Development of a Photodynamic Diagnosis Method for Oral Squamous Cell Carcinoma Using 5-Aminolevulinic Acid and a Luminescence Plate Reader

Purpose: To establish a simple and accurate photodynamic diagnosis (PDD) method for oral squamous cell carcinoma (OSCC). Methods: OSCC cell lines HSC-2, HSC-3, HSC-4, and Sa3, and normal human oral keratinocytes (HOK) were used. First, we examined the amount of cells needed to detect differences in fluorescence intensities for PDD. OSCC cell lines were adjusted to concentrations of 1 × 104 (104), 1 × 105 (105), and 1 × 106 (106) cells/ml. The experimental groups comprised a group with 5-aminolevulinic acid (5-ALA (+)), and a group without 5-ALA (5-ALA (-)). For each OSCC cell line, 100 μl of each concentration of cells of the 5-ALA groups was seeded onto fluorescence plates, and fluorescence intensity was measured at 60-min intervals for 240 min. Results are expressed as the ratio of fluorescence intensity in 5-ALA (+) to 5-ALA (-). As cells at the concentration of 106 cells/ml provided the clearest results, fluorescence intensities of all cell lines were measured using this concentration at 20-min intervals for 700 min using the same methods. Results: The 5-ALA (+) to (-) ratio increased in a cell concentration-dependent manner at 240 min;the ratio was highest with 106 cells/ml and lowest with 104 cells/ml. With 106 cells/ml in the 5-ALA (+) group, fluorescence intensity increased in a metabolic time-dependent manner;the increase was highest in HSC-2 cells, followed by HSC-4 cells, HSC-3 cells, Sa3 cells, and HOK. Fluorescence intensity was significantly enhanced after 40 min in HSC-2, HSC-3, and HSC-4 cells, after 60 min in Sa3 cells, and after 100 min in HOK compared to the 5-ALA (-) group (P < 0.05). Moreover, fluorescence intensity was significantly increased in OSCC cell lines compared to HOK after 40 min. Conclusion: Early detection of OSCC is possible by screening only microplate reader measurements of fluorescence intensity for PDD.

5-Amino-4-oxopentanoic acid photodynamic diagnosis guided microsurgery and photodynamic therapy on VX2 brain tumour implanted in a rabbit model

Background Complete tumour resection is important for improving the prognosis of brain tumour patients. However, extensive resection remains controversial because the tumour margin is difficult to be distinguished from surrounding brain tissue. It has been established that 5-amino-4-0xopentanoic acid （5-aminolevulinic acid, ALA） can be used as a photodynamic diagnostic marker and a photosensitizer for photodynamic therapy in surgical treatment of brain tumours. We investigated the efficacy of ALA photodynamically guided microsurgery and photodynamic therapy on VX2 brain tumour implanted in a rabbit model. Methods Eighty New Zealand rabbits implanted with VX2 brain tumours were randomly assigned to five groups： control, conventional white light microsurgery, a photodynamic therapy group, a photodynamically guided microsurgery group and a group in which guided microsurgery was followed by photodynamic therapy. The VX2 tumour was resected under a surgical microscope. The tumour resection was confirmed with histological analysis. All animals were examined with MRI for presence of any residual tumour tissue. The survival time of each rabbit was recorded. Results All treatment groups showed a significantly extended survival time compared with the control group. Photodynamically guided microsurgery combined with photodynamic therapy significantly prolonged survival time, compared with guided microsurgery alone. MRI and the autopsy results confirmed removal of most of the tumours. Conclusions Our results suggest that photodynamically guided surgery and photodynamic therapy significantly reduce or delay local recurrence, increase the effectiveness of radical resection and prolong the survival time of tumour bearing rabbits. Their combination has the potential to be used as a rapid and highly effective treatment of metastatic brain tumours.

Photodynamic diagnostics of early gastric cancer:Complexity measures of gastric microcirculation and new model of metastatic adenocarcinoma of rat stomach

The detection of early gastric cancer that often develops asymptomatically is crucial for improving patient survival.The photodynamic diagnosis(PDD)of gastric cancer using 5-aminolevulinic acid/protoporphyrin IX(5-ALA/PpIX)has been reported in several studies.However,the selectivity of PDD of gastric tumor is poor with often false-positive results that require the development of new methods to improve PDD for early gastric cancer.Therefore,a measure of the complexity of gastric microcirculation(multi-scale entropy,MSE)and the detrendedfluctuation analysis(DFA)were applied as additional tools to detect early gastric cancer in rats.In this experimental study,we used our original model of metastatic adenocarcinoma in the stomach of a rat.To induce a gastric tumor,we used a long-term combination(for 9 months,which is 1/2 of the life of rats)of two natural factors,such as chronic stress(overpopulation being typical for modern cities)and the daily presence of nitrites in food and drinks,which are common ingredients added to processed meat andfish to help preserve food.Our results clearly show that both methods,namely,PDD using 5-ALA/PpIX and complexity/correlation analysis,can detect early gastric cancer,which was confirmed by histological analysis.Pre-cancerous areas in the stomach were detected as an intermediatefluorescent signal or MSE level between normal and malignant lesions of the stomach.However,in some cases,PDD with 5-ALA/PpIX produced a false-positivefluorescence of exogenousfluorophores due to its accumulation in benign and inflammatory areas of the mucosa.This fact indicates that the PDD itself is not sufficient for the correct diagnosis of gastric cancer,and the use of additional characteristics,e.g.,complexity measures or scaling exponents,can significantly improve the diagnostic accuracy of PDD of gastric cancer that should be confirmed in further clinical studies and applications.

Clinical applications of 5-aminolevulinic acid-mediated fluorescence for gastric cancer

5-aminolevulinic acid(ALA) is a naturally occurring amino acid that is a protoporphyrin IX(Pp IX) precursor and a next-generation photosensitive substance. After exogenous administration of ALA, Pp IX specifically accumulates in cancer cells owing to the impaired metabolism of ALA to Pp IX in mitochondria, which results in a red fluorescence following irradiation with blue light and the formation of singlet oxygen. Fluorescence navigation by photodynamic diagnosis(PDD) using ALA provides good visualization and detection of gastric cancer lesions and is a potentially valuable diagnostic tool for gastric cancer for evaluating both the surgical resection margins and extension of the lesion. Furthermore, PDD using ALA might be used to detect peritoneal metastases during preoperative staging laparoscopy, where it could provide useful information for the selection of a therapeutic approach. Another promising application for this modality is in the evaluation of lymph node metastases. Photodynamic therapy(PDT) using ALA to cause selective damage based on the accumulation of a photosensitizer in malignant tissue is expected to be a non-invasive endoscopic treatment for superficial early gastric cancer. ALA has the potential to be used not only as a diagnostic agent but also as a therapeutic drug, resulting in a new strategy for cancer diagnosis and therapy. Here, we review the current use of PDD and PDT in gastric cancer and evaluate its future potential beyond conventional modalities combined with a light energy upconverter, a light-emitting diode and nearinfrared rays as light sources.

The Effectiveness of a Portable Fluorescence Spectrophotometer for Early Detection of Oral Cancer

Purpose: To investigate whether photodynamic diagnosis (PDD) using a portable fluorescence spectrophotometer (FC-1) can easily and objectively discriminate between normal and tumor cells at the dental chairside, and to further compare it with PDD that requires speculum examination by focusing on protoporphyrin IX (PPIX). Methods: Three cell lines (2 human oral squamous cell carcinoma-derived cell lines, HSC-2 and HSC-3 cells, and oral keratinocytes, HOK cells) were cultured. 5-Aminolevulinic acid hydrochloride (5-ALA) and deferoxamine mesylate (DFO) were mixed in DMEM, and the mixture was set to Control (DMEM only) and PDD (5-ALA+DFO) groups. And then, a fluorescence was measured under room temperature (RT) and 37°C (Incubation) by using FC-1. In this study, the two conditions were combined with the Control and PDD groups to form the Control/RT, Control/Incubate, PDD/RT, and PDD/Incubate groups. Additionally, the amount of singlet oxygen (1O2) generated by irradiation with 405 nm LED was measured using electron spin resonance spectroscopy to detect PPIX in the cell supernatant after 24 hours. Results: In HSC-2 and HSC-3, the fluorescence intensity values increased significantly at 2 hours between the Control/RT and PDD/RT groups. In addition, there was a significant difference between HSC-2 and HSC-3 compared to HOK. In all cell lines, the fluorescence intensity values of the PDD/Incubate group were significantly higher than those of the PDD/Control group. The amount of 1O2 generated by 405 nm LED irradiation was higher in the cell supernatants of all cell lines in the order of Control/RT < Control/Incubate < PDD/RT < PDD/Incubate group, and HSC-3 in the PDD/Incubate group showed a significant increase compared to HOK. Conclusion: It is suggested that PDD using FC-1 can clearly distinguish between normal cells and tumor cells in vitro studies using cell lines at 2 hours under 37°C, and it can detect not only intracellular PPIX, but also extracellular PPIX.