In vitro and in vivo porphyrin accumulation by C6 glioma cells after exposure to 5-aminolevulinic acid

Several malignant tissues synthesize endogenous porphyrins after exposure to 5-aminolevulinic acid (5-ALA). The present experiments have been designed to elucidate whether the C6 glioma cell, a model cell for human malignant glioma, similarly synthesizes porphyrins when exposed to 5-ALA, and whether specific synthesis occurs when C6 cells are inoculated into rat brains to form a tumor. In this situation the blood-brain barrier may interfere with 5-ALA availability, and spreading of porphyrins with edema outside the tumor may occur. Flow cytometry is used to determine the course of cell volume and porphyrin fluorescence intensities in cultured C6 cells which are incubated in 1 mM 5-ALA. For the induction of experimental brain tumors, 10⁴ untreated C6 cells are inoculated into the brains of rats. After 9 days animals receive 100 mg 5-ALA/kg body weight. Brains are removed after 3, 6, or 9 h and frozen coronal sections obtained for H/E staining or fluorescence spectography. Cultured C6 cells show a linear increase of protoporphyrin IX fluorescence after exposure to 5-ALA, which begins to plateau after 85 min. Marked fluorescence is also observed in solid and infiltrating experimental tumor. However, faint fluorescence also occurs in normal tissue, basal pia, choroid plexus, and, more obviously, in white-matter tracts bordering the tumor (maximal distance: 1.5 ± 0.7 mm). The observations demonstrate that C6 cells synthesize protoporphyrin IX after exposure to 5-ALA in vitro and in vivo. However, when utilizing 5-ALA for fluorescence detection or photodynamic therapy of brain tumors, attention should be paid to the possibility of protoporphyrin IX occurring outside the tumor.