Abstract
Cancer stem cells (CSCs) are dominantly responsible for tumor progression and chemo/radio-resistance, resulting in tumor recurrence. 5-aminolevulinic acid (ALA) is metabolized to fluorescent protoporphyrin IX (PpIX) specifically in tumor cells, and therefore clinically used as a reagent for photodynamic diagnosis (PDD) and therapy (PDT) of cancers including gliomas. However, it remains to be clarified whether this method could be effective for CSC detection. Here, using flow cytometry-based analysis, we show that side population (SP)-defined C6 glioma CSCs (GSCs) displayed much less 5-ALA-derived PpIX fluorescence than non-GSCs. Among the C6 GSCs, cells with ultralow PpIX fluorescence exhibited dramatically higher tumorigenicity when transplanted into the immune-deficient mouse brain. We further demonstrated that the low PpIX accumulation in the C6 GSCs was enhanced by deferoxamine (DFO)-mediated iron chelation, not by reserpine-mediated inhibition of PpIX-effluxing ABCG2. Finally, we found that the expression level of the gene for heme oxygenase-1 (HO-1), a heme degradation enzyme, was high in C6 GSCs, which was further up-regulated when treated with 5-ALA. Our results provide important new insights into 5-ALA-based PDD of gliomas, particularly photodetection of SP-defined GSCs by iron chelation based on their ALA-PpIX-Heme metabolism.
Highlights
Proposed to overcome these limitations, including inhibition of PpIX efflux by the suppression of ATP-binding cassette sub-family G member 2 (ABCG2) transporter[17,18,19,20], potentiation of PpIX synthesis by increasing the activity of enzymes and transporters that are involved in PpIX synthesis[21,22], and reduction of the PpIX to heme conversion by iron removal or relevant enzyme inhibition[23,24,25,26]
C6 cells were treated with 5-ALA for 4 hours to allow PpIX synthesis and excited with 488 nm laser due to the availability of the lasers equipped on FACS
17.5 ± 10.6% of C6 cells remained at low fluorescence, suggesting that C6 cells have a cellular heterogeneity of 5-ALA-mediated accumulation of fluorescent metabolites
Summary
Proposed to overcome these limitations, including inhibition of PpIX efflux by the suppression of ATP-binding cassette sub-family G member 2 (ABCG2) transporter[17,18,19,20], potentiation of PpIX synthesis by increasing the activity of enzymes and transporters that are involved in PpIX synthesis[21,22], and reduction of the PpIX to heme conversion by iron removal or relevant enzyme inhibition[23,24,25,26]. Clinical studies on 5-ALA-mediated PpIX accumulation in glioblastoma multiforme (GBM) were performed[27,28]. The relationship between PpIX accumulation and GSCs was still unclear. It remains to be fully provided that how we could overcome the heterogeneity of cancerous cells in terms of 5-ALA-mediated fluorescence intensities. The accurate evaluation of heterogeneous cancer cells and enhancement of PpIX accumulation in the GSCs need to be explored. Using flow cytometry (FACS)-based analysis, we assessed the levels of 5-ALA-mediated PpIX accumulation in C6 glioma CSCs and non-CSCs, and found that the former exhibits lower PpIX fluorescence intensity, among which cells with the poorer ability of PpIX accumulation are highly tumorigenic. We propose an improved method for 5-ALA-based fluorescence detection of SP-defined GSCs
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