Background

The hypothesis under investigation is that intratumoural (IT) H₂O₂ leads to tumour reoxygenation, via breakdown to H₂O and O₂. This is evidenced by O₂ bubbles 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.

Method

Subcutaneous HCT116 xenografts were formed in Foxn1^nu 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.

Results

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-H₂O₂ injection.

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)).

Conclusion

Hypoxia quantification in HCT116 xenografts demonstrated that H₂O₂ injection leads to significant reoxygenation. H₂O₂ represents a novel means of oxygen delivery and alleviating tumour hypoxia, which has implications for scheduling of H₂O₂ relative 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.