What's needed is a way to evaluate radiation response at a far earlier stage. With this aim, researchers from the University of Arkansas have used optical imaging to examine radiation resistance in cancer cells. In particular, they imaged the fluorescence from nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) to identify early metabolic changes in radiation-resistant lung cancer cells in response to irradiation (J. Biomed. Opt. 22 060502).

"The optical redox ratio of FAD/(FAD+NADH) is very sensitive to cellular metabolism," explained Narasimhan Rajaram. "There are no contrast agents that need to be injected as this technique relies only on the endogenous fluorescence from the cells. It can be used in vitro for basic biological studies and in vivo for clinical translation."

On a simple level, the optical redox ratio can quantify the ratio of oxidative phosphorylation to glycolysis (although other pathways can also contribute). Decreases in this ratio are associated with relative increases in glycolytic metabolism, which can occur under hypoxic conditions and is a possible cause of radiation resistance. Identifying such metabolic changes early on could help identify patients with radiation-resistant tumours.

In vitro testing

Rajaram and colleagues examined radiation-resistant human lung carcinoma A549 cells. The cells were generated and validated by Ruud Dings at UAMS, who made them radiation-resistant by repeated irradiation of radiation-sensitive A549 cells. Using a custom-built two-photon microscope to measure NADH and FAD fluorescence, they determined the optical redox ratio before, and 24 h after, exposure to a 2 Gy radiation dose, in both the parental and the resistant cell line.

Representative images of the parental and resistant cells revealed that the average optical redox ratio was not significantly different between the two cell lines at baseline. However, 24 h after a single radiation exposure, there was a significant decrease in the redox ratio of the radiation-resistant cells compared with the parental cells.

The researchers note that they did not observe any changes in cell confluency after radiation, and thus propose that the measurements reflect radiation-induced metabolic changes. Specifically, the decrease in redox ratio indicates increased glycolysis in the resistant cells.

Studying HIF-1

Fractionated radiotherapy is used to allow reoxygenation between fractions. This reoxygenation, however, can also generate tumour reactive oxygen species that lead to the stabilization of hypoxia-inducible factor (HIF-1) – a key promoter of glycolytic metabolism. With this in mind, the team investigated whether the observed shift to increased glycolysis was associated with upregulation of HIF-1, by determining the protein content of HIF-1α at baseline and 24 h after radiation exposure.

HIF-1α protein content in the radiation-resistant cells was significantly greater after radiation, compared with baseline cells and with parental cells after radiation. These results suggest a possible role for rapid activation of HIF-1α in decreasing oxygen consumption and increasing glycolytic metabolism in radiation-resistant cells after radiation.

The authors concluded that the optical redox ratio provides a non-invasive method for identifying early metabolic changes in cancer cells that may indicate radiation resistance. For clinical implementation, a tumour biopsy would be taken, followed by imaging of the cells grown as organoids.

"It is also possible to measure [the redox ratio] directly in vivo," added Rajaram. "In the past, I have been involved in clinical studies on skin cancer that directed laser light at 350 and 450 nm, respectively, using optical fibres to measure NADH and FAD fluorescence. The challenge in vivo is signal-to-noise ratio."

The researchers are continuing investigations of the radiation-resistant and radiation-sensitive cells to determine the time course of the observed metabolic changes. "We are also working to determine a causal role for specific metabolic pathways, and examining whether the redox ratio can be measured in excised human tumours that are stored as frozen tissue," said Rajaram.

Related stories

• Why does tumour hypoxia oscillate?
• We should strive to apply physics to the biology itself
• Biological data personalize radiotherapy
• Imaging cerebral energy metabolism