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Glioblastoma (GBM) is a high-grade, poor prognosis tumor that is resistant to standard treatment. The presence of a small number of glioma stem cells (GSCs) surviving in the harsh microenvironment is responsible for their refractoriness. This study aimed to investigate the effect of a hypoxic environment on the sensitivity of GSCs to photodynamic therapy with 5-aminolevulinic acid (ALA-PDT).Six human GSC lines, Mesenchymal types HGG13, HGG30, HGG1123, and Proneural types HGG146, HGG157, HGG528, were divided into two groups: normoxia (OHypoxia-GSCs showed higher mRNA levels of FECH (ferrochelatase), which is required for iron synthesis to convert PpⅨ to heme, compared with Normoxia-GSCs. Flow cytometry revealed that the accumulation of PpⅨ in Hypoxia-GSCs reduced upon incubation with ALA. However, Hypoxia-GSCs showed less reduction in sensitivity to ALA-PDT than Normoxia-GSCs.Hypoxia-GSCs had lower intracellular PpⅨ accumulation than Normoxia-GSCs due to increased gene expression of FECH, and that their sensitivity to ALA-PDT was reduced less, despite accumulating lower concentrations of PpⅨ. ALA-PDT is a potentially effective therapy for hypoxia-tolerant GSCs that exist in hypoxia at 5% oxygen concentration.

Cancer cells with stem cell-like features are generally more resistant to chemotherapy and radiotherapy than differentiated tumor cells. Thus, these cells tend to increase the propensity for tumor recurrence and metastasis. This study investigated the efficacy of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) in destructing glioma stem cells (GSCs), including the mesenchymal subtype (MES-GSCs) demonstrated to have the lowest radio- and chemosensitivity.Five high-grade glioma (HGG) GSC lines and derived differentiated glioma cell (DGC) lines were examined for protoporphyrin-IX (PpIX) expression using fluorescence-activated cell sorting (FACS) and then assessed for ALA-PDT sensitivity using cell viability assays. MES-GSCs surviving ALA-PDT were then isolated and evaluated for stem cell and mesenchymal marker expression levels (CD44, ALDH1A3, KLF4, nestin) by qRT-PCR. The ability of these surviving cells to form tumors was then examined using colony forming and by xenograft tumor assays in athymic mice. Finally, the relationship between PpIX expression level (high versus low) and ALA-PDT sensitivity was examined by FACS and colony forming assays.ALA-PDT was effective against all GSC lines including MES-GSCs. MES-GSC lines exhibited higher PpIX expression than derived DGCs. Surviving MES-GSCs demonstrated lower stem cell marker expression and tumor forming potential than naive MES-GSCs. Higher PpIX production capacity by MES-GSCs was associated with greater colony forming ability, and ALA-PDT was more effective against MES-GSCs with greater PpIX accumulation.ALA-PDT may be clinically effective against HGG by targeting GSCs, including MES-GSCs.

Background Glioblastoma (GBM) is a highly malignant lethal brain cancer. Accumulated evidence suggests that elevated resistance of GBM to both chemo- and radio-therapy is, at least in part, due to the presence of a small population of glioma stem cells (GSC). In the present study, we aimed to determine the sensitivity of GSCs to 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT). Methods For this purpose, we established GSC-enriched cell cultures (termed glioma stem-like cells or GSLCs) from A172 human GBM cell line. Under our cultivation conditions, GSLCs formed floating spheroid clusters that contained increased population of CD133/Sox2 expressing cells. Firstly, to compare the activity of protoporphyrin IX (PpIX) biosynthesis in the GSLCs and the parental A172 glioma cells, we examined the expression levels of biosynthesis enzymes and transporters for PpIX using qRT-PCR, and investigated the intracellular levels of PpIX with use of flow cytometry analysis. Then, we evaluated the sensitivity of these cells to ALA-PDT in vitro. Finally, to confirm the therapeutic impact of ALA-PDT on GSLCs with more clinically relevant model, we performed the same experiment using three different patient-derived glioma sphere lines, which cultivated them either in stem cell media or under differentiation conditions in the presence of serum. Results and Conclusion GSLCs expressed higher mRNA levels of PpIX biosynthesis enzymes and its transporters PEPT1/2 and ABCB6, when compared to the parental A172 glioma cells. Consistently, flow cytometry analysis revealed that upon incubation with ALA, GSLCs accumulate a higher level of PpIX. Finally, we showed that GSLCs were more sensitive to ALA-PDT than the original A172 cells, and confirmed that all patient-derived glioma sphere lines also showed significantly increased sensitivity to ALA-PDT if cultivated under the pro-stem cell condition. Our data indicate that ALA-PDT has potential as a novel clinically useful treatment that might eliminate GBM stem cells that are highly resistant to current chemo- and radio-therapy.

Background Oral 5-aminolevulinic acid (ALA) induces biosynthesis/accumulation of the natural photo-sensitizer protoporphyrin IX (PpIX) in cancer cells. ALA is used widely in photodynamic diagnosis (PDD) and therapy (PDT) during malignant glioma surgery, but few studies have examined the effects of photodynamics plus ALA on normal brain tissue in vivo. We investigated the effects of ALA-mediated PDD and PDT on normal brain tissue. Methods We established a rat model in which the brain surface was irradiated through the skull by light-emitting diode (635 nm) after ALA administration. Using this model, we investigated the effects of various amounts of light irradiation with various ALA doses on brain tissue. Results Neurological symptoms developed with administration of ALA at 240 or 120 mg/kg accompanied by irradiation at 100 or 400 J/cm2, respectively. Dye leakage occurred due to disruption of the blood–brain barrier (BBB) at 90 mg/kg and 100 J/cm2, respectively. Thickness of the cortex increased significantly at 240 mg/kg and 400 J/cm2, respectively. The number of neurons appeared to decrease at 200 mg/kg plus 400 J/cm2, respectively, and there was an increase in the number of cells that were positive for terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) staining. Conclusions ALA-mediated PDT is safe at doses of 90 mg/kg or less followed by light irradiation of 100 J/cm2 in rat brains. At doses above this threshold, ALA-PDT led to irreversible BBB and brain damage in rats.

Background Fluorescence image guided surgery (FIGS) with 5-aminolevulinic acid for malignant gliomas improves surgical outcome. One of the problems during FIGS is photobleaching under surgical microscopic white light. A solid laser-based white light source for neurosurgery that we developed does not include light with a wavelength of around 405 nm, which is strongly absorbed by protoporphyrin IX. In the present study, we examined the efficacy of this light source to prevent the photobleaching of protoporphyrin IX-induced fluorescence. Methods Filter papers transfused with protoporphyrin IX solution and a coronally sectioned F98 glioma rat model pretreated with 50 mg/kg 5-aminolevulinic acid were continuously exposed to white light. One group was exposed to conventional xenon-based white light and another group was exposed to laser-based white light. Fluorescence at a wavelength of 635 nm was measured with a radiospectrometer (in vitro study) and the relative fluorescence brightness was also measured in digital images (in vivo study) under excitation from violet blue light emitted from diodes every 5 min. Results and conclusion Estimated time for 50% photobleaching was prolonged about two times in the laser-based white light exposure group compared with that in the xenon-based white light exposure group (9.1/18.7 min). In the brain tumor rat model, it was also prolonged about 2.7 times (15.1/40.7 min). A laser-based white light source may inhibit photobleaching during FIGS for malignant gliomas. This light source for neurosurgical microscopy has the potential to prolong the prognosis of malignant glioma patients.