ROS production is the main factor conditioning cell death in response to 5-Aminolevulinic Acid-based Photodynamic Therapy in human lung adenocarcinoma spheroids

MEISS R.,; DIEZ B.,; FUKUDA H.; TEIJO MJ.,; BATLLE A.,

Simposio; Cancer Research & Oncology 2016, LabRoots Virtual Event Poster Hall.; 2016

Photodynamic therapy (PDT) is an approved anticancer treatment modality that eradicates tumor cells by the photochemical generation of reactive oxygen species (ROS) following absorption of visible light by a photosensitizer, which is selectively taken up by malignant cells. 5-Aminolevulinic acid (ALA) is the biological precursor of Protoporphyrin IX, the only endogenous photosensitizer.Multicellular spheroids (MCSs) are three-dimensional compact cellular aggregates that mimic micro-tumors with no vascular irrigation, representing a suitable model for the study of PDT-induced cell death, since tumor vasculature has a controversial role in tumor eradication by PDT. Previously, we found that cells cultured in MCSs appear to be more resistant to photodamage than cells cultured in monolayers, as the light dose yielding 50% cell death after PDT (LD50) determined by the MTT assay and the APH assay, was lower in cells growing in monolayers (7 min) than in spheroids (10 min).The aim of this study was to detect the limiting factors conditioning PDT efficacy in MCSs.ALA -PDT was performed in A549 human lung adenocarcinoma cells growing as monolayers or as MCSs. These were initiated by seeding 5.104 cells/ml on 3% agar:RPMI (1:1) coated wells and harvested after 7 days with periodic fresh medium renewal. Cells were incubated with 1 mM ALA for 3h followed by irradiation with a bank of two fluorescent lamps (Osram L36W/10) for different times and within 1h analyzed for PDT-induced events.Porphyrin synthesis was not significantly different between monolayers and MCS, neither quantitatively (monolayers: 0,109±0,1 pg/cell; MCSs: 0,09±0,1 pg/cell) nor qualitatively, since PpIX fluorescence distribution was uniform along all sections of the spheroids analyzed under confocal microscopy.With normal PpIX synthesis and distribution, the limiting factor on PDT efficacy could be light penetration into the inner section of the MCSs. To this end, spheroids were trypsinized and cells counted for apoptosis by acridine orange/ethidium bromide staining and AnnexinV-FITC/propidium iodide labelling. Results showed a higher percentage of viable cells when 20 min-irradiation PDT was performed on spheroids (66,57±6,5%) compared to monolayers (3,5±0,2%). Moreover, caspase-3 expression and cytochrome c release were also lower in MCSs.To visualize cell photodamage in intact spheroids, histological sections were stained for general morphology with hematoxylin/eosin, PAS staining for mucopolysaccharides, and Ki67 immunostaining as proliferation marker. Results showed that not only can light pass through the structure of the spheroid, but also that PDT can disrupt cellular morphology, secretory differentiation (which is consistent with the adenocarcinoma histology of the cell line) and cell proliferation.Another limiting factor on PDT efficacy could be a defective ROS production in MCSs, therefore, singlet oxygen (the main effector of PDT-induced cell damage) and superoxide anion generation were assessed by flow cytometry. It was found that both ROS production were significantly lower in MCSs (1O2: 5,5±2,7%; O2-: 4,8±0,3%) compared to monolayers (1O2: 83,5±1,9%; O2-: 70,6±8,1%).These results indicate that oxygen availability and not porphyrin synthesis or light accessibility might be the conditioning factor for the differential response of MCSs to ALA-based PDT.