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

Abstract

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.