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Studies using mouse cancer models are important to aid the translation of preclinical radiotherapy research to clinical implementation. Ankjærgaard et al., (2021 in Radiat. Meas. 147, p. 106636) demonstrated alanine to be an excellent in vivo dosimeter for murine subcutaneous flank tumors. Mice were positioned on their side, resulting in the contralateral side receiving approximately 3.5% (0.5 Gy) of the prescribed dose, which could be problematic for investigation of abscopal effects. Here we investigate a different setup, where mice are positioned on the stomach during irradiation. This position allows for irradiation of the hind leg and a larger distance between subcutaneous tumors in both flanks. The aim is to test whether the irradiation setup 1) can be used to irradiate subcutaneous hind leg tumors and 2) provides enhanced tissue sparing to the contralateral flank.

Seven C57BL/6 female mice (weights: 21.6 - 24.4 g) were anaesthetized using air with 2.8-4% isoflurane, positioned on their stomach and irradiated in two separate sessions with 1) 15 Gy to the right hind leg with an alanine pellet placed directly on the skin by the ankle, and 2) 11 x 15 Gy (=165 Gy) to the right flank with alanine pellets placed on both flanks. The large dose ensures measurable signal in the contralateral alanine pellet. Pellets were placed at the isocenter of a 3x3 cm² 10 MV FFF asymmetrical field delivered by a Varian TrueBeam LINAC, with the main part of the field outside the mouse to spare normal tissue at the contralateral side. Mice were covered in bolus on all sides, leaving minimal air between mice, alanine and bolus.

The measured average dose to the hind legs was (14.63 ± 0.43) Gy, and to the right flank (14.74 ± 0.23) Gy (Fig. 1), both at k=2, and in agreement with previously published data. The dose is about 2% lower than the planned 15 Gy, likely caused by the TPS dose was calculated from an assumption of dose deposited in water, not taking a possible small air gap between mouse and bolus into account, a possibly lower output of the LINAC on the day (it can vary ±2%), and setup uncertainties. The average dose from the pellets placed on the contralateral flank (Fig. 1c) gave (0.14 ± 0.04) Gy at k=2, which is approximately 1% of the prescribed dose. This is an improvement over the previous setup, giving a dose reduction of about 70%. This approach of using alanine as an in vivo dosimeter on live mice will be extended to proton-, electron- and l energy x-ray beams.

This work presents alanine dosimetry for validating a simple setup for small-field localized irradiations of subcutaneous tumors implanted in the flank or in the hind limbs in mice. The setup has a large degree of reproducibility between irradiations, and spares the contralateral side of the mouse, allowing investigation of abscopal effects. Alanine is easy and flexible to use, its size comparable to subcutaneous mouse tumors, which makes it ideal for dosimetric validation in vivo studies.