1.

Introduction

Nearly 50% of all cancer patients each year are treated with radiation therapy, either alone or in combination with surgery or other forms of therapy.¹ A critical challenge facing these patients is tumor resistance to radiation therapy. Currently, radiation response is determined based on tumor volume shrinkage using clinical imaging modalities 1 month after the treatment regimen. Because there is no present method to evaluate radiation response during the treatment period, patients who do not respond favorably are losing critical time when they could be switched to alternative forms of therapy, such as chemotherapeutics or surgery.

Conventional radiation therapy for solid tumors typically consists of fractionated dosing ($2\mathrm{Gy}/\mathrm{day}$) for a period of 5 to 6 weeks. Fractionated therapy is believed to improve the treatment outcome by promoting reoxygenation and, hence, radiosensitizing previously hypoxic cancer cells.² Reoxygenation due to fractionated radiotherapy is critically associated with treatment response.^3–5 However, reoxygenation after radiation can also lead to the accumulation of tumor reactive oxygen species (ROS), which leads to the stabilization of hypoxia-inducible factor (HIF-1) even under well-oxygenated conditions⁶ and induces aerobic glycolysis.⁷ Aerobic glycolysis or increased glucose utilization under well oxygenated conditions has been implicated as a potential cause of radiation and chemotherapy resistance.⁸ The switch to aerobic glycolysis can promote radiation resistance in two possible ways: (1) utilization within the pentose phosphate shunt to maintain the NADPH-glutathione buffer and, hence, scavenge radiation-induced ROS,⁹ and (2) increased production of lactate, an important ROS scavenger, leading to decreased radiation sensitivity.^10,11 Identifying such metabolic changes very early after commencing a radiation regimen would help identify patients with radiation-resistant tumors and allow them to avoid the toxic side effects of ineffective therapy.

Endogenous fluorescence from nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) presents a label-free and validated¹² method for quantifying cell metabolism.¹³ The optical redox ratio of $\mathrm{FAD}/(\mathrm{FAD}+\mathrm{NADH})$ can be used to quantify the relative ratio of oxidative phosphorylation to catabolism.¹² Specifically, increased oxidative phosphorylation is associated with an increase in the optical redox ratio due to oxidation of NADH to ${\mathrm{NAD}}^{+}$. Previous work has demonstrated a strong correlation between the optical redox ratio and the concentration ratio of ${\mathrm{NAD}}^{+}/({\mathrm{NAD}}^{+}+\mathrm{NADH})$ in various cell types.^12,14 Decreases in the optical redox ratio have been associated with relative increases in catabolism, which can occur under hypoxic conditions¹⁵ or upon an increase in macromolecule synthesis (e.g., fatty acids).¹² Furthermore, the optical redox ratio has been shown to be sensitive to a metabolic response to chemotherapy in tumor-derived organoids.^16,17 Here, our objectives were to determine the early metabolic changes in cancer cells in response to radiation treatment and if these changes were associated with the level of observed radiation resistance. To accomplish our objective, we obtained an isogenic radiation-resistant cell line by repeated exposure of a parental, radiation-sensitive cell line to radiation. We measured the optical redox ratio at baseline (preradiation) and 24 h after exposure to a single, clinically relevant radiation dose of 2 Gy in the parental and resistant cell line, and found statistically significant differences between the cell lines. Our results suggest a potential increase in glycolytic metabolism in radiation-resistant cells after radiation. This information could be useful to predict and customize treatment for improved response rates.

2.

Materials and Methods

2.1.

Cell Culture, Irradiation, and Clonogenic Assay

Human lung carcinoma A549 cells were purchased from American Type Culture Collection (ATCC; CCL185) and grown in Ham’s F-12K (Kaighn’s) medium mixed with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin–streptomycin. These cells were irradiated at an average dose of 2.2 Gy every three days for a cumulative dose of 55 Gy (25 fractions) to create a radiation-resistant cell population [A549-RR; Fig. 1(a)]. An orthovoltage x-ray irradiator (X-Rad 320, Precision X-Ray, Inc., North Branford, Connecticut) was used for radiation. To assess clonogenic survival, cells in the exponential growth phase were trypsinized, washed, counted, and seeded at a concentration of 50 cells per well into 6-well plates. The plates were incubated overnight at 37°C and 5% ${\mathrm{CO}}_2$. Approximately 16 h after seeding, the flasks were treated with radiation and incubated for 8 days. To quantify colony formation, cells were washed with normal saline and fixed in 90:10 methanol:acetic acid for 30 min. Next, cells were stained for 2 h with 1% crystal violet in the methanol/acetic acid mixture. The number of resulting colonies (defined as consisting of $>50\text{cells}$) per well was counted. The number of identified colonies at 0 Gy divided by the seeding density was used to normalize the number of colonies formed per cell line at the various doses, as described in our previous work.^18,19 All experiments were performed in triplicates.

Fig. 1

(a) Flowchart depicting the development of stable cultures of radiation-resistant A549 cells (A549-RR). (b) Clonogenic assay for survival of the A549-RR cells compared with the A549 cells when exposed to 2 Gy of radiation. Colonies ($>50\text{cells}$) were quantified by fixing cells in methanol/acetic acid and staining with crystal violet. The number of identified colonies at 0 Gy divided by the seeding density was used to normalize the number of colonies formed per cell line at the various doses.

3.

Results and Discussion

Figure 1 shows the results of clonogenic survival assays conducted on both cell lines. The A549-RR cells showed a nearly 2.5-fold increase in survival after exposure to 2 Gy compared with the A549 parental cells. The enhancement in radiation resistance is highest at 2 Gy, the dose used to develop radiation resistance.

Figure 2(a) shows representative images of A549 and A549-RR cells at baseline and 24 h after exposure to 2 Gy of radiation. The average redox ratio was not significantly different between the two cell lines at baseline [Fig. 2(b)]. However, 24 h after radiation, there was a statistically significant ($p=0.01$) decrease in the redox ratio of A549-RR cells compared with the parental A549 cells. For both cell lines, the change in redox ratio postradiation was not significantly different compared with their respective baseline measurements. We did not observe any changes in cell confluency 24-h postradiation, and therefore, these measurements reflect radiation-induced metabolic changes. The change in optical redox ratio after radiation is consistent with measurements of the n-OCR, as determined by the Seahorse assay [Fig. 2(c)]. Additionally, n-OCR for the two cell lines was also significantly different at baseline ($p<0.05$). The lack of significant difference in the optical redox ratio between the two cell lines at baseline may be related to differences in macromolecule biosynthesis or NADPH concentrations that can affect the redox ratio¹² but would not be reflected in the n-OCR. These results indicate that the A549-RR cells have decreased levels of oxygen consumption both at baseline and postradiation and resort to increased glycolysis after radiation to potentially reduce ROS-induced toxicity. Increased glucose catabolism under normoxic, well-oxygenated conditions is representative of an aerobic glycolysis phenotype⁷ and is consistent with a previous study that demonstrated elevated glucose uptake and decreased oxygen consumption in radiation-resistant head and neck cancer cells.⁹

Fig. 2

Radiation causes a decrease in the optical redox ratio after 24 h in the radiation-resistant cells, indicating increased glycolytic metabolism. (a) Representative redox images of parental and radiation-resistant A549 cells at baseline prior to radiation and 24 h after 2 Gy of radiation. (b) Quantification of redox ratio images indicates a statistically significant decrease in the optical redox ratio 24 h after radiation in the A549-RR cells compared with the parental A549 cells ($p=0.01$). (c) Differences in the n-OCR (calculated as OCR/PPR) are consistent with the optical redox ratio. Asterisks placed above bars indicate statistical significance. Error bars in panels (b) and (c) represent standard deviation of the mean plate value.

To determine whether the shift to increased glycolysis was associated with upregulation of HIF-1, we determined the protein content of $\mathrm{HIF}\text{-}1\alpha$ at baseline and 24 h after exposure to radiation. HIF-1 is a master regulator of oxygen homeostasis and directly targets several genes involved in glucose metabolism.²³ $\mathrm{HIF}\text{-}1\alpha$ protein content in the A549-RR cells was significantly greater after radiation compared to baseline ($p=0.004$) and compared with parental A549 cells after radiation ($p=0.01$; Fig. 3). Taken together with results from Fig. 2, these data point to a possible role for rapid activation of $\mathrm{HIF}\text{-}1\alpha$ in decreasing oxygen consumption and increasing glycolytic metabolism in the A549-RR cells under normoxic conditions. Further studies of the optical redox ratio in these cell lines using HIF-1 inhibitors will elucidate a causal role for $\mathrm{HIF}\text{-}1\alpha$ in promoting these metabolic changes.

Fig. 3

Radiation causes a significant increase in $\mathrm{HIF}\text{-}1\alpha$ in the radiation-resistant cells 24 h after radiation. Western blots of $\mathrm{HIF}\text{-}1\alpha$ protein expression demonstrate statistically significant differences between A549 and A549-RR cells at baseline and 24 h after radiation, indicating reoxygenation-induced $\mathrm{HIF}\text{-}1\alpha$ expression in the A549-RR cells. Representative images are from the same gel and the same image. The 45-kDa actin band in the Ponceau S stain was used as a loading control.

In summary, we have presented the optical redox ratio as a potential noninvasive method for identifying early metabolic changes in cancer cells that may be indicative of radiation resistance. The matched model used here provides an excellent model to study molecular and metabolic pathways associated with radiation resistance in isogenic cell lines. The metabolic changes identified here using endogenous fluorescence are consistent with a recent study by Campos et al.²⁴ that showed significantly lower mean NADH lifetime in UM-SCC-22B head and neck cancer cells exposed to 10 Gy radiation compared with normal oral keratinocytes, with a lower lifetime indicative of increased glycolysis. They also identified a significant increase in $\mathrm{HIF}\text{-}1\alpha$ expression in the UM-SCC-22B head and neck cancer cells as early as 30 min after radiation.²⁴ Other studies using models of radiation resistance have also identified increased HIF-1α expression in radiation-resistant cells²⁵ and increased uptake of glucose that is shunted through the pentose phosphate pathway.⁹ Furthermore, radiation-resistant cells were also found to have significantly higher levels of fatty acid synthesis,⁹ which is interesting given that an increase in fatty acid synthesis has been shown to result in a lower redox ratio.¹² In addition to glucose, glutamine has also been identified as a possible source of synthesis of glutathione, an ROS scavenger that helps inhibit radiosensitivity.²⁶ We plan to extend our studies to include earlier time points, beginning immediately (within minutes) after radiation and determine the specific pathways of glucose utilization in the radiation-resistant cells. These investigations could potentially guide the selection of optimal time points for measuring the optical redox ratio in patient biopsy-derived organoids to provide a rapid determination of tumor radiation resistance and guide treatment planning.