ALA- and ALA-hexylester-induced protoporphyrin IX fluorescence and distribution in multicell tumour spheroids

Synthesis of protoporphyrin IX (PpIX) in intact murine mammary cancer cell spheroids is reported from optical sections obtained using a laser scanning confocal fluorescence microscope. EMT6 spheroids 275-350 mum in diameter w ere incubated in 0.1-15 mM aminolevulinic acid (ALA) or 0.001-2 mM ALA-hexy lester (h-ALA) to test the ability of both pro-drugs to diffuse into the sp heroids and induce PpIX production. Spheroids incubated with ALA show signi ficant fluorescence nonuniformity for all concentrations, with the outermos t cells exhibiting greater porphyrin fluorescence. Comparable levels of flu orescence throughout the optical section are achieved with approximately 10 0-fold lower h-ALA concentrations, indicating that the interior cells maint ain esterase activity and porphyrin synthesis and that h-ALA diffuses effic iently to the spheroid interior. Fluorescence gradients are less pronounced with h-ALA incubation, in part because of apparent saturation of esterase activity in the spheroid perimeter. Proliferating (Ki67 positive) and quies cent cell populations exhibit remarkably different h-ALA concentration depe ndencies. The incubation concentration resulting in maximum fluorescence wi th ALA is 10 mM, while the optimal concentration for h-ALA is 200-fold lowe r at 0.05 mM. Exceeding these optimal concentrations for both pro-drugs lea ds to an overall loss of fluorescence. (C) 2001 Cancer Research Campaign.