Through protein expression analysis of developmental neural lineage markers, we have identified GSC classes resembling: I) oligodendrocyte progenitor cells (OPC) and neural progenitor cells (NPC), II) neural progenitor cells (NPC), and III) astrocyte progenitor cells (APC; ). Each of these GSC types exhibited distinct and particular hallmarks found in GBM, including varied cellular and nuclear morphologies, invasive potential, and survival (). Since only five cell lines were used for sampling different GSC classes, other GSC categories potentially exist with similar phenotypic features but different molecular markers. These neural lineage markers may not be specific to the stem-like cells and may be present in the other cells of the tumor. Further analysis of one OPC-specific marker demonstrated that CNP expression correlated with longer survival in mice harboring GSC-derived xenografts ( & ) and in human patients via assaying a clinically-annotated tissue microarray containing 115 GBM samples (), suggesting the predictive power of our classification scheme in GSCs. Utility of CNP as a prognostic marker will need to be validated on an additional clinically annotated GBM tissue microarray. Taken together, these data reveal molecular variation of patient-derived GSCs with the clinically applicable strategy of immunoassaying with neural lineage markers.
GSCs categorized into various classes based on differential expression of neural lineage markers, nuclear irregularities, xenograft invasiveness in vivo, and patient survival via human GBM microarray analysis.
Because of GBM’s phenotypic diversity between patients and the lack of efficacious treatment regimens, many groups previously report molecular classification of gross GBM specimens by gene expression analysis (5
). Each of these groups uncovered specific gene signatures to subclassify GBM that predict molecular phenotype or survival better than histological analysis alone. Some of these GBM subclasses, such as “proneural” (29
), “oligoneural” (5
), and other classifications (33
), suggest neurodevelopmental links in gliomagenesis (29
). In relationship to Verhaak and colleagues (32
), class I GSCs resemble the “proneural” subtype according to OLIG2 expression, class II GSCs may resemble the “neural” subtype with a preponderance of NPC markers, and class III GSCs resemble the “classical” GBM subtype with elevated EGFR expression. Genome-wide expression profiling is ongoing to cluster our GSC classes into these previously identified GBM subtypes. Instead of previous subtyping methodologies (5
), our approach was to evaluate patient-derived GSCs, which are hypothesized to drive gliomagenesis, without contamination from non-GSCs and other cell types present in clinical resection samples. A few groups have also reported gene expression analyses of GSCs, isolated either through sphere-forming capacity (8
) or AC/CD133 expression (39
), and these GSCs were found to segregate into distinct subgroups with some phenotypic correlation. Our study aimed to discover clinically applicable biomarkers using protein expression assays with a developmental array of neural lineage markers. We demonstrate that different GSC subtypes correlate with tumor invasiveness and likely has clinically relevant prognosis for survival in human patients. This study is also unique in that GSC protein marker expression rather than transcriptional analysis was performed. As previously posited by Brennan and colleagues (40
), total mRNA or microRNA levels do not always correlate with protein levels, therefore reducing the predictive ability of gene expression arrays to identify pathologically useful biomarkers. With their identification of EGFR and PDGFRA specific subclasses, GSC line 44 demonstrated an abundance of both EGFR and PDGFRA expression noting the potential differences in GSC cultures and probable mosaic molecular features characteristic of GBM (). Additional evidence for the advantage of protein expression analysis is reflected in comparing our tissue microarray results with an assay of the NCI REMBRANDT and TCGA databases. No significant survival difference was observed in CNP-expressing versus non-expressing GBMs with REMBRANDT or TCGA data despite a rather robust difference revealed with tissue microarray analysis. CNP expression may be a suitable classifier within the proneural or oligoneural group due to its link to oligodendrocyte related genes (5
), but the lack of adequate CNP up-regulated tumors (n=1) in this GBM subset with 56 cases discourages conjecture (28
). CNPs potential activity in RNA editing (12
), may explain differences in mRNA transcript and protein levels, yet these speculations remain to be resolved. The selective neural lineage marker approach is also advantageous to rapid clinical translation, since many of the proteins studied are already familiar to neuropathologists and highly validated detection tools (i.e. antibodies and immunoassay protocols) are already available.
Not surprisingly, extensive cellular and molecular GBM heterogeneity was found between and within our patient GSC samples (). Furthermore, when GSCs were implanted into immunodeficient mice, GSC lines that expressed and exhibited a preponderance of oligodendrocytic markers and features in vitro
shifted to an astrocytic morphology in vivo
(). This in vivo
data diminishes the likelihood that we isolated a lower-grade or even “oligodendroglioma stem cell” from a GBM sample, either because of the existence of multiple stem-like cell clones within a single GSC line (41
) or cell culture conditions (42
). Xenografts from one Class I GSC line (12.1) even showed new expression of EGFR (Supp. Fig. 1, A4 & A7
), an APC marker that correlated with the invasiveness of Class III GSCs. It is possible that although 12.1 GSC xenografts expressed EGFR in vivo
, EGFR is not critical for in vitro
propagation of the GSC line. Activation of EGFR expression in xenografts may explain why mice with 12.1 GSC-derived xenografts had markedly worse survival probability compared to another class I GSC line (12.1 versus 22) (Supp. Fig. 2
). Additionally, EGFRvIII which is found mutated in 40% of EGFR amplified GBMs has not been detected in any of our GSC lines to date (43
). Interestingly, 22 GSC xenografts (least infiltrative) had the highest survival probability along with the highest proliferative index at 83% Ki67 positivity, suggesting that invasiveness is more important for GBM mortality. Class III 99 GSC xenografts are more invasive but had the second best survival (Supp. Fig. 2
). Altogether, it seems that studies to identify anti-GBM therapies will need to factor in GSC heterogeneity demonstrated in this and other studies (44
Since interpreting survival data in GSC xenografts is limited to a small sample size, we used a large clinically annotated GBM tissue microarray to link GSC neural lineage marker protein expression findings with clinical patient outcomes, and found significantly improved survival in patients with CNP-positive GBM (). CNP expression in GBM and GSCs has been previously demonstrated in vitro
and in vivo
). We extend these studies by linking CNP expression to reduced invasiveness and survival with in vivo
mouse xenograft studies, and in human GBM patient specimens. Although protein or genetic expression in enriched GSCs does not necessarily correlate with fully developed GBM as represented by tissue microarray, it seems that in some cases, such as for CNP, the GSCs follow a predetermined “differentiation” program not unlike normal stem cells. Since CNP is associated with oligodendrocyte progenitor cell development and myelin formation (47
), CNP-positive tumors in GBM may be considered to contain an ‘oligodendroglioma-like’ component, currently not taken into account by the World Health Organization (WHO) classification scheme (48
). Additionally, CNP expression might be hypothesized to indicate an OPC cell-of-origin in as a GBM subset (46
). CNP may not be a GSC-specific marker, but its expression in mouse GSC xenografts and human GBM tumor specimens reliably correlates with decreased infiltration and improved patient survival.
This contrasts with the continuing controversy of using CD133 expression as a GSC-specific marker. Although nuclear elongation correlates with increased invasion in this study, little to no CD133 expression was found in our GSC lines - all were carefully validated for GSC properties of growth in minimal stem cell media, self renewal, multipotent differentiation and highly efficient tumor initiation ( and ). It is interesting that tumor cells with elongated nuclei were frequently observed and correlated with the Class III APC, highly invasive GSC-derived xenografts. In contrast, Chen and colleagues (36
) observed similar elongated nuclei in invasive mouse xenografts from CD133+
The phenotypic and histological aberrations found in the different GSC and GSC-derived xenograft classes also have much potential for clinical application. GBM cell invasion into normal brain parenchyma limits neurosurgeons’ ability to remove tumors, often leading to recurrence immediately adjacent to the resection cavity (50
). Better understanding and elucidating mechanisms of GSC invasion would aid in clinical treatment planning and defining prognosis.
In conclusion, with the emergence of molecular therapeutics and personalized medicine, understanding and identifying the various molecular pathologies of GBM and its intrinsic GSCs will be increasingly important in developing novel diagnostic and therapeutic strategies.