Acridine Orange Used for Photodynamic Therapy Accumulates in Malignant Musculoskeletal Tumors Depending on pH Gradient

Abstract

Background: Intra-operative photodynamic therapy has been applied with acridine orange (AO-PDT) to human musculoskeletal sarcomas for the past 4 years, resulting in a low local recurrence rate, within 10%, after intra-marginal tumor resection and excellent limb function. However, it is still unclear why acridine orange (AO) specifically accumulates in tumor cells, especially in malignant tumor cells. The purpose of this study was to clarify the mechanism of AO accumulation in malignant musculoskeletal tumors. Materials and Methods: Sixty-two musculoskeletal tumors, including 35 malignant and 27 benign tumors, were studied. Using freshly resected tumor material, the extracellular pH (pHe) was measured and the fluorescence intensity of AO accumulated in the tumors was measured by an image analyzer after ex vivo exposure to 1.0 μg/ml AO, followed by blue excitation. In the in vitro study, bafilomycin A1 was exposed to LM 8 mouse osteosarcoma cells, in order to inhibit V-ATPase, subsequently causing a decrease in the pH gradient (ΔpH) between the intracellular pH (pHi) and extracellular pH (pHe) or between the pHi and the pHe. AO accumulation and the cytocidal effect of AO were evaluated. Results: The results of the in vivo study, using human materials freshly resected at surgery, revealed that the pHe of the malignant musculoskeletal tumors was significantly lower than that of the benign tumors and normal muscles or adipose tissues and also showed that the AO fluorescence intensity of the malignant musculoskeletal tumors was significantly stronger than that of the benign tumors and normal muscles or adipose tissues. The results also revealed that the AO fluorescence intensity negatively correlated with the pHe in tumors and normal tissues. The in vitro study showed that bafilomycin A1 inhibited the accumulation of AO in acidic organelles, such as lysosomes, and that the cytocidal effect of AO-PDT was also remarkably inhibited. Discussion: Based on results of the in vivo and in vitro studies, it is suggested that malignant musculoskeletal tumors have a large ΔpH between the pHi and the pHe or between the pHi and the vaculolar pH and also that a large ΔpH increases AO accumulation in tumors. We, therefore, believe that AO-PDT may be more effective in highly malignant musculoskeletal tumors than low grade ones, because of their acidity. Conclusion: AO accumulation in musculoskeletal tumors was dependent on the ΔpH between the pHi and the pHe, or between the pHi and the vacuolar pH.