Development of a Portable Hypoxia Chamber for Ultra-high Dose Rate Laser-Driven Proton Radiobiology Applications

BackgroundThere is currently significant interest in assessing the role of oxygen in the radiobiological effects at ultra-high dose rates. Oxygen modulation is postulated to play a role in the enhanced sparing effect observed in FLASH radiotherapy, where particles are delivered at 40-1000 Gy/s. Furthermore, the development of laser-driven accelerators now enables radiobiology experiments in extreme regimes where dose rates can exceed 10^9 Gy/s, and predicted oxygen depletion effects on cellular response can be tested. Access to appropriate experimental enviroments, allowing measurements under controlled oxygenation conditions, is a key requirement for these studies. We report on the development and application of a bespoke portable hypoxia chamber specifically designed for experiments employing laser-driven sources, but also suitable for comparator studies under FLASH and conventional irradiation conditions.Materials and MethodsWe used oxygen concentration measurements to test the induction of hypoxia and the maintenance capacity of the chambers. Cellular hypoxia induction was verified using hypoxia inducible factor-1α immunostaining. Calibrated radiochromic films and GEANT-4 simulations verified the dosimetry variations inside and outside the chambers. We irradiated hypoxic human skin fibroblasts (AG01522B) and patient-derived glioblastoma (E2) cancer stem cells with laser-driven protons, conventional protons and reference 225 kVp X-rays to quantify DNA DSB damage and repair under hypoxia. We further measured the oxygen enhancement ratio for cell survival exposed to cyclotron-accelerated protons and X-rays in the normal fibroblast and radioresistant GBM stem cells. ResultsOxygen measurements showed that our chambers maintained a radiobiological hypoxic environment for at least 45 minutes and pathological hypoxia for up to 24 hrs after disconnecting the chambers from the gas supply. We observed a significant reduction in the 53BP1 foci induced by laser-driven protons, conventional protons and X-rays in the hypoxic cells compared to normoxic cells at 30 minutes post-irradiation. Under hypoxic irradiations, the Laser-driven protons induced significant residual DNA DSB damage in hypoxic AG01522 cells compared to the conventional dose rate protons suggesting an important impact of these extreme high dose-rate exposures. We obtained an oxygen enhancement ratio (OER) of 2.1 ± 0.108 and 2.501 ±0.125 respectively for the AG01522 and patient derived GBM stem cells for the X-rays using our hypoxia chambers for irradiation. ConclusionWe demonstrated the design and application of portable hypoxia chambers for studying cellular radiobiological endpoints after laser-driven protons at ultra-high dose, conventional protons and X-ray exposures. Good levels of reduced oxygen concentration could be maintained in the absence of external gassing to quantify hypoxic effects and the data obtained provided an indication of an enhanced residual DNA DSB damage under hypoxic conditions at ultra-high dose rate compared to the conventional protons or X-rays.

Impact of Hypoxia on Relative Biological Effectiveness and Oxygen Enhancement Ratio for a 62-MeV Therapeutic Proton Beam

This study uses the yields of double-strand breaks (DSBs) to determine the relative biological effectiveness (RBE) of proton beams, using cell survival as a biological endpoint. DSB induction is determined when cells locate at different depths (6 positions) along the track of 62 MeV proton beams. The DNA damage yields are estimated using Monte Carlo Damage Simulation (MCDS) software. The repair outcomes are estimated using Monte Carlo excision repair (MCER) simulations. The RBE for cell survival at different oxygen concentrations is calculated using the repair-misrepair-fixation (RMF) model. Using 60Co γ-rays (linear energy transfer (LET) = 2.4 keV/μm) as the reference radiation, the RBE for DSB induction and enzymatic DSB under aerobic condition (21% O2) are in the range 1.0–1.5 and 1.0–1.6 along the track depth, respectively. In accord with RBE obtained from experimental data, RMF model-derived RBE values for cell survival are in the range of 1.0–3.0. The oxygen enhancement ratio (OER) for cell survival (10%) decreases from 3.0 to 2.5 as LET increases from 1.1 to 22.6 keV/μm. The RBE values for severe hypoxia (0.1% O2) are in the range of 1.1–4.4 as LET increases, indicating greater contributions of direct effects for protons. Compared with photon therapy, the overall effect of 62 MeV proton beams results in greater cell death and is further intensified under hypoxic conditions.

Non-Homogeneous Tumor Growth and Its Implications for Radiotherapy: A Phenomenological Approach

Tumor regrowth and heterogeneity are important clinical parameters during radiotherapy, and the probability of treatment benefit critically depends on the tumor progression pattern in the interval between the fractional irradiation treatments. We propose an analytic, easy-to-use method to take into account clonal subpopulations with different specific growth rates and radiation resistances. The different strain regrowth effects, as described by Gompertz law, require a dose-boost to reproduce the survival probability of the corresponding homogeneous system and for uniform irradiation. However, the estimate of the survival fraction for a tumor with a hypoxic subpopulation is more reliable when there is a slow specific regrowth rate and when the dependence on the oxygen enhancement ratio of radiotherapy is consistently taken into account. The approach is discussed for non-linear two-population dynamics for breast cancer and can be easily generalized to a larger number of components and different tumor phenotypes.

Hyperthermia: The Optimal Treatment to Overcome Radiation Resistant Hypoxia

Regions of low oxygenation (hypoxia) are a characteristic feature of solid tumors, and cells existing in these regions are a major factor influencing radiation resistance as well as playing a significant role in malignant progression. Consequently, numerous pre-clinical and clinical attempts have been made to try and overcome this hypoxia. These approaches involve improving oxygen availability, radio-sensitizing or killing the hypoxic cells, or utilizing high LET (linear energy transfer) radiation leading to a lower OER (oxygen enhancement ratio). Interestingly, hyperthermia (heat treatments of 39–45 °C) induces many of these effects. Specifically, it increases blood flow thereby improving tissue oxygenation, radio-sensitizes via DNA repair inhibition, and can kill cells either directly or indirectly by causing vascular damage. Combining hyperthermia with low LET radiation can even result in anti-tumor effects equivalent to those seen with high LET. The various mechanisms depend on the time and sequence between radiation and hyperthermia, the heating temperature, and the time of heating. We will discuss the role these factors play in influencing the interaction between hyperthermia and radiation, and summarize the randomized clinical trials showing a benefit of such a combination as well as suggest the potential future clinical application of this combination.