Tumour reoxygenation by intratumoural injection of hydrogen peroxide (H₂O₂): a mechanism for enhanced radiosensitivity


Session type:

Samantha Nimalasena1, Selvakumar Anbalagan2, Carol Box2, Jessica K.R. Boult2, Nigel Bush2, Simon P. Robinson2, John Yarnold2, Navita Somaiah2
1Royal Marsden NHS Foundation Trust, 2Institute of Cancer Research (ICR)



The hypothesis under investigation is that intratumoural (IT) H2Oleads to tumour reoxygenation, via breakdown to H2O and O2. This is evidenced by Obubbles visible on ultrasound following IT injection. In vitro and in vivo studies were performed in a human colorectal tumour model to confirm efficacy, and test the reoxygenation hypothesis.


Subcutaneous HCT116 xenografts were formed in Foxn1nu mice (n=12). Dual hypoxia markers were used to measure the change in tumour hypoxia following IT injection of 0.05ml 0.5% H2O2 + 1% sodium hyaluronate gel. The gel mixture was injected under US guidance. A control group received no injection (n=3). CCI-103F recorded baseline hypoxia, followed 1-hour post-IT injection by pimonidazole. A thresholding technique was used on imaged sections to quantify fluorescence.

The combined effect of H2O2 + IR was tested in a 3D in vitro model. HCT116 spheroids were treated with 0.6mM H2O2, 3Gy ionising radiation (IR), or a combination (H2O2  administered 30 min pre-IR) and compared with untreated controls. Spheroid growth was monitored every 3 days.


HCT116 xenografts injected with H2O2 + sodium hyaluronate exhibited a reduction in hypoxia (expressed as percentage tumour area) from baseline, compared to non-injected controls. A significant decrease in hypoxia staining was demonstrated in H2O2-injected tumours (mean reduction 45%, range 25-61, p=0.02), compared to controls. Areas showing abundant CCI-103F adducts exhibited less pimonidazole adducts post-H2Oinjection.


Spheroid growth kinetics demonstrated growth inhibition by H2O2+ IR, compared with either agent alone at all time points (reaching statistical significance on day 15 and 19 (p=0.01)).


Hypoxia quantification in HCT116 xenografts demonstrated that H2O2 injection leads to significant reoxygenation. H2Orepresents a novel means of oxygen delivery and alleviating tumour hypoxia, which has implications for scheduling of H2Orelative to RT in the clinic. 3D spheroid data corroborate clinical reports demonstrating enhancement of radiation response by H2O2. Confirmation of the effect in vitro enables investigation of mechanisms of tumour response.

If efficacy of H2O2+ RT is confirmed in the randomised phase II trial (NCT03946202) in breast cancer, it may be applicable to other tumour types where hypoxia influences radiotherapy outcomes.

Impact statement

Targeting tumour hypoxia to enhance radiosensitivity.