Tumor hypoxia represents one of the most significant barriers to effective radiotherapy. When cancer cells exist in oxygen-poor environments, they become remarkably resistant to radiation damage, requiring up to three times the radiation dose to achieve the same therapeutic effect as well-oxygenated cells. This resistance stems from oxygen's critical role in fixing radiation-induced DNA damage: without sufficient oxygen, cancer cells can repair themselves and continue proliferating.1,2.4
The battle against hypoxic tumor resistance has taken a promising turn with the development of KORTUC (Kochi Oxydol-Radiation Therapy for Unresectable Carcinomas), an innovative radiosensitization method that delivers hydrogen peroxide directly to the tumor site through intratumoral injection in a sodium hyaluronate gel matrix (Figure 1).
A recent study by Nimalasena et al.1 demonstrated KORTUC's ability to reoxygenate hypoxic tumor regions. Crucially, the researchers used live imaging to monitor this process in real time, capturing the temporal dynamics of reoxygenation.
KORTUC's mechanism of action
When a tumor is treated with KORTUC, the hydrogen peroxide rapidly decomposes through two primary pathways. The first involves catalase and peroxidase enzymes breaking down H₂O₂ into water and oxygen. The second, occurring in the presence of ferrous iron, generates oxygen and hydroxyl radicals through Fenton and Haber-Weiss reactions. This dual mechanism not only provides direct oxygenation but also generates reactive oxygen species that can enhance radiation sensitivity through additional cytotoxic pathways.1,2.3,4
Figure 1. The concept of a new enzyme-targeting radiosensitization treatment (KORTUC II). Using a new radiosensitizer, various radioresistant tumors can be converted into radiosensitive tumors. Image credit: Yaogawa et al.3. Used under the Creative Commons by Attribution (CC-BY) license.
Live cell imaging for capturing reoxygenation dynamics
While ultrasound imaging can confirm oxygen bubble formation during injection (Figure 2), it cannot reveal the biological effects on tumor cells. To monitor oxygenation at the cellular level, the researchers turned to live cell imaging.
Figure 2. Ultrasound images of an HCT116 tumor (i) pre-injection, (ii) during injection, and (iii) 30 min and (iv) 60 min post-injection of KORTUC. Oxygen microbubbles were seen as a persistent white haze in images (ii)–(iv). The white arrow indicates the needle entry point, and the dashed box delineates the tumor region. Reverberation apparent below the needle is an ultrasound imaging artefact. Image credit: Nimalasena et al.1 Used under the Creative Commons by Attribution (CC-BY) license.
Using the Celigo™ image cytometer, the team acquired both brightfield and fluorescence images of tumor spheroids at regular intervals post-treatment using the ‘Spheroid Analysis’ suite of applications (Figure 3).2 They employed Image-iT™ Red Hypoxia Reagent, a live cell hypoxia marker that fluoresces specifically under low oxygen conditions (<5% O2).5 This reversible fluorescent probe allowed the dynamic monitoring of changes in cellular oxygenation over time, something that would have been impossible with traditional endpoint assays.
Figure 3. Merged brightfield and fluorescence images of (i) HCT116 and (ii) HN5 spheroids pre-treated with Image-iT™ Red (24 h) and then treated with H2O2 (0–9.6 mM). Representative spheroids are shown. The apparent increase in size of HCT116 spheroids when treated at higher H2O2 concentrations at 24 hours is due to spheroid disaggregation because of the cytotoxic effects of H2O2. Scale bar = 500 µm. Image credit: Nimalasena et al.1 Used under the Creative Commons by Attribution (CC-BY) license.
The use of live imaging offered several key advantages:
- Automated time-course monitoring: Multiple spheroids could be imaged across different treatment conditions at predetermined time points, providing temporal data on reoxygenation.
- Quantitative analysis: Software enabled precise quantification of spheroid diameter and average fluorescence intensity, capturing both morphological changes and oxygenation status simultaneously.
- High-throughput capability: Entire 96-well plates could be imaged, allowing researchers to test multiple hydrogen peroxide concentrations (0-9.6 mM) across different time points with statistical rigor.
- Non-invasive monitoring: The same spheroids could be monitored throughout the experiment, reducing variability and providing more robust data than terminal assays.
Complementing live imaging with immunohistochemistry assays
Notably, the study paired live imaging with traditional immunohistochemistry (IHC) approaches. While Celigo-based imaging provided real-time dynamics, pimonidazole staining offered spatial resolution and confirmed the live imaging findings at fixed timepoints.
This dual approach revealed that reoxygenation occurred rapidly (within 1 hour) at hydrogen peroxide concentrations ≥1.2 mM. However, the effect was transient at lower doses, with hypoxia re-emerging by 24 hours. This temporal pattern was sustained longer in HCT116 spheroids (up to 6 hours at ≥2.4 mM H2O2) compared to HN5 spheroids.2
By combining methods, the researchers addressed limitations inherent to each. Live imaging provided dynamic information but relied on a single fluorescent marker, while IHC offered spatial resolution and used well-established hypoxia detection methods. Together, they provided a comprehensive picture of KORTUC's reoxygenation effects.
Clinical implications and future directions
The live imaging data were particularly valuable for determining the optimal timing of radiotherapy relative to KORTUC injection. The finding that reoxygenation peaks about 1 hour post-injection and is sustained for at least this period could directly inform clinical protocols, where radiotherapy is provided within a certain time after KORTUC injection.
As KORTUC progresses through Phase II clinical trials, the mechanistic insights gained through this imaging approach will continue to inform optimal treatment protocols and patient selection strategies. Looking ahead, real-time monitoring may also support more personalized treatment approaches, where individual responses can be monitored and treatment adjusted accordingly.
This research highlights how pairing innovative therapies with advanced imaging technologies can transform our understanding of cancer biology, bringing us closer to more effective, and tailored cancer therapies.
For research use only. Not for use in diagnostic procedures.
References
- Tumour reoxygenation after intratumoural hydrogen peroxide (KORTUC) injection: a novel approach to enhance radiosensitivity. Nimalasena, S., Anbalagan, S., Box, C. et al. 2024, BJC Rep 2, p. 78.
- Intratumoral Hydrogen Peroxide With Radiation Therapy in Locally Advanced Breast Cancer: Results From a Phase 1 Clinical Trial. Samantha Nimalasena, Lone Gothard, Selvakumar Anbalagan, Steven Allen, Victoria Sinnett, Kabir Mohammed, Gargi Kothari, Annette Musallam, Claire Lucy, Sheng Yu, Gift Nayamundanda, Anna Kirby, Gill Ross, Elinor Sawyer, Fiona Castell, Susan Cleator, Imogen. 2020, International Journal of Radiation Oncology*Biology*Physics, Volume 108, Issue 4,, pp. Pages 1019-1029.
- Serial Assessment of Therapeutic Response to a New Radiosensitization Treatment, Kochi Oxydol-Radiation Therapy for Unresectable Carcinomas, Type II (KORTUC II), in Patients with Stage I/II Breast Cancer Using Breast Contrast-Enhanced Magnetic Resonance I. Yaogawa S, Ogawa Y, Morita-Tokuhiro S, Tsuzuki A, Akima R, Itoh K, Morio K, Yasunami H, Onogawa M, Kariya S, Nogami M, Nishioka A, Miyamura M. 2015, Cancers (Basel) 8(1), p. 1.
- Intratumoral Hydrogen Peroxide With Radiation Therapy in Locally Advanced Breast Cancer: Results From a Phase 1 Clinical Trial. Nimalasena, Samantha, et al. 2020, International Journal of Radiation Oncology, Biology, Physics, Volume 108, Issue 4,, pp. 1019 - 1029.
- Hypoxia measurements in live and fixed cells using fluorescence microscopy and high-content imaging. Bhaskar S. Mandavilli, Aimei Chen, and Yi-Zhen Hu. s.l. : Thermo Fisher Scientific, Eugene, OR 97402 .
