Abstract
Photodynamic diagnosis (PDD) is a form of cancer detection based on the administration of an exogenous photo-activated compound that accumulates in malignant cells, followed by appropriate photo irradiation. The authors describe a spectroscopic study of 5-aminolevulinic acid (5-ALA)-generated photosensitizer protoporphyrin IX (PpIX) fluorescence in human oral squamous cell carcinoma (OSCC) cell lines to validate its clinical use. 5-ALA-induced PpIX fluorescence intensity was measured in the presence and absence of deferoxamine mesylate (DFO). Two, one and two cell lines produced poorly, moderately and well differentiated carcinomas, respectively, on transplantation in scid mice. The fluorescence intensity was high in the poorly differentiated cell lines, followed by the moderately differentiated cell line; the intensity of the well differentiated cell lines was low and not significantly different from that of normal keratinocytes in the absence of DFO. It was elevated to the level of poorly differentiated cell lines following DFO treatment. This elevation was not observed in normal keratinocytes. The results indicate that DFO enhances the photodynamic sensitivity of 5-ALA in non-responsive carcinoma cells as a result of increased cellular accumulation by inhibiting haeme biosynthesis. This PDD system can be applied clinically to the detection of OSCC irrespective of the degree of differentiation.
Photodynamic diagnosis (PDD) has been used to examine brain, skin, colon and oral cancers . It involves administration of a photosensitizer to cells and tissues, subsequent exposure of the tissues to light of a specific wavelength and detection of characteristic emission fluorescence .
5-Aminolevulinic acid (5-ALA) has been advocated as a photosensitising agent for PDD and photodynamic therapy (PDT) . This compound selectively causes the accumulation of protoporphyrin IX (PpIX), an effective photosensitizer, in malignant tumour cells . After it is converted intracellularly to PpIX, the PpIX can be detected by its typical red fluorescence under illumination with blue light. With the introduction of deferoxamine mesylate (DFO), the photodynamic system is becoming widely used for a variety of malignancies, because it can enhance the fluorescence intensity in non- or weakly-fluorescence responsive malignant tumours. The primary advantages of using 5-ALA lie in its rapid elimination from the body and the short delay between administration of the agent and examination .
As the oral cavity is readily accessible with laser light, it is a suitable site for PDD. This technique has been a powerful tool for the diagnosis and treatment of oral squamous cell carcinoma (OSCC) . OSCC exhibit various histological features, indicating that they consist of cells with different natures. It is of value to study the 5-ALA-induced fluorescence in cells with different degrees of differentiation because it may vary depending on the nature of cells. There is no comparative study on 5-ALA-generated PpIX fluorescence using human oral cancer cell lines with varying differentiation. In the current study, the authors evaluated the response of five cell lines with different natures established from human OSCC to 5-ALA with or without DFO to examine the validity of the photodynamic system for the diagnosis and treatment of OSCC.
Materials and methods
Cell culture
Human OSCC cell lines, HSC-2, HSC-3, HSC-4 and Ca9-22, all originally obtained from the Japanese Collection of Research Bioresources (JCRB), and SAS, provided from Tokyo Dental University, were used. Human normal oral keratinocytes from the buccal mucosa of a healthy male were used as controls. Human OSCC cells were cultured at 37 °C under 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum and penicillin. Human normal oral keratinocytes were grown at 37 °C under 5% CO 2 in defined keratinocyte Serum Free Medium (SFM) with growth supplement and penicillin.
Transplantation of carcinoma cells
HSC-2, HSC-3, HSC-4, Ca9-22 and SAS were transplanted subcutaneously into the back of 4-week-old male scid mice ( n = 15) weighing 20–25 g. When the tumours reached a size of 10 × 10 mm, approximately 14 days after transplantation, they were removed and subjected to histological examination. The removed tissues were fixed with formalin and embedded in paraffin. The tumour tissue blocks were cut into 4-μm thick sections and stained with haematoxylin and eosin (H-E). The degree of tumour differentiation was determined according to the WHO classification .
Fluorescence measurement
Confluent cells were used for the experiment. 5-ALA (Wako Chemical Industries, Tokyo, Japan) was freshly dissolved in phosphate-buffered saline (PBS) at a concentration of 3 mg/ml before each experiment. Cells cultured in a 10-cm dish were exposed to 5-ALA (300 μg/ml) with or without DFO (50 μg/ml) for 4 h in the dark. After the cells were rinsed twice with PBS, they were dissociated from the dish using trypsin/EDTA and suspended in 10 ml of PBS. The fluorescence intensity at 635 nm was measured in a concentration of 1 × 10 4 cells/ml. A 100-W mercury light source was used in a dark room to excite PpIX at a wavelength of 400–440 nm. A fluorescence spectrometer (HITACHI 650-10, Japan) was used to evaluate the fluorescence intensity generated by 5-ALA treatment alone and by treatment with a mixture of 5-ALA and DFO. As a control, cultured cells exposed to medium alone were also evaluated. The fluorescence intensity of each cell line was calculated as a mean of three measurements and Student’s t -test was used for statistical analysis.
Results
Tumour differentiation in the five cell lines
Squamous cell carcinomas induced by transplantation of HSC-2 and Ca9-22 cells were poorly differentiated. SAS cells produced moderately differentiated carcinomas and HSC-3 and HSC-4 cells produced well differentiated carcinomas ( Fig. 1 ).
Fluorescence intensity
The fluorescence intensities of HSC-2 and Ca9-22 in medium alone were 0.33. The value for SAS was 0.22, significantly lower than those of HSC-2 and Ca9-22, but significantly higher than the 0.03 of HSC-3 and the 0.07 of HSC-4. The values for HSC-3 and HSC-4 were not statistically different from the 0.08 of the control normal oral keratinocytes. When treated with 5-ALA, the fluorescence intensities increased in all carcinoma cell lines and control keratinocytes, but the increases were not statistically different. There was no significant difference between the values for HSC-3 and HSC-4 and the control keratinocyte value after treatment with 5-ALA. The values for HSC-2 and Ca9-22 were 0.43 and 0.57, respectively, on further addition of DFO. The respective values for SAS, HSC-3 and HSC-4 were 0.39, 0.38 and 0.50. Compared with 5-ALA alone, all carcinoma cell lines except for HSC-2 showed significant increase. The increases of HSC-3 and HSC-4 were most prominent and reached levels similar to those of HSC-2 and SAS. No significant increase was observed in normal oral keratinocytes following DFO treatment ( Fig. 2 ).
