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Limited diffusion of oxygen into remote tumor areas and oxygen consumption through oxidative phosphorylation (OXPHOS) leads to hypoxia in most solid tumors. This scarcity of oxygen is known to induce radioresistance, but can also have a disrupting effect on the anti-tumor immune response. Therefore, we investigated the potential of OXPHOS inhibition to relieve tumor hypoxia by decreasing the oxygen consumption. Furthermore, we developed a radiolabeled antibody which recognizes murine CAIX ([¹¹¹In]In-DTPA-mCAIX), an enzyme upregulated by cancer cells under chronic hypoxic conditions, to monitor chronic hypoxia in syngeneic mouse models. 

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 before and after treatment with the OXPHOS inhibitor IACS-010759. The in vivo tumor microenvironment of B16ova and MOC1 tumors, treated (10mg/kg IACS-010759) and vehicle-treated (0.5% methylcellulose), was characterized by immunohistochemistry. The biodistribution of [¹¹¹In]In-DTPA-mCAIX was measured by ex vivo radioactivity counting and in vivo SPECT imaging comparing different antibody doses and time points post injection. Intratumoral distribution of tracer uptake was visualized using autoradiography. Image analysis was performed by parametric mapping and zonal analysis in ImageJ.

The data show that mitochondrial complex I inhibitor IACS-010759 inhibited oxygen consumption in a dose dependent manner in several tumor cell lines in vitro. Furthermore, diffusion limited hypoxia in vivo is reduced up to 200μm from perfused blood vessels by IACS-01079 treatment (10mg/kg) in MOC1 and B16ova tumors (fig 1). This can be visualized by staining pimonidazole as well as CAIX. In vitro, [In¹¹¹]-DTPA-mCAIX showed specific binding to B16ova cells when cultured at 1% O₂ (9.3±1.2%), but not to cells cultured at 20% O₂ (0.8±0.04%). In vivo, CAIX expression could be visualized by SPECT using [In¹¹¹]-DTPA-mCAIX. Radiotracer uptake in the tumor was significantly higher compared with uptake of isotype control tracer [In¹¹¹]-DTPA-IgG1 (34.6±5.8 vs. 13.4±2.7 %ID/g) (fig 2F). Autoradiography and immunohistochemistry of tumor sections showed a strong spatial correlation of CAIX with [In¹¹¹]-DTPA-mCAIX (r=0.72±0.11) and not with [In¹¹¹]-DTPA-IgG1 (-0.16±0.33)(fig 2A-E). 

OXPHOS inhibition decreases oxygen consumption in several tumor cell lines in vitro and decreases diffusion limited hypoxia in vivo. Furthermore, the hypoxia related marker CAIX can be used to visualize hypoxic areas in syngeneic mouse models using the SPECT-radiotracer [In¹¹¹]-DTPA-mCAIX. In the future, this technique could be used to distinguish hypoxic from non-hypoxic tumors before or during OXPHOS inhibition treatment and thereby help optimizing this strategy to relieve tumor hypoxia and improve immuno- and radiotherapy efficacy in preclinical models.