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. Author manuscript; available in PMC: 2014 Feb 12.
Published in final edited form as: Cancer Cell. 2012 Dec 11;22(6):765–780. doi: 10.1016/j.ccr.2012.11.005

The EphA2 Receptor Drives Self-Renewal and Tumorigenicity in Stem-Like Tumor-Propagating Cells from Human Glioblastomas

Elena Binda 1, Alberto Visioli 1, Fabrizio Giani 1, Giuseppe Lamorte 2, Massimiliano Copetti 3, Ken L Pitter 4, Jason T Huse 5, Laura Cajola 6, Nadia Zanetti 6, Francesco DiMeco 7,8, Lidia De Filippis 1, Annunziato Mangiola 9, Giulio Maira 9, Carmelo Anile 9, Pasquale De Bonis 9, Brent A Reynolds 10, Elena B Pasquale 11, Angelo L Vescovi 1,3,6
PMCID: PMC3922047  NIHMSID: NIHMS552560  PMID: 23238013

SUMMARY

In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2High and EphA2Low populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both, ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications.

INTRODUCTION

Human glioblastoma multiforme (hGBM) is the most malignant among gliomas. Also due to their very heterogeneous cellular, genetic, epigenetic and molecular make-up (Maher et al., 2011), hGBMs have a dismal prognosis and almost inevitably recur (Krex et al., 2007; Chen et al., 2012).

Recent findings have demonstrated the existence of a subpopulation of hGBM cells, called cancer stem cells, whose idiosyncratic properties make them resilient to standard therapies. First identified in acute myeloid leukemia (Bonnet and Dick, 1997), cancer stem cells, better defined as tumor-propagating cells (TPCs; Kelly et al., 2007), have then been isolated from a variety of solid tumors (Ponti et al., 2005)(Ricci-Vitiani et al., 2009)(Buzzeo et al., 2007). TPCs with both stem cells characteristics and tumor-initiation and propagation ability have now emerged as key players in hGBM pathogenesis (Hadjipanayis and Van Meir, 2009; Galli et al., 2004).

While the origin and nature of hGBM TPCs remain to be unraveled, alterations of G1 arrest regulatory pathways in Nestin- or GFAP-positive cells can cause the onset of high-grade gliomas (Alcantara Liaguno et al., 2011). Thus, hGBM TPCs might derive from the transformation of neural stem cells or their transit amplifying precursor progeny (Alcantara-Llaguno et al., 2009; Vescovi et al., 2006). Oligodendroglial precursors might also be the cell of origin in some hGBMs (Sukhdeo et al., 2011). The peculiar characteristics of TPCs encompass a relative quiescent nature, unlimited self-renewal, the clonal capacity to found a tumor (Galli et al., 2004) and resistance to conventional and multimodal treatments (Bao et al., 2006).

Owing to their stem-like nature, TPCs might be regulated by the same cues that control the activity of normal neural precursors and stem cells (NSCs). In fact, pathways that impinge on self-renewal and cell fate in normal NSCs are also active in brain tumors (Alcantara-Llagumo et al., 2011; Dell’Albani, 2008). Also, therapeutic agents targeting Wnt, Hedgehog or Notch deplete the TPC population in hGBMs (Takebe et al., 2011) and tumor suppressor genes can regulate TPC self-renewal (Zheng et al., 2008). The connection of TPCs with NSCs is reinforced by the discovery that critical effectors of NSC activity in the brain stem cell niche, such as bone morphogenetic proteins, suppress the growth of hGBM TPCs, enforcing their differentiation into astroglia (Lee et al., 2008; Piccirillo et al., 2006; Zhang et al., 2006). Another key regulator in the adult NSC niche, nitric oxide, can also drive TPC proliferation in hGBMs (Eyler et al., 2011).

Eph receptor tyrosine kinases and their ephrin ligands influence central nervous system development, stem cell niches and cancer cells (Goldshmit et al 2006; Genander and Frisen, 2010; Pasquale, 2010). Deregulation of the Eph receptor/ephrin system is associated with acquisition of tumorigenic properties, tumor growth, angiogenesis and metastasis in human cancers. In particular, EphA2 receptor is overexpressed in many human epithelial malignancies and hGBMs, where it can promote proliferation and invasiveness through mechanisms that are not well understood and may be independent of ephrin ligand binding (Wykosky and Debinski, 2008; Miao et al., 2009; Pasquale, 2010; Gopal et al., 2011; Nakada et al., 2011; Miao and Wang, 2012). High EphA2 expression also correlates with tumor stage, progression and patient survival (Wykosky et al., 2005; Liu et al 2006; Wang et al., 2008; Miao et al., 2009; Li et al., 2010; Wu et al., 2011).

In this study, we have investigated the putative regulatory role and function of the EphA2 receptor in TPCs from hGBMs.

RESULTS

High EphA2 expression in hGBMs and their TPCs

Analysis of hGBM surgery specimens showed high EphA2 mRNA expression as compared to other EphA and EphB receptors (Fig. S1A). Real-time PCR (qPCR) revealed how EphA2 mRNA levels were up to 100 fold higher in hGBMs than in normal human brain tissue, as compared to a 10 fold upregulation in low-grade gliomas, epitheliomas and primitive neuroectodermal tumors (Fig. S1B).

Strong EphA2 immunoreactivity was found in many cells of the non-necrotic hGBM core (Fig.1A) versus a few positive cells in the tumor periphery (Fig.1B) and normal brain (Fig. S1C). Accordingly, immunolabeling of hGBM tissues showed co-expression of EphA2 and antigens of normal and transformed neural precursors, namely Nestin, Sox2 and Olig2 (Fatoo et al., 2011; Ligon et al., 2007) (Fig.1C). In contrast, the signal for ephrinA1, an EphA2 preferred ligand (Wykosky et al., 2008; Miao et al 2009), was variable in intensity and distribution in the hGBM core and undetectable in the periphery (Fig. S1C).

Figure 1. The EphA2 receptor in human glioblastomas tissues and TPCs.

Figure 1

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(A–B) Example of strong and frequent EphA2 immunoreactivity (brown) in the tumor core (A; arrows) as compared to infrequent, weaker labeling in the periphery (B) of the same hGBM specimen (6 hGBMs yielded similar results). Insets: higher magnification. Bottom: no primary antibody. Blue, hematoxylin counterstain. Bar, 20 μm. See also Figs. S1A–C.

(C) Immunolabeling of hGBM tissue shows that cells positive for the putative neural stem cell markers Nestin, Sox2 or Olig2 (red) co-express (arrows) the EphA2 receptor (green; 5 hGBMs yielded similar results). Bar, 20 μm.

(D) Co-expression of EphA2 (green) and ephrinA1 (red) on the surface of cells in hGBM tissues (left) and TPC cultures (right). Arrows mark examples of co-expression (yellow). Bars, 10 μm.

(E) Higher EphA2 expression as detected by qPCR in cultured TPCs from the core (TPCs; n=14 independent cultures) versus cells from the periphery of the same hGBM (non-TPCs; n=6 independent cultures; Student’s t test, p=0.017). Controls: vhNSCs and human fibroblasts (HF). Error bars: SEM. See also Figs. S1D–J.

(F) TPCs differentiated by mitogen starvation show upregulation of astroglial- (GFAP), oligodendroglial (GalC) and neuronal (β Tub, MAP2, tyrosine hydroxylase (TH), glutamate and GABA) markers (left), while loosing EphA2 expression (right).

We also found high EphA2 mRNA and protein levels in cells acutely dissociated from hGBMs or cultured as neurospheres and enriched for the putative TPC markers SSEA-1 or CD44 (Figs. S1D–F). Analysis of hGBM TPCs confirmed this overexpression (Fig.1E), which was from 2 to 300 fold that of their original hGBM tissue (Fig. S1G). EphA2 and ephrinA1 were detected in both hGBMs and their TPCs (Fig.1D). Notably, EphA2 was heavily downregulated when TPCs were differentiated, losing stemness and tumorigenicity (Piccirillo et al., 2006; Zhang et al., 2006) (Fig.1F), reinforcing a correlation between high EphA2 levels and the TPC state.

EphA2 mRNA levels in individual TPC neurosphere cultures also correlated with their specific growth kinetics (Figs. S1H–I), i.e. with their self-renewal activity (Rietze and Reynolds, 2006), and with EPHA2 gene copy number at the 1p36.12 locus (p<0.0001) (Fig. S1H, S1J).

Enriching for cells with enhanced tumor-propagating ability based on EphA2 expression

To reinforce the correlation between EphA2 enhanced levels and the TPC state we FACS-sorted acutely isolated hGBM cells into two distinct pools, expressing either high (EphA2High) or low (EphA2Low) EphA2 levels, and then assessed their in vitro clonogenicity and intracranial tumorigenic capacity. As expected, the EphA2High fraction contained more clonogenic cells than the EphA2Low fraction (Fig.2A) and mice implanted with EphA2High cells showed a higher mortality (median survival 4 months) than those receiving EphA2Low cells (median survival 7 months) (Fig.2B). Limiting dilution intracranial transplantation using acutely dissociated EphA2High and EphA2Low cells (Fig. S2A–C) confirmed that high EphA2 levels are a hallmark of TPCs and can be used for their enrichment. In addition, when primary hGBM cells were sorted based on their combined expression of EphA2 and the putative TPC antigen SSEA-1 (Figs.2C–D), intracranial tumorigenicity was significantly higher in EphA2High SSEA-1High than in EphA2Low SSEA-1Low cells (Fig.2E). Similar results were obtained with EphA2High CD44High and EphA2Low CD44Low cells (Figs. S2D–F). By applying the extreme limiting dilution analysis approach we were able to show that the frequency of tumor initiating cells (TICs) was always significantly higher in cell fractions with an increased EphA2 expression, alone or in combination with SSEA-1 or CD44 (Fig. S2G). Finally, when EphA2High and EphA2Low cultured TPCs were assayed for intracranial tumorigenicity in a limiting dilution assay, as few as 100 EphA2High cells established large gliomas in 60 days, as compared to a minimum requirement of 5,000 cells when using EphA2Low, below which level tumorigenicity was negligible, even after 7 months (Figs.2F–G).

Figure 2. Enrichment of the stem-like tumorigenic pool based on EphA2 levels.

Figure 2

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(A) Viable (propidium iodide negative) tumor cells acutely isolated from hGBM specimens (top left) were sorted into EphA2High and EphA2Low fractions (bottom left). The EphA2High fraction displayed higher clonogenic index than the EphA2Low fraction (right) (n=4 tumors). Error bars: SEM; **p=0.0004 for EphA2High vs. EphA2Low by Student’s t test.

(B) Intracranial transplantation of 6×104 EphA2High or EphA2Low cells confirmed the much higher tumor-propagating capacity of the former (MC test, log-rank p-value <0.0001 for EphA2High vs. EphA2Low; n=8).

(C) Confocal images show widespread co-localization (arrowheads; yellow) of EphA2 (red) and SSEA-1 (green) in hGBM tissue. Bar, 20 μm. (D) Cells from the same hGBM were sorted and gated according to EphA2 and SSEA-1 levels. (E) Kaplan-Meier survival curves show that mice receiving intracranially 2×104 and 1×104 EphA2High SSEA-1High purified TPCs die earlier (median survival: 135 and 164 days, respectively) than mice receiving EphA2Low SSEA-1Low cells (56% and 67% survival at 230 days, respectively). MC and GBW tests, log-rank p-value <0.0001 EphA2High SSEA-1High vs. EphA2Low SSEA-1Low; n=9). Survival was also shorter when implanting 4×104 EphA2High SSEA-1High as compared to EphA2Low SSEA-1Low TPCs. See also Fig. S2.

(F) Limiting dilution intracranial transplant of cultured, luciferase-tagged TPCs sorted into EphA2High and EphA2Low pools (top). Light emission imaging analysis (bottom; 5,000, 1,000 and 100 cells per mouse) shows a higher tumor-initiating ability of EphA2High versus EphA2Low TPCs. Error bars, SEM; ***p<0.0001, **p=0.002, EphA2High vs. EphA2Low.

(G) Kaplan-Meier analysis shows that mice receiving EphA2High TPCs die earlier than mice receiving EphA2Low cells (MC and GBW tests, log-rank p-value <0.0001 EphA2High vs. EphA2Low; n=9).

EphrinA1-Fc downregulates EphA2 and inhibits TPC growth and expression of neural stem markers

Due to the emerging correlation between high EphA2 expression and TPC state, we used ephrinA1-Fc (a soluble ephrinA1 dimer fused to Fc) to downregulate EphA2 (Wykosky et al., 2005; Liu et al., 2007) and examined its effects on hGBM TPCs. A dose-dependent EphA2 downregulation ensued, which peaked at ephrinA1-Fc concentrations of 1 to 5 μg/ml, producing up to 90% receptor depletion (Fig. S3A). EphrinA1-Fc induced a negligible EphA2 downregulation both in cells derived from the hGBM periphery (non-TPCs) – bearing stem-like features but negligible tumorigenicity (Piccirillo et al., 2009) – and v-myc-immortalized, non-transformed hNSCs (vhNSCs; De Filippis et al., 2007) (Fig. 3A). Immunofluorescence assays confirmed EphA2 loss in both acutely isolated and cultured TPCs upon ephrinA1-Fc treatment (Fig. 3B).

Figure 3. EphA2 downregulation by ephrinA1-Fc inhibits in vitro proliferation and depletes the stem cell-like pool in hGBM TPCs.

Figure 3

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(A) (Top) Cells were treated with ephrinA1-Fc for 24 hours. EphA2 was heavily downregulated in TPCs but not vhNSCs and non-TPCs. (Bottom) FACS analysis and flow cytometry quantitative data showing EphA2 downregulation in TPCs based on equivalent molecules of phycoerythrin (ME-PE) (n=10 independent cultures); error bars, SEM; **p<0.005 by Student’s t test. See also Fig. S3A.

(B) EphrinA1-Fc (5 μg/ml for 24 hours) downregulates EphA2 expression in TPCs acutely isolated from patients or from neurospheres (TPCs #8 shown as an example), but not in vhNSCs. Bar, 20 μm.

(C–F) EphrinA1-Fc (5 μg/ml for 48 hours) triggers obvious morphological changes (arrowheads) in acutely isolated TPCs (C, control; D, treated) or serially subcultured TPC neurospheres (E, control; F, treated), promoting cell adhesion. Bar, 50 μm.

(G–I) Acutely isolated TPCs cannot establish stable TPC primary lines if exposed to ephrinA1-Fc (G, red line), which also inhibits steady growth in pre-established TPCs (G); p<0.0001 vs. Control-Fc. Negligible inhibition of ephrinA1-Fc was observed in non-TPCs (H) and vhNSCs (I) (p<0.005 vs. Control-Fc; TPCs#8 and non-TPCs#8 are shown as representative examples); error bars, SEM. See also Figs. S3B–C.

(J) Co-expression of EphA2 (red) with the putative stem antigens Nestin, Sox2 or Olig2 (green) in TPC spheroids treated for 24 hours with Control-Fc or ephrinA1-Fc. EphrinA1-Fc nearly abolishes expression of both EphA2 and the stem antigens. Insets: higher magnification. Bar, 40 μm. See also Figs. S3D–E.

Unless otherwise indicated, the data are representative of three independent experiments giving similar results.

TPCs acutely isolated from hGBM specimens adhered to the dish when treated with ephrinA1-Fc, losing their capacity to grow and to generate stable TPC lines in the neurosphere assay (Figs. 3C–D, G), an effect also seen in 14 established TPC cultures (Figs. 3E–F, G and Figs. S3B–C). In contrast, ephrinA1-Fc had negligible effects on non-TPCs (Fig. 3H) and vhNSCs (Fig. 3I). Hence, ephrinA1-Fc dowregulates EphA2 and hinders the expansive growth of cells that possess both stem cell characteristics and tumor-propagating ability.

EphrinA1-Fc depletes the TPC pool, inhibits self-renewal and induces astroglial differentiation

The effects of ephrinA1-Fc suggested that it might inhibit the expansion of the TPC pool. This was emphasized by the ability of ephrinA1-Fc to downregulate both EphA2 and the neural precursor markers Nestin, Sox2 or Olig2 (Fig. 3J) that are co-expressed in TPC spheroids. Also, FACS analysis showed loss of the putative TPC markers SSEA-1, CD44 and CD133 (Dell’Albani, 2008; Fatoo et al., 2011) (Fig. S3D), but not of BMI-1 (Fatoo et al., 2011). Finally, ephrinA1-Fc depleted the TPC side population (SP), thought to comprise stem-like cells) (Fukaya et al., 2010) (Fig. S3E).

To confirm depletion of the TPC stem-like pool by ephrinA1-Fc, we measured self-renewal ability in a clonogenic assay, whereby single cells from acutely dissociated or from established TPC lines were plated in single wells by automated FACS and grown as neurospheres. EphrinA1-Fc drastically decreased clonogenicity in TPCs but not in non-TPCs, slightly increasing vhNSC clonogenicity (Fig.4A). Loss of self-renewal was not caused by changes in cell cycling (Fig.4B) or viability (Fig.4C). Notably, ephrinA1-Fc induced a marked, time-dependent upregulation of the astroglial antigen GFAP, without affecting the neuronal βIII-tubulin and oligodendroglial GalC markers (Fig.4D). Thus, ephrinA1-Fc hinders self-renewal in TPCs in a non-cytotoxic way and increases astroglial differentiation.

Figure 4. EphrinA1-Fc inhibits TPC self-renewal by inducing a differentiated phenotype.

Figure 4

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(A) Clonogenic assays show a dose-dependent inhibition of self-renewal by ephrinA1-Fc in TPCs but not in non-TPCs and vhNSCs; error bars, SEM; ***p<0.0001 vs. Control-Fc cells.

(B) Up to 5 μg/ml ephrinA1-Fc does not alter TPC cell cycling, as determined by FACS analysis of BrdU incorporation; error bars: SEM.

(C) Cytofluorimetric Tunel analysis shows no induction of apoptosis in TPCs or non-TPCs treated with ephrinA1-Fc; error bars: SEM; **p<0.005, *p<0.05 vs. Control-Fc.

(D) Quantitative FACS analysis shows a time-dependent increase in equivalent molecules of fluorescein (ME-FITC) for astroglial GFAP but not neuronal βIII tubulin (β–Tub) or oligodendroglial GalC markers in ephrinA1-Fc-treated TPCs; error bars, SEM; *p=0.0095, **p=0.0005 vs. Control-Fc. (Inset) Western blotting confirms a marked, time-dependent increase in GFAP under the same settings.

(E) (Top) Tyrosine phosphorylation of EphA2 immunoprecipitated from whole hGBM lysates and from TPCs treated with ephrinA1-Fc. (Bottom) Western blots for EphA2, ERK, Akt and FAK (Tyr397) expression and phosphorylation in TPCs treated with increasing ephrinA1-Fc concentrations over a 24 hours time course. Lower ephrinA1-Fc concentrations preferentially stimulate signaling, higher ones more rapidly downregulate the receptor.

(F–J) (F) TPCs spread on Cultrex show an organized actin cytoskeleton and F-actin assembled in stress fibers (arrows). Typical ring-like actin bundles are seen at higher magnification (H, I; arrowheads). (G, J) EphrinA1-Fc-treatment (5 μg/ml for 5 min) causes TPC elongation and actin concentration at cell-cell junctions (arrowhead). Bar, 20 μm.

Unless otherwise indicated, the data are representative of three independent experiments giving similar results.

Molecular regulation of TPCs stemness by EphA2

We studied the molecular events underlying the changes induced in hGBM TPCs by ephrinA1-Fc. EphrinA1-Fc transiently increased EphA2 tyrosine phosphorylation, in a dose-dependent manner, causing a strong and persistent downregulation of EphA2 expression (Fig.4E). We also observed marked ERK phosphorylation, which returned to basal levels after 24 hours, a moderate increases in Akt and FAK phosphorylation (Fig.4E, bottom panels), and reorganization of the actin cytoskeleton (Figs.4F–J).

Altogether, this suggested that EphA2 expression sustains TPC self-renewal and that receptor loss played a causal role in the depletion of the hGBM stem-like pool. To prove this, we used a mixture of siRNAs to assess the effects of direct EphA2 downregulation (Fig.5A). EPHA2 silencing caused changes consistent with loss of stemness and increased differentiation, i.e. i) loss of clonogenicity (Fig.5B) and amplification rate (Fig.5C), ii) downregulation of putative stem cell markers (Fig.5E) and iii) increased GFAP expression (Fig.5F). These changes were quite similar to those induced by ephrinA1-Fc (Figs. 3G, 3J, 4A and 4D). Per se, EphA2 downregulation activated ERK in TPCs, as shown by a prominent increase in its phosphorylation (Figs.5F–G). Feeble Akt and FAK activation were detected. Furthermore, when EphA2 levels began to normalize, due to the elapsing effect of siRNAs transfection (Fig.5A, arrow), TPCs growth and ERK phosphorylation also began to normalize (Figs.5C, arrow and 5F). siRNA-mediated knockdown of EPHA2 expression in vhNSCs also inhibited growth (Fig.5D), in contrast to ephrinA1-Fc treatment, which neither inhibited growth nor downregulated EphA2 in these cells (Figs. 3A–B and 3I). Transduction of TPCs with individual EPHA2 siRNA, and rescue with a siRNA-resistant EPHA2 construct, confirmed these effects as the result of the EphA2 mRNA knockdown (Fig.5G). Importantly, inhibition of ERK activation partially rescued loss in clonogenicity as induced by EPHA2 siRNA, implicating ERK activation in the loss of TPC stemness, as caused by EphA2 downregulation (Fig.5H).

Figure 5. EPHA2 siRNA knockdown in hGBM TPCs inhibits self-renewal and increases differentiation, concomitant with ERK and Akt activation.

Figure 5

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(A) TPCs were treated with an EPHA2 siRNA pool, non-targeting control (NTC) or GAPDH control pool siRNAs. Three days post-transfection, only the EPHA2 siRNAs caused a substantial decrease in EphA2 mRNA, as detected by qPCR. Between 12 and 16 days post-transfection (DIV), EphA2 levels began to normalize (arrow); error bars, SEM.

(B) siRNA-mediated knockdown of EPHA2 expression causes loss of TPC clonogenicity; error bars, SEM; **p<0.005 EphA2 vs. NTC siRNAs.

(C) TPC growth decrease is concomitant with siRNA-mediated EphA2 downregulation and normalizes when EphA2 levels begin recover (arrow); error bars, SEM; p<0.0001 EPHA2 siRNAs vs. NTC.

(D) siRNA-mediated EphA2 downregulation decreased vhNSCs growth; error bars, SEM; ***p<0.0001 EPHA2 siRNAs vs. NTC. Inset: Western blot for EphA2 upon EPHA2 siRNA treatment.

(E) Confocal analysis shows that siRNA-mediated EphA2 downregulation in TPC spheroids is associated with depletion of putative stem markers, as compared to NTC-treated spheroids (NTC siRNAs). Insets: magnification. Bar, 40 μm.

(F–G) Western blot analysis show increased GFAP but not β–Tub or GalC levels in TPCs treated with EPHA2 siRNAs versus NTC or GAPDH siRNAs (F). ERK phosphorylation is also strongly increased concomitant with EphA2 downregulation, with more prominent effects at 72 hours than at 5 days, when EphA2 levels begin to recover. (G) Representative Western blot showing that only the siRNA sequences that effectively reduce EphA2 expression increase GFAP expression and activate ERK. An EPHA2 construct lacking the 3’UTR sequence targeted by EPHA2 siRNA #3 was used to transfect TPCs in a control rescue experiments.

(H) The reduced TPC clonogenicity caused by knockdown of EPHA2 expression was partially restored by 10 μM UO126, which inhibits ERK; error bars, SEM; ***p<0.0001, **p<0.005 vs. untreated TPCs.

Unless otherwise stated, data are representative of three independent experiments giving similar results.

Notably, EphA2 phosphorylation on Serine897 (Ser897) promotes oncogenic activities of the receptor that do not depend on its interaction with ephrin ligands (Miao et al., 2009). We detected a strong signal for Ser897-phosphorylated EphA2 in the hGBM core and in TPCs but not in the hGBM periphery, non-TPCs or vhNSCs (Fig.6A). Furthermore, treatment with high doses of soluble monomeric EphA2 extracellular domain that block endogenous EphA2-ephrin interaction did not affect GFAP levels or ERK activity in TPCs (Fig.6B). Thus, the effects of EphA2 in TPCs appear to be independent from activation by endogenous ephrinA ligands.

Figure 6.

Figure 6

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EphA2 does not appear to be activated by ephrins in hGBM TPCs is phosphorylated on Ser897 in hGBM TPCs and treatment with the soluble EphA2 extracellular domain to inhibit ephrin binding to endogenous EphA2 does not affect TPC signaling pathways.

(A) Top: EphA2 is constitutively phosphorylated on Ser897 in the core of hGBMs (left) but not in the periphery of hGBMs (centre) or normal brain (right). Bottom: EphA2 is highly phosphorylated on Ser897 in hGBM TPCs but not non-TPCs or vhNSCs. EphA2 Ser897 phosphorylation suggests ephrin-independent oncogenic activities.

(B) Representative Western blots showing that treatment of TPCs with 10 μg/ml EphA2 extracellular domain to inhibit possible interactions with endogenous ephrin ligands does not affect GFAP levels or ERK phosphorylation as compared to Fc protein used as a control. Data are representative of three independent experiments giving similar results.

EphrinA1-Fc and EPHA2 silencing by siRNAs suppress TPC tumorigenicity, in vivo

The main effect of ephrinA1-Fc treatment or siRNA-mediated EPHA2 silencing is the depletion of the tumorigenic hGBM TPC pool, which ought to reduce their tumorigenic capacity, in vivo. To examine the in vivo effects of ephrinA1-Fc we used three experimental paradigms. hGBM TPCs were 1) treated with ephrinA1-Fc in culture, prior to transplantation (pre-treatment), 2) treated starting immediately after transplantation (co-treatment), or 3) allowed to establish sizeable tumors before beginning treatment with ephrinA1-Fc (post-treatment). All three protocols were evaluated in a subcutaneous xenograft model. Furthermore, pre- and post-treatment protocols were also evaluated in an intracranial (orthotopic) xenograft model (Galli et al., 2004; Piccirillo et al., 2006).

When TPCs were injected subcutaneously, ephrinA1-Fc inhibited growth in all three protocols (Fig.7A). Similar results were obtained with the more complex, yet clinically more relevant, orthotopic model. In the intracranial hGBM model, tumor growth was greatly reduced in the pre-treatment settings, as shown by quantitative imaging of luciferase-tagged hGBM TPCs (luc-TPCs; Fig.7B left panels, C, left, E, F) and by the increase in overall survival (Fig.7C, right). Importantly, ephrinA1-Fc infusion into the brain for 14 days by means of osmotic mini-pumps also effectively suppressed the growth of well pre-established hGBMs (Fig.7B middle and right panels, D, left, G, H). Kaplan-Meier analysis revealed a median survival of 130 days for mice receiving ephrinA1-Fc versus 72 days for controls, infused with Fc (Fig.7D, right) – confirming the therapeutic efficacy of ephrinA1-Fc administration.

Figure 7. EphA2 downregulation by ephrinA1-Fc or by siRNA-mediated knockdown inhibit TPC tumorigenicity in immunodeficient mice.

Figure 7

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(A) Treating TPCs with ephrinA1-Fc (5 μg/ml for 48 hours) prior to subcutaneous implantation (PRE-treatment) lessens their tumor-initiating capacity (left). Similar results were obtained by co-injecting TPCs and ephrinA1-Fc (CO-treatment) or injecting ephrinA1-Fc (10 μg/day) around the tumor starting 11 days after cell transplant (POST-treatment). (Right) Volumes of subcutaneous tumors 35 days after TPCs injection. Histograms, mean volume ± SEM; **p=0.0002 vs. Control-Fc mice, n=6.

(B) Imaging of luciferase-tagged TPCs (luc-TPCs) injected into the brain of Scid/bg mice. After 42 days, untreated TPCs established larger tumors (vehicle, top left) than ephrinA1-Fc PRE-treated TPCs (bottom left). Luc-TPC tumors established for 7 days (7 DPT, top center) grew quickly when a mini-pump delivered Control-Fc for 14 days starting at 11 days post-transplant (27 DPT, bottom center). In contrast, tumor growth was markedly inhibited by ephrinA1-Fc (top and bottom right panels).

(C) (Left) Quantitative analysis of luc-TPC signals for the PRE-treatment intracranial transplants. Histograms, mean ± SEM; ***p<0.0001 vs. Control-Fc mice; n=6. (Right) Kaplan-Meier survival curves showing that mice receiving ephrinA1-Fc treated TPCs have a significant longer life span than mice injected with Control-Fc cells (MC and GBW tests, log-rank p-value=0.0005 and 0.0013 respectively; n=9).

(D) (Left) Quantitative time course analysis of the luc-TPC signal for POST-treatment paradigm (arrows mark the time of mini-pumps implantation). Histograms, mean ± SEM; *p<0.05 vs. Fc-treated mice; n=8. (Right) Kaplan-Meier survival curves are shown (MC and GBW tests, log-rank p-value <0.0001 and p=0.0002 vs. Control-Fc mice; n=9).

(E–H) Mouse brain sections immunolabeled for luciferase show that tumors established from luc-TPCs PRE-treated with Control-Fc (E) spread through the brain parenchyma more than those established from cells PRE-treated with ephrinA1-Fc (F) at 42 DPT. Similarly, ephrinA1-Fc infused into the brain for 2 weeks starting 11 days after tumor establishment (H) inhibits the growth of luc-TPC tumors more than Control-Fc (G). Arrowheads mark the edges of the tumors. CC: corpus callosum; LV: lateral ventricle; St: Striatum. See also Fig. S4. Bar, 1 mm.

(I) Loss of intracranial tumorigenicity in luc-TPCs treated with EPHA2 siRNAs for 72 hours prior to transplantation as compared to NTC siRNA-treated or untreated TPCs. Tumor growth was monitored by quantitative imaging analysis (top). Histograms, mean ± SEM; ***p<0.0001 EPHA2 siRNAs vs. NTC; n=6. (Bottom) Kaplan-Meier survival analysis. Mice receiving EPHA2 siRNA-transfected TPCs die significantly later than those receiving untreated TPCs or TPCs transfected with non-targeting control siRNAs (MC and GBW tests, log-rank p-value <0.0001 vs. NTC siRNAs treatment; n=9).

Immunohistochemistry confirmed that EphA2, which was high in TPC-derived, control intracranial hGBMs (Fig. S4A), strongly downregulated upon ephrinA1-Fc infusion (Fig. S4B). Control tumors contained numerous malignant cells (Fig. S4C), whereas ephrinA1-Fc-treated ones contained few neoplastic cells and many more differentiated elements (Fig. S4D). Accordingly, ephrinA1-Fc-treated tumors embodied a lower percentage of mitotic cells (Figs. S4E–F) and were significantly less vascularized than Fc-treated controls (2-fold reduction in total vascular area, with less and smaller blood vessels; Figs. S4G–H and data not shown).

To determine if decreasing EphA2 receptor levels was sufficient to inhibit hGBM tumorigenicity, we analyzed the effects of siRNAs-mediated EPHA2 silencing on the in vivo tumor-propagating ability of luc-TPCs, in comparison with luc-TPCs treated with control-siRNAs or untreated ones. Knockdown of endogenous EPHA2 suppressed the growth of TPC-derived intracranial tumors (Fig.7I, top) and substantially increased overall survival (Fig.7I, bottom).

Given the prominent involvement of EphA2 in the pathogenic mechanisms of hGBMs documented above, we analyzed the TCGA data set (Network, 2008) for relative EphA2 mRNA expression in hGBM sub-categories (Verhaak et al., 2010). We found that the classical and mesenchymal subtypes had the highest EphA2 expression (Fig.8). In addition, when the mesenchymal or proneural subtypes were divided into two groups based on median EphA2 expression, high EphA2 expression trended with poor patient survival (mesenchymal: 10.9 vs. 15.03 months, p=0.0415 Mantel-Cox (MC), p=0.0215 Gehan-Breselow-Wilcoxon (GBW); proneural: 9.9 vs.16.73 months, p=0.2026 MC test, p=0.0411 GBW test). There was no significant correlation between EphA2 and patient survival in the neural and classical subtypes.

Figure 8. EphA2 is differentially expressed in GBM subtypes and its abundance correlates with patient survival.

Figure 8

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(A) Relative EphA2 mRNA expression in the four GBM sutbypes of the TCGA dataset. (***p<0.0001 by ANOVA; n=495). (B) Kaplan-Meier survival curves for the Classical, Mesenchymal, Proneural and Neural subtypes. High and low expression are defined as above and below the median expression value for each subtype, log rank p-values were determined by MC and GBW tests.

DISCUSSION

We describe a specific role for the EphA2 receptor in the pathogenesis of hGBMs. We report how enhanced EphA2 expression is a property of the TPCs in these cancers and show a causal relationship between high EphA2 expression and the capacity of these cells to expand their pool size and form hGBMs. Accordingly, high levels of EphA2 expression can be used to enrich for TPCs by FACS. Furthermore, treatment with a soluble form of the EphA2 ligand, ephrinA1-Fc, hinders the self-renewal ability of hGBM TPCs causing a drastic loss in their capacity to establish and propagate hGBM phenocopies subcutaneously or intracranially. We demonstrate that EphA2 receptor downregulation is a causal event in the suppression of the tumor-propagating capacity of TPCs. Intracranial administration of ephrinA1-Fc causes EphA2 downregulation in hGBM orthotopic xenografts, significantly hindering their growth and expansion in the brain, even if these tumors are established ahead of ephrinA1-Fc administration.

Aberrant expression of Eph receptors has been reported in various cancers, including gliomas, and correlates with malignancy (Wykosky and Debinski, 2008; Pasquale, 2010; Nakada et al., 2011; Miao and Wang, 2012). In our survey of twelve hGBM patients’ specimens, we found EphA2 to be the most upregulated of the Eph receptors. This is consistent with the EphA2 overexpression previously reported in most high-grade human gliomas, which correlates with poor prognosis (Wykosky et al., 2005; Liu et al 2006; Wang et al., 2008; Miao et al., 2009; Li et al., 2010; Wu et al., 2011), suggesting a role for EphA2 in the pathogenesis of hGBMs.

While studies with glioma cell lines have implicated EphA2 in cell growth and invasiveness (Wykosky et al., 2005; Liu et al., 2006; Liu et al., 2007; Miao et al., 2009; Li et al., 2010; Gopal et al., 2011), the identity and nature of the actual target cells in the patients’ own GBM remain unclear. In addition, the cellular functions affected by EphA2, the regulatory mechanisms underpinning EphA2’s actions in hGBMs pathophysiology and the possibility of manipulating this system to suppress glioma growth are not well characterized. hGBMs contain subpopulations of cells that act as stem-like TPCs, which have now been proven to be crucial therapeutic targets (Chen et al., 2012). We found that the EphA2 receptor is co-expressed with neural precursor markers in hGBMs, being concentrated in regions rich in TPCs. Furthermore, hGBM cell preparations enriched in TPCs display enhanced EphA2 levels which, conversely, are low in low-grade gliomas (Figure S1B) that contain few TPCs (Galli et al., 2004). Altogether, these findings suggest that high EphA2 levels are a distinguishing feature of hGBM TPCs.

Conclusive evidence to this idea came from fractionating TPCs into EphA2Low and EphA2High populations, also in combination with high levels of the putative TPC markers. The highest tumorigenic potential was associated with high EphA2 or combined, high EphA2 SSEA-1 or EphA2 CD44 expression. Thus, in vitro and in vivo limiting dilution experiments show that high EphA2 expression can be exploited to obtain preparations highly enriched in TPCs, which are capable of establishing intracranial hGBMs by injection of as few as 100 cells.

Overexpression of EphA2 in TPCs suggests a role for this receptor in hGBM pathogenesis. A first indication that EphA2 might drive the expansion of the TPC pool came from the significant correlation between the rate of self-renewal of multiple TPC preparations and their EphA2 mRNA levels and EPHA2 gene copy number. This was reinforced by the fact that EphA2 expression decreases dramatically when hGBM TPCs lost their stemness upon differentiation.

To verify the role of EphA2 in TPC self-renewal and amplification, we first perturbed EphA2 by using the ephrinA1-Fc ligand (Noblitt et al., 2004; Lee et al., 2011; Khodayari et al., 2011). This treatment decreased the proliferation of both acutely isolated and of serially subcultured hGBM TPCs, was coupled to the failure of primary hGBM TPCs to generate stably expanding lines and caused both loss of expansion capacity and clonogenic ability in well established TPC cultures.

The ephrinA1-Fc action was associated to decreased expression of putative hGBM TPC antigens (such as Olig2, Nestin, Sox2, CD133, CD44 and SSEA-1), occurred in the absence of detectable changes in cell cycle or apoptosis and produced increased astroglial differentiation and the depletion of the chemoresistant side population. Notably, these effects were specific to TPCs, since stem-like cells from the same tumors but lacking tumor-propagating ability (non-TPCs) and non-transformed neural stem cells (vhNSCs) were resilient to ephrinA1-Fc administration. Thus, in vitro exposure to ephrinA1-Fc leads to the depletion of those stem-like cancer cells that are responsible for hGBM initiation, growth and recurrence after therapy.

EphrinA1-Fc caused prolonged EphA2 downregulation in TPCs, suggesting a critical role for this phenomenon in this ligand’s tumor suppressor activities. Although ephrin Iigands trigger Eph receptor tyrosine phosphorylation and activation, thereby eliciting complex downstream signaling, they also cause Eph receptor downregulation (Wykosky et al., 2005; Liu et al., 2007; Pasquale, 2010). We observed only negligible inhibition of TPC clonogenicity (less than 10%; not shown) at the lower ephrinA1-Fc concentrations tested, which induced strong intracellular signaling but limited EphA2 downregulation. Conversely, at higher ephrinA1-Fc concentrations, signaling was weaker, but EphA2 downregulation, inhibition of TPC clonogenicity and growth were much more evident. Indeed, while signaling was transient and returned to basal levels within 24 hours, the inhibition of TPC tumorigenicity and EphA2 downregulation persisted well beyond 24 hours. The importance of EphA2 downregulation was further supported by the observation that in vhNSCs ephrinA1-Fc failed to both induce EphA2 downregulation and inhibit cell growth and self-renewal. Furthermore, siRNA-mediated knockdown of EPHA2 expression closely mimicked the effects of ephrinA1-Fc on TPC self-renewal, expansion, differentiation in vitro and tumorigenic ability in vivo. Interestingly, EPHA2 expression knockdown also inhibited the growth and self-renewal of the “ephrinA1-Fc-resilient” vhNSCs. Therefore, EphA2 downregulation alone can inhibit TPC tumor-propagating activity although it remains possible that ephrinA1-Fc-induced EphA2 signaling may also contribute to the suppression of TPC tumorigenicity (Wykosky and Debinski, 2008; Nakada et al., 2011; Miao and Wang, 2012). Thus, EphA2 is prominently expressed in TPCs and is required for sustained self-renewal and expansion of this tumor-propagating cell pool in hGBMs.

The depletion of the TPC stem-like pool induced by ephrinA1-Fc treatment and EphA2 downregulation seems to arise from increased cell differentiation. First, loss of the undifferentiated state is demonstrated by the depletion of Sox2, Olig2, Nestin, CD133, CD44 and SSEA-1, as well as the increased expression of the astroglial marker GFAP, as induced by both ephrinA1-Fc and knockdown of EPHA2 expression. Second, both manipulations also inhibit the typical undifferentiated stem-like functions of TPCs, such as self-renewal and extensive amplification capacity. Given the unchanged cell cycling and death rate, these data imply that ephrinA1-Fc and EphA2 downregulation cause the loss of undifferentiated functional and antigenic properties as well as the acquisition of a more mature cell phenotype by TPCs.

The activities of EphA2 in hGBM TPCs appear to involve unconventional ephrin-independent activities of EphA2, which are not yet well understood but may also involve crosstalk with other signaling systems (Miao et al, 2009; Pasquale, 2010; Gopal et al 2011; Miao and Wang, 2012). This is supported by the prominent phosphorylation of EphA2 at the Ser897 residue (Miao et al., 2009) that we detected in the TPC-enriched core but not the periphery of hGBMs, and in cultured TPCs but not non-TPCs or vhNSCs. Phosphorylation of EphA2 Ser897 by Akt is known to promote EphA2’s oncogenic activities independently of ephrin binding, and in fact interaction of EphA2 with ephrinA1 inhibits phosphorylation at this site (Miao et al, 2009). Consistent with a role of EphA2 in TPCs that does not depend on activation by endogenous ephrins, using the soluble EphA2 extracellular domain to block EphA2-ephrin interaction did not affect GFAP levels or ERK activation in TPCs. In contrast, EPHA2 siRNA-mediated knockdown depleted the stem cell pool concomitantly with ERK, suggesting that this pathway may mediate the effects of EphA2. This was confirmed by using the ERK inhibitor UO126, which re-established self-renewal ability in TPCs with downregulated EphA2 expression. This suggests that ERK can promote differentiation in TPCs that have lost EphA2 expression.

The hindrance of self-renewal capacity induced by ephrinA1-Fc predicts that this ligand will lessen the ability of TPCs to establish tumors in vivo. In fact, a 48 hours exposure to soluble ephrinA1-Fc reduced the capacity of TPCs to establish hGBM phenocopies, both subcutaneously and intracranially. A similar effect was observed when we implanted TPCs subcutaneously and began ephrinA1-Fc infusion either at the same time or after sizeable tumors had been established. Treatment of intracranial tumors with ephrinA1-Fc significantly reduced the tumor mass, vascularization and proliferation and dramatically decreased EphA2 levels. When EPHA2 expression in TPCs was knocked down with siRNAs, intracranial tumorigenicity was nearly abolished. Thus, EphA2 is critical to maintain TPC properties and plays a pivotal role in the pathogenesis of hGBMs in the brain, as driven by TPCs.

Our findings are consistent with previous studies suggesting that EphA2 expression correlates with malignancy in hGBMs (Liu et al., 2006; Wang et al., 2008; Li et al., 2010; Wu et al., 2011). Hence, we examined the relationship between EphA2 expression and patient survival in relation to the recent stratification of hGBMs into different subtypes (Verhaak et al., 2010). We found that EphA2 is most highly expressed in the classical and mesenchymal groups and that, at least in the mesenchymal subtype, patients with EphA2 levels above the median trended towards poorer survival. This trend was also present and statistically significant in the proneural subtype, although this subtype has lower mean levels of EphA2 than the mesenchymal and classical ones. The correlation with poor survival in these two groups further underscores the importance of EphA2 expression in aggressive tumor behavior, which may be more relevant in the mesenchymal and proneural hGBM subtypes.

We were able to drastically inhibit the growth of pre-existing orthotopic hGBM xenografts under experimental conditions that mimic those needed for experimental human therapy, such as protracted delivery through intra-parenchymal infusion. This supports the value of devising anti-GBM therapies that harness the Eph/ephrin system to target the TPC compartment. Hence, the concept emerges of impinging in non-toxic fashion on specific molecular targets, such as EphA2, to tackle the subset of tumor cells that plays the most critical role in hGBM pathogenesis.

We show that a signaling system that is operational in the neural stem cell niche also regulates self-renewal and tumorigenicity in hGBM TPCs, in agreement with previous observations (Ying et al., 2011). Eph receptors and ephrins regulate the migration, survival, proliferation, cell fate and differentiation of neural precursors during development (Goldshmit et al., 2006; Genander and Frisen, 2010). In the adult, such activities persist in stem cell niches, including those of the adult central nervous system, although our understanding of Eph/ephrin regulatory functions in adult neurogenesis remains limited. Since other Eph receptors besides EphA2 are expressed in adult neural stem cell niches (Goldshmit et al., 2006; Genander and Frisen, 2010), by using agents that selectively target EphA2 we may be able to target stem-like cells in hGBMs without affecting the normal brain stem cell population.

These findings emphasize the importance of approaches that exploit fundamental similarities in the physiology of normal neural stem cells and their stem-like, tumor-propagating counterpart in brain tumors (Vescovi et al., 2006). Such approaches can make use of the wealth of information derived from studies on regulatory systems in normal neural stem cells to identify candidate effectors capable to affect TPCs, thus helping to design more effective and specific anti-GBM therapies.

EXPERIMENTAL PROCEDURES

Primary culture, culture propagation, population analysis and cloning

Adult human glioblastoma (hGBM), low grade gliomas, ependimomas, PNET tissues and normal human brain samples were obtained and classified according to the World Health Organization guidelines. All of the tumors were banked in accordance with research ethics board approval from the Institute of Neurosurgery, Catholic University of the Sacred Heart (Prot. RBAP10KJC5) and patients gave informed consent prior to surgery. hGBM non-necrotic core and periphery tissues were dissected and digested in a papain solution (Worthington Biochemical Corp, Lakewood, NJ, USA). Primary stem-like tumor propagating cells (acutely isolated cells) were plated in NeuroCult NS-A medium (Stemcell Technologies Inc, Vancouver, BC, Canada) containing 20 ng/ml of epidermal- and 10 ng/ml of fibroblast-growth factor (Peprotech Inc, Rocky Hill, NJ, USA) (culture medium; Galli et al., 2004). EphA2 was stimulated with ephrinA1-Fc (R&D Systems Inc, Minneapolis, MN, USA) for the indicated concentrations and times. Mouse monoclonal IgG1 isotype (R&D Systems) was used as control. For population analysis, clonogenic assays and differentiation experiments see the matching section in the Supplemental Experimental Procedures.

Statistical analysis for the correlation

Growth curves were analyzed with a hierarchical linear model for repeated measurements to assess trend over time (Diggle et al., 1994; Singer and Willett, 2003). Log-transformed cell number was considered as outcome. Association with EphA2 mRNA expression levels and EPHA2 copy number were assessed including them into the statistical models (Figs. S1H–J). A spatial power correlation type was used to account for unequally spaced time occasions during the experiment (Singer and Willett, 2003). Unless indicated otherwise, comparisons between ephrinA1-Fc (also with different doses) treated cells and Control-Fc treated cells were carried out with a hierarchical linear model for repeated measurements. p-values <0.05 were considered statistically significant. All analyses were performed using SAS Statistical Package Release 9.1 (SAS Institute, Cary, NC, USA) or GraphPad Prism.

Evaluation of tumorigenicity by subcutaneous or orthotopic implantation

Tumorigenicity was studied by TPCs subcutaneous or orthotopic injections (Galli et al., 2004). All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees (IACUCs) at the University of Milan Bicocca. See the matching section in the Supplemental Experimental Procedures for details.

Molecular analysis

TCGA data analysis was performed as described by Squatrito et al., 2010: survival and expression data were based on clinical TCGA data and relative mRNA expression obtained from the TCGA data portal (http://cancergenome.nih.gov/dataportal/data/about) and the MSKCC cBio Genome Data Analysis Portal (http://cbio.mskcc.org/gdac-portal/index.do) respectively. Tumor subtype classification was previously described (Verhaak et al., 2010). Relative mRNA results are based on relative distribution of the expression values for diploid tumor samples and were subjected to statistical analysis by one-way ANOVA (GraphPad Prism v5.0 software). Survival curves were analyzed using the Kaplan-Meier method, with groups compared by respective median survival of number of days taken to reach 50% morbidity; log rank p value was measured using both MC and GBW tests.

Supplementary Material

Supplementary data

SIGNIFICANCE.

Identification and characterization of key regulatory mechanisms in TPCs is crucial for the development of specific therapies for hGBMs. We show that TPCs overexpress EphA2 in hGBMs, which underpins their inherent ability to maintain an undifferentiated state, supporting their self-renewal and tumorigenicity. EphA2 abundance provides a measure of the stem-like potential and tumor-propagating ability of TPCs from hGBMs. Thus, high EphA2 levels can be used to enrich for TPCs by cell sorting. EphrinA1-Fc ligand-induced downregulation or siRNA-mediated knockdown of EPHA2 expression both cause loss of self-renewal as well as induce differentiation and loss of tumor-initiating capacity in hGBM TPCs. Sustained intracranial infusion of ephrinA1-Fc under settings that resemble putative therapeutic conditions elicits effective anti-tumorigenic activity.

HIGHLIGHTS.

  • Stem-like tumor-propagating cells (TPCs) in hGBMs highly express the EphA2 receptor.

  • High EphA2 expression supports the undifferentiated state and self-renewal in TPCs.

  • TPC content and tumorigenicity are higher in EphA2High than EphA2Low hGBM cells.

  • EphrinA1-Fc treatment or EPHA2 silencing lessen TPC self-renewal and tumorigenicity.

Acknowledgments

This research was supported by grants from McDonnell Foundation (220020207), AIRC (IG-10141), MIUR (RBAP10KJC5, RF-INN-2008-1220368) and NIH (P01CA138390). We thank Lucia Sergisergi for providing the luciferase lentivirus.

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