Related Experiment Video
Updated: Jun 27, 2025
Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids
Published on: April 6, 2022
Characterizing OXPHOS inhibitor-mediated alleviation of hypoxia using high-throughput live cell-imaging
Anne P M Beerkens1,2, Daan F Boreel3,4, James A Nathan5
1Radiotherapy & OncoImmunology Laboratory, Department of Radiation Oncology, Radboud University Medical Center, Nijmegen, 6525GA, The Netherlands. anne.beerkens@radboudumc.nl.
Background:
Hypoxia is a common feature of many solid tumors and causes radiotherapy and immunotherapy resistance. Pharmacological inhibition of oxidative phosphorylation (OXPHOS) has emerged as a therapeutic strategy to reduce hypoxia. However, the OXPHOS inhibitors tested in clinical trials caused only moderate responses in hypoxia alleviation or trials were terminated due to dose-limiting toxicities. To improve the therapeutic benefit, FDA approved OXPHOS inhibitors (e.g. atovaquone) were conjugated to triphenylphosphonium (TPP+) to preferentially target cancer cell's mitochondria. In this study, we evaluated the hypoxia reducing effects of several mitochondria-targeted OXPHOS inhibitors and compared them to non-mitochondria-targeted OXPHOS inhibitors using newly developed spheroid models for diffusion-limited hypoxia.
Methods:
B16OVA murine melanoma cells and MC38 murine colon cancer cells expressing a HIF-Responsive Element (HRE)-induced Green Fluorescent Protein (GFP) with an oxygen-dependent degradation domain (HRE-eGFP-ODD) were generated to assess diffusion-limited hypoxia dynamics in spheroids. Spheroids were treated with IACS-010759, atovaquone, metformin, tamoxifen or with mitochondria-targeted atovaquone (Mito-ATO), PEGylated mitochondria-targeted atovaquone (Mito-PEG-ATO) or mitochondria-targeted tamoxifen (MitoTam). Hypoxia dynamics were followed and quantified over time using the IncuCyte Zoom Live Cell-Imaging system.
Results:
Hypoxic cores developed in B16OVA.HRE and MC38.HRE spheroids within 24 h hours after seeding. Treatment with IACS-010759, metformin, atovaquone, Mito-PEG-ATO and MitoTam showed a dose-dependent reduction of hypoxia in both B16OVA.HRE and MC38.HRE spheroids. Mito-ATO only alleviated hypoxia in MC38.HRE spheroids while tamoxifen was not able to reduce hypoxia in any of the spheroid models. The mitochondria-targeted OXPHOS inhibitors demonstrated stronger anti-hypoxic effects compared to the non-mito-targeted OXPHOS inhibitors.
Conclusions:
We successfully developed a high-throughput spheroid model in which hypoxia dynamics can be quantified over time. Using this model, we showed that the mitochondria-targeted OXPHOS inhibitors Mito-ATO, Mito-PEG-ATO and MitoTam reduce hypoxia in tumor cells in a dose-dependent manner, potentially sensitizing hypoxic tumor cells for radiotherapy.
Insights
Mitochondria-targeted oxidative phosphorylation (OXPHOS) inhibitors effectively reduced tumor hypoxia in a novel spheroid model. These targeted therapies show promise for enhancing radiotherapy in hypoxic tumors.
Area of Science:
- Oncology
- Cancer Biology
- Pharmacology
Background:
- Hypoxia is prevalent in solid tumors, contributing to resistance against radiotherapy and immunotherapy.
- Oxidative phosphorylation (OXPHOS) inhibition is a strategy to combat tumor hypoxia, but clinical trials faced challenges with efficacy and toxicity.
- Mitochondria-targeted drug delivery, using conjugates like triphenylphosphonium (TPP+), aims to improve the therapeutic benefit of OXPHOS inhibitors.
Purpose of the Study:
- To evaluate the hypoxia-reducing effects of various mitochondria-targeted OXPHOS inhibitors.
- To compare the efficacy of mitochondria-targeted inhibitors against their non-targeted counterparts.
- To utilize a newly developed spheroid model for assessing diffusion-limited hypoxia dynamics.
Main Methods:
- Generated B16OVA and MC38 murine cancer cell lines with a hypoxia-responsive element (HRE) reporter system.
- Treated spheroids with various OXPHOS inhibitors, including mitochondria-targeted versions (Mito-ATO, Mito-PEG-ATO, MitoTam).
- Quantified hypoxia dynamics over time using live-cell imaging (IncuCyte Zoom).
Main Results:
- Mitochondria-targeted OXPHOS inhibitors (Mito-PEG-ATO, MitoTam) demonstrated dose-dependent hypoxia reduction in both melanoma and colon cancer spheroids.
- Mito-ATO showed efficacy in colon cancer spheroids but not melanoma.
- Targeted inhibitors exhibited superior anti-hypoxic effects compared to non-targeted OXPHOS inhibitors.
Conclusions:
- A high-throughput spheroid model was established for quantifying hypoxia dynamics.
- Mitochondria-targeted OXPHOS inhibitors effectively reduce tumor cell hypoxia in a dose-dependent manner.
- These findings suggest potential for sensitizing hypoxic tumors to radiotherapy.
