Abstract P264: Targeting oxygen metabolism reduces hypoxia in the tumor microenvironment of a syngeneic mouse model
Authors: Boreel Daan F. , Span Paul , Peters Hans , van den Bijgaart Renske J.E. , Heskamp Sandra , Adema Gosse J. , Bussink Johan 
Published: December 2021

Abstract
When tumors outgrow their chaotic vasculature, limited diffusion of oxygen into remote tumor areas combined with increased oxygen consumption leads to hypoxia in most solid tumors. This oxygen scarcity is known to induce radioresistance, but may also interfere with the anti-tumor immune response. Hypoxia inhibits immune effector cell function, while immune cells with a more suppressive phenotype become more active. Using the novel mitochondrial complex I inhibitor IACS-010759, we aim to pharmacologically attenuate oxidative phosphorylation (OXPHOS) and subsequently decrease tumor oxygen consumption, hereby relieving hypoxia and sensitizing tumors to both radio- and immunotherapy. Several syngeneic murine cell lines and tumor models on a C57Bl/6 background were used (B16ova, MOC1, MC38 and GL261). In vitro oxygen consumption of these tumor cells was measured using the Agilent XF Seahorse Analyzer and cell growth was measured using the Incucyte ZOOM Live Cell Imaging system. Hypoxia and immune marker expression by these cells in vitro, and in vivo in the tumor microenvironment (TME), were determined using flow cytometry and immunohistochemistry. Tumor hypoxia was calculated as the fraction of tumor area in different zones at increasing distances around vessels on tumor sections. In vitro, IACS-010759 treatment potently inhibits oxygen consumption in a dose dependent manner. Under physiological glucose concentrations (1.5mM) IACS-010759 reduces cell growth at least 2-fold more potently than under high glucose culture conditions. On the other hand, growth is only marginally or not at all inhibited under low oxygen conditions (1.0%). This shows that cancer cells use oxygen when it is available, and tend to rely on glycolysis under hypoxia. This contradicts the longstanding notion that cancer cells primarily use glycolysis, also under aerobic conditions, and suggests that cancer cells are often metabolically plastic. Daily IACS-010759 treatment (10mg/kg) of mice bearing B16ova tumors for 4 or 9 days reduces the hypoxic fraction at increased distance from vessels, presumably by the inhibition of oxygen consumption closer to vessels, allowing oxygen to diffuse further into the tissue. No growth reduction by IACS-010759 treatment was observed in these tumors, showing they are able to use glycolysis for their energy supply. Flow cytometry analysis of these tumors showed no changes in abundance of lymphocyte and myeloid cell populations, suggesting inhibition of OXPHOS has no direct inhibitory effect on immune cell presence. We show that IACS-010759 decreases oxygen consumption in several tumor cell lines in vitro while it also causes growth inhibition if cancer cells rely on oxygen. In vivo, diffusion limited hypoxia can be reduced by the inhibition of OXPHOS, while the abundance of immune cells within the TME was not affected. The observed changes might lead to a more immune permissive TME when induced by radio- and immunotherapy, potentially increasing the efficacy of this treatment combination.
Citation Format: Daan F. Boreel, Paul Span, Hans Peters, Renske J.E. van den Bijgaart, Sandra Heskamp, Gosse J. Adema, Johan Bussink. Targeting oxygen metabolism reduces hypoxia in the tumor microenvironment of a syngeneic mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P264.