Accumulation of protoporphyrin IX in medulloblastoma cell lines and sensitivity to subsequent photodynamic treatment.

BACKGROUND

Medulloblastoma (MB) is the most common malignant primary brain tumor of childhood. High risk patients still have a poor outcome, and especially young patients suffer from standard therapy induced sequelae. Therefore, other therapeutic options need to be explored. In glioblastoma (GBM), application of 5-aminolaevulinic acid (5-ALA) results in selective accumulation of protoporphyrin IX (PPIX) in the tumor cells, which can be exploited during fluorescence-guided surgery to increase the extent of resection or for photodynamic therapy (PDT) induced phototoxicity. It is not entirely clear, whether MB cells accumulate PPIX and are sensitive to PDT.

METHODS

Human MYC-amplified (Med8A and D283) and non-amplified (UW228-2 and ONS76) MB cell lines were incubated for 2, 4 or 6 h with increasing doses (0-100 μg/ml) of 5-ALA, and PPIX accumulation was determined by flow cytometry. To assess sensitivity to 5-ALA/PDT, cells were incubated with 5-ALA and subsequently exposed to laser light of 635 nm wavelength (18.75 J/cm). After an additional 24 h culture period, viability of cells was quantified using the WST-1 assay. Expression of ferrochelatase was detected by reverse transcription and quantitative polymerase chain reaction. Ferrochelatase activity was quantified by measuring the enzymatic conversion of PPIX to zinc-protoporphyrin. Expression of the ABCG2 transporter protein CD338 was detected by flow cytometry.

RESULTS

All MB cell lines showed a time- and dose-dependent accumulation of PPIX after exposure to exogenous 5-ALA and became sensitive to 5-ALA/PDT-induced phototoxicity. PPIX accumulation was reduced compared to U373 GBM cells at shorter incubation periods and limiting 5-ALA doses. Moreover, not all MB cells became PPIX positive and overall phototoxicity was lower in the MB cell lines. Notably, the MYC-amplified MB cells demonstrated a more pronounced photosensitivity compared to their non-amplified counterparts. There was no difference in expression of ferrochelatase, but enzymatic activity appeared to be reduced in the MB cells compared to U373 GBM cells, whereas CD338 was expressed on the MB cells only.

CONCLUSION

Medulloblastoma cell lines accumulate PPIX after application of 5-ALA and become sensitive to PDT, associated with low ferrochelatase expression and activity. Photosensitivity is more pronounced in MYC-amplified cell lines. In contrast to GBM cells, however, PPIX accumulation appears to be reduced, restricted to a subset of cells and associated with lower photosensitivity of the MB cell lines, possibly due to expression of the ABCG2 transporter protein CD338 on MB cells.