ESTRO 2024 - Abstract Book

S5357

Radiobiology - Tumour biology

ESTRO 2024

Purpose/Objective:

Hypoxia is a common feature of solid tumors and has a negative impact on radiotherapy and immunotherapy outcome. An emerging therapeutic strategy to alleviate hypoxia is inhibition of the oxidative phosphorylation (OXPHOS), to reduce the oxygen consumption rate of tumor cells. Several OXPHOS inhibitors have already been tested in clinical studies for their anti-hypoxic effects. However, these inhibitors are not specifically targeting cancer cells. We aim to alleviate hypoxia through inhibition of OXPHOS using mitochondria-targeted triphenylphosphonium (TPP+) conjugated OXPHOS inhibitors, which are selectively taken up by cancer cells and thereby sensitize tumor cells for radiotherapy, immune checkpoint blockade and their combination.

Material/Methods:

B16ova murine melanoma cells and MC38 murine colon carcinoma cells containing a HIF-Responsive Element (HRE)- induced Green Fluorescent Protein (GFP) with an oxygen-dependent degradation domain (HRE-eGFP-ODD), were used to assess hypoxia dynamics in spheroids. 24h after spheroid formation, the spheroids were treated with atovaquone (ATO), mitochondria-targeted atovaquone (Mito-ATO) and a PEGylated form of Mito-ATO (Mito-PEG-ATO) at a range of concentrations (30, 15, 7.5, and 3.75 µM). Diffusion-limited hypoxia was followed over time using the Incucyte Zoom Live Cell Imaging system. The total GFP integrated intensity (total sum of the object’s mean fluorescent intensity in the image) was used to quantify hypoxia.

Results:

Inhibition of OXPHOS in B16ova spheroids using 30 µM Mito-PEG-ATO resulted in reduction of diffusion-limited hypoxia within 24h after start of treatment. This reduction in hypoxia persisted for at least 48h and was not observed in control spheroids. (Fig 1A) Furthermore, treatment with ATO and Mito-PEG-ATO caused a dose-dependent alleviation of hypoxia in B16ova spheroids, while treatment with Mito-ATO did not result in reduction of hypoxia. In MC38 spheroids, ATO, Mito-ATO and Mito-PEG-ATO all showed a dose-dependent reduction of hypoxia. The anti hypoxic effect was strongest after treatment with Mito-PEG-ATO. In both spheroid models, treatment with 7.5 µM Mito-PEG-ATO resulted in a statistically significant reduction (p<0.0001 for both B16ova and MC38) of hypoxia compared to treatment with 7.5 µM ATO (p=0.1604 for B16ova and p=0.2379 for MC38), showing the potency of this compound (Fig 1B).