The institutional review board at Geisinger Clinic approved the collection of patient-derived glioblastoma multiforme (GBM) tissue. Primary hGSC spheres were cultured from human glioma samples as previously described [13]. The molecular background of the glioblastomas used in this study are included in Supplementary Table 3. All hGCs used in this study were authenticated by ATCC using short tandem repeat (STR) analysis. All human primary cells used were between passages 5 and 10. All cultures were routinely tested for mycoplasma contamination using the LookOut Mycoplasma PCR Detection kit (Sigma).
The cortices from P2 rat pups were dissected and diced with a scalpel followed by dissociation by papain (Worthington) and DNase I (Sigma) at 37 °C for 80 min. Papain buffer was removed and tissue triturated in media containing 10%FBS (Life Technologies) three times, until completely dissociated. Cells were pelleted and resuspended in DMEM including 0.5% BSA and ITS (Life Technologies), filtered through a 30 μm mesh filter, then incubated at 37 °C for 15 min on a non-cell-culture treated 100 mm dish to allow microglia attachment. Floating cells were collected, centrifuged, and anti-A2B5-magnetic bead labeling was conducted according to manufacturer’s protocol (Miltenyi Biotec). Purified cells were resuspended in N2B2 media and seeded on rat DRG neurons isolated as previously described [16, 50], and maintained in N2B2 + T3 (R&D Systems) media for 10–14 days to allow for myelination.
The following primary antibodies were applied overnight at 4 °C: Nestin and NeuN (Millipore), GFAP (DAKO), A2B5 (R&D Systems), Mushashi1, Nanog, and Sox2 (Cell Signaling). Slides were examined using a Zeiss Axiovert fluorescent microscope.
Cells were seeded on fibronectin-coated (10 μg/ml, Sigma) glass chamber slides (NUNC) in the presence of 10% FBS for 2.5 h. Cultures were treated with 500 nM of Lck inhibitor (A770041, Axon Medchem) or DMSO vehicle for 2 h and then fixed in 4% formaldehyde. The samples were stained overnight at 4 °C with Phospho-Paxillin antibody (CellSignaling). The following day, secondary antibodies were applied followed by application of Rhodamine RedX Phalloidin (Life Technologies) and Hoechst stain (Life Technologies). Slides were imaged using a Zeiss LSM 750 confocal microscope.
Tissue microarrays containing paraffin-embedded samples of normal brain, GBM, astrocytoma, oligodendroglioma, and meningioma tissues were obtained from US Biomax, Inc. Tissue sections were incubated overnight with phospho-Lck (Y394) and phospho-Lck (S158) at a concentration of 10 μg/ml (our own pLck-Y394 antibody [25] and Abcam, respectively). Images were captured with a Zeiss Axiovert inverted microscope at ×20 magnification.
Primary antibodies against phosphorylated and total Lck, Src, Fyn, Lyn, or Yes (Cell Signaling) and the lysates were incubated overnight at 4 °C with gentle rotation. The following primary antibodies were used: p(Y418)-Src family, Lck (D88), p(Y188)-paxillin, pCrkII, Histone H3, Lamin A/C (Cell Signaling), and β-actin (Sigma). Densitometric analysis was conducted with FluorChem SP analytical software.
Electrical impedance due to cell migration is represented as cell index. hGCs were seeded on fibronectin-coated wells of the RTCA CIM-plate 16 (ACEA Biosciences) and allowed to attach and spread for 1 h at room temperature. After 1 h, wells were treated with DMSO vehicle or 500 nM Lck-I. As cells migrated towards the lower chamber containing 10% FBS with DMSO vehicle or 500 nM Lck-I, measurements were captured every 5 min. Cell index data was plotted overtime and the slope between 1 and 10 h was calculated.
RNA transcripts within the hGC pseudopodia were determined via qRT-PCR. Total RNA was isolated from pseudopodia that had migrated through 1 μM pores of 10 μg/ml human fibronectin-coated six-well inserts (BD Falcon) for 24 h. 900 ng of total RNA from each hGC pseudopodia sample was reverse-transcribed using the RT2 First Strand Kit (Qiagen). Quantitative PCR was performed using the Human Cell Motility RT2 PCR Array (Qiagen). The relative abundance of each transcript (Ct < 35) is represented by a gene heat map from GAPDH normalized Ct values using GENE-E software (Broad Institute).
Polysome-associated RNA from hGSc was isolated as described by Gandin et al. [26]. Briefly, following sucrose gradient centrifugation, polysomes were fractionated using a gradient station (BioComp) for continuous fractionation. Polysome-associated RNA was then isolated from the polysome containing fragments using standard RNA extraction protocol. Approximately 100 ng of total RNA was reverse-transcribed using the RT2 PreAMP cDNA Synthesis Kit (Qiagen) followed by qPCR for Lck, CrkII, Paxillin, and Rac1 (Qiagen). The expression level of each RNA transcript (CT < 40) was normalized to GAPDH.
To determine the number of animals (n = 10 animals per group) we performed a power analysis assuming results with confidence level above 90%. Intracranial injections were performed using a stereotaxic apparatus (Kopf) on 8-week-old Nu/J male mice (Jackson Laboratories) initially sedated with 4% isoflurane and maintained with 2% isoflurane. After leveling the skull, a hole was drilled with a #72 micro drill bit (Kyocera) at coordinates −2.0 mm AP and +1.5 mm ML relative to Bregma. A 75 RN Hamilton syringe was then lowered to a depth of −2.5 mm DV at a rate of .5 mm per minute, and 200,000 primary hGSCs resuspended in a total volume of 4 μl were injected at a rate of 0.5 μl per minute using a Stoelting Quintessiential Stereotaxic Injector. To reduce backflow, the syringe rested for 2 min post-injection, before it was withdrawn at a rate of 0.5 mm per minute, and the cavity was immediately sealed with bonewax (Ethicon). A subcutaneous pocket was created, and an Alzet osmotic pump 1004 fitted with brain infusion kit 3 and either 1.175 mg of Lck-I (Axon Medchem) or DMSO/Kolliphor ELP vehicle. A second hole was drilled at coordinates of +0.5 mm and +1.1 mm ML relative to Bregma with the same micro-drill bit to place the brain infusion catheter in the right lateral ventricle. The catheter was lowered into the ventricle and glued to the skull using Loctite 454 cyanoacrylate adhesive. After 4 weeks, brains were harvested, post-fixed in 4% formaldehyde, paraffin embedded, sliced, and stained with HuNu antibody (Abcam). Since the ALZET pumps can deliver the Lck inhibitor for a maximum of 4 weeks, we could not perform meaningful survival studies. It is theoretically possible to exchange or refill the pumps but when we tried this, we noticed various technical issues with the intracranial catheters that had adverse effects on the survival and overall health of the mice. Hence, we decided not to pursue survival studies
HuNu-stained brain slices from the Nu/J mice used in the stereotactic experiments were imaged at 10× (Zeiss) and stacked. Each mouse brain was represented by 30 consecutive 40 µM-thick slices at an interval of 120 µM between slices and were stacked and aligned using Brainmaker software (MBF Biosciences). HuNu-stained contours within this 3D stack were then manually traced for each slice, and a 3D reconstruction of each tumor was created using Neurolucida (MBF Biosciences). Quantification of each tumor was achieved using Neurolucida Explorer (MBF Biosciences). Statistical significance was determined by the two-tailed Student’s t-test.
RNA-seq was performed using Illumina HiSeq2500 (GeneWiz). The 50-nucleotide sequence reads were aligned to the hg19 build of the human genome using gsnap [51]. The genomic locations of genes and exons defined in Refseq were extracted from the refGene.txt (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refGene.txt.gz). Read summarization at the gene level was done with in-house scripts using reads with a mapping quality of 20 or greater. Gene expression was analyzed in R with the limma package [52, 53], applying voom precision weights to account for the mean-variance dependency observed in the standardized read counts [54]. Differential gene expression analysis was done by applying the decideTests routine to the eBayes fit of the contrast of the samples treated or not treated with the Lck-I, using the Benjamini–Hochberg method to control the false discovery rate [55]. Genes with an adjusted p-value < 0.05 were collected for functional analysis, done using the Gene Set Enrichment Analysis [56] at http://software.broadinstitute.org/gsea/index.jsp
Data for the figures were obtained from a dataset of RNA sequencing of 667 glioblastoma and low-grade glioma samples in The Cancer Genome Atlas hosted on the GlioVis data visualization portal. Figures were plotted using R version 3.4.4 (R Foundation for Statistical Computing, Vienna, Austria)
100,000 primary glioma stem cells per well were seeded in a non-treated 24-well plate and grown in suspension over the first 24 h without any treatment. Subsequently, neurospheres were treated with a final A77 concentration of 500 nM or the equivalent volume of DMSO once per day for 5 days. Total RNA was isolated at day 1 and day 5 post-treatment using the Arcturus PicoPure RNA Isolation Kit (ThermoFisher), and RT-qPCR was performed on 700 ng of each RNA sample using the RT2 First Strand Kit and RT2 Primer Assays (Qiagen) for stemness markers NANOG, OCT4, and SOX2.
Our goal is to obtain results with greater than 90% confidence level. We used the D’Agostino and Pearson normality test and the Shapiro–Wilk normality test to determine the distribution of our individual data sets, which determined that our data follow a Gaussian distribution. The homogeneity of variances was confirmed with Brown and Forsythe test and assuming that the standard deviation for measurements is no more than 3/4 of the mean, a two-tailed Student’s t-test was employed to compare between two sets. Significance was determined when p < 0.05. For determination of Stemness using StemChecker, the significance was calculated by the hypergeometric test, which assess the enrichment of stemness signature genes against the full annotated human genome. The adjusted p-value was calculated by the Bonferroni correction.
