GF-independent GSCs are an intrinsic Wnt5aHigh/EphA2Low invasive subset within GBM
To investigate as to whether GF-independent GSCs (I-GSCs) might embody an intrinsic component of the tumor itself, acutely isolated cells from IDH1-wild-type GBM specimen were plated at clonal density in serum-free medium, either in the presence of EGF and FGF2 [14, 49] or avoiding the classical mitogenic stimulation. Following exposure to GFs, typical neurospheres formed in culture and, even in the mitogen-free cultures, primary neurospheres displaying protrusion and elongation of cell shape could be detected (Fig. 1A). Interestingly, when compared to their cognate cells isolated in the presence of GFs (D-GSCs), I-GSCs displayed a peculiar functional phenotype, regardless of the subtype, i.e. TCGA-CL, TCGA-MES and TCGA-PRO [22, 54, 55]. As clearly shown in Fig. 1B-C, I-GSCs’ global growth trend (Fig. 1B and Supplementary Fig.S1A) and clonal efficiency (Fig. 1C) was somewhat lower than that of their matched D-GSCs. Upon growth factors removal, human neural stem cells (NSCs), used as negative control, were confirmed to die rapidly (Fig. 1B). Remarkably, when cultured in the presence of mitogens I-GSCs cells acquired the typical growth rate of their siblings D-GSCs (Fig. 1D). The size of neurospheres generated in mitogen-free cultures appeared smaller than that of D-GSCs, suggesting differences in the cell cycle length. This was demonstrated by the definition of I- and D-GSCs’ cell cycle signature. Yet, the former tends to display a higher percentage of cells gated in G0/G1 phase and a lesser percentage of cells gated in S phase as compared to the latter as a whole (Supplementary Fig.S1B).
Regardless of the subtype tested, I-GSCs seemed to display a more “astrocyte-like” phenotype, with a significant increase in the frequency of the astroglial differentiation marker GFAP but not in neuronal or oligodendroglial ones (Fig. 1E and Supplementary Fig.S1C). Yet, the more mature makeup of these cells, versus their GF-dependent counterpart, emerged to be sustained by a relative lower expression of markers associated to GSCs state in GBM, including SSEA-1 and EphA2 (Fig. 1E-F and Supplementary Fig.S1D). Meanwhile, I-GSCs upregulated the tissue invasiveness mediator Wnt5a, which has a key role in GSCs dispersion [27, 33], CD44 and Bone Morphogenetic Protein Receptors (BMPRs) [56] (Fig. 1E-F and Supplementary Fig.S1E). Both GSCs populations inherently displayed a different pattern of EphA2 and Wnt5a expression across subtypes [27, 32]. In any case, EphA2 and Wnt5a proteins were infrequently co-expressed. Strikingly, the Wnt5aHigh/EphA2Low profile was shown to be a predictor of poor prognosis in the TCGA dataset (Fig. 1G).
We next assessed whether the highest level of Wnt5a in I-GSCs was related to their invasive potential observing that these cells, once more irrespective of the subtype, infiltrated more efficiently than their cognate D-GSCs (Fig. 1H). A key role for Wnt5a in modulating GSCs and even NSCs ability to extensively infiltrate was also confirmed (Fig. 1I and Supplementary Fig.S1F) [27, 33].
Altogether, these data report the identification and the in vitro characterization of a subset of mitogen-independent GSCs isolated from patient’s tumor specimens, by exploiting their inherent ability to self-maintain and to infiltrate and the unique aggressive Wnt5aHigh/EphA2Low profile.
I-GSCs establish tumors in vivo endowed with exacerbated lethality and intracranial invasion
To verify as to whether mitogen withdrawal might affect also the overall in vivo tumorigenic and invasive capacity of GSCs, I- and D-GSCs were infused orthotopically into immunocompromised SCID mice [14, 27]. As expected, upon intracranial transplantation, both GSCs subpopulations were shown to give rise to prototypical human GBM. Strikingly, Wnt5aHigh/EphA2Low I-GSCs’ tumorigenicity was exacerbated and so did their lethal capacity, irrespectively of the subtype (Fig. 2 and Supplementary Fig.S2). We found that, as early as 30–80 days post-transplantation (DPT), depending on the median end-stage peculiar of each GSCs line, tumors from I-GSCs-bearing mice were much more expanded and able to very rapidly spread all throughout the brain parenchyma, as compared to those from D-GSCs-injected mice (Fig. 2A-C and Supplementary Fig.S2A-B). Yet, the rate of proliferation was higher in I-GSCs-derived tumors and so did the rate of vascularization, with a significant increased vessel density ranging from 4 to 10-fold with respect to those from D-GSCs-carrying mice (Supplementary Fig.S2C). Consistently, mice receiving I-GSCs exhibited more lethal tumors with an overall survival that was more than two times shorter than those of animal carrying D-GSCs-derived tumors. Yet, a median survival window of only 39, 66 and 88 days was peculiar of TCGA-CL, TCGA-PRO and TCGA-MES I-GSCs-receiving mice, respectively, whereas D-GSCs-implanted counterpart survived for 72, 129 and 210 days (Kaplan-Meier survival analysis; P < 0.01, Log-rank and Gehan-Breslow-Wilcoxon tests, n = 5 mice/group) (Fig. 2D).
Data so far demonstrate that isolating GSCs from patient’s tumor specimens under more physiological condition, i.e. avoiding artificial GF-stimulation, exposes one of the most dangerous “hostile” traits of GBM, that is its tumorigenicity and invasiveness, as exacerbated.
I-GSCs faithfully resemble the tissue of origin and display a distinctive “motile” transcriptomic fingerprint
To pinpoint the inherent GSCs’ critical regulators, with particular emphasis on those involved in spreading and tumorigenic potential, we next carried out a side-by-side comparative transcriptomic, genomic and genetic analysis of I- and D-GSCs as well as their tissue of origin. As shown in Fig. 3A, hierarchical cluster analysis of global gene expression profiles clearly distinguished GBM tissues and both GSCs subpopulations, regardless of the subtypes. Yet, a similar transcriptional signature was retrieved in GBM patient’s specimens and I-GSCs, suggesting that these cells faithfully resembled the functional characteristics of the tissue of origin (Fig. 3A-B and Supplemental Table S5) [57]. Remarkably, a distinctive expression program emerged for the GF-independent GSCs, which clearly underlined enrichment for genes controlling extracellular matrix (ECM) remodeling, cell migration and metastasis (CTNND2, ANOS1, ENPP2, MMP15, DCLK1, ITGB4, CDH1, EPHB1, BCAN, PRELP, CXCL10, EGFR-AS1, AQP9), calcium ion binding (PCP4, ADCY2, EDNRB, PLSCR4, ANXA8L1) cell focal adhesion (CNTN1, MID1, CADM3, NCAM, L1CAM) and angiogenesis (VCAM, EDNRB) (Fig. 3C-D and Supplemental Table S6). High level of monocyte chemotactic factor (CCL) and of genes associated with coagulation and immune/complement responses (CD74, HLA-DRA, TRGV4, IGKV1–6) also indicates a pro-inflammatory state. Several genes were almost confirmed to regulate differentiation (GFAP, OLIG3, BMPR1B, MYCNUT, DLX5, DNER) and to encode for RTK activity, critical in the oncogenesis of GBM, including NTRK, REPS2, ERBB4 and DOK5, confirming an “hybrid” astrocyte-like/mesenchymal-like (AC-like/MES-like) state within this subset of GSCs [58]. Nevertheless, the GF-dependent counterpart was defined by genes controlling mitotic cell cycle and cytokinesis-associated genes (CCNB1, ATM, CDKN3, KIF14, KIF20B), cell growth, proliferation, cycling and stemness (CDK1, DUSP6, LAMC1, MAPK10, YES1, EPHA2, EPHB2) (Fig. 3C-D). These data were also confirmed when the emerging profile of I- and D-GSCs across subtypes was compared to each other (Supplementary Fig.S3A-B). Consistently, significant enhancements were found in the expression of selective biological signaling including apoptosis and necrosis, cellular invasion and immune cell trafficking in the GF-independent cells versus D-GSCs counterpart (Fig. 3E, Supplementary Fig.S3C and Supplementary Table S1), which was confirmed to embody the subset of cycling and proliferating cells.
When the distribution of somatic mutations, including SNVs and indels, and copy number changes were evaluated focal subtype-specific aberrations typically associated to GBM were similarly identified between the two culture conditions [22, 54]. Yet, mutation-calling analysis revealed that samples from TCGA-CL subtype harboured mutations in EGFR and TP53 genes, whereas the higher rate of somatic variant mutations in NF1, PTEN, EPHA2 and MET genes occurred predominantly in TCGA-MES and TCGA-PRO cases, respectively (Fig. 3F and Supplementary Tables S2–3). Several of the typical well-defined GBM-related, arm-level changes were also found, as emerged from analysis of copy number variations. Well in line with the transcriptomic fingerprint (Fig. 3C), focal amplifications typical of D-GSCs cells, regardless of the subtype, were detected at 5q34 (q-value = 0.037, two-sided Fisher’s exact test) (TERT, CCNB1) and 1p36.13 (q-value = 0.011) (LAMC1, KIF14, EPHA2, EPHB2), whereas focal amplification at 9q31.1 (q-value = 0.015) (INVS, MURC) was specific for the I-GSCs counterpart (Supplementary Fig.S3D and Supplemental Table S4).
Taken together, all of these findings confirmed that the relatively in vitro slow-propagating subset of I-GSCs is defined by a peculiar “mesenchymal-like” molecular signature with specific genes controlling the enhanced invasive phenotype and tumorigenic potential, as compared to GF-dependent counterpart.
Clinical potential of the combined modulation of Wnt5a and EphA2 activity
Having observed a unique, lethal Wnt5aHigh/EphA2Low profile specific for the highly invasive I-GSCs cells as opposed to that Wnt5aLow/EphA2High of the proliferative D-GSCs siblings, to address the therapeutically cogent issue of how to tackle GSCs and along the line of developing combinatorial approaches, we next explored the combined effects of modulating Wnt5a and EphA2 in vitro and in vivo on both GSCs subpopulations.
As depicted by invasion assay in Fig. 4A-B, a counteraction of Wnt5a activity by the previously described Wnt5a antagonistic hexapeptide PepA [27] significantly hindered in vitro invasiveness of I-GSCs and, to a lesser extent, of D-GSCs cells. Furthermore, in vitro administration of ephrinA1-Fc alone, the EphA2 cognate ligand [59], reduced more effectively D-GSCs’ growth potential, the major site of EphA2 overexpression, as compared to I-GSCs counterpart (Fig. 4C). Meanwhile, the highest suppressive effect on GSCs proliferation ability of PepA as single-agent was observed in the latter, upregulating Wnt5a. Remarkably, as clearly outlined in Fig. 4C, when both GSCs were exposed simultaneously to ephrinA1-Fc and PepA, the combination of the two molecules treatment was observed superior to the single treatments alone with an additive effect in terms of lessening GSCs’ proliferation.
Next, we verified whether PepA infused in combination with ephrinA1-Fc into the brain of GBM orthotopic xenografts for 14 days by means of osmotic mini-pumps further reduced GSCs’ in vivo invasive and tumorigenic ability, as compared to the single treatments alone. Consistent with the in vitro observation, PepA and ephrinA1-Fc treatment alone significantly inhibited D-GSCs’ tumorigenicity, while a more significant suppressive effect of the same single-agent on I-GSCs’ growth and invasiveness were observed (Fig. 5A-C and Supplementary Fig.S4). Remarkably, the combination of PepA with ephrinA1-Fc treatment on both GSCs cells-derived tumors was superior to the single treatments alone in terms of suppressing the growth and the capacity for brain dispersal (Fig. 5A-C and Supplementary Fig.S4). Consistently, Kaplan-Meier survival analysis reported that mice receiving PepA and ephrinA1-Fc as single-agent have a significant longer life span than mice treated with vehicle (P < 0.0001, Log-rank and GBW tests, n = 5 mice/group) (Fig. 5D). Most important, intracranial simultaneous administration of the two molecules under putative therapeutic conditions, was able to hinder either tumor propagating ability (extending the overall survival from 29 and 62 days to 41 and 84 days in I- and D-GSCs controls vs. treated mice, respectively; P < 0.0001, Log-rank and GBW tests, n = 5 mice/group) or invasiveness in an additive manner and, importantly, without substantial cytotoxic effects.
These data show that Wnt5a antagonistic peptide PepA in combination with ephrinA1-Fc is able to hinder in vivo GSCs’ tumorigenicity and invasiveness in an additive manner and, as such, suggest a new potential combinatorial therapy against GSCs, under experimental settings that are conducive to clinical applications.