Vienna, Austria

ESTRO 2023

Session Item

Tumour radiobiology
Poster (Digital)
Reintroducing free radical hydrogen peroxide as potent radiosensitizer in solid tumors
Febe Geirnaert, Belgium


Reintroducing free radical hydrogen peroxide as potent radiosensitizer in solid tumors

Febe Geirnaert1, Inès Dufait2, Lisa Kerkhove1, Amir Rifi1, Hugo Vandenplas3, Ka Lun Law2, Cyril Corbet4, Thierry Gevaert2, Mark De Ridder2

1Vrije Universiteit Brussel, Radiotherapy, Brussels, Belgium; 2Universitair Ziekenhuis Brussel, Radiotherapy, Brussels, Belgium; 3Universitair Ziekenhuis Brussel, Medical Oncology, Brussels, Belgium; 4UCLouvain, Pharmacology and Therapeutics, Brussels, Belgium

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Purpose or Objective

The hypoxic microenvironment is the main cause of radioresistance in solid tumors. Free radicals, including hydrogen peroxide (H2O2), have formerly been demonstrated to increase radiosensitivity of cancer cells. Despite being described as potent radiosensitizers, most studies on directing free radicals towards cancer patients were put to a halt due to severe toxicity issues associated with intravenous administration. While recent advances in controlled drug delivery may circumvent this issue, comprehensive studies investigating the mechanistical pathways are still lacking. In this project, we aim to investigate the underlying mechanisms responsible for the radiosensitizing effects of H2O2.

Material and Methods

Murine CT26 colorectal cancer cells and 4T1 breast cancer cells were exposed to H2O2 for 1 hour. The toxicity and radio-modulatory effects of H2O2 were determined under static (0.1% oxygen in a hypoxic chamber) and metabolic hypoxia (micropellets) by colony formation assay. Reactive oxygen species (ROS) levels (DCFDA), DNA damage (γH2AX), apoptosis (Annexin-V/7-AAD) and ferroptosis (C11BODIPY) were examined by flow cytometry. Oxygen consumption rate (OCR) and mitochondrial electron transport chain (ETC) complex activity was measured by Seahorse analyzer. The oxygen pressure after H2O2 injection was analyzed in real-time under hypoxic conditions (tissue mimetic culture system (TMCS)) using an oxygen monitor.


Non-toxic concentrations of H2O2 were determined per cell line and used subsequently. Treatment with H2O2 radiosensitized CT26 and 4T1 cells under both static and metabolic hypoxic conditions. Enhancement ratios achieved were respectively 2.38 and 1.67 under static hypoxic conditions and 1.44 and 2.21 under metabolic hypoxic conditions. ROS levels did not increase following 1 hour treatment with H2O2 whereas increased levels of ferroptosis, apoptosis and DNA damage were observed with H2O2 alone, yet did not result in an enhanced augmentation in combination with radiation. A dose-dependent decrease in OCR was observed in both cell lines after H2O2 treatment correlating to an inhibition of the activity of ETC complex I, and to a lesser extent of complex II. Injection of H2O2 in our TMCS resulted in an immediate and significant surge in oxygen levels.


The radiosensitizing effect of H2O2 has been primarily attributed to the induction of apoptosis. However, our preliminary results suggest that the main radiosensitizing mechanism of H2O2 under hypoxic conditions can be ascribed to reoxygenation of cells after the conversion of H2O2 to water and oxygen along with inhibition of cellular respiration through a complex I blockade. Both the radiosensitizing and oxygen-releasing effects of packaged H2O2 in vivo are currently under investigation. Gaining knowledge on the underlying working mechanisms of H2O2 as a radiosensitizer is vital in designing future combination therapies for cancer patients.