Growth factor independence underpins a paroxysmal, aggressive Wnt5aHigh/EphA2Low phenotype in glioblastoma stem cells, conducive to experimental combinatorial therapy

Background Glioblastoma multiforme (GBM) is an incurable tumor, with a median survival rate of only 14–15 months. Along with heterogeneity and unregulated growth, a central matter in dealing with GBMs is cell invasiveness. Thus, improving prognosis requires finding new agents to inhibit key multiple pathways, even simultaneously. A subset of GBM stem-like cells (GSCs) may account for tumorigenicity, representing, through their pathways, the proper cellular target in the therapeutics of glioblastomas. GSCs cells are routinely enriched and expanded due to continuous exposure to specific growth factors, which might alter some of their intrinsic characteristic and hide therapeutically relevant traits. Methods By removing exogenous growth factors stimulation, here we isolated and characterized a subset of GSCs with a “mitogen-independent” phenotype (I-GSCs) from patient’s tumor specimens. Differential side-by-side comparative functional and molecular analyses were performed either in vitro or in vivo on these cells versus their classical growth factor (GF)-dependent counterpart (D-GSCs) as well as their tissue of origin. This was performed to pinpoint the inherent GSCs’ critical regulators, with particular emphasis on those involved in spreading and tumorigenic potential. Transcriptomic fingerprints were pointed out by ANOVA with Benjamini-Hochberg False Discovery Rate (FDR) and association of copy number alterations or somatic mutations was determined by comparing each subgroup with a two-tailed Fisher’s exact test. The combined effects of interacting in vitro and in vivo with two emerging GSCs’ key regulators, such as Wnt5a and EphA2, were then predicted under in vivo experimental settings that are conducive to clinical applications. In vivo comparisons were carried out in mouse-human xenografts GBM model by a hierarchical linear model for repeated measurements and Dunnett’s multiple comparison test with the distribution of survival compared by Kaplan–Meier method. Results Here, we assessed that a subset of GSCs from high-grade gliomas is self-sufficient in the activation of regulatory growth signaling. Furthermore, while constitutively present within the same GBM tissue, these GF-independent GSCs cells were endowed with a distinctive functional and molecular repertoire, defined by highly aggressive Wnt5aHigh/EphA2Low profile, as opposed to Wnt5aLow/EphA2High expression in sibling D-GSCs. Regardless of their GBM subtype of origin, I-GSCs, are endowed with a raised in vivo tumorigenic potential than matched D-GSCs, which were fast-growing ex-vivo but less lethal and invasive in vivo. Also, the malignant I-GSCs’ transcriptomic fingerprint faithfully mirrored the original tumor, bringing into evidence key regulators of invasiveness, angiogenesis and immuno-modulators, which became candidates for glioma diagnostic/prognostic markers and therapeutic targets. Particularly, simultaneously counteracting the activity of the tissue invasive mediator Wnt5a and EphA2 tyrosine kinase receptor addictively hindered GSCs’ tumorigenic and invasive ability, thus increasing survival. Conclusion We show how the preservation of a mitogen-independent phenotype in GSCs plays a central role in determining the exacerbated tumorigenic and high mobility features distinctive of GBM. The exploitation of the I-GSCs' peculiar features shown here offers new ways to identify novel, GSCs-specific effectors, whose modulation can be used in order to identify novel, potential molecular therapeutic targets. Furthermore, we show how the combined use of PepA, the anti-Wnt5a drug, and of ephrinA1-Fc to can hinder GSCs’ lethality in a clinically relevant xenogeneic in vivo model thus being conducive to perspective, novel combinatorial clinical application. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-022-02333-1.

--3 with a spacer having sequence Gly-Pro-Gly insert between the ectodomain and Fc region. (6). Here, we used a mouse ephrinA1-Fc (R&D) that shares 85% and 94% aa sequence identity with human and rat ephrinA1, respectively. The inhibited efficacy of ephrin-A1 on gliomas is accompanied by the down-regulation of EphA2 and FAK (7) and in GBM CSCs ephrinA1-Fc transiently increases EphA2 tyrosine phosphorylation, in a dose-dependent manner, causing a strong and persistent downregulation of EphA2 expression (8).
Flow cytometry analysis GBM cells were collected, washed twice with PBS and then stained for 30 minutes at room temperature (RT) with zombie dye violet (1:500) (Biolegend). Subsequently, cells were incubated for 30 minutes at RT with blocking solution (PBS+2mM EDTA+ 5%FBS). Cells were then washed and stained with goat anti-EphA2 (R&D System) antibody (0.5ug/5x10^5 cells, 1h RT) and next with anti-goat AlexaFluor 647 (1:250, 1h RT, Invitrogen). After one wash with PBS cells were fixed and permeabilized with cytofix/cytoperm reagent kit (Becton Dickinson) and stained with rabbit anti-Wnt5a (ThermoFisher) antibody (1:25, 1h RT) and then with anti-rabbit AlexaFluor 488 (1:250, 1h RT), according to the manufacturer's instructions. Cells were acquired with Gallios instrument (Beckman Culter) and analysed with Flow jo X software. For cell cycle analysis, cells were collected by centrifuge, rinsed twice with phosphate buffered saline (PBS pH 7.4) and ricollected by centrifugation. Pellets were resuspended in ice cold 70% ethanol and stored at 4°C for a minimum of 2 hours. Cells were then collected by centrifugation, rinsed twice in PBS and resuspended in 25 ug/ml propidium iodide (PI) in PBS with 20 ug/ml RNase A. Cells were stored (protected from light) at room temperature and Fluorescence (FL2) was measured using a Beckman Coulter Gallios cytometer. Cells cycle phase was then estimated using kaluza software.

Microarray analysis of gene expression Gene expression profiling was obtained by the Affymetrix
GeneChip® Human Transcriptome Array 2.0 (Affymetrix) as previously described (1,3). Quality control steps and expression data analysis of .CEL files was performed using R (ver. 3.6.0) (1,3).
--4 mRNA abundance levels were analysed with Partek Genomics Suite package ver. 6.6 and P-values corrected by the Benjamini-Hochberg false discovery rate (FDR; q-values). Q-value <0.05 was considered significantly differentially expressed. Analysis of the biological functions, canonical pathways and regulatory networks of differently expressed mRNAs was performed by Ingenuity Pathway Analysis (IPA; Qiagen, http://www.ingenuity.com/) and R software (1,3). To identify canonical pathway and biological functions a right-tailed Fischer's exact test was performed and Pvalues were further adjusted using FDR. Cutoff for significance was set as q-value < 0.05, z-scores > 2 (minimum activation threshold) and z-score < -2 (minimum inhibition score) (1,3,4).  Inc) and results written to FASTQ files. Read mapping and variant calling analysis were performed as previously described (3,12). Somatic SNVs and indels were identified in tissue samples with matched blood DNA by integrating the results from GATK and VarScan (13,14). To filter out germline variants, nucleotide variants in samples with unavailable blood DNA were identified using GATK HaplotypeCaller corrected with a panel of normal samples as a substitute for the missing matched normal. Somatic variants were annotated using AnnoVar algorithm (15), which aggregates information from genomic and protein resources (GENECODE, UniProt, dbNSFP) with cancer (COSMIC, ClinVar) and non-cancer variant databases (dbSNP, 1000 Genomes, Kaviar, Haplotype Reference Consortium, Exome Aggregation Consortium, NHLBI Exome Variant Server). Among the annotated variants, we selected only those producing a direct effect on the protein sequence (missense, truncating, stoploss, splicing variants, frameshift, and in-frame indels). Variants reported in the non-cancer databases with a minor allele frequency ≥ 0.05 were classified as germline polymorphisms and then excluded. The functional effect of missense SNVs and in-frame indels was determined using multiple prediction algoritms. MutationTaster2, Polyphen2, Provean, and SIFT were applied to predict the pathogenicity of missense SNVs (16)(17)(18)(19). The pathogenic effect of inframe indels was determined by FATHMM-Indel, Provean, SIFT-Indel, and VEST-Indel (18,(20)(21)(22).
Variants predicted as damaging by two or more algorithms were classified as pathogenic mutations.
All the selected variants were then validated by Sanger sequencing using the ABI Prism BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and results analysed as previously described (3). All primer used are listed in the Supplementary Table S3. PCR was initiated at 94°C for 5 min, followed by 40 cycles of 94°C for 45s, 68°c for 45s, 72°c for 2 min whit a final extension of 72°c for 10min. and EGFRvIII (243bp product) respectively, according to (25). Long range PCR amplification.