Abstract
1147
Objectives Nitric Oxide Synthase (NOS) activities in various types of tumor cells are uncoupled. The underlying mechanism for uncoupling is a reduced ratio between the tetrahydrobiopterin (BH4) and dihydrobiopterin (BH2) levels leading to reduced NO bioavailability and corresponding enhanced superoxide and peroxynitrite generation. One consequence of this is endothelial dysfunction leading to poor tumor vascularization and reduced oxygenation. This study aims to improve normalization of tumor vasculature and tumor oxygenation by increasing the BH4:BH2 ratio.
Methods We used A549 lung cancer xenograft and MMTVneu spontaneous breast cancer tumor models in mice. These tumor bearing animals were treated, by oral gavage (1mg/kg), with sepiapterin (SP), a metabolic precursor of BH4. We then measured tumor oxygenation by multi-spectral optoacoustic tomography (MSOT). In MSOT, differences in the absorption properties of oxyhemoglobin and deoxyhemoglobin were exploited to measure the oxygenation of tumor hemoglobin in SP-treated animals to control animals (n=8 mice per group). Tumor bearing athymic mice were treated with SP daily for six days. The animals were imaged as at three time points: prior to the first SP treatment, day 3 after the first SP treatment, and day 6 after the first SP treatment. The same experiments were repeated with the spontaneous MMTVneu tumor model also. The mice were treated with SP daily for six days. The animals were imaged at four time points: prior to the first SP treatment, day 5 after the first SP treatment, day 8 after the first SP treatment, and day 16 after the first SP treatment.
Results MSOT data demonstrated that SP treatment increased the percentage of oxygenated hemoglobin in xenograft tumors when compared to vehicle treated control tumors. The percentage of oxygenated hemoglobin in the control tumors decreased over time. This was expected, since hypoxia increases as tumors grow. There is a significantly higher (p<0.05) percentage of oxygenated hemoglobin in the SP-treated tumors versus control tumors after six days of SP treatment. The SP curve remains relatively constant - suggesting that the SP-treated tumors are not outgrowing their vasculature like the control tumors. Spontaneous orthotopic MMTV tumors displayed an increased percentage of oxygenated hemoglobin, with a significant increase after eight days of SP treatment. There was also a decrease in hemoglobin oxygenation in control MMTV tumors. These results from the spontaneous tumor model provide additional evidence that SP treated tumors are not outgrowing their vasculature the way control tumors are, and are better oxygenated than untreated tumors. The increase in oxygenation, measured by MSOT imaging, relative to untreated controls was 30% compared to only 5% with the xenograft tumors. Staining the tumor sections for endothelial cell marker (CD31) and vascular smooth muscle cells (SMA) revealed considerable more staining in the central regions of the spontaneous tumors while the xenografts stained more in the periphery. SP treatment enhanced the staining intensity in the spontaneous tumors but not so in the xenografts.
Conclusions The results proved the hypothesis that recoupling NOS in tumors enhances tumor oxygenation. These results also highlight the spontaneous tumors having more normal vasculature than the subcutaneous xenograft tumors. The outcomes could have potential applications in enhancing radio- and chemo-sensitivities.