Glioblastoma multiforme (GBM), an aggressive tumor that typically exhibits treatment failure with high mortality rates, is associated with the presence of cancer stem cells (CSCs) within the tumor. CSCs possess the ability for perpetual self-renewal and proliferation, producing downstream progenitor cells that drive tumor growth. Studies of many cancer types have identified CSCs using specific markers, but it is still unclear as to where in the stem cell hierarchy these markers fall. This is compounded further by the presence of multiple GBM and glioblastoma cancer stem cell subtypes, making investigation and establishment of a universal treatment difficult. This review examines the current knowledge on the CSC markers SALL4, OCT-4, SOX2, STAT3, NANOG, c-Myc, KLF4, CD133, CD44, nestin, and glial fibrillary acidic protein, specifically focusing on their use and validity in GBM research and how they may be utilized for investigations into GBM’s cancer biology.

AimTo identify and characterize cancer stem cells (CSC) in glioblastoma multiforme (GBM).MethodsFour-micrometer thick formalin-fixed paraffin-embedded GBM samples from six patients underwent 3,3-diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers NANOG, OCT4, SALL4, SOX2, and pSTAT3. IF IHC staining was performed to demonstrate co-expression of these markers with GFAP. The protein expression and the transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, and STAT3 were investigated using Western blotting (WB) and NanoString gene expression analysis, respectively.ResultsDAB and IF IHC staining demonstrated the presence of a CSC population expressing NANOG, OCT4, SOX2, SALL4, and pSTAT3 with the almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4, within GBM. The expression of NANOG, SOX2 and, pSTAT3 but, not OCT and SALL4, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of NANOG, OCT4, SALL4, STAT3, and SOX2 in GBM.ConclusionThis study demonstrated a population of CSCs within GBM characterized by the expression of the CSC markers NANOG, SALL4, SOX2, pSTAT3 and OCT4 at the protein and mRNA levels. The almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4 would support the putative existence of a stem cell hierarchy within GBM.

AimTo investigate the expression of the renin–angiotensin system (RAS) in cancer stem cells (CSCs), we have previously characterized in glioblastoma multiforme (GBM).Methods3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for the stem cell marker, SOX2, and components of the RAS: angiotensin converting enzyme (ACE), (pro)renin receptor (PRR), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) on 4 μm-thick formalin-fixed paraffin-embedded sections of previously characterized GBM samples in six patients was undertaken. Immunofluorescent (IF) IHC staining was performed to demonstrate expression of GFAP, SOX2, PRR, ACE, ATIIR1, and ATIIR2. The protein expression and the transcriptional activities of the genes encoding for ACE, PRR, ATIIR1, and ATIIR2 were studied using Western blotting (WB) and NanoString gene expression analysis, respectively.ResultsDAB and IF IHC staining demonstrated the expression SOX2 on the GFAP+ GBM CSCs. Cytoplasmic expression of PRR by the GFAP+ CSCs and the endothelium of the microvessels was observed. ACE was expressed on the endothelium of the microvessels only, while nuclear and cytoplasmic expression of ATIIR1 and ATIIR2 was observed on the endothelium of the microvessels and the CSCs. ATIIR1 was expressed on the GFAP+ CSCs cells, and ATIIR2 was expressed by the SOX2+ CSCs. The expression of ACE, PRR, and ATIIR1, but not ATIIR2, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of ACE, PRR, and ATIIR1, but not ATIIR2.ConclusionThis study demonstrated the expression of PRR, ATIIR1, and ATIIR2 by the SOX2 CSC population, and ACE on the endothelium of the microvessels, within GBM. ACE, PRR, and ATIIR1 were expressed at the protein and mRNA levels, with ATIIR2 detectable only by IHC staining. This novel finding suggests that the CSCs may be a novel therapeutic target for GBM by modulation of the RAS.