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Tumour hypoxia is a major factor contributing to radiotherapy resistance. We have previously shown that the antimalarial drug atovaquone increases tumour oxygen levels and synergises with radiotherapy in preclinical models. We have also recently demonstrated that atovaquone effectively and safely alleviates hypoxia at radiotherapy-relevant levels in lung cancer patients. Radiotherapy is increasingly combined with immunotherapy, which is also negatively affected by tumour hypoxia. Since radiotherapy boosts the antitumour immune response, increasing tumour oxygenation during combination treatment with radiotherapy and immunotherapy may provide further clinical benefit. Our purpose was to evaluate the efficacy and safety of atovaquone plus immune checkpoint blockade prior to testing the viability of this combination with radiotherapy in preclinical models.

Balb/c mice implanted with the syngeneic colorectal cancer cell line CT26 were treated with atovaquone and/or anti-PD-L1. Tumour hypoxia was determined by microscopy in tumour sections stained for the hypoxia probe EF5. Treatment efficacy was assessed by monitoring tumour growth. An anti-CD8 depleting antibody was used to ascertain if cytotoxic lymphocytes were involved in the antitumour response. Mice experiencing complete tumour eradication were re-inoculated with CT26 cells and monitored for tumour development to assess memory response. Finally, mouse weight and haematological (white blood cell count, platelet count and haemoglobin) and biochemical (bilirubin, alanine aminotransferase and albumin) blood markers were measured to evaluate treatment toxicity.

Atovaquone alone efficiently reduced hypoxia but did not affect tumour growth. In contrast, treatment with atovaquone plus anti-PD-L1 resulted in higher rates of tumour regression than treatment with anti-PD-L1 alone. CD8+ T lymphocyte depletion completely abrogated the effect of the combination treatment, suggesting that this synergy is ultimately dependent on T cell-mediated killing. We also demonstrated that mice with complete tumour regression after atovaquone and anti-PD-L1 combination treatment did not develop tumours after re-challenge with CT26 cells, unlike those inoculated with a different syngeneic cancer cell line, demonstrating that mice had developed a memory antitumour response. No differences were found in mouse weight or any of the haematological and biochemical markers analysed, suggesting that the combination of atovaquone and anti-PD-L1 was not associated with toxicity.

Atovaquone synergises with anti-PD-L1, favouring the development of a memory anti-tumour immune response, and without causing any toxicity. Our results support future testing of atovaquone in combination with immune checkpoint blockade and radiotherapy.