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Following a Phase I trial confirming safety, a multicentre Phase II trial is underway comparing the efficacy of radiotherapy (RT) vs. intratumoural (IT) H₂O₂ + RT in breast cancer patients.

The hypothesis under test is that H₂O₂ leads to tumour reoxygenation, via breakdown into H₂O and O₂ within tumours. 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 the effect, and to investigate the reoxygenation hypothesis. 

Subcutaneous HCT116 xenografts were established in Foxn1^numice (n=12). Dual hypoxia markers were used to measure the difference in tumour hypoxia pre- and post- IT injection of 0.05ml 0.5% H₂O₂+ 1% sodium hyaluronate gel. IT injections were into the centre of the tumour under US guidance. A control cohort received no injection (n=3). CCI-103F was administered to provide a record of baseline tumour hypoxia, followed by pimonidazole (1 hour following IT injection). The experimental schema is shown in Fig.1A. A thresholding technique was used to quantify fluorescence on imaged tumour sections.

In order to test the combined effect of H₂O₂+ IR in a 3D in vitro model, HCT116 spheroids were treated with 0.6mM H₂O₂, 3Gy ionising radiation (IR), or a combination (0.6mM H₂O₂ administered 30 min pre-IR) and compared with untreated controls. Spheroid diameter was measured at 3-day intervals (until day 23) using an automated cytometer (Celigo) to monitor growth kinetics.

HCT116 xenografts injected with H₂O₂ + sodium hyaluronate exhibited a reduction in hypoxia adduct formation (expressed as percentage of tumour area) from baseline (Fig.1B-C), in comparison to non-injected controls. A significant decrease (paired t-test) in hypoxia staining was demonstrated in H₂O₂-injected tumours (mean reduction 45%, range 25-61, p=0.02), compared to control tumours. Areas within the tumour showing abundant CCI-103F adducts were associated with less pimonidazole adducts following H₂O₂injection. 

Growth inhibition was demonstrated in spheroids treated with the combination of H₂O₂+ IR, compared with H₂O₂ or IR alone, at all time points, and reaching statistical significance on day 15 and 19 (p=0.01) (Fig. 2). 

Hypoxia quantification in HCT116 xenografts demonstrated significant reoxygenation following H₂O₂ injection. 3D spheroid data are in keeping with clinical reports demonstrating enhancement of radiation response by H₂O₂. Recapitulation of the clinical effect in vitro allows for further investigation of mechanisms of tumour response.

IT H₂O₂ represents a novel method of oxygen delivery and alleviating tumour hypoxia, which has implications for scheduling of H₂O₂ relative to RT in the clinic. If efficacy of IT H₂O₂+ RT is confirmed in the randomised phase II trial in breast cancer, it could improve treatment of several other tumour types where hypoxia is known to affect outcomes following RT.