lineage assays. The MSC-derived secretome, collected under hypoxic conditions, was analyzed for its cytotoxic effects on cancer cells using MTT, TMRM staining, and scratch assays. NanoLC-MS/MS profiling and STRING-DB analysis identified key proteins involved in apoptosis, tumor suppression, and DNA damage response. Results: P2 secretomes exhibited the highest protein concentration and strongest cytotoxic effects. UCSC-derived secretomes showed superior anti-cancer activity, significantly reducing cancer cell viability, mitochondrial membrane potential, and migration. Dose-dependent studies revealed up to 95% cancer cell death with UCSC secretome, while ADSC secretome achieved 80%. In contrast, HEK293-derived secretomes had minimal effects. LC-MS profiling identified tumor-suppressive and apoptosis-inducing proteins, with unique protein signatures distinguishing UCSC and ADSC secretomes. STRING analysis mapped key regulatory pathways in tumor inhibition. Conclusion: MSC-derived secretomes, particularly from UCSCs, effectively target aggressive breast cancer cells while sparing normal cells, offering a promising alternative to conventional therapies. Future research should optimize secretome formulations, identify key bioactive components, and validate their efficacy in in vivo models and clinical trials to advance this therapy toward clinical applications. Key words: Mesenchyal stem cells, umbilical cord tissue, gene network, secretomes, cytotoxicity, chemotherapeutic drugs