Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Hospital, Moorenstrasse 5, 40225 Düsseldorf, Germany; Department of Neurosurgery, Heinrich Heine University Hospital, Moorenstrasse 5, 40225 Düsseldorf, Germany.
Department of Neurosurgery, Heinrich Heine University Hospital, Moorenstrasse 5, 40225 Düsseldorf, Germany.
J Photochem Photobiol B. 2018 Dec;189:298-305. doi: 10.1016/j.jphotobiol.2018.11.002. Epub 2018 Nov 7.
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.
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.
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.
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.
髓母细胞瘤（MB）是儿童中最常见的恶性原发性脑肿瘤。高危患者的预后仍然很差，特别是年轻患者会遭受标准治疗引起的后遗症。因此，需要探索其他治疗选择。在胶质母细胞瘤（GBM）中，应用 5-氨基酮戊酸（5-ALA）可导致原卟啉 IX（PPIX）在肿瘤细胞中选择性积累，可在荧光引导手术中利用该特性来增加切除范围或用于光动力疗法（PDT）诱导的光毒性。目前尚不完全清楚 MB 细胞是否会积累 PPIX 并对 PDT 敏感。
人 MYC 扩增（Med8A 和 D283）和非扩增（UW228-2 和 ONS76）MB 细胞系在不同剂量（0-100μg/ml）的 5-ALA 孵育 2、4 或 6 小时后，通过流式细胞术确定 PPIX 积累情况。为了评估对 5-ALA/PDT 的敏感性，将细胞用 5-ALA 孵育，然后用 635nm 波长的激光照射（18.75J/cm）。在另外 24 小时培养期后，使用 WST-1 测定法定量测定细胞活力。通过逆转录和定量聚合酶链反应检测亚铁螯合酶的表达。通过测量 PPIX 向锌原卟啉的酶促转化来定量测定亚铁螯合酶活性。通过流式细胞术检测 ABCG2 转运蛋白 CD338 的表达。
所有 MB 细胞系在暴露于外源性 5-ALA 后均表现出时间和剂量依赖性的 PPIX 积累，并对 5-ALA/PDT 诱导的光毒性敏感。与 U373 GBM 细胞相比，在较短的孵育时间和限制 5-ALA 剂量时，PPIX 积累减少。此外，并非所有 MB 细胞均变为 PPIX 阳性，并且 MB 细胞系的总体光毒性较低。值得注意的是，与非扩增型细胞系相比，MYC 扩增型 MB 细胞表现出更明显的光敏感性。亚铁螯合酶的表达没有差异，但与 U373 GBM 细胞相比，酶活性似乎在 MB 细胞中降低，而 CD338 仅在 MB 细胞上表达。
髓母细胞瘤细胞系在应用 5-ALA 后积累 PPIX，并对 PDT 敏感，与低铁螯合酶表达和活性有关。在 MYC 扩增型细胞系中，光敏感性更为明显。然而，与 GBM 细胞不同，PPIX 积累似乎减少，仅限于细胞亚群，并且与 MB 细胞系的低光敏感性相关，这可能是由于 MB 细胞上表达了 ABCG2 转运蛋白 CD338。
