Proof-of-Concept and Characterization of Photosynthetic Bacteria as Radiation Enhancers

Abstract

Molecular O2 is a potent chemical radiosensitizer. Re-sensitizing hypoxic tumor tissues with O2 can enhance the therapeutic efficacy of ionizing radiation. Photosynthetic cyanobacteria (S. elongatus) or cyanos are currently being explored for various bioengineering applications due to their ability to produce oxygen when exposed to light. Here, we explore the use of these bacteria to replenish molecular O2 content inside hypoxic tumors, which in turn will enhance the effect of radiation. To assess the feasibility of this approach, we used A549 lungs cancer cell spheroids as models, which are three-dimensional (3D) in vitro cell aggregates that mimic tumors in terms of oxygen and nutrient gradients. The cyanos were grown in 12:12h light /dark conditions in a bacterial shaker incubator (37ºC and 150 rpm). Biocompatibility of cyanos co-cultured withA549 monolayers and spheroids was estimated using the MTT assay and Calcein AM staining. The level of hypoxia content in the spheroids were measured using the Image IT green hypoxia dye. Spheroids co-cultured with cyanos were subjected to full spectrum lights for different time periods. After light incubation, different dosages of radiation (4, 6 and 12 Gy) were delivered to the spheroids using an X-ray irradiator (225 kBp, 13 mA). Post irradiation, the spheroids were harvested and analyzed based on Nuc green dye staining to estimate the dead cell population and clonogenic survival to estimate the post-therapy survival fraction. Under exposure to full spectrum LED light, cyanos increased the concentration of dissolved O2 in the medium, eventually reaching fourfold higher levels (i.e., ∼240% of dissolved O2). Addition of cyanos to mammalian cell cultures did not result in detectable toxicity for either cellular monolayers or 3D spheroids. Cell viability was 100% even at the highest tested concentration of 1 × 107 number of cyanos cultured with 10,000 A549 cells. This confirmed the biocompatibility of the cyanos in cell culture environment. Post irradiation, as depicted from the Nuc green dye staining, was twofold higher (p<0.01) in spheroids co-cultured with cyanos and exposed to light. Surprisingly, in the clonogenic assay, this group of spheroids exhibited a higher survival fraction compared to the control group. This unexpected outcome warrants further investigation of the potentially radioprotecting effect of cyanos. Altogether, this supports the potential therapeutic efficacy of the cyanos in hypoxic tumors. Cyanos in the presence of light are effective in enhancing radiation-induced cell death. While clinical translation is still far down the road, such a method could be employed in the context of brachytherapy or superficial radiotherapy to overcome tumor hypoxia.