Abstract
5-Aminolevulinic acid (5-ALA) induced fluorescence to augment surgical resection for high grade glioma has become a standard of care. Protoporphyrin IX (PpIX) visibility is however subject to the variability of the single tumor expression and to the interobserver interpretation. We therefore hypothesized that in different glioma cell lines with variable 5-ALA induced fluorescence, the signal can be pharmacologically increased. We therefore analyzed in three different GBM cell lines, with different expression of epidermal growth factor receptor (EGFR), the variability of 5-ALA induced PpIX fluorescence after the pharmacological blockade at different steps of PpIX breakdown and influencing the outbound transport of PpIX. Using flow cytometry, fluorescence microplate reader, and confocal microscopy the PpIX fluorescence was analyzed after exposure to tin protoporphyrin IX (SnPP), deferoxamine (DFO), and genistein. We furthermore constructed a microscope (Qp9-microscope) being able to measure quantitatively the concentration of PpIX. These values were compared with the extraction of PpIX in tumor biopsy taken during the GBM surgery. Although all three cell lines showed an increase to 5-ALA induced fluorescence their baseline activity was different. Treatment with either SnPP, DFO and genistein was able to increase 5-ALA induced fluorescence. Qp9-microscopy of tumor sample produced a color coded PpIX concentration map which was overlaid on the tumor image. The PpIX extraction from tumor sample analyzed using the plate reader gave lower values of the concentration, as compared to the expected values of the Qp9-microscope, however still in the same decimal range of μg/mL. This may be due to homogenization of the values during extraction and cell disaggregation. In conclusion pharmacological augmentation in GBM cell lines of PpIX signal is possible. A quantitative PpIX map for surgery is feasible and may help refine surgical excision. Further correlations of tumor tissue samples and Qp9-microscopy is needed, prior to develop an intraoperative surgical adjunct to the already existing 5-ALA induced surgery.
Highlights
It is widely accepted that the extent of surgical resection plays an important role in overall survival in patients with glioma, both in IDH wild type and IDH mutated, and both in high grade and low-grade glioma [1, 2]
Having previously established the final concentration of 5-Aminolevulinic acid (5-ALA) at 1 mM for 8 h as the optimal conditions for 5-ALA treatment and quantification of PpIX fluorescence [16], we proceeded to study the effect of drugs that modulate proteins involved in PpIX conversion into non-fluorescent metabolites
Based on two independent experiments performed by flow cytometry, we showed that the HO-1 inhibitor selected three compounds: tin protoporphyrin IX (SnPP) is able to significantly improve 5-ALAinduced PpIX fluorescence in U87MG and U87vIII, alone or in combination with 5-ALA
Summary
It is widely accepted that the extent of surgical resection plays an important role in overall survival in patients with glioma, both in IDH wild type and IDH mutated, and both in high grade and low-grade glioma [1, 2]. The amount of PpIX in tumor cells is favored by both the activity of cytosolic porphobilinogen deaminase (PBGD) during the replication phase, and by the down-regulation of ferrochelatase (FECH) [6] Other factors such as protein transporters, tyrosine kinase activity and its downstream effect on hemoxygenase-1 (HO-1), availability of free Fe2+-ions and its effect on FECH, are related with PpIX fluorescence [7, 8]. Because of the above mentioned factors, the final enhancing results are highly variable and difficult to predict both in clinical and laboratory setting, and might depend on EGFR expression status in glioma cell lines [7]. Proteins transporters such as ATP-binding cassette subfamily G member 2 (ABCG2) regulate intracellular concentration of PpIX. These proteins may be differently expressed or activated in relation with the epigenetic status of GBM [9] (Figure 1)
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