To determine the function of Lrig1 in GSCs, we first engineered genetically normal cultured adult mouse NSCs into tumour-initiating GSCs. To achieve this, we used previously reported adult mouse NSCs containing an improved Fucci reporter (Mort et al., 2014; Marqués-Torrejón et al., 2021). These were transformed via CRISPR deletion of Trp53 (encoding P53) and PiggyBac-mediated delivery of an EGFRvIII over-expression plasmid (Supplementary Figures S1A–D). These cells, termed PE, were confirmed as fully transformed and tumour initiating by in vivo transplantation into immunocompromised (NSG) mice. The resulting tumours could respond to treatment with cytotoxic therapy, with TMZ abolishing Venus signal (cells in S/G2/M phase) and decreasing both Ki67 and CldU (Supplementary Figures S2A–D). PE cells therefore provide a useful glioma-initiating cell line in which we could monitor the cycling cell population using the Fucci cell cycle reporter.

Next, to determine Lrig1’s role in GSC proliferation we deleted Lrig1 from PE cells using CRISPR/Cas9. Lrig1 knock-out (Lrig1-KO) cells were isolated using FACS, using a cell surface antibody to enrich for the ablated cells (Supplementary Figures S3A–C). Fucci reporter levels were assessed in the Lrig1 wild-type (WT or Lrig1^+/+) and Lrig1 KO PE populations to determine the proportion of cells in distinct phases of the cell cycle. In WT Lrig1^+/+ GSCs under proliferative conditions (EGF/FGF-2), a full range of cell cycle stages was observed with the Fucci reporter (Figure 1A). However, Lrig1 KO GSCs were found to display a significant increase in S/G2/M phase (Venus-positive, Q4) cells (Figure 1A), indicative of an increase in cycling cells. Consistently, using single cell colony-forming assays we found that, although colony formation by Lrig1 WT and KO cells occurred with similar frequency, Lrig1 KO colonies were on average larger in size (Figures 1B–D). Together with our previous study (Marqués-Torrejón et al., 2021), these data suggest that Lrig1 restricts proliferation of both normal NSCs and their transformed derivatives (PE-GSCs).

To assess Lrig1’s role in quiescence, the PE mouse GSCs were treated with replacement of growth factors with high levels of BMP4 in culture (Carén et al., 2015). Both Lrig1 WT and Lrig1 KO GSCs underwent morphological changes from a proliferative bipolar phase-bright appearance in EGF/FGF to a stellate astrocytic-like morphology upon BMP treatment (Figure 2A). Quiescence (B cell) markers, such as CD9 and Id1, and proliferative (C cell) markers, such as EGFR and Olig2, were then assessed by immunoblotting (Figure 2B). In WT Lrig1^+/+ GSCs, BMP4 treatment reduces EGFR and OLIG2 expression and upregulates CD9 and ID1 compared to EGF/FGF (Figure 2B). Following Lrig1 KO, we still observed downregulation of the proliferation and NSC marker Olig2 upon BMP treatment. As we found previously (Marqués-Torrejón et al., 2021), endogenous EGFR protein was reduced in Lrig1 KO cells compared to Lrig1 WT, even under proliferative conditions (EGF/FGF) (we have observed this lack of EGFR band to occur in other tumour lines when the receptor is very active). Assessment of the proliferative markers, PCNA and MCM-2, expressed in all phases of the cell cycle except G0, indicated higher levels in Lrig1 KO cells than their WT counterparts in EGF/FGF-2 (Figures 2C,D). Upon BMP treatment, PCNA and MCM-2 levels decreased in both Lrig1 WT and Lrig1 KO cells. Thus, Lrig1 KO GSCs can respond to the replacement of growth factors with high levels of BMP.

To determine if the cytostatic response to BMP was dose-dependent, we next plated GSCs with different BMP doses and assessed BMP signalling by immunoblotting (Figure 2E). Interestingly, this revealed impaired BMP signalling in Lrig1 KO GSCs via both canonical (pSmad1/5) and non-canonical (pERK1/2) pathways (Figure 2E). Re-introduction of Lrig1 into Lrig1 KO GSCs was achieved by transfection with a mouse Lrig1 overexpression construct using the PiggyBac transposase system (FLAG-tagged mouse Lrig1 expressed from the CMV promoter) and subsequent sorting (Figure 2F). This overexpression was able to rescue the impaired BMP signalling of Lrig1 KO GSCs, as shown by rescued pSMAD1/5 levels (Figures 2G,H). Analysis of Bmpr1A/1B/2 gene expression revealed no significant changes between Lrig1 WT and Lrig1 KO GSCs (Supplementary Figure S4), indicating a change in BMP receptor expression was not the cause of this signalling impairment, as has been observed in some human GSCs (Carén et al., 2015). Thus, mouse GSCs have increased proliferative capacity following loss of Lrig1 and display reduced responsiveness to cytostatic BMP signalling.

To analyse the effect of Lrig1 loss on the tumour-initiating capacity of GSCs we employed two approaches: first, ex vivo GSC transplantation of Lrig1 WT or Lrig1 KO GSCs into organotypic slice cultures (Marques-Torrejon et al., 2018) (Figure 3A); and second, in vivo transplantation into immunocompromised (NSG) mice. Using the slice culture assay we could track acute responses of GSCs to the tissue microenvironment. GSCs were microinjected into the striatum of adult mouse coronal brain slices, and after 7 days the co-culture tissue was fixed. Some GSCs expressed GFAP (astrocyte and type B cell marker) and mCherry-Cdt1 in both co-cultures. GSC proliferation was then assessed using CellTrace™ and the proliferative marker Ki67 (Figures 3B–D). Lrig1 KO GSCs showed a lower CellTrace™ intensity than Lrig1 WT cells, indicating more cell divisions had occurred during the 7 days of co-culture (diluting the CellTrace™ signal) (Figures 3B,C). Similarly, a higher percentage of Ki67 cells was found in the Lrig1 KO population (Figures 3B,D).

Following in vivo transplantation of either Lrig1 WT or Lrig1 KO GSCs into the striatum of NSG mice, we found that Lrig1 KO GSCs have greater tumour-initiating capacity and consequently mice showed reduced survival (Figures 3E,F). Immunostaining was performed to assess both proliferative/stem cell (Nestin, Sox2, Ki67, Vimentin, SSEA1, CD133) and quiescent (Cd9 and Gfap) markers within the tumours (Supplementary Figure S5). We detected significantly higher KI67 and significantly lower GFAP expression in the Lrig1 KO tumours than WT controls. While not significant, higher levels of the stem cell markers Nestin, Vimentin and SSEA1 were also seen in the Lrig1 KO tumours. This confirms that Lrig1 KO enhances GSC proliferation in vivo and ex vivo, consistent with our in vitro cell culture data.

We next utilised a distinct highly aggressive GSC model cell line, termed NPE-IE (Gangoso et al., 2021). NPE-IE cells contain the common GBM mutations Nf1 loss, Pten loss, and EGFRvIII overexpression. They were generated from secondary tumours following serial transplantation of NPE cells in BL6 host mice and have undergone epigenetic immunoediting to generate a highly aggressive and immune evasive (NPE-IE) GSC model reminiscent of the ‘mesenchymal’ subtype of GBM.

Using NPE-IE cells we explored the consequences of Lrig1 overexpression, to assess if this would be sufficient to suppress proliferation. NPE-IE cells were transfected with a mouse Lrig1 overexpression construct using the PiggyBac transposase system (FLAG-tagged mouse Lrig1 expressed from the CMV promoter, as in Figures 2F–H). Cells with the highest Lrig1 levels were selected by antibody-mediated FACS and expanded (Supplementary Figure S6). Single cell colony forming assays in NPE-IE mLrig1 and control NPE-IE cells revealed that while Lrig1 overexpression does not alter colony-forming capacity, the average colony size is decreased (Figures 4A–C). As expected, immunoblotting analysis found BMP treatment enhances Lrig1 expression in NPE-IE cells, consistent with elevated levels of quiescence markers Id1 and Cd9 (Figure 4D). Additionally, under proliferative conditions (EGF/FGF) NPE-IE mLrig1 cells show higher Id1 and Cd9 levels compared to control NPE-IE in EGF/FGF, supporting evidence that Lrig1 overexpression enhances quiescence in vitro.

To explore the effect of Lrig1 overexpression on the tumour-initiating capacity of aggressive NPE-IE GSCs, we transplanted NPE-IE mLrig1 and parallel NPE-IE controls into the striatum of NSG mice (Figure 5). Tumour progression could be tracked in vivo with bioluminescent (IVIS) imaging due to a luciferase reporter in NPE-IE cells. Two weeks after transplantation, IVIS imaging of luciferase signal revealed tumour formation in both mice transplanted with NPE-IE GSCs or NPE-IE mLrig1 GSCs (Figure 5A). Survival analysis over a 20-day period confirmed that mice transplanted with NPE-IE mLrig1 GSCs showed a delayed onset of symptoms and thus increased survival compared to NPE-IE control GSCs (Figure 5B). This suggests that Lrig1 overexpression reduces tumour progression in vivo.

Altogether, our investigations suggest that the cell surface protein Lrig1 is an important regulator of not only EGFR signalling, but also BMP responsiveness. This has important implications for our understanding of the proportions of quiescent and proliferative subpopulations in GBMs and suggests Lrig1 agonists may have value in suppressing the proliferative GSC state.

NSCs and GSCs were maintained in vitro in serum-free basal medium supplemented with N2 and B27 (Life Technologies), Laminin-1 (Sigma, 1 μg/ml) and growth factors EGF and FGF-2 (Peprotech # 100–18b and #315–09, 10 ng/ml each) (Pollard et al., 2009). For single cell colony-forming assays NSCs were plated as single cells in 96 multi-well plates. Treatments with BMP-4 were for 3 days (5,10 and 20 ng/ml, Peprotech, AF-120–05 ET-100).

Mice were maintained on a regular diet in a pathogen-free facility on a 12-h light/dark cycle with unlimited access to food and water. Intracranial transplantation of GSCs was performed using a stereotaxic frame to inject 2,00,000 cells resuspended in 2 μL of NSC media into the striatum of 6- to 8-week-old male NOD/SCID/GAMMA (NSG) mice, following administration of isoflourane general anesthesia and Rimadyl analgesic. Coordinates were 0.6 mm anterior and 1.5 mm lateral to the bregma and 2.4 mm deep. Cell preparation and procedures were performed as previously described (Pollard et al., 2009). Bioluminescent imaging was performed 20 min after D-Luciferin (50 mg/kg) subcutaneous injection, using the IVIS Lumina LT Series III instrument (Perkin Elmer). Animals were fixed in a stereotactic apparatus Stoelting (SKU S51725) and an automatic injector KD Scientific product, Model KDS-311-CE, catalog number 78-9311UU. Animals were anaesthetised and transcardially perfused with 4% paraformaldehyde in 0.1 M PBS and brains processed for vibratome sectioning (Leica VT1,000S vibratome). For in vivo detection of slowly proliferating cells, CldU (invitrogen) was dissolved in PBS at 5 mg/ml and injected (IP) at 1 mg/20 g mouse, 200 ul once daily for 3 days. After 3 weeks, mice were sacrificed and CldU was assessed by IHC. All animal procedures were performed under United Kingdom Government Home Office approved Procedure Project License (PC0395462) and following approval of the local Animal Welfare and Ethical Review Body (AWERB).

For organotypic co-culture, young adult (5 weeks old) C57BL/6 SCRM (BL6) mice were used. They were sacrificed by cervical dislocation and processed as previously described in (Marques-Torrejon et al., 2018). Samples were blocked in 10% normal goat serum and 0.2% Triton X-100 in PBS for 1 h and incubated for 48 h in blocking buffer with the appropriate primary antibodies: GFAP (1:500, Z0334 DAKO) KI67 (1:100 Thermo RM9106), SOX2 (1:100, AB5603, Millipore), NESTIN (1:10, Rat 401, Developmental Studies Hybridoma Bank), p53 (1:500, ab6326, Cell signalling), LRIG1 (1:100, R&D, AF3688), CD9 (1:100, 14–0091–82, eBioscience, 1:500), RFP (1:500, Abcam 62,341), BrdU (1; 1,000 Abcam, ab6326), SSEA1 (1:500, Biolegend, ab63260), Vimentin-40E (1:50, DSHB), CD133 (1:500, Milllipore, ab6326), CD9 (1; 500,14–0091–82, eBioscience), and pSMAD1/5 (1:500, Cell signalling, 9,516).

Cells were fixed with 4% paraformaldehyde for 20 min, incubated in blocking buffer (10% normal goat serum and 0.2% Triton X-100 in 0.1 M phosphate buffer saline) for 30 min, and incubated overnight at 4 C with the indicated primary antibodies. After several washes with PBS, immunoreactivity was detected using appropriate Alexa Fluor-conjugated (Life Technologies) secondary antibodies (1:500) diluted in blocking buffer. Cells were counterstained with 4′,6′, -diamidino-2-phenylindole (DAPI) or DRAQ5 and mounted with Fluorsave (Calbiochem). Manufacturer’s instructions were followed for both EdU [Click it Thermo Fisher (C10337)] and for protein synthesis [Click it Thermo Fisher OP-Puro (C10456)] assays. Images were taken and analyzed using Confocal (Leica SP8, four and five detectors) and Nikon TiE microscopes. CellTrace™ quantification was measured using FIJI software (Fiji version:2.0.0-rc-69). A negative control with no fluorescence was used.

For cell sorting using antibodies, cells were split and washed with PBS. The cell pellet was incubated with the primary antibody LRIG1 (1:200, R&D AF3688) diluted in PBS with 4% FCS at 37 C for 1 h. After washing with PBS, the pellet was incubated in secondary Alexa Fluor-conjugated (Life Technologies) antibodies diluted (1:500) in PBS-4%FCS for 10 min. BD LSR Fortessa (SORP) and BD Fusion Cell Sorters were used for analysis and sorting, respectively.

Immunoblotting was performed using standard protocols. Antibodies were diluted in 5% milk powder in PBS Triton 0.1%, and protein detection was carried out with HRP-coupled secondary antibodies and X-ray films. The following primary antibodies were used: LRIG1 (1:100, R&D, AF3688), EGFR (1:1,000, D38B1, Cell Signaling, #4267), EGFR-p (1:1,000, Tyr 1,068, Cell Signaling, #3,777), pSMAD1-5 (1:1,000, Cell Signaling, #9,516), SMAD1 (1:1,000, Cell Signaling, #9,743), ppERK1/2 (1:1,000, Cell Signaling #9101), ERK1/2 (1:1,000, Cell Signaling 4,695), Olig2 (1:3,000, Millipore, #9610), Id1 (1:2,500; Biocheck # BCH-1/195–14), CD9 (1:100; Millopore, #CBL 162), BLBP (Abcam, 1: 1,000; #3,24,230, GAPDH (1:50,000; GenTex, GTX627408). No accutase was used when collecting cell pellets. Western blot quantification was performed in ImageJ. Each band was selected, and the mean intensity measured in arbitrary units. The intensity of each band was then divided by the intensity of the respective GAPDH band to normalise the loading. Normalised values were then divided by the normalised intensity of the control band to give the fold change relative to the control for each blot.

Synthetic Alt-R crRNA and tracrRNA were manufactured by Integrated DNA Technologies. dsDNA block oligonucleotides were manufactured by Twist Bioscience and recombinant Cas9 protein was made in-house. NSCs were transfected using PiggyBac-GFP-Luc BSD plasmid with EGFRvIII and the guides for p53 (Gangoso et al., 2021). For Lrig1 mutant cells, design and construction of CRISPR sgRNAs are described in (Bressan et al., 2017). We disrupted Exon1 based on (Suzuki et al., 2002). We used the 4D Amaxa nucleofector and DN-100 program. For Lrig1 deletion we used two RNA guides with the following sequences: AAG​GCG​ACT​CTC​AGC​GCG​GC and TAC​TCA​CAG​GCT​GCG​CGT​CC (Marqués-Torrejón et al., 2021). To overexpress Lrig1, we used the mLrig1-CFLAG plasmid (mouse LRIG1 expressed from CMV promoter; gift from Prof Kim Jensen, University of Copenhagen).

For genomic DNA isolation, each well of a confluent 24-well plate was lysed with 40 µL of lysis buffer (0.45% NP40, 0.45% Tween20, 1x NEB LongAmp PCR buffer) containing 0.2 mg ml−1 proteinase K (Sigma). After a 2 h digestion at 55 C, samples were heated to 95 C (10 min) and 1–2 µL of the lysate was used in a 10 µL PCR reaction. PCR mix consisted of 0.2 µL DMSO (100% v/v, Sigma), 0.3 µL dNTPs (10 mM, Thermo Fisher Scientific), 2.0 µL 5x LongAMP buffer (NEB), 0.4 µL LongAMP Taq DNA polymerase (NEB), and 12 pmol of each primer. Thermal cycling was performed using the following conditions: one cycle 94 C for 3 min; 35 cycles (94 C for 30 s, 60 C for 30 s, 65 C for 2 min); followed by a final extension at 65 C (Gangoso et al., 2021).

RNA was extracted using the RNeasy spin column kit (Qiagen), plus DNase treatment to eliminate gDNA. cDNA was generated with SuperScript III (Invitrogen), and quantitative RT-PCR was performed using Taqman universal PCR Master Mix (Applied Biosystems). The following Taqman assays (Life Technologies) were used: Bmpr1a Mm00477650_m1, Bmpr1b Mm03023971_m1, Bmpr2 Mm00432134_m1, Id1 Mm00775963_g1, Gapdh Mm00775963_g1.

Results are presented as mean ± SD of a number (n) of independent experiments. Statistical significance was determined by two-tailed Student’s t-tests using GraphPad (version 9.0.0). Treatment experiments were analysed using paired t-tests. Significance of Kaplan Meier survival curves determined by Log-rank (Mantel-Cox) test. When comparisons were performed with relative values (normalised values and percentages), data were normalised by using an arc-sen transformation. Values of p < 0.05 were considered statistically significant. Box and whisker plots show the mean, and maximum and minimum values.