Transmembrane CLIC1 sustains EGFRvIII–STAT3 oncogenic signaling in Glioblastoma through interaction with OSMR

Transmembrane CLIC1 sustains EGFRvIII–STAT3 oncogenic signaling in Glioblastoma through interaction with OSMR | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Transmembrane CLIC1 sustains EGFRvIII–STAT3 oncogenic signaling in Glioblastoma through interaction with OSMR Arezu Jahani-Asl, Amir Hossein Mansourabadi, Kamaldeep Randhawa, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6537096/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oncostatin M receptor (OSMR) plays diverse and important roles in several human malignancies, including brain, breast, and pancreatic cancer 1-4 . Glioblastoma (GB) is the most malignant genetically diverse brain tumour, with no cure. The most common genetic mutation in GB is a truncated active mutant of epidermal growth factor receptor (EGFR), the EGFRvIII. OSMR orchestrates a feedforward signaling mechanism with EGFRvIII and the signal transducer and activator of transcription 3 (STAT3), to drive GB progression 4 . Beyond EGFRvIII, OSMR promotes brain tumour stem cells (BTSCs) via upregulation of mitochondrial oxidative phosphorylation and contributes to therapy resistance 5 . The molecular mechanisms underlying the multifaceted roles of OSMR in different contexts are largely unclear. Here, we systematically mapped the OSMR interactome using Mammalian Membrane Two-Hybrid High-Throughput Screening (MaMTH-HTS). This unbiased approach led to the identification of OSMR-specific and OSMR/EGFRvIII-specific binding proteins, revealing context-dependent OSMR functions. Among a subset of common interactors, we uncovered chloride intracellular channel 1 (CLIC1) as a critical regulator of both OSMR-STAT3 signaling and the OSMR/EGFRvIII complex in GB. CLIC1 physically associates with both OSMR and EGFRvIII and plays a key role in EGFRvIII packaging into extracellular vesicles (EVs). Genetic deletion of CLIC1 disrupts the OSMR/EGFRvIII interaction, impairs STAT3 activation, reduces EGFRvIII EV content, and slows GB progression. Using whole-cell patch-clamp recordings and a monoclonal antibody that selectively targets transmembrane CLIC1 (tmCLIC1omab), we establish a distinct pharmacologically and biophysically tmCLIC-mediated current in GB indispensable for sustaining EGFRvIII/STAT3 signaling. Importantly, we show that OSMR is required for maintaining CLIC1-mediated ionic balance at the plasma membrane (PM). Our study uncovers a bidirectional cross-talk between OSMR and tmCLIC1 in GB, which is essential for fueling its malignant growth. Biological sciences/Stem cells/Cancer stem cells Biological sciences/Cancer/Cancer genetics Biological sciences/Cell biology/Cell signalling Biological sciences/Molecular biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 MAIN GB is the most aggressive incurable brain tumour with a diverse genetic profile. The oncogenic EGFRvIII mutant is present in more than 30% of GB patients and is initially assigned to the classical GB subtype. The mesenchymal GB subtype has also been described as one of the most malignant brain tumours. The aggressive nature of classical and mesenchymal subtypes is attributed in large part to the activity of STAT3, endowing the cells with the ability to enter an epithelial-to-mesenchymal (EMT)-like state, maintain stemness, grow, and metastasize 6 – 9 . Recent studies have revealed that the cytokine receptor, OSMR, is a central player in propelling classical and mesenchymal GB subtypes. OSMR plays a crucial role in mediating oncogenic signal transduction initiated by the ligand OSM 1 , 10 . The OSM-OSMR induces the activation of STAT3 as well as key oncogenic pathways including the phosphoinositide 3-kinase (PI3K)–AKT, c-Jun N-terminal kinases/mitogen-activated protein kinase (JNK/MAPK), and RAS/MAPK pathways 11 – 13 . OSMR functions as a co-receptor for EGFRvIII in GB, inducing a positive feedback loop with STAT3 4 . It also plays a crucial role in upregulating mitochondrial respiration, and conferring resistance to ionizing radiation (IR) therapy 5 . Additionally, OSMR is a key regulator of brain tumour stem cell (BTSC) self-renewal 5 and the immune microenvironment 14 , suggesting that its function is highly context-dependent. Here, we employed MaMTH-HTS, a technology uniquely suited to identifying interacting partners of integral membrane proteins. Our screening revealed that OSMR physically and functionally interacts with tmCLIC1, positioning it as a central player in both OSM/OSMR cytokine signaling and the OSMR/EGFRvIII co-receptor complex. We demonstrate that the genetic deletion of tmCLIC1 and pharmacological inhibition of tmCLIC1 impairs BTSCs, and oncogenic EGFRvIII/STAT3 signaling. Furthermore, our data show that OSMR is essential for the function of the membrane configuration of CLIC1, highlighting a previously unrecognized crosstalk between a cytokine receptor and an ion channel in driving key oncogenic pathways in GB. Our finding has important implications for the design of novel therapeutic strategies to suppress GB. Mapping OSMR interactome via Mammalian Membrane Two-Hybrid High Throughput Screening (MaMTH-HTS) To address the question of how OSMR networks with alternate molecules to confer its different functions, we performed MaMTH-HTS in HEK293T cells expressing OSMR bait, in the presence or absence of co-expressed EGFRvIII, alongside a prey library of ~ 8000 open reading frames from the Human ORFeome V8.1 collection 15 . We identified 334 high-confidence candidate binding partners for OSMR (Fig. 1 ), following rigorous analyses and exclusion of MaMTH-HTS ‘frequent flyers’, which was established from repeated MaMTH-HTS results of 9 ‘individual bait’ protein control experiments, as previously described 16 . Among the identified candidates, 204 hits, including 59 PM proteins, were detected when OSMR was used as bait in the absence of EGFRvIII, and 160 candidate binding partners, including 40 PM proteins, were identified when OSMR was used as a bait in the presence of EGFRvIII. Importantly, our data revealed 30 candidate binding partners of OSMR that were commonly shared by both groups, 7 of which were PM proteins. The PM proteins encompassed proteins that are integral to the membrane as well as proteins that are associated with the membrane. GO term functional analysis of protein clusters in each group revealed molecules engaged in metabolic processes and signalling as top categories underscored in each group (Fig. 1 ). Furthermore, these analyses revealed that the GO term for immune system processes was highly enriched in OSMR-unique interactome, whereas the regulation of biogenesis processes was among top GO terms for OSMR-EGFRvIII category. This novel OSMR interactome map highlights its role in different biological processes with unique variations depending on a cell’s genetic signature. Analysis of OSMR common binding partner and the involvement of CLIC1 To unravel the mechanism by which OSMR operates to promote tumour growth across different GB subtypes, we focused on the common candidate binding partners ( Supp. Table 1 ), which encompassed different classes of proteins including metabolic processes, signalling, biogenesis, development, and immune system processes. To validate MaMTH data in the context of GB, we employed four different patient-derived BTSCs that naturally harbour EGRFvIII mutation (BTSC73 and BTSC147) or lack the mutation (BTSC12, BTSC30) ( Supp. Table 2 ). We conducted a counter-screen in which we employed siRNA ( Supp. Table 3 ) targeting each of the genes encoding these proteins, followed by assessing cell viability. Our results revealed that knockdown (KD) of 12 genes demonstrated a substantial reduction in cell viability exceeding 50% across different BTSCs (Fig. 2 a-e). These included CLEC2B, CLIC1, CREB3, CSGALNACT2, DCN, EIF2C3, GC, GPBP1, HISTH3A, IMMP2L, NAT8, and ST3GAL4. Prior to follow up investigation on select candidates, we applied additional screening criteria pertaining to the known role of these proteins in BTSCs and GB as well as their known roles in regulation of different hallmarks of cancer. These criteria led us to focus on the CLIC1, as the top candidate. CLIC1 is highly expressed in various cancers including GB, with its expression significantly correlating with poor prognosis 17 . CLIC1 exists as a soluble cytoplasmic protein, however, under specific cellular conditions that are favored in cancer cells 18 , it can translocate to PM where it functions as an ion channel 19 , 20 . Interestingly, similar to OSMR 13 , CLIC1 is highly expressed in the mesenchymal GB subtype and enriched in the cancer stem cells 17 where it promotes proliferation 21 . To begin with, we electroporated a pool of four siRNAs targeting CLIC1 in different BTSCs and assessed cell viability and stem cell frequency using PrestoBlue and extreme limiting dilution assay (ELDA) 22 , respectively. RT-qPCR and immunoblotting analyses showed efficient siRNA-mediated knockdown of CLIC1 by greater than 85% and a significant decline in CLIC1 protein expression level relative to BTSCs electroporated with a non-targeting RNAi (siCTL) ( Supp. Figure 1 , Fig. 2 f-i, Supp. Figure 2 a,b). Our data revealed a significant decrease in BTSCs viability ( Supp. Figure 2 c,d), BTSC sphere size ( Supp. Figure 2 e,f), and stem cell frequency (Fig. 2 j-q) upon KD of CLIC1. CLIC1 interacts with both OSMR and EGFRvIII in patient derived BTSCs and is required for OSMR/EGFRvIII complex Via conducting co-immunostaining studies using antibodies to OSMR and CLIC1, we observed that CLIC1 colocalizes with OSMR in different BTSCs (Fig. 3 a,d). We thus asked whether CLIC1 interacts with OSMR endogenously. We conducted Proximity Ligation Assay (PLA) using OSMR and CLIC1 antibodies in different BTSCs. Our data showed in situ interaction of CLIC1 with OSMR across all BTSC lines that were examined (Fig. 3 e-h), with EGFRvIII-expressing BTSCs that harbour elevated OSMR expression, exhibiting stronger signal (Fig. 3 e,f) compared to BTSCs lacking the EGFRvIII (Fig. 3 g,h). In parallel, we also conducted co-immunoprecipitation (co-IP) experiments using an antibody against endogenous OSMR followed by Western blotting (WB) with a CLIC1 antibody. We confirmed the results obtained with PLA via IP-WB in which we showed that OSMR interacts with CLIC1 endogenously in BTSCs (Fig. 3 i,j). Next, we generated transgenic BTSCs in which we induced genetic deletion of CLIC1 using CRISPR in two different EGFRvIII-expressing BTSC73 and BTSC147 (Fig. 3 k,l, Supp. Figure 3 a,b). Similar to our findings with the transient KD experiments, monoallelic (CRISPR-a) or biallelic (CRISPR-b) genetic deletion of CLIC1 induced a robust reduction in BTSC stem cell frequency (Fig. 3 m-p), cell viability ( Supp. Figure 3 c,d), and sphere size ( Supp. Figure 3 e,f). We next employed BTSCs harbouring monoallelic or biallelic deletion of CLIC1 and conducted PLA to confirm the specificity of the interaction signal. Our results showed that the PLA interaction signal of OSMR-CLIC1 was completely diminished in CLIC1 CRISPR BTSCs compared to control BTSCs (Fig. 3 q-t). Given that OSMR forms a co-receptor with EGFRvIII, we next asked whether CLIC1 is a component of the same complex with EGFRvIII. We conducted co-immunostaining analysis in different BTSCs and found colocalization of EGFRvIII with CLIC1 (Fig. 4 a,b). Thus, we performed PLA experiments using antibodies to EGFRvIII and CLIC1 in control and CLIC1 CRISPR BTSCs. Our results showed in situ interaction of CLIC1 with EGFRvIII (Fig. 4 c,d). Together, we established that CLIC1 forms a complex with both OSMR and EGFRvIII in patient-derived BTSCs. CLIC1 is required for OSMR/EGFRvIII interaction and STAT3 phosphorylation OSMR is found in complex with EGFRvIII to amplify receptor tyrosine kinase (RTK) signalling 4 . Given our results that CLIC1 interacts with both OSMR and EGFRvIII, we asked whether CLIC1 is required for the OSMR/EGFRvIII interaction. First, we confirmed the interaction of OSMR and EGFRvIII in BTSC73 and 147 (Fig. 3 e,f). Next, we subjected CLIC1-CRISPR and corresponding control BTSCs to PLA analysis using antibodies to OSMR and EGFRvIII. Strikingly, genetic deletion of CLIC1 significantly impaired the interaction of OSMR with EGFRvIII in each of BTSC147 and BTSC73 (Fig. 4 g-j), revealing that CLIC1 is required for OSMR/EGFRvIII complex. These data raised the question of whether CLIC1 is functionally required for maintaining the oncogenic OSMR or OSMR/EGFRvIII signalling. We conducted immunoblotting experiments in CLIC1 CRISPR and control BTSC73 and BTSC147 using antibodies to STAT3-Y-705 and EGFRvIII. Strikingly, we found significant attenuation in the expression of the phospho-STAT3 and EGFRvIII in CLIC1 CRISPR BTSCs (Fig. 4 k-r). Our data suggest that CLIC1 tightly maintains the activation of EGFRvIII and STAT3. CLIC1 drives GB tumourigenesis and maintains EGFRvIII/EGFR phosphorylation in vivo Our findings on a central role of CLIC1 in regulation of OSMR/EGFRvIII interaction and EGFRvIII and STAT3 phosphorylation led us next to examine whether CLIC1 contributes to GB tumourigenesis in vivo . We conducted intracranial tumour assay using BTSC73, BTSC147, and corresponding CLIC1 CRISPR BTSCs lines. Our data revealed that deletion of CLIC1 significantly impaired tumourigenesis, and prolonged survival (Fig. 5 a-h). We next subjected tumour sections obtained from the control mice as well as the smaller tumours from the CLIC1 CRISPR group to H&E staining (Fig. 5 i,j) and immunostaining analysis using a phospho-Y1068-EGFR antibody that can detect both the phosphorylated wild type (WT) EGFR and EGFRvIII (Fig. 5 k). Strikingly, we found that the phosphorylation of EGFR/EGFRvIII was robustly attenuated in the mice group xenografted with CLIC1 CRISPR BTSCs. Our analysis established that the genetic deletion of CLIC1 significantly reduces the tumor size and impairs the activation of EGFR/EGFRvIII in vivo . Pharmacological inhibition of the transmembrane conformation of CLIC1 (tmCLIC1) using a monoclonal antibody impairs the phosphorylation of EGFRvIII and STAT3 Our data raised the question of how CLIC1 maintains EGFRvIII-STAT3 oncogenic pathway. CLIC1 is found as a soluble cytoplasmic protein as well as a transmembrane protein that forms an ion channel 21 . We conducted cell fractionation experiments in different BTSCs and found that CLIC1 is localized to both the membrane and cytoplasm ( Supp. Figure 4 ), although, the majority of CLIC1 was found in the cytoplasm. CLIC1 is shown to be packaged into extracellular vesicles (EVs) in order to target neighbouring cells 23 , promoting tumour growth 23 , 24 . Interestingly, similar to CLIC1 17,23,25 , EGFRvIII impacts various aspects of tumourigenesis not only in the cell of origin, but in the neighbouring cells, also shown to be mediated via EVs 26 . Thus, we questioned whether CLIC1 is required for the horizontal propagation of EGFRvIII to the neighboring cells. By extracting EVs from CTL BTSCs and their corresponding CLIC1 CRISPR cells, we evaluated EGFRvIII protein expression levels in the cell lysates and EV fractions. Immunoblotting analysis revealed that EGFRvIII levels were significantly reduced in CLIC-deleted EVs, suggesting a role for CLIC1 in mediating the horizontal propagation of EGFRvIII to the neighboring cells (Fig. 6 a-c, Supp. Figure 5 a,b ) . At the same time, these data raised the question of whether tmCLIC1contributes to maintaining intracellular EGFRvIII/STAT3 signalling via altering CLIC1-mediated ionic conductance. tmCLIC1 is proposed to regulate cell cycle, pH, cell volume, and cell proliferation 27 . Given the smaller fraction of tmCLIC1 in BTSCs ( Supp. Figure 4 ), we first aimed to conduct immunostaining on non-permeabilized BTSCs using a CLIC1 antibody. Imaging analysis revealed a strong CLIC1 signal in non-permeabilized BTSCs (Fig. 6 d-f). Thus, we set out to investigate the membrane localization and electrophysiological properties of tmCLIC1 in three distinct human BTSC lines including BTSC73, BTSC147, and BTSC30. To assess basal level tmCLIC1 levels, we used IAA94, a known blocker of CLIC1. We conducted an electrophysiological evaluation of tmCLIC1 levels at + 40mV on the membranes of CLIC1 CRISPR and control BTSCs. Our results revealed that the tmCLIC1 currents were significantly reduced in the CLIC1 CRISPR lines compared to the CTL BTSCs (Fig. 6 g), leading to the question of whether tmCLIC1 impacts EGFRvIII/STAT3 signalling. To address this question, we generated and employed a monoclonal CLIC1 antibody (tmCLIC1omab®) that specifically inhibits tmCLIC1 and its associated ionic currents. We treated different glioma cells and BTSCs with the tmCLIC1omab or IgG control, followed by an analysis of EGFRvIII and STAT3 phosphorylation in response to the ligand OSM. The tmCLIC1 current was measured as the IAA94-sensitive current, defined as the difference between the initial current and the current after drug perfusion. Our data revealed that both the EGFR phosphorylation at tyrosine (Y) 1068 and STAT3 phosphorylation at Y705 were significantly attenuated upon inhibition of the CLIC1-mediated Cl- current by a tmCLIC1 antibody in EGFRvIII expressing BTSC 147 and 73. Importantly, tmCLIC1omab® had no impact on EGFRvIII-STAT3 signalling in BTSC30 that naturally does not harbour the EGFRvIII or elevated STAT3 expression (Fig. 6 h-m, Supp. Figure 6 ). Impact of OSMR on CLIC1-Mediated Cl Current We have established that OSMR physically and functionally interacts with CLIC1 to maintain EGFRvIII/STAT3 oncogenic signalling in BTSC and GB and a PM CLIC1 is engaged in regulating this process. Next, we set out to investigate whether OSMR is required for CLIC1-mediated Cl − current. We induced KD of OSMR using short hairpin RNA (shOSMR) in different cell lines and subjected the OSMR knockdown (KD) and control (CTL) Scrambled (SCR) cells to patch clamp experiments to evaluate whether reduced OSMR expression levels alter Cl − currents. Cell currents were assessed both prior to (baseline) and following the addition of 100 µM of CLIC1 inhibitor, IAA94 to the bath solution (Fig. 6 n-s). The tmCLIC1 current was measured as the IAA94-sensitive current, defined as the difference between the initial current and the current after drug perfusion. The corresponding current-voltage relationship was recorded in each group. Strikingly, our data revealed that inhibition of OSMR significantly attenuated the tmCLIC1-associated Cl − current (Fig. 6 r-t). Taken together, we provide data that tmCLIC1 regulates OSM/OSMR-STAT3 and OSMR/EGFRvIII oncogenic signalling and at the same time OSMR promotes tmCLIC1 function. We establish that the cooperation of OSMR with tmCLIC1 is required to maintain key oncogenic pathways in GB. Our study provides new opportunities to develop novel therapeutic strategies for GB. DISCUSSION In this study, we comprehensively mapped the OSMR interactome to identify its high confidence binding partners under different conditions leading to a better understanding of OSMR’s multifaceted roles in GB progression. Through systematic protein-protein interaction mapping using MaMTH-HTS, followed by counter-screening and loss- and gain-of-function studies, we demonstrate that tmCLIC1 interacts with OSMR physically, independently of cellular identity, and acts as a critical regulator of diverse oncogenic pathways driven by OSMR, EGFRvIII, and STAT3. We demonstrate a bidirectional relationship for tmCLIC1-OSMR interaction, which plays a pivotal role in regulating ion conductance, thereby driving GB progression. CLIC1 has been implicated in multiple malignancies, including lung cancer 28 , pancreatic adenocarcinoma 29 , epithelial ovarian cancer 30 , and medulloblastoma 31 . It is associated with poor prognosis in GB and is abundantly expressed in cancer stem cells (CSCs) 17 , 32 . Previous studies have highlighted the role of tmCLIC1 in cellular proliferation and viability, with genetic deletion leading to cell swelling, mitotic defects, and reduced proliferation in medulloblastoma cells 31 . tmCLIC1 may act as a cell cycle accelerator by mediating cell volume changes. For instance, during the prophase to metaphase transition, cells undergo a significant volume decrease, reaching a minimum size at metaphase. This reduced volume is preferred by the cell and is referred to as pre-mitotic condensation 33 , 34 . Pre-mitotic condensation requires the efflux of Cl-, which may be mediated by tmCLIC1. tmCLIC1 has also been shown to play a prominent role in ROS production 18 , 35 , 36 , and cell cycle regulation 37 . For example, IAA94-mediated inhibition of tmCLIC1 is shown to prolong the G1 phase, thereby extending the overall cell cycle duration 38 . In agreement with these findings, our data show that CLIC1 deletion suppresses BTSC self-renewal and proliferation, underscoring its role in cell cycle regulation. Given tmCLIC1's established function in maintaining redox balance 39 , its knockdown likely elevates oxidative stress and disrupts redox homeostasis, thereby impairing BTSC proliferative capacity and self-renewal potential. These findings reinforce the idea that CLIC1 is a critical regulator of GB cell division and tumour aggressiveness. The interplay between ROS, tmCLIC1-generated chloride currents, combined with evidence of tmCLIC1/OSMR interaction, highlights the need to investigate CLIC1's role in in cancer stem cells’ metabolism in the context of GB. Whether similar to OSMR 5 , tmCLIC1 regulates oxidative phosphorylation (OXPHOS) to maintain BTSCs and buffer ROS in the mitochondria remains to be investigated. We further demonstrate a crucial role for CLIC1 in maintaining EGFRvIII intracellularly. This effect may also be attributed to CLIC1's role in tumour microenvironment modulation, as it is positively associated with stromal activation, epithelial-mesenchymal transition (EMT), and angiogenesis, hallmarks of GB progression 40 . Chloride channels, including CLIC1, have been implicated in innate and adaptive immune responses, particularly in phagocyte-mediated immunity. CLIC1 is required for phagosomal acidification and antigen processing, which are crucial for CD4⁺ T cell activation 40 – 42 . CLIC1-mediated regulation of macrophage-driven inflammation and ROS production may facilitate the establishment of a tumour-promoting microenvironment conducive to tumour growth and metastasis. Furthermore, CLIC1 plays a role in the innate immune system through its involvement in the activation of NLRP3 inflammasome 43 . Aberrant activity of the NLRP3 inflammasome has been implicated in the progression of several cancers including glioma 44 , 45 . Additionally, tmCLIC1 modulates Cl⁻ currents in activated microglia, particularly in response to β-amyloid protein (Aβ) stimulation 36 . Interestingly, OSMR and its ligand OSM operate in a paracrine fashion in which OSM secreted via immune cells typically binds the receptor on the tumour cells to direct oncogenic signalling. These findings suggest that OSMR-CLIC1 cross talk may influence immune surveillance, an area that requires future investigation. It is also possible that loss of CLIC1 induces a cell shift towards a more differentiated phenotype. Previous research on cell fate dynamics supports this notion. Stockhausen et al. demonstrated that induced differentiation of BTSCs with serum leads to loss of EGFRvIII expression and decreased tumourigenic potential 46 . In another study by Gritti et al., CLIC1 expression was lost upon induction of differentiation with FBS 47 . These results suggest that both EGFRvIII and CLIC1 are important in maintaining stemness. Similarly, in another study by He et al., the chloride channel, Ano1, was found to limit the differentiation of epithelial progenitors towards cells of the secretory lineage 48 . Further studies are required to determine if the interaction of CLIC1 with EGFRvIII aids in establishing a cellular network that suppresses the BTSCs differentiation. In this study, we provide data showing that CLIC1 regulates the horizontal transfer of EGFRvIII via EV. We observed a striking attenuation of EGFRvIII in EVs derived from CLIC1-CRISPR cells, suggesting that CLIC1 may regulate EGFRvIII packaging, thereby influencing neighboring cells. In support of this model, tmCLIC1 vesicle transfer has been implicated in glioma stem cell proliferation 23 . Furthermore, blocking of the chloride current in stem-like cells through the re-purposing of known pharmacological drugs, suggests that tmCLIC1 could be a promising target for therapy 47 , 49 . Similarly, tumour cells in GB are shown to impact neighboring cells via transferring EGFRvIII through EV 26 . Another study in gastric cancers revealed that the presence of CLIC1 in EVs are associated with resistance to vincristine by transferring resistance traits to neighboring cells 24 . Whether tmCLIC1 functional expression represents an adaptive mechanism enabling drug resistance in GB remains an exciting area of research to be explored. Our findings have established that tmCLIC1 interaction with OSMR and EGFRvIII is essential for sustaining oncogenic signaling. Using a monoclonal CLIC1 antibody that can only target tmCLIC1, we establish the significance of tmCLIC1, however, the functional contribution of cytoplasmic CLIC1 and its involvement in mitochondrial and metabolic pathways remains to be investigated in GB. In conclusion, our findings establish tmCLIC1 as a central player in GB pathogenesis, orchestrating cytokine networks and enhancing the RTK signalling. Its interaction with OSMR and EGFRvIII is critical for sustaining BTSC self-renewal and GB tumourigenesis. These results highlight tmCLIC1 as a promising therapeutic target, suggesting that its inhibition, in combination with standard GB treatments, may lead to improved patient outcomes. Methods and Materials Brain tumour Stem Cell (BTSC) and generation of transgenic cell lines The human BTSC lines 73, 147, 12, and 30 were generously provided by Dr. Samuel Weiss at the University of Calgary. They were generated in accordance with the University regulation from excess or discarded tissue collected during surgery from adult patients following informed consent from patients. BTSCs were characterised for major mutations ( Supp. Table 2 ) and were cultured and maintained, as described in Supplementary methods. Transgenic CRISPR BTSCs were generated, using methods previously described 50 . To design the gRNA, Off-Spotter software, version 0.2.2 ( https://cm.jefferson.edu/Off-Spotter/ ), was used. Two guide RNA strands (forward and reverse complement) were generated to target exons 5–9 of the CLIC1 gene. To generate the construct, the Golden Gate Assembly Cloning strategy 51 was used in which gRNA1 and gRNA2 were cloned into pL-CRISPR.EFS.GFP (Addgene plasmid #57818), and pL-CRISPR.EFS.tRFP (Addgene plasmid, #57819), plasmids, respectively. Plasmids were sequenced (Genome Quebec), verified and electroporated [1300 volts by the AMAXA nucleofector 2b device (Lonza, #AAB1001) to deliver 3 µg of each plasmid construct (gRNA1-GFP and gRNA2-RFP) into ~ 2 million BTSC147 or BTSC73]. Electroporated cells were cultured in a T-75 low attachment flask at 37°C in a 5% CO 2 . 48 hours after electroporation, BTSC spheres were dissociated into a single cell suspension using Accumax dissociation solution (Innovative Cell Technologies, #AM105) and subjected to Fluorescent Activated Cell Sorting (FACS) analysis using the BD FACSAriaTM Fusion (BD Biosciences) to sort for double positive GFP and RFP cells. Sorted cells were plated at a density of 1 cell/well into two 96-well plates containing 100 \(\:{\mu\:}\) l of BTSC media. Wells were monitored every two days to assess sphere formation and clonal samples were collected from multiple positive clones and were subject to genomic DNA isolation. Isolated DNA was analysed by PCR using internal primers and external primers to the gRNA-guided CAS9 cut site, designed using Primer3Plus software, version 3.3.0 ( https://www.primer3plus.com/ ), to determine monoallelic, biallelic, or non-deletion control clones. Monoallelic deletion clones in both BTSC73 and BTSC147 cell lines were determined by PCR in which the presence of one internal and one external band on a 2% Agarose gel, was observed, while a biallelic deletion was determined by a single external band. Knockout or knockdown of CLIC1 was validated using RT-qPCR and WB to assess gene and protein expression. Since there we no clones identified that presented no cuts in the CLIC1 gene following incubation with the CRISPR-CAS9 construct, which could have been used as a control for CAS9, the chosen control for experiments using the CRISPR-CAS9-CLIC1 cells was that of the parental BTSC73 or BTSC147 line. The gRNA and primer sequences used in this experiment are listed in Supp. Table 4 . Finally, short Interfering RNA (siRNA) was used to generate transient knockdown (KD) in patient-derived BTSCs. BTSCs were processed into a single-cell suspension. ON TARGET-plus SMART pool human CLIC1 siRNA (Dharmacon, #L-009530-00-0005) at a concentration of 100 nM, and ON TARGET-plus non-targeting pool (Dharmacon, #D-001810-10-05), were employed, as described in supplemental methods. Mammalian Membrane Two-Hybrid-High Throughput Screen (MaMTH-HTS) To identify binding partners of OSMR in the presence and absence of EGFRvIII, Mammalian Membrane Two-Hybrid (MaMTH) High Throughput Screen (HTS) technology was used as described 16 , 52 . Briefly, plasmid expressing OSMR ‘Bait’ protein with C- terminally fused MaMTH-HTS Bait tag (Cub-GAL4TF-P2A-tagBFP) alone or alongside plasmid expressing EGFRvIII (fused with 3xFLAG at its N-terminus) was transfected into a pooled ‘Prey’ library of HEK293T MaMTH-HTS reporter cell lines. The library of reporter cell lines stably expressed members of the Human ORFeome V8.1 collection (~ 8000 open reading frames - ORF’s) fused to MaMTH-HTS Prey tag (Nub) at their N-terminus and P2A-mCherry at their C-Terminus and contained chromosomally integrated GFP reporter under the control of the GAL4 transcription factor (GAL4TF) promoter. Transfections were performed using X-tremeGene™ 9 Transfection Reagent (Roche, XTG9-RO) as specified by the manufacturer protocol. In order to induce Bait ( Supp. Figure 7 ) and Prey expression, cells were grown for 2–3 days in the presence of 0.5 µg/ml Tetracycline in the following conditions: 37°C, 5% CO 2 in DMEM containing 10% FBS, and 1% Penicillin/Streptomycin media. Cells were then harvested by trypsinization and resuspended at a concentration of 1–2 x 10 6 cell/ml in Basic Sorting Buffer (1X PBS, 5 mM EDTA, 25 mM HEPES pH 7.0, 1% BSA) and subjected to sorting by Flow Cytometry using BD FACSMelody (BD Biosciences). Cells were sequentially selected according to the following: tag-BFP fluorescence indicating ‘Bait’ expression, mCherry fluorescence indicating ‘Prey’ expression, and GFP fluorescence indicating Bait-Prey interaction. Cells were collected for analysis in DMEM containing 25% FBS and centrifuged pellets were processed in Phire Tissue Direct Dilution Buffer (ThermoFisher Scientific). Amplification of ORFs was done using Phire Tissue Direct PCR Master Mix (ThermoFisher Scientific) and products were purified using QIAquick PCR Purification Kit (Qiagen). Purified PCR products were subjected to Nextera XT library preparation and deep sequencing using the Illumina HiSeq 2500 system (150 bp single read). Sequencing data was processed and hits were identified using custom software developed using R programming language and integrated Bowtie2 alignment tool, version 2.5.2 53 . Counter-screen assay BTSCs were transfected with siRNA using RNAiMax in a 96-well plate. Briefly, 1 pmol of siRNA and 0.1 µl of RNAiMax were diluted in 5 µl of Opti-MEM and incubated separately for 5 minutes. The solutions were then gently combined and incubated at room temperature for 15 minutes to form transfection complexes. A total of 1,000 BTSCs per well were seeded in 80 µl of NeuroCult medium, followed by the addition of 10 µl of the siRNA-RNAiMax complex with gentle shaking. After incubation at 37°C for 5 hours, 90 µl of NeuroCult medium was added to each well, and cells were further incubated at 37°C for 48 hours. Transfected cells were then subjected to the PrestoBlue assay to assess cell viability. Duolink Proximity Ligation Assay (PLA) Proximity Ligation Assay was conducted following the manufacturer's protocol using the Duolink In Situ Red Starter Kit (Sigma, #DUO92101) as optimized by our group 54 , and described in the supplemental Methods section. Extreme Limiting Dilution Assay (ELDA) ELDA was performed as previously described 55 , with detailed methods presented in the supplemental Methods section. Stereotaxic injections and bioluminescent imaging Stereotaxic surgeries were conducted by injecting ~ 3 x 10 5 luciferase expressing CLIC1-CRISPR-BTSCs and control BTSCs into the right striata (0.8 mm lateral to the bregma, 1 mm dorsal, and 2.5 mm from the pial surface). Prior to injection, BTSCs were dissociated into single-cell suspensions in serum-free, antibiotic-free medium and tested for luciferase activity using IVIS imaging system. Kaplan-Meier survival plots were generated by collecting mice at the point of reaching the end stage (major body weight loss, dehydration, hunched back, piloerection, and lethargy). Median survivals were calculated using a log-rank test with GraphPad Prism, following previously described methods 56 . Briefly, the Kaplan-Meier approach estimated the probability of survival at each time point. Before any time elapses, all mice were considered “at risk”, however, no deaths occurred. Therefore, the probability of survival was input as a value of 1. Subsequently, information about the next elapsed time (day 7) included information about the how many participants experienced the event of interest (death). From this information, a survival probability was calculated using the formula: S t+1 = S t *((N t+1 -D t+1 )/N t+1 ), where S t represents survival probability, N t represents number at risk, D t represents number of events. The survival probability for each subsequent time point is calculated in a similar fashion and a stair-step survival curve is plotted. For tumour volume assessment, the mice received intraperitoneal injections of 200 µl of 15 mg/ml d-luciferin (ThermoFisher Scientific, #88292), underwent anesthesia via isoflurane inhalation, and were then subjected to weekly bioluminescence imaging using a CCD camera (IVIS, Xenogen). Subsequent collection and analysis of all bioluminescent data were performed utilizing Living Image 2.0 software (PerkinElmer, MA, USA). Briefly, after initialization of the IVIS Spectrum system and acquiring an image, the images were analyzed using “ROI tools” in the tool palette, selecting the area of interest. The IVIS imager captures pixels with specific photon intensity values, with brighter areas corresponding to higher photon detection, indicative of tumour induction. Patch-Clamp Experiment Patch electrodes (BB150F-8P with filaments, Science Products), with a diameter of 1.5 mm, were pulled from hard borosilicate glass on a Brown-Flaming P-87 puller (Sutter Instrument, Novato, CA) and fire polished to a tip diameter of 1-1.5 µm and an electrical resistance of 3–4 Mꭥ. Cells were voltage-clamped using an Axopatch 200B amplifier (Axon Intrument) in whole-cell configuration in which After formation of giga-seal, the membrane patch is disrupted providing a direct low resistance access to cell interior, allowing recordings from ion channels of whole cell. The voltage step protocol used to isolate current/voltage relationships consisted of 800 ms pulses from − 60 mV to + 60 mV (20 mV voltage steps). The holding potential was set according to the resting potential of the single cell (between − 40 and − 80 mV). tmCLIC1-mediated chloride currents were isolated from other ionic currents by perfusing IAA94 (100 µM) dissolved in the bath solution and by mathematical subtraction of the residual current from the control. Solution used are the following: bath solution (mM): 125 NaCl, 5.5 KCl, 24 HEPES, 1 MgCl2, 0.5 CaCl2, 5 D-Glucose, 10 NaOH; pH 7.4; Pipette solution (mM): 135 KCl, 10 Hepes, 10 NaCl; pH 7.4. Analysis was performed using Clampfit 10.2 (Molecular Devices) and OriginPro 9.1. tmCLIC1-mediated current (IAA94-sensitive current) was measured by analytical subtraction of residual ionic current after addition of inhibitor from total current (I TOT ) of the cell at each membrane potential tested. Current/voltage relationship were constructed plotting the averaged current density of the least 100 ms of the pulse against the corresponding membrane potential. Current density (pA/pF) results from the ratio between the ionic current (pA) and cells capacitance (pF). Statistical analyses were performed comparing the slopes (proportional to channel conductance) of the I/V curves of the different groups. Generation of monoclonal tm-CLIC1 antibody The tmCLIC1omab® antibody was designed (license# 11665M), targeting the N-terminal of the CLIC1 protein derived from mice. Polyclonal antibodies were obtained through a 138-day immunization protocol in which mice are immunized against a NH 2 -CLIC1 synthetic peptide conjugated to OVA (ovalbumin) (NH 2 -EQPQVELFVKAGSDGAKIGNC-COOH) (Glycine 0.1 M pH 3, Tris-HCl 1 M pH 8). Hybridoma cells that produced antibodies were sorted in single cells to obtain monoclonal antibodies. tmCLIC1omab® was used at a final concentration of 3.5 µg/ml. Extracellular vesicle isolation and validation To sequentially isolate EVs, the cell supernatant was centrifuged at 200 x g for 10 minutes, 300 x g for 10 minutes, 2,000 x g for 10 minutes, 10,000 x g for 30 minutes, and 100,000 x g for 2 hours, respectively (Optima XPN-100 ultracentrifuge, Beckman Coulter, USA). A beige/white pellet of EVs and a clear supernatant were evident post-centrifugation. After removing the supernatant and resuspending the small EV pellet in 1 ml PBS, the resulting pellet was then washed and centrifuged at 49,000 rpm for 30 minutes (Optima MAX 130K Refrigerated Benchtop Ultracentrifuge, Beckman Coulter, USA). Isolated EVs were then visualized, measured, counted, and characterized in the range of 10 − 2,000 nm using a Nanoparticle Tracking Analyzer (NTA) (Particle Metrix ZetaView® Nanoparticle Tracking Analysis) 57 , 58 . We used size-mode analysis with ZetaView software (version 8.02.28) following calibration with polystyrene beads (105 and 500 nm). Samples were analyzed at a minimum of 9 camera positions with 2-second video length at 21°C. Bioinformatic analysis Novel interactions from MAMTH screen were integrated with the known interactions from the Integrated Interaction Database IID ver. 2021 − 05 59 and further annotated with Gene Ontology Biological Process and Cellular Component (release 2022-11-03), and a subset of pathways from the PathDIP database ver. 4 60 . Resulting network was built, annotated and analyzed using NAViGaTOR ver. 3.0.19 61 . Statistical analysis Statistical analysis between conditions was performed using a Student t -test or one-way ANOVA in which the mean of n = 3 replicates were compared between two conditions to assess significance. Analysis was undertaken with the aid of GraphPad software 7. Data is shown as mean with standard deviation (mean ± SD). p-values less than 0.05 were considered significant and were marked with an asterisk as follows, * P < 0.05; ** P < 0.01; *** P < 0.001. Precise p values are provided in the figure legends, unless noted otherwise. Declarations ACKNOWLEDGEMENT This work was supported by CIHR grants to A.J.-A and I.S., and J.R. (# 162198, #519474) and a AIRC IG grant (#24758) to M.M.; A.J.-A. is a Canada Research Chair at the University of Ottawa and is supported by Canada Foundation for Innovation (CFI) and Ontario Research Fund (ORF). I.S. is supported by CFI (#225404, #30865), ORF (RDI #34876, RE010-020), and Natural Sciences Research Council (NSERC RGPIN-2024-04314). IJ was supported in part by funding from Natural Sciences Research Council (NSERC RGPIN-2024-04314), CIHR (#519474), Canada Foundation for Innovation (CFI #225404, #30865), and Ontario Research Fund (RDI #34876, RE010-020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Hara, T. et al. 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Supplementary Files mamthppisandinterpreyppis241217.xlsx Supplementary Data Corresponding to Figure 1 SupplementaryAPR242025Natcellbio.docx Supplementary Figures and Tables Related to the Manuscript Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6537096","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449281970,"identity":"a4cd8747-62ac-480c-8c47-d424b637ea14","order_by":0,"name":"Arezu 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Biosciences, University of Milan","correspondingAuthor":false,"prefix":"","firstName":"Michele","middleName":"","lastName":"Mazzanti","suffix":""},{"id":449281984,"identity":"a27b646e-6055-42c7-a9f9-0535274986d1","order_by":14,"name":"Igor Stagljar","email":"","orcid":"https://orcid.org/0000-0002-5260-3327","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Igor","middleName":"","lastName":"Stagljar","suffix":""}],"badges":[],"createdAt":"2025-04-26 23:10:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6537096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6537096/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81954262,"identity":"76d0149a-6809-4426-94bd-896e1f08f8c2","added_by":"auto","created_at":"2025-05-05 09:41:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8440945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMapping global binding partners of OSMR\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eUsing MaMTH-HTS, we mapped the OSMR interactome in the absence and presence of EGFRvIII. The interactome is represented with green edges for the novel MaMTH candidates and gray edges highlighting known interactions identified using the Integrated Interaction Database (IID). Purple edges indicate interactions both identified in MaMTH and predicted by IID. The candidate binding partners of OSMR in the absence of EGFRvIII (OSMR-specific interactors) are represented in blue. The candidate binding partners of OSMR in the presence of EGFRvIII (OSMR/EGFRvIII-specific interactors) are represented in red. The candidate binding partners of OSMR that were commonly shared by both groups are shown in the center. Go term functional annotation of the potential candidates are shown with different coloured squares.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6537096/v1/c04a5c85146535ffb809c3ae.png"},{"id":81954258,"identity":"a42c90dd-a8f1-41f2-bead-ac97b88240b6","added_by":"auto","created_at":"2025-05-05 09:41:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2118431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell viability evaluation following gene silencing of candidate binding protein targets.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-e\u003c/strong\u003e, Cell viability was assessed in four different patient-derived BTSCs (#12, #30, #73, and #147), transfected with siRNAs targeting different genes encoding candidate binding proteins. Each graph (\u003cstrong\u003ea-d\u003c/strong\u003e) is normalized to the control group transfected with a non-targeting siRNA. Data are presented as means ± SEM, n=3 biological replicates in a-d with Heatmap plot (\u003cstrong\u003ee\u003c/strong\u003e) comparing the differences across four BTSCs. The colour gradient, represented by column Z-scores, illustrates relative cell viability assessed by PrestoBlue analysis. \u003cstrong\u003ef-i\u003c/strong\u003e, Knockdown of CLIC1 was induced in BTSCs as described, and whole cell lysates were subjected to immunoblotting using a CLIC1 antibody. ACTIN was used as a loading control. \u003cstrong\u003ej-q\u003c/strong\u003e, CLIC1 knockdown and control cells were subjected to Extreme Limiting Dilution Assay n=3 biological replicates. Statistical analysis was performed using a student-t test. Data are represented as the means ± SD. BTSC73 (\u003cstrong\u003ek\u003c/strong\u003e): ***\u003cem\u003eP\u003c/em\u003e = 0.0011; BTSC147 (\u003cstrong\u003em\u003c/strong\u003e): **\u003cem\u003eP\u003c/em\u003e = 0.0017; BTSC12 (\u003cstrong\u003eo\u003c/strong\u003e): **\u003cem\u003eP\u003c/em\u003e = 0.0077; BTSC30 (\u003cstrong\u003eq\u003c/strong\u003e): ***\u003cem\u003eP\u003c/em\u003e = 0.0006 for each pairwise comparison.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6537096/v1/dc2d4153d649246a29e78f60.png"},{"id":81956657,"identity":"fd211ce8-640e-482d-9e0e-c339bff181e5","added_by":"auto","created_at":"2025-05-05 09:57:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4100318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLIC1 and OSMR interact endogenously in BTSCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-d\u003c/strong\u003e, BTSCs were subjected to co-immunostaining using antibodies to CLIC1 (green) and OSMR (red). Nuclei were stained with Hoechst. Merged images depict co-localization of OSMR and CLIC1 (yellow). n=3 biological replicates. \u003cstrong\u003ee-h\u003c/strong\u003e, BTSCs 73 and 147 were subjected to proximity ligation assay (PLA) using antibodies to CLIC1 and OSMR. Each red puncta represents PLA signal. Primary antibodies were omitted for control group. Nuclei were stained with Hoechst. Scale bar, 10 µm. \u003cstrong\u003ei,j\u003c/strong\u003e, BTSCs 147 or 77 were subjected to immunoprecipitation (IP) using an OSMR antibody or IgG control and pull downs were analyzed by immunoblotting using CLIC1 antibody. \u003cstrong\u003ek,l\u003c/strong\u003e, mRNA expression of CLIC1 was confirmed by RT-qPCR in CLIC1 CRISPR BTSCs 147 and 73. Gene expression was normalized to the housekeeping gene GUSB. Data for two different clones (CRISPR-a and CRISPR-b) are shown for each BTSCs and represented as the means ± SD, n = 3. Statistical analysis was performed using one-way ANOVA. BTSC147 (\u003cstrong\u003ek\u003c/strong\u003e): ****\u003cem\u003eP\u003c/em\u003e = 0.000000006 and 0.000000005, respectively, from left to right; BTSC73 (\u003cstrong\u003el\u003c/strong\u003e): ****\u003cem\u003eP\u003c/em\u003e = 0.00003 and 0.00001, respectively, for each pairwise comparison from left to right. \u003cstrong\u003em-p\u003c/strong\u003e, CLIC1 CRISPR and control BTSCs were subjected to ELDA and SCF was plotted. n=3 biological replicates/ Statistical analysis was preformed using one-way ANOVA. Data are represented as the means ± SD. BTSC147 (\u003cstrong\u003en\u003c/strong\u003e): ***\u003cem\u003eP\u003c/em\u003e = 0.0002; BTSC73 CRISPR-a and CRISPR-b (\u003cstrong\u003ep\u003c/strong\u003e): **\u003cem\u003eP\u003c/em\u003e= 0.0084, **\u003cem\u003eP\u003c/em\u003e = 0.0011 for each pairwise comparison respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eq-t\u003c/strong\u003e, The interaction of CLIC1 and OSMR was assessed by PLA in CLIC1 CRISPR and control BTSCs as described in e-h. Nuclei were stained with Hoechst. The number of PLA puncta (red) per nucleus was quantified in CRISPR and control BTSC147 and BTSC73 (\u003cstrong\u003er,t\u003c/strong\u003e). Data are presented as the means ± SEM. Each dot represents the number of PLA dots per nucleus. A minimum of n=3 biological replicate images was assessed for statistical analysis. Statistical analysis was performed using one-way ANOVA. BTSC147 (\u003cstrong\u003er\u003c/strong\u003e): ****\u003cem\u003eP\u003c/em\u003e = 0.00000003 and 0.00000001 respectively, from left to right; BTSC73 (\u003cstrong\u003et\u003c/strong\u003e): ****\u003cem\u003eP\u003c/em\u003e =0.000002 and 0.0000009 respectively, for each pairwise comparison from left to right. Scale bar, 10 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6537096/v1/d63eb5ec32ea39b7874a5c27.png"},{"id":81955229,"identity":"540dda98-859f-409a-a9e7-df7b7ea20280","added_by":"auto","created_at":"2025-05-05 09:49:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2674212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLIC1 is required for the OSMR/EGFRvIII interaction and STAT3 activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,b\u003c/strong\u003e, BTSCs 73 and 147 were subjected to co-immunostaining using antibodies to CLIC1 and EGFRvIII. Representative images represent n=3 independent biological replicates. \u003cstrong\u003ec,d\u003c/strong\u003e, CLIC1 CRISPR and control BTSCs were subjected to PLA analysis using antibodies to CLIC1 and EGFRvIII. \u003cstrong\u003ee,f\u003c/strong\u003e, BTSCs 147 and 73 were subjected to PLA analysis using antibodies to OSMR and EGFRvIII (\u003cstrong\u003ee\u003c/strong\u003e) and quantification of # of PLA per nuclei are presented (\u003cstrong\u003ef\u003c/strong\u003e). \u003cstrong\u003eg-j\u003c/strong\u003e, Different CLIC1 CRISPR and BTSCs were subjected to PLA analysis using antibodies to OSMR and EGFRvIII (\u003cstrong\u003eg,i\u003c/strong\u003e). Quantification of PLA dots per nucleus per group is presented (\u003cstrong\u003eh,j\u003c/strong\u003e). Nuclei were stained with Hoechst. Scale bar, 10 μm. Statistical analysis was performed using one-way ANOVA. BTSC73 (\u003cstrong\u003eh\u003c/strong\u003e): ****\u003cem\u003eP\u003c/em\u003e = 0.000000001 and 1 x 10\u003csup\u003e-13 \u003c/sup\u003erespectively, from left to right; BTSC147 (\u003cstrong\u003ej\u003c/strong\u003e): ****\u003cem\u003eP\u003c/em\u003e = 1 x 10\u003csup\u003e-15\u003c/sup\u003e for each pairwise comparison \u003cstrong\u003ek-r\u003c/strong\u003e, CLIC1 CRISPR and control BTSCs were subjected to immunoblotting using antibodies to P-STAT3-Y705, total STAT3, EGFRvIII, and total EGFR. ACTIN was used as loading control. \u0026nbsp;Densitometric quantification of p-STAT3 to total STAT3 and (\u003cstrong\u003el,n\u003c/strong\u003e) and EGFRvIII to total EGFR (\u003cstrong\u003ep,r\u003c/strong\u003e) are presented. Statistical analysis was performed using a student t-test. n=3 biological replicates. BTSC73 (\u003cstrong\u003el\u003c/strong\u003e): *\u003cem\u003eP\u003c/em\u003e = 0.0121; BTSC147 (\u003cstrong\u003en\u003c/strong\u003e): *\u003cem\u003eP\u003c/em\u003e = 0.0423; BTSC73 (\u003cstrong\u003ep\u003c/strong\u003e): ***\u003cem\u003eP\u003c/em\u003e = 0.0003; BTSC147 (\u003cstrong\u003er\u003c/strong\u003e): ***\u003cem\u003eP\u003c/em\u003e = 0.0001 for each pairwise comparison.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6537096/v1/dfeec141874c7bd5dfd6d497.png"},{"id":81954264,"identity":"229cfe32-b6b5-49e1-af26-1b4db2587680","added_by":"auto","created_at":"2025-05-05 09:41:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6393464,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic deletion of CLIC1 impairs tumourigenesis, expands lifespan and suppresses EGFR/EGFRvIII activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-h, \u003c/strong\u003eCLIC1 CRISPR BTSCs (#73,) control BTSC73, CLIC1 CRISPR BTSCs (#147), and control BTSC147 were xenografted intracranially into 8 weeks old SCID male mice. IVIS imaging was conducted to evaluate tumour volume (\u003cstrong\u003ea,d\u003c/strong\u003e) and signal was quantified (\u003cstrong\u003eb,e\u003c/strong\u003e). Kaplan-Meier survival plots were graphed to evaluate animal lifespan in each group (\u003cstrong\u003ec,f\u003c/strong\u003e). Log-rank test (BTSC73) (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001, \u003cem\u003en\u003c/em\u003e ³ 5 mice), BTSC#73 vs. CLIC1 CRISPR-a (\u003cem\u003eP \u003c/em\u003e= 0.0011), BTSC#73 vs. CLIC1 CRISPR-b (\u003cem\u003eP \u003c/em\u003e= 0.001), CLIC1 CRISPR-a vs. CLIC1 CRISPR-b (\u003cem\u003eP \u003c/em\u003e= 0.0014). Log rank test for BTSC#147, (\u003cem\u003eP \u003c/em\u003e= 0.0078, n=3), BTSC#147 vs. CLIC1 CRISPR-a (\u003cem\u003eP \u003c/em\u003e= 0.0246), BTSC#147 vs CLIC1 CRISPR-b (\u003cem\u003eP \u003c/em\u003e= 0.0246), CLIC1 CRISPR-a vs CLIC1 CRISPR-b (non-significant). Representative panels for changes in body weight following BTSC implantation are shown (\u003cstrong\u003eg-h\u003c/strong\u003e). \u003cstrong\u003ei-k\u003c/strong\u003e, Brain sections depicting tumour in control BTSC73 and CLIC1-CRISPR BTSC73 were subjected to H\u0026amp;E staining (\u003cstrong\u003ei,j\u003c/strong\u003e) or immunostaining with a p-EGFR-Y1068 antibody (\u003cstrong\u003ek\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6537096/v1/2566683b117cb9b1508d9cee.png"},{"id":81956658,"identity":"97038c32-0aaf-46fc-a261-e97d36487545","added_by":"auto","created_at":"2025-05-05 09:57:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3015249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAn OSMR-CLIC1 cross talk regulates key oncogenic processes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-c, \u003c/strong\u003eCLIC1 CRISPR and control BTSCs were subjected to extracellular vesicle (EV) isolation. EV-digested lysates and whole cell lysates were subjected to immunoblotting analysis using antibodies indicated on the blot (\u003cstrong\u003ea\u003c/strong\u003e). Quantification of EGFRvIII in EV were conducted (\u003cstrong\u003eb,c\u003c/strong\u003e). Statistical analysis was performed using one-way ANOVA. n=3 biological replicates. BTSC147 (\u003cstrong\u003eb\u003c/strong\u003e): ***\u003cem\u003eP\u003c/em\u003e = 0.00025, ****\u003cem\u003eP\u003c/em\u003e = 0.000027; BTSC73 (\u003cstrong\u003ec\u003c/strong\u003e): ***\u003cem\u003eP\u003c/em\u003e = 0.00098, ****\u003cem\u003eP\u003c/em\u003e= 0.000059 for each pairwise comparison. \u003cstrong\u003ed-f\u003c/strong\u003e, Non-permeabilized BTSCs were subject to CLIC1 staining to depict plasma membrane. Nuceli are stained with Hoechst. Scale bar, 10 µm, n=3 biological replicates. \u003cstrong\u003eg\u003c/strong\u003e, Electrophysiological evaluation of transmembrane (tm)-CLIC1 was conducted at +40 mV on CLIC1 CRIPSR and control BTSCs. WT (white bar), CLIC1 +/- (monoallelic deletion, grey bar) and CLIC1 -/- (biallelic deletion, black bar). *\u003cem\u003eP\u003c/em\u003e = 0.024, **\u003cem\u003eP \u003c/em\u003e= 0.0091. \u003cstrong\u003eh-m\u003c/strong\u003e, CLIC1 CRISPR and control BTSCs were treated with a monoclonal antibody that specifically inhibit the membrane configuration of CLIC1 (tm-CLIC1-omab) for 72 hours and were analyzed by immunoblotting using antibodies to p-STAT3, STAT3, p-EGFR and EGFR. Densitometric analysis of p-STAT3 to total STAT3 \u003cstrong\u003e(h,j,k\u003c/strong\u003e) and p-EGFR to total EGFR (\u003cstrong\u003ei,l,m\u003c/strong\u003e) in tm-CLIC1-mab treated antibodies versus control are shown. Statistical analysis was performed using unpaired students’ t test. n = 3 or \u0026gt; 3 biological replicates. BTSC73 (\u003cstrong\u003eh\u003c/strong\u003e): **\u003cem\u003eP\u003c/em\u003e = 0.0022; BTSC73 (\u003cstrong\u003ei\u003c/strong\u003e): *\u003cem\u003eP\u003c/em\u003e = 0.0335; BTSC147 (\u003cstrong\u003ek\u003c/strong\u003e): **\u003cem\u003eP\u003c/em\u003e = 0.0038. \u003cstrong\u003en-q\u003c/strong\u003e, Representative whole cell current for cells were conducted at several voltages ranging from -60 to +60 mV with 20 mV increments in control (\u003cstrong\u003en,p\u003c/strong\u003e) and in OSMR knockdown BTSCs (\u003cstrong\u003eo,q\u003c/strong\u003e). Currents were recorded in resting conditions (\u003cstrong\u003en,o\u003c/strong\u003e) and after IAA94 administration (\u003cstrong\u003ep,q\u003c/strong\u003e). \u003cstrong\u003er\u003c/strong\u003e, I/V plot for control (SCR, black circles) and after perfusion of IAA94 (empty circles), and \u003cstrong\u003es\u003c/strong\u003e, I/V plot for shOSMR cells in CTL condition (black triangles) and after perfusion of IAA94 (empty triangles) are shown. Average current-voltage recordings at different test potentials are shown. \u003cstrong\u003et\u003c/strong\u003e, tmCLIC1 current for four different GB cell lines were analyzed at +40 mV in shOSMR and SCR control groups. The average current for the SCR (grey) and shOSMR (black) cells are shown. BTSC73 *\u003cem\u003eP\u003c/em\u003e = 0.0179; BTSC30 *\u003cem\u003eP\u003c/em\u003e = 0.0173, L1312 *\u003cem\u003eP\u003c/em\u003e = 0.0205.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6537096/v1/6af16aea6c559e3a447625c5.png"},{"id":82108723,"identity":"df42aa1e-df3f-4d2f-b1d8-5d59fabbb85e","added_by":"auto","created_at":"2025-05-06 22:58:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27572312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6537096/v1/c76216da-3b18-481a-97cd-90d3b7bd09a4.pdf"},{"id":81954260,"identity":"f8409543-fca9-4c53-b245-c9389574439a","added_by":"auto","created_at":"2025-05-05 09:41:58","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":49538,"visible":true,"origin":"","legend":"Supplementary Data Corresponding to Figure 1","description":"","filename":"mamthppisandinterpreyppis241217.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6537096/v1/95b733be127dcba21ace206d.xlsx"},{"id":81954281,"identity":"ae016bd4-7c1f-4808-bb9c-15d1e9e09fe8","added_by":"auto","created_at":"2025-05-05 09:41:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27841770,"visible":true,"origin":"","legend":"Supplementary Figures and Tables Related to the Manuscript","description":"","filename":"SupplementaryAPR242025Natcellbio.docx","url":"https://assets-eu.researchsquare.com/files/rs-6537096/v1/3da21d0f2fcc73bf582d783a.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Transmembrane CLIC1 sustains EGFRvIII–STAT3 oncogenic signaling in Glioblastoma through interaction with OSMR","fulltext":[{"header":"MAIN","content":"\u003cp\u003eGB is the most aggressive incurable brain tumour with a diverse genetic profile. The oncogenic EGFRvIII mutant is present in more than 30% of GB patients and is initially assigned to the classical GB subtype. The mesenchymal GB subtype has also been described as one of the most malignant brain tumours. The aggressive nature of classical and mesenchymal subtypes is attributed in large part to the activity of STAT3, endowing the cells with the ability to enter an epithelial-to-mesenchymal (EMT)-like state, maintain stemness, grow, and metastasize\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Recent studies have revealed that the cytokine receptor, OSMR, is a central player in propelling classical and mesenchymal GB subtypes. OSMR plays a crucial role in mediating oncogenic signal transduction initiated by the ligand OSM\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The OSM-OSMR induces the activation of STAT3 as well as key oncogenic pathways including the phosphoinositide 3-kinase (PI3K)\u0026ndash;AKT, c-Jun N-terminal kinases/mitogen-activated protein kinase (JNK/MAPK), and RAS/MAPK pathways\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. OSMR functions as a co-receptor for EGFRvIII in GB, inducing a positive feedback loop with STAT3\u003csup\u003e4\u003c/sup\u003e. It also plays a crucial role in upregulating mitochondrial respiration, and conferring resistance to ionizing radiation (IR) therapy\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Additionally, OSMR is a key regulator of brain tumour stem cell (BTSC) self-renewal\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and the immune microenvironment\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, suggesting that its function is highly context-dependent.\u003c/p\u003e \u003cp\u003eHere, we employed MaMTH-HTS, a technology uniquely suited to identifying interacting partners of integral membrane proteins. Our screening revealed that OSMR physically and functionally interacts with tmCLIC1, positioning it as a central player in both OSM/OSMR cytokine signaling and the OSMR/EGFRvIII co-receptor complex. We demonstrate that the genetic deletion of tmCLIC1 and pharmacological inhibition of tmCLIC1 impairs BTSCs, and oncogenic EGFRvIII/STAT3 signaling. Furthermore, our data show that OSMR is essential for the function of the membrane configuration of CLIC1, highlighting a previously unrecognized crosstalk between a cytokine receptor and an ion channel in driving key oncogenic pathways in GB. Our finding has important implications for the design of novel therapeutic strategies to suppress GB.\u003c/p\u003e\n\u003ch3\u003eMapping OSMR interactome via Mammalian Membrane Two-Hybrid High Throughput Screening (MaMTH-HTS)\u003c/h3\u003e\n\u003cp\u003eTo address the question of how OSMR networks with alternate molecules to confer its different functions, we performed MaMTH-HTS in HEK293T cells expressing OSMR bait, in the presence or absence of co-expressed EGFRvIII, alongside a prey library of ~\u0026thinsp;8000 open reading frames from the Human ORFeome V8.1 collection\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. We identified 334 high-confidence candidate binding partners for OSMR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), following rigorous analyses and exclusion of MaMTH-HTS \u0026lsquo;frequent flyers\u0026rsquo;, which was established from repeated MaMTH-HTS results of 9 \u0026lsquo;individual bait\u0026rsquo; protein control experiments, as previously described\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Among the identified candidates, 204 hits, including 59 PM proteins, were detected when OSMR was used as bait in the absence of EGFRvIII, and 160 candidate binding partners, including 40 PM proteins, were identified when OSMR was used as a bait in the presence of EGFRvIII. Importantly, our data revealed 30 candidate binding partners of OSMR that were commonly shared by both groups, 7 of which were PM proteins. The PM proteins encompassed proteins that are integral to the membrane as well as proteins that are associated with the membrane. GO term functional analysis of protein clusters in each group revealed molecules engaged in metabolic processes and signalling as top categories underscored in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, these analyses revealed that the GO term for immune system processes was highly enriched in OSMR-unique interactome, whereas the regulation of biogenesis processes was among top GO terms for OSMR-EGFRvIII category. This novel OSMR interactome map highlights its role in different biological processes with unique variations depending on a cell\u0026rsquo;s genetic signature.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of OSMR common binding partner and the involvement of CLIC1\u003c/h2\u003e \u003cp\u003eTo unravel the mechanism by which OSMR operates to promote tumour growth across different GB subtypes, we focused on the common candidate binding partners (\u003cb\u003eSupp. Table\u0026nbsp;1\u003c/b\u003e), which encompassed different classes of proteins including metabolic processes, signalling, biogenesis, development, and immune system processes. To validate MaMTH data in the context of GB, we employed four different patient-derived BTSCs that naturally harbour EGRFvIII mutation (BTSC73 and BTSC147) or lack the mutation (BTSC12, BTSC30) (\u003cb\u003eSupp. Table\u0026nbsp;2\u003c/b\u003e). We conducted a counter-screen in which we employed siRNA (\u003cb\u003eSupp. Table\u0026nbsp;3\u003c/b\u003e) targeting each of the genes encoding these proteins, followed by assessing cell viability. Our results revealed that knockdown (KD) of 12 genes demonstrated a substantial reduction in cell viability exceeding 50% across different BTSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-e). These included CLEC2B, CLIC1, CREB3, CSGALNACT2, DCN, EIF2C3, GC, GPBP1, HISTH3A, IMMP2L, NAT8, and ST3GAL4. Prior to follow up investigation on select candidates, we applied additional screening criteria pertaining to the known role of these proteins in BTSCs and GB as well as their known roles in regulation of different hallmarks of cancer. These criteria led us to focus on the CLIC1, as the top candidate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCLIC1 is highly expressed in various cancers including GB, with its expression significantly correlating with poor prognosis\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. CLIC1 exists as a soluble cytoplasmic protein, however, under specific cellular conditions that are favored in cancer cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, it can translocate to PM where it functions as an ion channel\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Interestingly, similar to OSMR\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, CLIC1 is highly expressed in the mesenchymal GB subtype and enriched in the cancer stem cells\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e where it promotes proliferation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To begin with, we electroporated a pool of four siRNAs targeting CLIC1 in different BTSCs and assessed cell viability and stem cell frequency using PrestoBlue and extreme limiting dilution assay (ELDA)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, respectively. RT-qPCR and immunoblotting analyses showed efficient siRNA-mediated knockdown of CLIC1 by greater than 85% and a significant decline in CLIC1 protein expression level relative to BTSCs electroporated with a non-targeting RNAi (siCTL) (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-i, \u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b). Our data revealed a significant decrease in BTSCs viability (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,d), BTSC sphere size (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee,f), and stem cell frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej-q) upon KD of CLIC1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCLIC1 interacts with both OSMR and EGFRvIII in patient derived BTSCs and is required for OSMR/EGFRvIII complex\u003c/b\u003e \u003c/p\u003e \u003cp\u003eVia conducting co-immunostaining studies using antibodies to OSMR and CLIC1, we observed that CLIC1 colocalizes with OSMR in different BTSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,d). We thus asked whether CLIC1 interacts with OSMR endogenously. We conducted Proximity Ligation Assay (PLA) using OSMR and CLIC1 antibodies in different BTSCs. Our data showed \u003cem\u003ein situ\u003c/em\u003e interaction of CLIC1 with OSMR across all BTSC lines that were examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-h), with EGFRvIII-expressing BTSCs that harbour elevated OSMR expression, exhibiting stronger signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee,f) compared to BTSCs lacking the EGFRvIII (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg,h). In parallel, we also conducted co-immunoprecipitation (co-IP) experiments using an antibody against endogenous OSMR followed by Western blotting (WB) with a CLIC1 antibody. We confirmed the results obtained with PLA via IP-WB in which we showed that OSMR interacts with CLIC1 endogenously in BTSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei,j).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we generated transgenic BTSCs in which we induced genetic deletion of CLIC1 using CRISPR in two different EGFRvIII-expressing BTSC73 and BTSC147 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek,l, \u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b). Similar to our findings with the transient KD experiments, monoallelic (CRISPR-a) or biallelic (CRISPR-b) genetic deletion of CLIC1 induced a robust reduction in BTSC stem cell frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em-p), cell viability (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d), and sphere size (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee,f). We next employed BTSCs harbouring monoallelic or biallelic deletion of CLIC1 and conducted PLA to confirm the specificity of the interaction signal. Our results showed that the PLA interaction signal of OSMR-CLIC1 was completely diminished in CLIC1 CRISPR BTSCs compared to control BTSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eq-t).\u003c/p\u003e \u003cp\u003eGiven that OSMR forms a co-receptor with EGFRvIII, we next asked whether CLIC1 is a component of the same complex with EGFRvIII. We conducted co-immunostaining analysis in different BTSCs and found colocalization of EGFRvIII with CLIC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). Thus, we performed PLA experiments using antibodies to EGFRvIII and CLIC1 in control and CLIC1 CRISPR BTSCs. Our results showed \u003cem\u003ein situ\u003c/em\u003e interaction of CLIC1 with EGFRvIII (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,d). Together, we established that CLIC1 forms a complex with both OSMR and EGFRvIII in patient-derived BTSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCLIC1 is required for OSMR/EGFRvIII interaction and STAT3 phosphorylation\u003c/h3\u003e\n\u003cp\u003eOSMR is found in complex with EGFRvIII to amplify receptor tyrosine kinase (RTK) signalling\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Given our results that CLIC1 interacts with both OSMR and EGFRvIII, we asked whether CLIC1 is required for the OSMR/EGFRvIII interaction. First, we confirmed the interaction of OSMR and EGFRvIII in BTSC73 and 147 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee,f). Next, we subjected CLIC1-CRISPR and corresponding control BTSCs to PLA analysis using antibodies to OSMR and EGFRvIII. Strikingly, genetic deletion of CLIC1 significantly impaired the interaction of OSMR with EGFRvIII in each of BTSC147 and BTSC73 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-j), revealing that CLIC1 is required for OSMR/EGFRvIII complex. These data raised the question of whether CLIC1 is functionally required for maintaining the oncogenic OSMR or OSMR/EGFRvIII signalling. We conducted immunoblotting experiments in CLIC1 CRISPR and control BTSC73 and BTSC147 using antibodies to STAT3-Y-705 and EGFRvIII. Strikingly, we found significant attenuation in the expression of the phospho-STAT3 and EGFRvIII in CLIC1 CRISPR BTSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek-r). Our data suggest that CLIC1 tightly maintains the activation of EGFRvIII and STAT3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCLIC1 drives GB tumourigenesis and maintains EGFRvIII/EGFR phosphorylation\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur findings on a central role of CLIC1 in regulation of OSMR/EGFRvIII interaction and EGFRvIII and STAT3 phosphorylation led us next to examine whether CLIC1 contributes to GB tumourigenesis \u003cem\u003ein vivo\u003c/em\u003e. We conducted intracranial tumour assay using BTSC73, BTSC147, and corresponding CLIC1 CRISPR BTSCs lines. Our data revealed that deletion of CLIC1 significantly impaired tumourigenesis, and prolonged survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-h). We next subjected tumour sections obtained from the control mice as well as the smaller tumours from the CLIC1 CRISPR group to H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei,j) and immunostaining analysis using a phospho-Y1068-EGFR antibody that can detect both the phosphorylated wild type (WT) EGFR and EGFRvIII (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek). Strikingly, we found that the phosphorylation of EGFR/EGFRvIII was robustly attenuated in the mice group xenografted with CLIC1 CRISPR BTSCs. Our analysis established that the genetic deletion of CLIC1 significantly reduces the tumor size and impairs the activation of EGFR/EGFRvIII \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePharmacological inhibition of the transmembrane conformation of CLIC1 (tmCLIC1) using a monoclonal antibody impairs the phosphorylation of EGFRvIII and STAT3\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur data raised the question of how CLIC1 maintains EGFRvIII-STAT3 oncogenic pathway. CLIC1 is found as a soluble cytoplasmic protein as well as a transmembrane protein that forms an ion channel\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. We conducted cell fractionation experiments in different BTSCs and found that CLIC1 is localized to both the membrane and cytoplasm (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), although, the majority of CLIC1 was found in the cytoplasm. CLIC1 is shown to be packaged into extracellular vesicles (EVs) in order to target neighbouring cells\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, promoting tumour growth\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Interestingly, similar to CLIC1\u003csup\u003e17,23,25\u003c/sup\u003e, EGFRvIII impacts various aspects of tumourigenesis not only in the cell of origin, but in the neighbouring cells, also shown to be mediated via EVs\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Thus, we questioned whether CLIC1 is required for the horizontal propagation of EGFRvIII to the neighboring cells. By extracting EVs from CTL BTSCs and their corresponding CLIC1 CRISPR cells, we evaluated EGFRvIII protein expression levels in the cell lysates and EV fractions. Immunoblotting analysis revealed that EGFRvIII levels were significantly reduced in CLIC-deleted EVs, suggesting a role for CLIC1 in mediating the horizontal propagation of EGFRvIII to the neighboring cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-c, \u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b\u003cb\u003e)\u003c/b\u003e. At the same time, these data raised the question of whether tmCLIC1contributes to maintaining intracellular EGFRvIII/STAT3 signalling via altering CLIC1-mediated ionic conductance. tmCLIC1 is proposed to regulate cell cycle, pH, cell volume, and cell proliferation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Given the smaller fraction of tmCLIC1 in BTSCs (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), we first aimed to conduct immunostaining on non-permeabilized BTSCs using a CLIC1 antibody. Imaging analysis revealed a strong CLIC1 signal in non-permeabilized BTSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-f). Thus, we set out to investigate the membrane localization and electrophysiological properties of tmCLIC1 in three distinct human BTSC lines including BTSC73, BTSC147, and BTSC30. To assess basal level tmCLIC1 levels, we used IAA94, a known blocker of CLIC1. We conducted an electrophysiological evaluation of tmCLIC1 levels at +\u0026thinsp;40mV on the membranes of CLIC1 CRISPR and control BTSCs. Our results revealed that the tmCLIC1 currents were significantly reduced in the CLIC1 CRISPR lines compared to the CTL BTSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg), leading to the question of whether tmCLIC1 impacts EGFRvIII/STAT3 signalling. To address this question, we generated and employed a monoclonal CLIC1 antibody (tmCLIC1omab\u0026reg;) that specifically inhibits tmCLIC1 and its associated ionic currents. We treated different glioma cells and BTSCs with the tmCLIC1omab or IgG control, followed by an analysis of EGFRvIII and STAT3 phosphorylation in response to the ligand OSM. The tmCLIC1 current was measured as the IAA94-sensitive current, defined as the difference between the initial current and the current after drug perfusion. Our data revealed that both the EGFR phosphorylation at tyrosine (Y) 1068 and STAT3 phosphorylation at Y705 were significantly attenuated upon inhibition of the CLIC1-mediated Cl- current by a tmCLIC1 antibody in EGFRvIII expressing BTSC 147 and 73. Importantly, tmCLIC1omab\u0026reg; had no impact on EGFRvIII-STAT3 signalling in BTSC30 that naturally does not harbour the EGFRvIII or elevated STAT3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh-m, \u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eImpact of OSMR on CLIC1-Mediated Cl Current\u003c/h3\u003e\n\u003cp\u003eWe have established that OSMR physically and functionally interacts with CLIC1 to maintain EGFRvIII/STAT3 oncogenic signalling in BTSC and GB and a PM CLIC1 is engaged in regulating this process. Next, we set out to investigate whether OSMR is required for CLIC1-mediated Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e current. We induced KD of OSMR using short hairpin RNA (shOSMR) in different cell lines and subjected the OSMR knockdown (KD) and control (CTL) Scrambled (SCR) cells to patch clamp experiments to evaluate whether reduced OSMR expression levels alter Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e currents. Cell currents were assessed both prior to (baseline) and following the addition of 100 \u0026micro;M of CLIC1 inhibitor, IAA94 to the bath solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003en-s). The tmCLIC1 current was measured as the IAA94-sensitive current, defined as the difference between the initial current and the current after drug perfusion. The corresponding current-voltage relationship was recorded in each group. Strikingly, our data revealed that inhibition of OSMR significantly attenuated the tmCLIC1-associated Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e current (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003er-t). Taken together, we provide data that tmCLIC1 regulates OSM/OSMR-STAT3 and OSMR/EGFRvIII oncogenic signalling and at the same time OSMR promotes tmCLIC1 function. We establish that the cooperation of OSMR with tmCLIC1 is required to maintain key oncogenic pathways in GB. Our study provides new opportunities to develop novel therapeutic strategies for GB.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we comprehensively mapped the OSMR interactome to identify its high confidence binding partners under different conditions leading to a better understanding of OSMR\u0026rsquo;s multifaceted roles in GB progression. Through systematic protein-protein interaction mapping using MaMTH-HTS, followed by counter-screening and loss- and gain-of-function studies, we demonstrate that tmCLIC1 interacts with OSMR physically, independently of cellular identity, and acts as a critical regulator of diverse oncogenic pathways driven by OSMR, EGFRvIII, and STAT3. We demonstrate a bidirectional relationship for tmCLIC1-OSMR interaction, which plays a pivotal role in regulating ion conductance, thereby driving GB progression.\u003c/p\u003e \u003cp\u003eCLIC1 has been implicated in multiple malignancies, including lung cancer\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, pancreatic adenocarcinoma\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, epithelial ovarian cancer\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and medulloblastoma\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It is associated with poor prognosis in GB and is abundantly expressed in cancer stem cells (CSCs)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Previous studies have highlighted the role of tmCLIC1 in cellular proliferation and viability, with genetic deletion leading to cell swelling, mitotic defects, and reduced proliferation in medulloblastoma cells\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. tmCLIC1 may act as a cell cycle accelerator by mediating cell volume changes. For instance, during the prophase to metaphase transition, cells undergo a significant volume decrease, reaching a minimum size at metaphase. This reduced volume is preferred by the cell and is referred to as pre-mitotic condensation\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Pre-mitotic condensation requires the efflux of Cl-, which may be mediated by tmCLIC1. tmCLIC1 has also been shown to play a prominent role in ROS production\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and cell cycle regulation\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. For example, IAA94-mediated inhibition of tmCLIC1 is shown to prolong the G1 phase, thereby extending the overall cell cycle duration\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In agreement with these findings, our data show that CLIC1 deletion suppresses BTSC self-renewal and proliferation, underscoring its role in cell cycle regulation. Given tmCLIC1's established function in maintaining redox balance\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, its knockdown likely elevates oxidative stress and disrupts redox homeostasis, thereby impairing BTSC proliferative capacity and self-renewal potential. These findings reinforce the idea that CLIC1 is a critical regulator of GB cell division and tumour aggressiveness. The interplay between ROS, tmCLIC1-generated chloride currents, combined with evidence of tmCLIC1/OSMR interaction, highlights the need to investigate CLIC1's role in in cancer stem cells\u0026rsquo; metabolism in the context of GB. Whether similar to OSMR\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, tmCLIC1 regulates oxidative phosphorylation (OXPHOS) to maintain BTSCs and buffer ROS in the mitochondria remains to be investigated.\u003c/p\u003e \u003cp\u003eWe further demonstrate a crucial role for CLIC1 in maintaining EGFRvIII intracellularly. This effect may also be attributed to CLIC1's role in tumour microenvironment modulation, as it is positively associated with stromal activation, epithelial-mesenchymal transition (EMT), and angiogenesis, hallmarks of GB progression\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Chloride channels, including CLIC1, have been implicated in innate and adaptive immune responses, particularly in phagocyte-mediated immunity. CLIC1 is required for phagosomal acidification and antigen processing, which are crucial for CD4⁺ T cell activation\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. CLIC1-mediated regulation of macrophage-driven inflammation and ROS production may facilitate the establishment of a tumour-promoting microenvironment conducive to tumour growth and metastasis. Furthermore, CLIC1 plays a role in the innate immune system through its involvement in the activation of NLRP3 inflammasome\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Aberrant activity of the NLRP3 inflammasome has been implicated in the progression of several cancers including glioma\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Additionally, tmCLIC1 modulates Cl⁻ currents in activated microglia, particularly in response to β-amyloid protein (Aβ) stimulation\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Interestingly, OSMR and its ligand OSM operate in a paracrine fashion in which OSM secreted via immune cells typically binds the receptor on the tumour cells to direct oncogenic signalling. These findings suggest that OSMR-CLIC1 cross talk may influence immune surveillance, an area that requires future investigation.\u003c/p\u003e \u003cp\u003eIt is also possible that loss of CLIC1 induces a cell shift towards a more differentiated phenotype. Previous research on cell fate dynamics supports this notion. Stockhausen et al. demonstrated that induced differentiation of BTSCs with serum leads to loss of EGFRvIII expression and decreased tumourigenic potential\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In another study by Gritti et al., CLIC1 expression was lost upon induction of differentiation with FBS\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. These results suggest that both EGFRvIII and CLIC1 are important in maintaining stemness. Similarly, in another study by He et al., the chloride channel, Ano1, was found to limit the differentiation of epithelial progenitors towards cells of the secretory lineage\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Further studies are required to determine if the interaction of CLIC1 with EGFRvIII aids in establishing a cellular network that suppresses the BTSCs differentiation.\u003c/p\u003e \u003cp\u003eIn this study, we provide data showing that CLIC1 regulates the horizontal transfer of EGFRvIII via EV. We observed a striking attenuation of EGFRvIII in EVs derived from CLIC1-CRISPR cells, suggesting that CLIC1 may regulate EGFRvIII packaging, thereby influencing neighboring cells. In support of this model, tmCLIC1 vesicle transfer has been implicated in glioma stem cell proliferation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Furthermore, blocking of the chloride current in stem-like cells through the re-purposing of known pharmacological drugs, suggests that tmCLIC1 could be a promising target for therapy\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Similarly, tumour cells in GB are shown to impact neighboring cells via transferring EGFRvIII through EV\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Another study in gastric cancers revealed that the presence of CLIC1 in EVs are associated with resistance to vincristine by transferring resistance traits to neighboring cells\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Whether tmCLIC1 functional expression represents an adaptive mechanism enabling drug resistance in GB remains an exciting area of research to be explored.\u003c/p\u003e \u003cp\u003eOur findings have established that tmCLIC1 interaction with OSMR and EGFRvIII is essential for sustaining oncogenic signaling. Using a monoclonal CLIC1 antibody that can only target tmCLIC1, we establish the significance of tmCLIC1, however, the functional contribution of cytoplasmic CLIC1 and its involvement in mitochondrial and metabolic pathways remains to be investigated in GB. In conclusion, our findings establish tmCLIC1 as a central player in GB pathogenesis, orchestrating cytokine networks and enhancing the RTK signalling. Its interaction with OSMR and EGFRvIII is critical for sustaining BTSC self-renewal and GB tumourigenesis. These results highlight tmCLIC1 as a promising therapeutic target, suggesting that its inhibition, in combination with standard GB treatments, may lead to improved patient outcomes.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBrain tumour Stem Cell (BTSC) and generation of transgenic cell lines\u003c/h2\u003e \u003cp\u003eThe human BTSC lines 73, 147, 12, and 30 were generously provided by Dr. Samuel Weiss at the University of Calgary. They were generated in accordance with the University regulation from excess or discarded tissue collected during surgery from adult patients following informed consent from patients. BTSCs were characterised for major mutations (\u003cb\u003eSupp. Table\u0026nbsp;2\u003c/b\u003e) and were cultured and maintained, as described in Supplementary methods. Transgenic CRISPR BTSCs were generated, using methods previously described\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. To design the gRNA, Off-Spotter software, version 0.2.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cm.jefferson.edu/Off-Spotter/\u003c/span\u003e\u003cspan address=\"https://cm.jefferson.edu/Off-Spotter/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), was used. Two guide RNA strands (forward and reverse complement) were generated to target exons 5\u0026ndash;9 of the CLIC1 gene. To generate the construct, the Golden Gate Assembly Cloning strategy\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e was used in which gRNA1 and gRNA2 were cloned into pL-CRISPR.EFS.GFP (Addgene plasmid #57818), and pL-CRISPR.EFS.tRFP (Addgene plasmid, #57819), plasmids, respectively. Plasmids were sequenced (Genome Quebec), verified and electroporated [1300 volts by the AMAXA nucleofector 2b device (Lonza, #AAB1001) to deliver 3 \u0026micro;g of each plasmid construct (gRNA1-GFP and gRNA2-RFP) into ~\u0026thinsp;2\u0026nbsp;million BTSC147 or BTSC73]. Electroporated cells were cultured in a T-75 low attachment flask at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e. 48 hours after electroporation, BTSC spheres were dissociated into a single cell suspension using Accumax dissociation solution (Innovative Cell Technologies, #AM105) and subjected to Fluorescent Activated Cell Sorting (FACS) analysis using the BD FACSAriaTM Fusion (BD Biosciences) to sort for double positive GFP and RFP cells. Sorted cells were plated at a density of 1 cell/well into two 96-well plates containing 100 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\mu\\:}\\)\u003c/span\u003e\u003c/span\u003el of BTSC media. Wells were monitored every two days to assess sphere formation and clonal samples were collected from multiple positive clones and were subject to genomic DNA isolation. Isolated DNA was analysed by PCR using internal primers and external primers to the gRNA-guided CAS9 cut site, designed using Primer3Plus software, version 3.3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.primer3plus.com/\u003c/span\u003e\u003cspan address=\"https://www.primer3plus.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), to determine monoallelic, biallelic, or non-deletion control clones. Monoallelic deletion clones in both BTSC73 and BTSC147 cell lines were determined by PCR in which the presence of one internal and one external band on a 2% Agarose gel, was observed, while a biallelic deletion was determined by a single external band. Knockout or knockdown of CLIC1 was validated using RT-qPCR and WB to assess gene and protein expression. Since there we no clones identified that presented no cuts in the CLIC1 gene following incubation with the CRISPR-CAS9 construct, which could have been used as a control for CAS9, the chosen control for experiments using the CRISPR-CAS9-CLIC1 cells was that of the parental BTSC73 or BTSC147 line. The gRNA and primer sequences used in this experiment are listed in \u003cb\u003eSupp. Table\u0026nbsp;4\u003c/b\u003e. Finally, short Interfering RNA (siRNA) was used to generate transient knockdown (KD) in patient-derived BTSCs. BTSCs were processed into a single-cell suspension. ON TARGET-plus SMART pool human CLIC1 siRNA (Dharmacon, #L-009530-00-0005) at a concentration of 100 nM, and ON TARGET-plus non-targeting pool (Dharmacon, #D-001810-10-05), were employed, as described in supplemental methods.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMammalian Membrane Two-Hybrid-High Throughput Screen (MaMTH-HTS)\u003c/h3\u003e\n\u003cp\u003eTo identify binding partners of OSMR in the presence and absence of EGFRvIII, Mammalian Membrane Two-Hybrid (MaMTH) High Throughput Screen (HTS) technology was used as described\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Briefly, plasmid expressing OSMR \u0026lsquo;Bait\u0026rsquo; protein with C- terminally fused MaMTH-HTS Bait tag (Cub-GAL4TF-P2A-tagBFP) alone or alongside plasmid expressing EGFRvIII (fused with 3xFLAG at its N-terminus) was transfected into a pooled \u0026lsquo;Prey\u0026rsquo; library of HEK293T MaMTH-HTS reporter cell lines. The library of reporter cell lines stably expressed members of the Human ORFeome V8.1 collection (~\u0026thinsp;8000 open reading frames - ORF\u0026rsquo;s) fused to MaMTH-HTS Prey tag (Nub) at their N-terminus and P2A-mCherry at their C-Terminus and contained chromosomally integrated GFP reporter under the control of the GAL4 transcription factor (GAL4TF) promoter. Transfections were performed using X-tremeGene\u0026trade; 9 Transfection Reagent (Roche, XTG9-RO) as specified by the manufacturer protocol. In order to induce Bait (\u003cb\u003eSupp. Figure\u0026nbsp;7\u003c/b\u003e) and Prey expression, cells were grown for 2\u0026ndash;3 days in the presence of 0.5 \u0026micro;g/ml Tetracycline in the following conditions: 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e in DMEM containing 10% FBS, and 1% Penicillin/Streptomycin media. Cells were then harvested by trypsinization and resuspended at a concentration of 1\u0026ndash;2 x 10\u003csup\u003e6\u003c/sup\u003e cell/ml in Basic Sorting Buffer (1X PBS, 5 mM EDTA, 25 mM HEPES pH 7.0, 1% BSA) and subjected to sorting by Flow Cytometry using BD FACSMelody (BD Biosciences). Cells were sequentially selected according to the following: tag-BFP fluorescence indicating \u0026lsquo;Bait\u0026rsquo; expression, mCherry fluorescence indicating \u0026lsquo;Prey\u0026rsquo; expression, and GFP fluorescence indicating Bait-Prey interaction. Cells were collected for analysis in DMEM containing 25% FBS and centrifuged pellets were processed in Phire Tissue Direct Dilution Buffer (ThermoFisher Scientific). Amplification of ORFs was done using Phire Tissue Direct PCR Master Mix (ThermoFisher Scientific) and products were purified using QIAquick PCR Purification Kit (Qiagen). Purified PCR products were subjected to Nextera XT library preparation and deep sequencing using the Illumina HiSeq 2500 system (150 bp single read). Sequencing data was processed and hits were identified using custom software developed using R programming language and integrated Bowtie2 alignment tool, version 2.5.2\u003csup\u003e53\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eCounter-screen assay\u003c/h3\u003e\n\u003cp\u003eBTSCs were transfected with siRNA using RNAiMax in a 96-well plate. Briefly, 1 pmol of siRNA and 0.1 \u0026micro;l of RNAiMax were diluted in 5 \u0026micro;l of Opti-MEM and incubated separately for 5 minutes. The solutions were then gently combined and incubated at room temperature for 15 minutes to form transfection complexes. A total of 1,000 BTSCs per well were seeded in 80 \u0026micro;l of NeuroCult medium, followed by the addition of 10 \u0026micro;l of the siRNA-RNAiMax complex with gentle shaking. After incubation at 37\u0026deg;C for 5 hours, 90 \u0026micro;l of NeuroCult medium was added to each well, and cells were further incubated at 37\u0026deg;C for 48 hours. Transfected cells were then subjected to the PrestoBlue assay to assess cell viability.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDuolink Proximity Ligation Assay (PLA)\u003c/h2\u003e \u003cp\u003eProximity Ligation Assay was conducted following the manufacturer's protocol using the Duolink \u003cem\u003eIn Situ\u003c/em\u003e Red Starter Kit (Sigma, #DUO92101) as optimized by our group\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, and described in the supplemental Methods section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExtreme Limiting Dilution Assay (ELDA)\u003c/h2\u003e \u003cp\u003eELDA was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, with detailed methods presented in the supplemental Methods section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStereotaxic injections and bioluminescent imaging\u003c/h2\u003e \u003cp\u003eStereotaxic surgeries were conducted by injecting\u0026thinsp;~\u0026thinsp;3 x 10\u003csup\u003e5\u003c/sup\u003e luciferase expressing CLIC1-CRISPR-BTSCs and control BTSCs into the right striata (0.8 mm lateral to the bregma, 1 mm dorsal, and 2.5 mm from the pial surface). Prior to injection, BTSCs were dissociated into single-cell suspensions in serum-free, antibiotic-free medium and tested for luciferase activity using IVIS imaging system. Kaplan-Meier survival plots were generated by collecting mice at the point of reaching the end stage (major body weight loss, dehydration, hunched back, piloerection, and lethargy). Median survivals were calculated using a log-rank test with GraphPad Prism, following previously described methods\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Briefly, the Kaplan-Meier approach estimated the probability of survival at each time point. Before any time elapses, all mice were considered \u0026ldquo;at risk\u0026rdquo;, however, no deaths occurred. Therefore, the probability of survival was input as a value of 1. Subsequently, information about the next elapsed time (day 7) included information about the how many participants experienced the event of interest (death). From this information, a survival probability was calculated using the formula: S\u003csub\u003et+1\u003c/sub\u003e = S\u003csub\u003et\u003c/sub\u003e*((N\u003csub\u003et+1\u003c/sub\u003e-D\u003csub\u003et+1\u003c/sub\u003e)/N\u003csub\u003et+1\u003c/sub\u003e), where S\u003csub\u003et\u003c/sub\u003e represents survival probability, N\u003csub\u003et\u003c/sub\u003e represents number at risk, D\u003csub\u003et\u003c/sub\u003e represents number of events. The survival probability for each subsequent time point is calculated in a similar fashion and a stair-step survival curve is plotted. For tumour volume assessment, the mice received intraperitoneal injections of 200 \u0026micro;l of 15 mg/ml d-luciferin (ThermoFisher Scientific, #88292), underwent anesthesia via isoflurane inhalation, and were then subjected to weekly bioluminescence imaging using a CCD camera (IVIS, Xenogen). Subsequent collection and analysis of all bioluminescent data were performed utilizing Living Image 2.0 software (PerkinElmer, MA, USA). Briefly, after initialization of the IVIS Spectrum system and acquiring an image, the images were analyzed using \u0026ldquo;ROI tools\u0026rdquo; in the tool palette, selecting the area of interest. The IVIS imager captures pixels with specific photon intensity values, with brighter areas corresponding to higher photon detection, indicative of tumour induction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePatch-Clamp Experiment\u003c/h2\u003e \u003cp\u003ePatch electrodes (BB150F-8P with filaments, Science Products), with a diameter of 1.5 mm, were pulled from hard borosilicate glass on a Brown-Flaming P-87 puller (Sutter Instrument, Novato, CA) and fire polished to a tip diameter of 1-1.5 \u0026micro;m and an electrical resistance of 3\u0026ndash;4 Mꭥ. Cells were voltage-clamped using an Axopatch 200B amplifier (Axon Intrument) in whole-cell configuration in which After formation of giga-seal, the membrane patch is disrupted providing a direct low resistance access to cell interior, allowing recordings from ion channels of whole cell. The voltage step protocol used to isolate current/voltage relationships consisted of 800 ms pulses from \u0026minus;\u0026thinsp;60 mV to +\u0026thinsp;60 mV (20 mV voltage steps). The holding potential was set according to the resting potential of the single cell (between \u0026minus;\u0026thinsp;40 and \u0026minus;\u0026thinsp;80 mV). tmCLIC1-mediated chloride currents were isolated from other ionic currents by perfusing IAA94 (100 \u0026micro;M) dissolved in the bath solution and by mathematical subtraction of the residual current from the control. Solution used are the following: bath solution (mM): 125 NaCl, 5.5 KCl, 24 HEPES, 1 MgCl2, 0.5 CaCl2, 5 D-Glucose, 10 NaOH; pH 7.4; Pipette solution (mM): 135 KCl, 10 Hepes, 10 NaCl; pH 7.4. Analysis was performed using Clampfit 10.2 (Molecular Devices) and OriginPro 9.1. tmCLIC1-mediated current (IAA94-sensitive current) was measured by analytical subtraction of residual ionic current after addition of inhibitor from total current (I\u003csub\u003eTOT\u003c/sub\u003e) of the cell at each membrane potential tested. Current/voltage relationship were constructed plotting the averaged current density of the least 100 ms of the pulse against the corresponding membrane potential. Current density (pA/pF) results from the ratio between the ionic current (pA) and cells capacitance (pF). Statistical analyses were performed comparing the slopes (proportional to channel conductance) of the I/V curves of the different groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of monoclonal tm-CLIC1 antibody\u003c/h2\u003e \u003cp\u003eThe tmCLIC1omab\u0026reg; antibody was designed (license# 11665M), targeting the N-terminal of the CLIC1 protein derived from mice. Polyclonal antibodies were obtained through a 138-day immunization protocol in which mice are immunized against a NH\u003csub\u003e2\u003c/sub\u003e-CLIC1 synthetic peptide conjugated to OVA (ovalbumin) (NH\u003csub\u003e2\u003c/sub\u003e-EQPQVELFVKAGSDGAKIGNC-COOH) (Glycine 0.1 M pH 3, Tris-HCl 1 M pH 8). Hybridoma cells that produced antibodies were sorted in single cells to obtain monoclonal antibodies. tmCLIC1omab\u0026reg; was used at a final concentration of 3.5 \u0026micro;g/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eExtracellular vesicle isolation and validation\u003c/h2\u003e \u003cp\u003eTo sequentially isolate EVs, the cell supernatant was centrifuged at 200 x g for 10 minutes, 300 x g for 10 minutes, 2,000 x g for 10 minutes, 10,000 x g for 30 minutes, and 100,000 x g for 2 hours, respectively (Optima XPN-100 ultracentrifuge, Beckman Coulter, USA). A beige/white pellet of EVs and a clear supernatant were evident post-centrifugation. After removing the supernatant and resuspending the small EV pellet in 1 ml PBS, the resulting pellet was then washed and centrifuged at 49,000 rpm for 30 minutes (Optima MAX 130K Refrigerated Benchtop Ultracentrifuge, Beckman Coulter, USA). Isolated EVs were then visualized, measured, counted, and characterized in the range of 10\u0026thinsp;\u0026minus;\u0026thinsp;2,000 nm using a Nanoparticle Tracking Analyzer (NTA) (Particle Metrix ZetaView\u0026reg; Nanoparticle Tracking Analysis)\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. We used size-mode analysis with ZetaView software (version 8.02.28) following calibration with polystyrene beads (105 and 500 nm). Samples were analyzed at a minimum of 9 camera positions with 2-second video length at 21\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic analysis\u003c/h2\u003e \u003cp\u003eNovel interactions from MAMTH screen were integrated with the known interactions from the Integrated Interaction Database IID ver. 2021\u0026thinsp;\u0026minus;\u0026thinsp;05\u003csup\u003e59\u003c/sup\u003e and further annotated with Gene Ontology Biological Process and Cellular Component (release 2022-11-03), and a subset of pathways from the PathDIP database ver. 4\u003csup\u003e60\u003c/sup\u003e. Resulting network was built, annotated and analyzed using NAViGaTOR ver. 3.0.19\u003csup\u003e61\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis between conditions was performed using a Student \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA in which the mean of n\u0026thinsp;=\u0026thinsp;3 replicates were compared between two conditions to assess significance. Analysis was undertaken with the aid of GraphPad software 7. Data is shown as mean with standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). p-values less than 0.05 were considered significant and were marked with an asterisk as follows, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Precise p values are provided in the figure legends, unless noted otherwise.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eThis work was supported by CIHR grants to A.J.-A and I.S., and J.R. (# 162198, #519474) and a AIRC IG grant (#24758) to M.M.; A.J.-A. is a Canada Research Chair at the University of Ottawa and is supported by Canada Foundation for Innovation (CFI) and Ontario Research Fund (ORF). I.S. is supported by CFI (#225404, #30865), ORF (RDI #34876, RE010-020), and Natural Sciences Research Council (NSERC RGPIN-2024-04314). IJ was supported in part by funding from Natural Sciences Research Council (NSERC RGPIN-2024-04314), CIHR (#519474), Canada Foundation for Innovation (CFI #225404, #30865), and Ontario Research Fund (RDI #34876, RE010-020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHara, T.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Interactions between cancer cells and immune cells drive transitions to mesenchymal-like states in glioblastoma. \u003cem\u003eCancer cell\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 779-792. e711 (2021).\u003c/li\u003e\n \u003cli\u003eAraujo, A. 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R.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e NAViGaTOR: network analysis, visualization and graphing Toronto. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 3327-3329 (2009).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6537096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6537096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOncostatin M receptor (OSMR) plays diverse and important roles in several human malignancies, including brain, breast, and pancreatic cancer\u003csup\u003e1-4\u003c/sup\u003e. Glioblastoma (GB) is the most malignant genetically diverse brain tumour, with no cure. The most common genetic mutation in GB is a truncated active mutant of epidermal growth factor receptor (EGFR), the EGFRvIII. OSMR orchestrates a feedforward signaling mechanism with EGFRvIII and the signal transducer and activator of transcription 3 (STAT3), to drive GB progression\u003csup\u003e4\u003c/sup\u003e. Beyond EGFRvIII, OSMR promotes brain tumour stem cells (BTSCs) via upregulation of mitochondrial oxidative phosphorylation and contributes to therapy resistance\u003csup\u003e5\u003c/sup\u003e. The molecular mechanisms underlying the multifaceted roles of OSMR in different contexts are largely unclear. Here, we systematically mapped the OSMR interactome using Mammalian Membrane Two-Hybrid High-Throughput Screening (MaMTH-HTS). This unbiased approach led to the identification of OSMR-specific and OSMR/EGFRvIII-specific binding proteins, revealing context-dependent OSMR functions. Among a subset of common interactors, we uncovered chloride intracellular channel 1 (CLIC1) as a critical regulator of both OSMR-STAT3 signaling and the OSMR/EGFRvIII complex in GB. CLIC1 physically associates with both OSMR and EGFRvIII and plays a key role in EGFRvIII packaging into extracellular vesicles (EVs). Genetic deletion of CLIC1 disrupts the OSMR/EGFRvIII interaction, impairs STAT3 activation, reduces EGFRvIII EV content, and slows GB progression. Using whole-cell patch-clamp recordings and a monoclonal antibody that selectively targets transmembrane CLIC1 (tmCLIC1omab), we establish a distinct pharmacologically and biophysically tmCLIC-mediated current in GB indispensable for sustaining EGFRvIII/STAT3 signaling. Importantly, we show that OSMR is required for maintaining CLIC1-mediated ionic balance at the plasma membrane (PM). Our study uncovers a bidirectional cross-talk between OSMR and tmCLIC1 in GB, which is essential for fueling its malignant growth.\u003c/p\u003e","manuscriptTitle":"Transmembrane CLIC1 sustains EGFRvIII–STAT3 oncogenic signaling in Glioblastoma through interaction with OSMR","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 09:41:54","doi":"10.21203/rs.3.rs-6537096/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"753c6940-bf8e-41d7-a530-6f52eed3eca0","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47810893,"name":"Biological sciences/Stem cells/Cancer stem cells"},{"id":47810894,"name":"Biological sciences/Cancer/Cancer genetics"},{"id":47810895,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":47810896,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-05-06T22:50:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 09:41:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6537096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6537096","identity":"rs-6537096","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}