TH-302 (evofosfamide) monotherapy exerts anticancer activity in Ewing's sarcoma cells under hypoxia

  • Clin Transl Oncol. 2025 Jun 14. doi: 10.1007/s12094-025-03956-4.
Marie Kühne  1  2  3 ,  Sabine Becker  1  4  3 ,  Jürgen Sonnemann  1  4  3 ,  Christian Marx  5  6
Affiliations
  • 1. Department of Pediatric and Adolescent Medicine, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany.
  • 2. UniversitätsTumorCentrum Jena, Jena University Hospital, Jena, Germany.
  • 3. Comprehensive Cancer Center Central Germany (CCCG), Campus Jena, Jena, Germany.
  • 4. Research Center Lobeda, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany.
  • 5. Center for Pandemic Vaccines and Therapeutics (ZEPAI), Paul-Ehrlich-Institute (PEI), Paul-Ehrlich-Str. 51-59, 63225, Langen, Germany. [email protected].
  • 6. Institute for Micro and Nanotechnologies (MacroNano®), Nano-Biosystem Technology, Ilmenau University of Technology, Ilmenau, Germany. [email protected].
PMID: 40515777 DOI: 10.1007/s12094-025-03956-4
Abstract

Background: Tumor hypoxia is a significant factor in Cancer progression, metastasis, and therapy resistance, leading to poor patient outcomes. Hypoxia-activated prodrugs (HAPs) are a class of agents that selectively target these hypoxic environments. They remain inactive under normal oxygen conditions but are activated by low oxygen levels. TH-302 (evofosfamide), a nitroimidazole mustard, is a clinically advanced HAP. This study aimed to investigate the effects of TH-302 in three Ewing's sarcoma (ES) cell lines.

Methods: TH-302 was assessed for its effects on DNA damage, cell proliferation, cell death, mitochondrial depolarization, Caspase-3/7 activation, and the emergence of sub-G1 cell populations using flow cytometry, fluorescence microscopy and quantitative real time (qRT)-PCR. These effects were compared under different oxygen concentrations.

Results: TH-302 induced DNA damage and reduced cell number in ES cells by over 60%, preferentially under hypoxic conditions, independent of the cellular p53 status. TH-302 caused cell death in up to 40% of cells and mitochondrial depolarization in up to 60% of cells. Additionally, TH-302-induced Caspase-3/7 activation and increased sub-G1 cell populations predominantly under hypoxia, with the most pronounced effects occurring at 0.2% environmental oxygen compared to 1% O2.

Conclusion: These in vitro findings suggest that TH-302 may be efficacious in ES. This provides a rationale for further in vivo investigations into the potential of TH-302 as a treatment for ES.

Keywords
Cancer therapy; Ewing's sarcoma; Hypoxia-activated prodrug (HAP); P53; TH-302 (evofosfamide); Tumor hypoxia.
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