MARCKS as a Target for Pathological Tunneling Nanotubes in Glioblastoma

MARCKS as a Target for Pathological Tunneling Nanotubes in Glioblastoma | 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 MARCKS as a Target for Pathological Tunneling Nanotubes in Glioblastoma Christopher Willey, Lauren Nassour-Caswell, Mayada Ahmed, Shane Rich-New, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6479274/v2 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 2 posted 7 You are reading this latest preprint version Show more versions Abstract During glioblastoma (GBM) progression, therapeutic resistance is influenced by a heterogeneous network of tumor- and tumor-promoting cells in the tumor microenvironment. Biological attacks against tumor cells (i.e. chemoradiotherapy) induce tumoral defense mechanisms bolstered by sophisticated communication mechanisms and aberrant signaling pathways. Tunneling nanotubes (TNTs) have been well documented to mediate this process by aiding the metabolic rescue of tumor cells or facilitating the recruitment and reprogramming of normal cells to become tumor-supportive. GBM brain tumor-initiating cells (BTIC) target normal human astrocytes (NHA) using TNTs, therefore investigating this interaction and the potential mediators involved is critical. Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) has never been investigated as a potential regulator of TNTs despite several overlapping signaling pathways. In the present study, we demonstrate a role for the MARCKS effector domain (ED) and PKC activation in the formation and functionality of TNTs between GBM BTICs and NHAs. We employ a cell-penetrable peptide derived from MARCKS effector domain (MED2), PKC-targeting drugs, and an inducible MARCKS ED U87 model to elucidate a potential role for MARCKS and PKC in TNT regulation between GBM cells (i.e. BTICs or U87s) and NHAs. Biological sciences/Cancer/CNS cancer Biological sciences/Cancer/Cancer microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The development of therapeutic resistance in glioblastoma (GBM) is a significant obstacle to improve patient outcomes and increase survival times. GBM forms interconnected networks comprised of heterogeneous populations of tumor and non-tumor cells, which in turn, establishes a resilient tumor microenvironment (TME) that promotes the development of treatment resistance and diffusion from the origin site [ 1 – 5 ]. Rapid communication and organelle exchange amongst intertwined cell populations mediates metabolic rescue of tumor cells, infiltration of normal cell regions, and acquired resistance over time [ 4 , 6 – 8 ]. However, targeting these structures through novel mediators remains to be explored. Several forms of communication ensue, including but not limited to paracrine signaling via vesicles, tumor microtubes (TMs) predominantly found extending from tumor cells and tunneling nanotubes (TNTs), which are a universal transport highway between multiple cell types [ 4 , 6 , 8 – 10 ]. Tunneling nanotubes were first identified in rat pheochromocytoma PC12 cells in the early 2000’s from the Rustom, et al. group and were shown to also be an in vivo phenomenon shortly after [ 11 – 13 ]. Since their discovery, TNTs have been identified in cellular communication and treatment resistance across several diseases, including GBM [ 3 – 6 , 8 , 9 ]. While these membrane-extending structures originate from normal physiological events such as embryonic development, their prevalence decreases with age. In adults, TNTs are more associated with metabolic challenges, where TNTs can help mediate metabolic rescue [ 3 , 4 , 6 , 8 , 10 , 14 – 16 ]. When cells become metabolically stressed, such as a neuron during an ischemic stroke or when oxygen capacity is exceeded due to rapid proliferation of GBM, TNTs can mediate the transfer of mitochondria from healthy cells to damaged ones [ 16 – 21 ]. Astrocytes are ideal targets for rescue due to their abundance and engagement in normal brain processes. The TNT-mediated exchange of mitochondria between astrocytes and neurons is intrinsic and is considered a useful stress-related TNT event [ 16 , 18 , 21 ]. GBM cells on the other hand are suspected of self-preserving by plundering mitochondria from astrocytes that have become tumor-supportive [ 16 – 19 , 22 , 23 ]. GBM cells have been documented to distribute mitochondria with mutated mitochondrial DNA (mtDNA) to surrounding NHAs to resist chemotherapy [ 3 , 24 ]. This accounts for one of the ways astrocytes become adapted to TME by reprogramming into tumor-associated astrocytes (TAA) [ 3 , 4 , 9 , 17 , 19 ]. Several groups have shown TAA’s can decrease GBM sensitivity to temozolomide (TMZ) and radiotherapy, enhance their invasive capacity, and recruit other cells to the TME in a TNT-mediated manner [ 22 , 25 – 27 ]. Though “normal” stressful TNT events are mediated by tightly controlled signaling, GBM harbors aberrant signaling mechanisms that manipulate these normal processes leading to pathological TNTs [ 4 , 6 – 8 ]. However, our understanding of key mediators of TNTs warrants further investigation in GBM. TNTs are rich in filamentous actin (F-actin), therefore actin-modulating dynamics are crucial to consider in addition to known, associated pathways. One pathway of interest is the PI3K-AKT-mTOR pathway, a known driver of TNTs and one of the most dysregulated and overactive pathways in GBM [ 4 , 6 , 8 – 10 , 14 ]. This pathway is activated by the phosphorylation and conversion of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), by phosphoinositide 3-kinase (PI3K) which activates downstream mediators such as protein kinase B (AKT) and mammalian target of rapamycin (mTOR), resulting in increased TNT formation, proliferation, migration, and resistance [ 28 – 30 ]. The pathway is negatively regulated by the tumor suppressor, phosphatase and tensin homolog (PTEN), however, 40% of GBM cases present a loss of PTEN function [ 28 , 31 – 34 ]. Moreover, PTEN deletion has been shown to elicit an injury-like response and promote self-renewal, both of which involve TNT utilization [ 8 , 35 , 36 ]. Moreover, PTEN loss is associated with increased TMZ resistance in addition to unmethylated MGMT promoter status [ 37 ]. In the absence of PTEN, PIP 2 substrate availability largely becomes reliant on the protein Myristoylated Alanine-Rich C Kinase Substrate (MARCKS), yet MARCKS signaling has never been investigated in the context of TNT dynamics [ 32 – 34 ]. MARCKS is implicated in GBM treatment resistance, stemness, migration, and other tumor progression factors [ 38 ]. MARCKS is known for its regulation of polarized signaling networks and the cytoskeleton through actin modulation, much of which is regulated through its electrostatically charged effector domain (ED) [ 31 , 39 – 44 ]. MARCKS cycles between binding PIP 2 at the membrane and translocating to the cytosol upon phosphorylation by protein kinase C (PKC), Rho-associated kinase (ROCK), or by binding to other substrates such as calmodulin or phosphatidylserine [ 38 , 41 , 42 , 45 ]. Following these modifications, MARCKS releases PIP 2 , leaving PIP 2 available for PI3k [ 38 , 41 – 43 , 45 ]. Interestingly, we have previously published that U87 lines modified to overexpress a pseudo-phosphorylated MARCKS ED exhibit increased resistance to radiation compared to those that overexpress a non-phosphorylated MARCKS ED [ 41 , 46 ]. Moreover, PKC is highly influential in MARCKS ED phosphorylation and has been implicated in adrenocorticoid carcinoma cell TNT formation, but its role in GBM-TNTs has not been reported [ 47 ]. Our group has previously investigated the cytotoxic benefits of utilizing a peptide derived from the MARCKS ED, termed MED2 [ 40 ]. In addition to inducing cytotoxicity, MED2 induced the retraction of the cytoplasmic extensions in GBM patient-derived xenograft (PDX) brain tumor-initiating cells (BTICs). Because TNTs are products of the cell membrane, we hypothesized that MARCKS is influencing TNTs as well through its ED. Moreover, due to the strong linkage between MARCKS phosphorylation events and the reciprocal effect on the PI3K-AKT-mTOR pathway in the absence of PTEN, we aimed to determine if MARCKS phosphorylation and upstream PKC activity may be regulating aberrant TNT function. Since tumor cells alone typically favor the use of tumor microtubes, we utilized a TNT- promoting co-culture model of GBM cells (BTICs or U87s) and normal human astrocytes (NHAs). Importantly, both GBM models possess null PTEN status, favoring MARCKS regulation of the downstream pathway. PKC stimulation results in increased TNT structures and the transfer of mitochondria from GBM cells to NHAs, while PKC inhibition and MED2 administration demonstrated contrasting results. We then investigated co-cultures of astrocytes and lentivirus-modified, doxycycline-inducible, mutated-MARCKS ED U87 models, validated as previously described [ 38 , 48 ]. Immortalized PTEN-null U87 cells that contain inducible differential mutations that overexpress endogenous MARCKS ED as wild-type (U87-WT), pseudo-phosphorylated (U87-PP), or non-phosphorylated (U87-NP) upon induction revealed distinct alterations in TNTs, phenocopying what we observed with PKC manipulation drugs. These data reveal a new role for PKC and MARCKS in TNT dynamics between NHAs and GBM cells. RESULTS Astrocytes increase TMZ resistance in JX14 JX14 cells naturally possess an epigenetically silenced unmethylated MGMT promoter and are PTEN-null. Previous studies have reported that although JX14 has unmethylated MGMT promoter, it is partially sensitive to TMZ [ 49 ]. We investigated if JX14 co-cultured with NHAs exhibited increased resistance to TMZ. We began culturing JX14 and NHAs as monocultures and indirect co-cultures designed to favor TNT-mediated events to assess for cytotoxicity after 5 days. NHA monocultures and co-cultures were both sensitive to TMZ when cultured alone, resulting in 15% viability ( \(\:\pm\:\:0.0366)\:\) and 18.5% viability ( \(\:\pm\:\:0.0109)\:\:\) respectively (P = 0.1157) (Fig. 1 A). Monocultured JX14 resulted in 18.5% viability ( \(\:\pm\:\:0.0078)\:\) following TMZ treatment, however, JX14 demonstrated a survival advantage when exposed to NHAs, with increased viability of 37.9% ( \(\:\pm\:\:0.0376)\:\) (P < 0.0001) (Fig. 1 A). Our next goal was to determine if our protein of interest was present in TNT structures. MARCKS is present in functional TNTs between JX14 and NHAs To determine if MARCKS is involved in TNT dynamics, we performed immunofluorescence on NHAs co-cultured with JX14 cells for 18h at a low cell density. We identified MARCKS in heterotypic TNT-like structures that stained positive for F-actin and the TNT marker, TNFα-induced protein 2 (TNFAIP2) (Fig. 2 A). Next, we sought to determine the degree of overlap of these proteins by plotting the line intensity profile across the numerous TNT structures present. Pearson’s correlation revealed positive correlations amongst all proteins (Fig. 2 B). To increase specificity, we performed the same analysis on a single TNT-ROI, for which we observed similar positive correlation trends across all proteins (Fig. 2 C). To determine if the TNTs were functional, we investigated if mitochondria were present in heterotypic TNTs by staining JX14 with a MitoTracker Red and NHAs differentially with a cell tracker then incubating the two cell types for 3.5h at a higher cell density. Using high-resolution confocal microscopy, we identified mitochondria within a clearly defined TNT structure (Fig. 2 D, subpanel D2). Using 3D-reconstruction and processing in Imaris, we visualized MARCKS co-localizing with mitochondria in the TNT (Fig. 2 D, subpanel D6). A MitoTracker map overlayed with anti-MARCKS and phalloidin staining suggests a close relationship between TNT transport and MARCKS expression (Fig. 2 D, subpanel D7-9). MARCKS effector domain peptide alters TNT formation and functionality Our aforementioned MED2, generated by conjugating the MARCKS ED to a cell-penetrating HIV-TAT sequence, induced the retraction of cellular extensions in BTIC xenoline, XD456, when adhered to Geltrex [ 40 ]. Due to the differential PTEN transcription profiles of XD456 (PTEN-WT) and JX14 (PTEN-null), we aimed to determine if this same result occurred in JX14. We observed retraction of cellular membrane extensions in JX14 infected with mCherry lentivirus upon exposure to 3µM MED2 for 1.5h in 30-minute increments (data not shown). Next, we aimed to determine how MED2 affected the integrity and function of heterotypic TNTs between JX14 and NHAs. We noted a decrease in TNTs and abnormal structures in any TNT-like structures (Fig. 3 A, gray box/arrows). TNT-like fragments were released into the extracellular space (Fig. 3 A, orange box/arrows). MED2-treated cells exhibited decreased average TNT-connected cells, TNTs per cell, and TNT length compared to control (Fig. 3 B-D). To investigate functionality, mitochondrial transfer in direct contact was quantified based on MitoTracker Red accumulation in acceptor cells, which is dependent on membrane potential. MED2 treatment reduced NHAs uptake of JX14-derived mitochondria and NHAs’ mitochondrial intensity (Fig. 3 E-F). Next, we aimed to determine the effects of MED2 on transient TNTs in real-time using live cell confocal imaging. We observed that TNTs readily form between JX14 and NHAs under normal conditions (Fig. 3 G). Extension of the TNT, contact with the opposing cell, and retraction into the originating cell occurs over a short period of approximately 6 minutes, with each event occurring 3 minutes apart (Fig. 3 G, white and orange arrows). Upon addition of MED2, the time to form, extend, and retract a TNT increased to approximately 12 minutes, indicating MED2 might slow the TNT formation process (Fig. 3 H, white box). It also appeared that the TNT attempting to form failed to maintain canonical structure and linearity upon extension. A nearby TNT (tethered to the membrane) was pinched off (Fig. 3 E, yellow box), and we suspect this may account for the TNT-like fragments we observed in the fixed sample noted earlier (Fig. 3 A, yellow box/arrows). These results strongly suggest that MARCKS ED influences heterotypic TNTs. PKC stimulation increases TNTs between NHAs and JX14 Since MARCKS phosphorylation is largely regulated by PKC, we investigated the effect of PKC stimulation on TNTs. Cells were treated with drugs for a total of 3.5h before fixation. Phorbol 12-myristate 13-acetate (PMA) was utilized to stimulate PKC, while Enzastaurin (EZ) inhibited PKC. We observed increased heterotypic TNT-like structures positive for anti-MARCKS between JX14 and NHAs under vehicle conditions and upon induction of PKC activation, which promotes MARCKS phosphorylation (Fig. 4 A-D). Contrastingly, we observed abnormal directionality of TNT-like structures when treated with EZ, resulting in decreased TNT-connected cells, TNTs per cell, and average TNT length compared to control (Fig. 4 A-D). Interestingly, we could rescue the TNT phenotype observed during PMA treatment with dual administration with the TNT-inhibitor, Cytochalasin B (Fig. 4 A-D). Kinetic measurements of mitochondrial uptake by NHAs over 18h were obtained every 30 minutes, during which we observed the peak of transfer around 2h. PMA-treated cells exhibited a significant increase in mitochondria uptake compared to all other conditions (Fig. 4 E). MED2 resulted in the least amount of mitochondria uptake, followed closely by EZ and PMA + CB (Fig. 4 E). We also quantified mitochondrial intensity in NHAs at 3.5h showing similar trends (Fig. 4 F). These results support PKC activity to be a key element in TNT formation. JX14 was also assessed in monoculture under these conditions, however, JX14 exhibited less homotypic TNT-related alterations, suggesting the potential primary utilization of TMs over TNTs (Fig. S1 ). U87 MARCKS Effector Domain mutants have altered TNT phenotypes To further investigate MARCKS ED function in TNT formation in JX14 and NHAs, we utilized U87-MARCKS ED mutant cells under PDX (serum-free) conditions to promote TNT dynamics. We first sought to characterize homotypic TNTs in the modified MARCKS ED U87s (full validation of mutations detailed previously) [ 38 , 41 , 48 ]. Anti-V5 was probed to ensure sufficient mutation induction and visualization of mutated MARCKS localization. WT conditions demonstrate MARCKS overexpression, in which phosphorylation is not altered. U87-WT exhibited a plethora of TNT-like structures, connecting most if not all cells present (Fig. 5 A). When pseudophosphorylated MARCKS (U87-PP) was induced, multiple TNT-like structures maintaining structure and appearance were observed, phenocopying PMA conditions (Fig. 5 B). Contrastingly, non-phosphorylatable MARCKS (U87-NP) induction exhibited cellular aggregation by adhering membranes, with TNT numbers greatly reduced and exhibiting compromised directionality and stability (Fig. 5 C). Next, we used negative stain transmission electron microscopy (TEM) on each cell line to investigate TNT morphology. U87-WT exhibited long branched TNT-like filaments, while U87-PP filaments appeared shorter, though still maintaining integrity (Fig. 5 D-E). Strikingly, U87-NP exhibited TNT-like filaments with clubbed tips (white arrow) or deformed structure (Fig. 5 F, white arrow). In line with these results, we quantified fewer average TNTs per cell, TNT-connected cells, and TNT length in U87-NP compared to both U87-PP and U87-WT (Fig. 5 G-I). These results phenocopy the morphological and functional aspects observed with PKC activation and inhibition, supporting a potential role for MARCKS ED in TNT dynamics. NHA co-cultures with U87 MARCKS mutants phenocopy pharmacological findings Next, we aimed to characterize heterotypic TNT dynamics between U87 MARCKS ED mutants co-cultured with NHAs. Co-cultures were maintained in the same conditions as JX14 and NHAs. We noted increased TNT-like structures and TNT-connected cells in the -WT and –PP, but not in the –NP co-culture group (Fig. 6 A-D). Interestingly, average TNT length was not significantly different in all groups, suggesting astrocytes may partially rescue this phenotype in –NP groups (Fig. 6 D). Next, TNT functionality was assessed by examining mitochondrial transfer from U87 cells to NHAs. NHAs received the most mitochondria from U87-WT cells, followed by U87-PP, and least from U87-NP, possibly indicating endogenous MARCKS activity contributes to increased transfer (Fig. 6 E-F). We then stimulated PKC in all co-cultures to detect any further changes in mitochondrial dynamics. PMA-treatment increased mitochondrial transfer to NHAs compared to vehicle and EZ-treatment conditions (Fig. 6 G-I). This suggests PKC activity may promote MARCKS phosphorylation in NHAs, increasing NHA-derived TNTs which may also function to uptake mitochondria. MARCKS co-expression in public database To further support the relevance of MARCKS in TNT regulation in GBM, we investigated public patient data available on GlioVis using the Chinese Glioma Genome Atlas dataset [ 50 ]. For relevance purposes, we set the parameters to include only CGGA data for primary classical GBM since JX14 is a primary, classically derived GBM xenoline [ 40 , 51 ]. We determined the correlation between MARCKS and other genes that are implicated in TNT formation and TNT-mediated mitochondrial trafficking: MyosinX (motor), ACTB and ACTG1 (actin-binding), and RHOT1 (mitochondria trafficking) [ 19 , 23 , 52 , 53 ]. MARCKS exhibited strong positive correlations with all these protein-encoding genes, suggesting some potentially overlapping signaling dynamics that could aid in TNT regulation (MARCKS vs. MyosinX (R 2 = 0.741); vs. ACTB (R 2 = 0.699); vs. ACTG1 (R 2 = 0.746); vs. RHOT1 (R 2 = 0.740); P < 0.0001) (Fig. 7 ). Discussion The current study provides evidence supporting an important role for MARCKS in heterotypic TNT regulation between GBM cells and NHAs. PKC stimulation, well-known to phosphorylate MARCKS ED, appears to be critical for proper TNT formation and function. Lentivirus overexpression of WT and pseudo-phosphorylated MARCKS in U87s supported these findings by demonstrating increased TNT formation and function compared to non-phosphorylatable MARCKS ED. In addition, we show strong positive correlations between MARCKS and TNT-related protein-encoding genes in classical, primary GBM patient data, obtained from the Chinese Glioma Genome Atlas (CGGA) using GlioVis [ 50 , 51 ]. PKC and downstream regulation of TNTs have been minimally investigated previously and are not well explored in the context of the brain or GBM, especially in relation to MARCKS [ 47 ]. Importantly, we demonstrate MARCKS’ localization in TNTs, and manipulation of PKC or MARCKS ED phospho-mutant expression alters TNT phenotypes. Interestingly, we were able to rescue increases in PMA-treated TNTs when dual-treating with the TNT-inhibitor, Cytochalasin B. We observed increased TNT-connected cells and mitochondrial transfer to NHAs when stimulating PKC with PMA, overexpressing WT or pseudo-phosphorylated MARCKS. Using fixed and live cell confocal imaging, we were able to determine that TNTs form readily between our stem-like GBM model, JX14, and NHAs, however, the timing of this process is significantly delayed when treated with MED2. MED2-treated TNTs presented with altered morphology and functionality, further supporting the role of MARCKS ED in TNT dynamics. Decreased TNT dynamics following EZ treatment or dual PMA and Cytochalasin B treatment were also observed. Based on MARCKS known role in f-actin bundling, we suspect membrane-bound There are several limitations in the present study. The usage of pharmacological agents over genetic or lentivirus modification in our most physiologically relevant GBM model, JX14, can be perceived as a weakness. However, we were able to support our findings using U87-MARCKS ED mutants that possess the same null PTEN status as the JX14 model, making both models suitable for MARCKS exploration studies. Additionally, by using PKC-targeting drugs, we were able to discern a role for PKC upstream of MARCKS. Another drawback is the current state of the TNT field, which lacks a single biomarker for TNTs, resulting in TNT assessments being largely based on three-dimensional (3D) morphology, proper staining, and organelle-passaging abilities [ 54 , 55 ]. Much debate still exists regarding the semantics of membrane-derived tubes. “Thin” and “thick” TNTs versus TMs have reported similarities and differences, further complicating the discovery of a single biomarker. Recent studies in B-Lymphoma cells have indicated that “thick” TNTs contain microtubules which raises debate over what constitutes a TNT compared to a TM, or if they are transient products of each other [ 56 ]. Nonetheless, the current state of the field considers them to be distinct entities and because TNTs are prevalent in several diseases, proper terminology could be cell-type or disease-specific [ 57 ]. Branching off this, it is important to acknowledge that our co-culture system does not represent the complexity of the heterogeneous TME, therefore, TNT-related signaling pathways may differ amongst various non-tumor cell populations when in contact with tumor cells. Though limiting, it was necessary to focus only on tumor cell outcome in the current study, therefore it will be important to determine the reciprocal effect on NHA-derived mito-transfer and NHAs that have received GBM-derived mitochondria from cells with altered MARCKS phosphorylation in the future. Additional tumor models can also be examined to determine whether variations in MARCKS levels and its endogenous phosphorylation influence TNTs. Indeed, prior studies have identified MARCKS as a potential gene biomarker for GBM “stemness” using-single cell cultures from GBM patients [ 58 ]. Due to clonal evolution of stem-like brain tumor-initiating cells, there is a possibility that clinical therapies (e.g. radiation and temozolomide) will lead to clonal selection such that the originally active TNT-related pathway may change to other alternative pathways during the selection process [ 59 ]. Future studies using our acquired therapeutic resistance models could help identify these potential alterations [ 60 , 61 ]. Despite these limitations, we anticipate that MARCKS-related therapies, such as MED2, could be successfully combined with other cytotoxic therapies for GBM treatment. In the current study, JX14 exposed to NHAs had increased viability following TMZ treatment compared to monoculture, therefore we are eager to investigate if this survival advantage is diminished upon dual treatment with MED2. We have previously shown MED2 is not cytotoxic to NHAs unless a considerably high concentration is administered, however, GBM xenolines are sensitive at low to moderate doses [ 40 ]. We postulate that coupling MED2 with therapies that are suggested to spare normal cells (i.e. tumor treating fields, FLASH radiation) will result in increased normal cell protection and tumoral cytotoxicity due to the reduction in TNT-mediated cellular interactions between GBM cells and NHAs [ 62 , 63 ]. Our findings suggest MARCKS expression can increase TNTs, but ED phosphorylation status and PKC activation seem imperative for proper formation and functionality (Fig. S2 ). We believe the field of cellular communication and GBM will greatly benefit from these insights and other groups will be able to build upon our findings in the future. Importantly, elucidating the role of MARCKS ED and upstream PKC in GBM-NHA TNT regulation may reveal a new potential target or combinatorial therapy for overcoming treatment resistance in GBM. To the best of our knowledge, this is the first study investigating MARCKS' role in TNT regulation between NHAs and GBM cells (i.e. BTICs or U87s). We anticipate our work will contribute to advancing the existing TNT and cellular communication field in GBM and other brain-related diseases. Materials and Methods Cell Culture Conditions Patient Derived Xenograft (PDX) media Dulbecco's Modified Eagle Medium (DMEM)/F12 50/50 (Corning, Cat. #10-090-CV), B27 supplement (50X) Gibco, Cat.#17504044), 1% penicillin-streptomycin (Corning, Cat.#30-001-CI), 1% sodium pyruvate (Corning Cat.#5000CI), EGF (20ng/mL), FGF (20ng/mL). Serum-Free Astrocyte (SFA) media Neurobasal medium (Gibco, Cat. #12349015) and Dulbecco's Modified Eagle Medium (DMEM)/F12, 50/50 (Corning, Cat. #10-090-CV) (1:1 v/v) supplemented with 1% Penicillin-streptomycin (Corning, Cat.#30-001-CI), 1 mM sodium pyruvate (Corning, Cat.#5000CI), bovine serum albumin (100mg/L) (Sigma Cat.# A9418-5G), N2 supplement (100X) (Gibco, Cat.#17502048), N-Acetyl Cysteine (2.5 mg/L) (Thermofisher, Cat. #160280250), Human EGF-basic (20ng/mL) (Gibco,, Cat. #PHG0311), and Human FGF (20ng/mL) (Gibco, Cat. #PHG0261). Model validation [ 64 ] Cell Lines JX14P (also known as “GBM14” or “14”) was purchased from Mayo Clinic and maintained in the UAB Brain Tumor Model Core. JX14P was kept in PDX media and re-passaged in the flank of athymic nude mice after passage 10 in vitro. PDX Generation described previously[ 65 ]. Their use was approved by the UAB IRB (IRB-300002910). Normal human astrocytes (NHA) were purchased from Lonza (cat. #CC-2565) and kept in SFA media. U87 MARCKS mutants were established as previously described [ 38 , 46 ]. In long-term culture, U87 cells were maintained in a 1:1 mixture of PDX media and DMEM/F12 50/50 (Corning, Cat. #10-090-CV) supplemented with 10% fetal bovine serum (FBS) (Sigma, Cat. #F0926). U87 cells were placed in complete PDX media 3 days prior to induction. 300 000 cells were plated in a 60mm petri dish and induced with 2 µg/ml of doxycycline in PDX media for 72h. For experiments, cells were maintained in only PDX media +/- doxycycline to promote stem-like properties and TNT formation. U87 MARCKS mutant and normal human astrocyte co-cultures were maintained in a 1:1 medium of PDX +/- doxycycline and SFA for experimental purposes. Drug Experiments Drug Concentration Vendor Temozolomide (TMZ) 150µM Sandoz, Basel, Switzerland, cat. # 0781-2694-44 Phorbol-Myristate Acetate (PMA) 1µM Sigma Aldrich, Cat #P8139 Enzastaurin (EZ) 3µM Selleckchem, Cat. #S1055 MARCKS Effector Domain Peptide (MED2) 3µM Established previously [ 40 ] CytochalasinB 1µM Sigma Aldrich, Cat # 250233 Immunocytochemistry (Nikon A1R-HD25 Ti2) 200 000 cells were seeded in monoculture or in co-culture at 1:1 ratio onto Geltrex-coated (Gibco, Cat. #A1413302) 12mm coverslips (Neuvitro, Cat. #NC0706236). Cells were fixed with a 1:1 ratio of 4% PFA and 0.05% glutaraldehyde for 15min. Cells were quenched for 10min with 100mM NH4Cl and then permeabilized with 0.01% triton-X for 3min. Protocol adapted from: Inés Sáenz-De-Santa-María, J Michael Henderson, Anna Pepe, Chiara Zurzolo. Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking. Current Protocols, 2023, 3 (11), pp.e939. ff10.1002/cpz1.939ff. ffpasteur-04315571f Cells were stained with anti-MARCKS (Abcam, Cat. #1446Y), anti-TNFAIP2 (Invitrogen, Cat. #PA5-34708 or sc-28318), anti-V5 (Invitrogen, Cat. #A01805-100), Phalloidin (Invitrogen, Cat. # A12379 or Cat. #R415), according to the manufacturer protocol. Cells were incubated with secondary antibodies conjugated to AF-488 (Thermofisher, Cat # A32731) or AF-647 (Thermofisher, Cat # A32733) respectively. Images were obtained on a Nikon A1 Confocal. Indirect Co-culture Viability Assay 500 000 GBM cells (JX14 or U87) were seeded onto Geltrex-coated (Gibco, Cat. #A1413302) 6 well plates. A 1.0 µm Transparent PET Membrane (Corning, Cat. #353102) was placed in the well and 500 000 NHAs were seeded onto the insert. Samples were then incubated for 5 days then individually assessed for viability using CellTiter-Glo 2.0 (Promega Cat: G9241). Protocol adapted from [ 66 ]: Thayanithy, V., O’Hare, P., Wong, P. et al. A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication. Cell Commun Signal 15, 46 (2017). https://doi.org/10.1186/s12964-017-0201-2 [ 67 ] Mitochondria Transfer Assay Direct contact, Cytation5 5 000 NHAs were stained with 5 µg/ml cell tracker blue according to the manufacturer protocol and designated “Acceptor cells”. 5 000 JX14 or induced U87 cells were separately stained with 500nM MitoTrackerRed according to the manufacturer protocol and were designated “Donor cells”. All groups were plated in a black-walled 96 well plate and were imaged immediately after plating on the Cytation5 very 30min for 18h. Double positive (acceptor cells containing mito-tracker) were quantified using the Gen5 software. Values were normalized by dividing the later timepoints by time 0h. Statistical analysis was conducted in GraphPad Prism as a two-way ANOVA with repeated measures and multiple comparisons. Direct contact, Nikon A1R 200 000 GBM cells (JX14 or U87) and NHAs per well were seeded at a 1:1 ratio and stained as described above. Cells were fixed as described in the immunocytochemistry section. Z-stacked images were acquired with a Nikon A1R confocal microscope. MitoTracker Red accumulation in at least 40 acceptor cells (NHA) was quantified in Bitplane Imaris 10.1. (details in supplemental methods). TNT Quantification 200 000 cells were seeded in monoculture or in co-culture at 1:1 ratio as described above. Cells were stained with appropriate markers described above. Homotypic and heterotypic TNTs and TNT-connected cells were identified using ICY Bioimage software (version 2.4.3.0.) TNT manual plug-in https://icy.bioimageanalysis.org/ . Average length was quantified using FIJI (version 2.14.0). At least 100 cells were assessed per condition. Cytation5 Cell Imaging Multimode Reader Samples were imaged on an Agilent Cytation5 fluorescence widefield microscope, equipped with xenon flash lamp (Agilent), using an Olympus 4x/0.13 NA air lens, 4x Phase/0.1 NA air lens, 20x/20x Phase 0.45 NA air lens. EGFP was detected using a 469/35nm EX (Agilent), 525/39nm EM (Agilent), and 497nm dichroic mirror (Agilent). Texas Red was detected using a 586/15nm EX (Agilent), 647/57nm EM (Agilent), and 605nm dichroic mirror. The samples were acquired with a Sony IMX 264 CMOS WFOV camera (1992 x 1992 pixels), pixel size: 3.45µm. Confocal Microscopy Image Acquisition Samples were acquired on a Nikon Ti2 inverted fluorescence microscope with a tandem galvano and Nikon A1R-HD25 resonance scanner. Images were acquired with 1024x1024 or 2048x2048 Nyquist pixel dimensions using an Apo 60x/1.4 NA gamma oil DIC wd 140 objective. EGFP was detected using a 488nm laser, TexasRed was detected using a 567nm laser, DAPI was detected using a 405nm laser, and far red (Cy5) was detected using a 637nm laser. Nis Elements 5.0 imaging software was used to acquire Z-stack images. Live Cell Confocal (Nikon A1R-HD25 Ti2) NHAs were stained with 1 µM CellMask™ Orange Actin Tracking stain based on the manufacturer’s protocol (Thermofisher Cat. #A57244). 200,000 cells were seeded at a 1:1 concentration (100,000 of each cell type) onto geltrex-coated 35mm coverglass petri dishes (Mattek Cat. # P35G-1.5-14-C) and allowed to settle for 2h. Cells were imaged at 60x/1.4NA on Nikon A1R-HD25 Ti2 eclipse inverted microscope. During imaging, cells were housed in a live cell temperature- (37C) and gas-controlled (5% CO2) Tokai Hit incubation stage chamber to mimic incubator conditions. Nis Elements 5.0 imaging software was used to acquire Z-stack images at 1024x1024 pixels every 3 minutes for 1.5h. Timelapses were then 3D-rendered in IMARIS or NIS Nikon Elements. Negative Stain Transmission Electron Microscopy (TEM) Samples were induced for 72h in PDX media then collected for to assess morphological changes present in mutant TNTs with negative stain TEM. 300 mesh carbon film, copper EM grids (EMS) were glow-discharged using a PELCO easiGlow 9000 system (Ted Pella). 4.5 mL of sample was applied to each grid and incubated for 1 min. Excess liquid was blotted away on blotting paper. One drop of 2% uranyl acetate (UA) solution (EMS) was applied to the grid followed by blotting excess. This step was repeated three times in total. The grids were then allowed to dry for 5 minutes prior to storage. Negative stain TEM images were collected on a 120 kV Technai T12 transmission electron microscope (TEM) (FEI) with a 4k CCD camera and total dose of 20–30 e-/Å2. Declarations Conflict Of Interest Statement The authors indicate no direct conflicts of interest for the manuscript but for full disclosure: CDW has received funding from AACR-Novocure, Varian Medical Systems, and OMS Foundation and has been a consultant and/or received honorarium from EMD Serono, LifeNet Health, and Guidepoint Global. Funding Statement Research reported in this publication was supported by The National Cancer Institute/National Institutes of Health (U01CA223976 to CDW, 3U01 CA223976-03S1 to CDW); National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award number T32NS121721 (to TLS); O’Neal Invests Young Supporters Board Predoctoral and Postdoctoral NextGen Scholars Award (to LCN-C, Predoctoral, and MK, Postdoctoral); the National Cancer Institute Cancer Center Support Grant P30 CA013148. Acknowledgements The UAB High-Resolution Imaging Facility was used to acquire all high-resolution images. We would like to give special thanks to Dr. Robert Grabski and Shawn Williams for their attentive help with protocol optimization for high-resolution imaging and IMARIS analysis. We would like to acknowledge Dr. Leti Beltran for collecting the TEM images at the University of Virginia. Data Availability All data related to the manuscript are available in the main, or supplementary figures. Data can be made available upon request. References Fine, H.A., Glioblastoma: Not Just Another Cancer . Cancer Discov, 2024. 14(4): p. 648–652. Seker-Polat, F., et al., Tumor Cell Infiltration into the Brain in Glioblastoma: From Mechanisms to Clinical Perspectives . Cancers (Basel), 2022. 14(2). Valdebenito, S., et al., Tunneling nanotubes, TNT, communicate glioblastoma with surrounding non-tumor astrocytes to adapt them to hypoxic and metabolic tumor conditions . Scientific Reports, 2021. 11(1): p. 14556. Venkataramani, V., et al., Disconnecting multicellular networks in brain tumours . Nature Reviews Cancer, 2022. 22(8): p. 481–491. DeCordova, S., et al., Molecular Heterogeneity and Immunosuppressive Microenvironment in Glioblastoma . Frontiers in Immunology, 2020. 11. Pinto, G., et al., Patient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids . Biochemical Journal, 2021. 478(1): p. 21–39. Qin, Y., et al., The Functions, Methods, and Mobility of Mitochondrial Transfer Between Cells . Front Oncol, 2021. 11: p. 672781. Roehlecke, C. and M.H.H. Schmidt, Tunneling Nanotubes and Tumor Microtubes in Cancer . Cancers, 2020. 12(4): p. 857. Lou, E., et al., Tunneling Nanotubes Provide a Unique Conduit for Intercellular Transfer of Cellular Contents in Human Malignant Pleural Mesothelioma . PLoS ONE, 2012. 7(3): p. e33093. Lou, E., A Ticket to Ride: The Implications of Direct Intercellular Communication via Tunneling Nanotubes in Peritoneal and Other Invasive Malignancies . Frontiers in Oncology, 2020. Chinnery, H.R., E. Pearlman, and P.G. McMenamin, Cutting edge: Membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea . J Immunol, 2008. 180(9): p. 5779–83. Rustom, A., et al., Nanotubular highways for intercellular organelle transport . Science, 2004. 303(5660): p. 1007–10. Osswald, M., et al., Brain tumour cells interconnect to a functional and resistant network . Nature, 2015. 528(7580): p. 93–8. Abounit, S., E. Delage, and C. Zurzolo, Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking. Current Protocols in Cell Biology, 2015. 67(1): p. 12.10.1–12.10.21. Zhang, S., M.G. Kazanietz, and M. Cooke, Rho GTPases and the emerging role of tunneling nanotubes in physiology and disease . American Journal of Physiology-Cell Physiology, 2020. 319(5): p. C877-C884. Hayakawa, K., et al., Transfer of mitochondria from astrocytes to neurons after stroke . Nature, 2016. 535(7613): p. 551–555. Boyineni, J., et al., Prospective Approach to Deciphering the Impact of Intercellular Mitochondrial Transfer from Human Neural Stem Cells and Brain Tumor-Initiating Cells to Neighboring Astrocytes . Cells, 2024. 13(3). Zhou, C., et al., Tunneling nanotubes: The transport highway for astrocyte-neuron communication in the central nervous system . Brain Res Bull, 2024. 209: p. 110921. Guan, F., et al., Mitochondrial transfer in tunneling nanotubes—a new target for cancer therapy . Journal of Experimental & Clinical Cancer Research, 2024. 43(1): p. 147. Borcherding, N. and J.R. Brestoff, The power and potential of mitochondria transfer . Nature, 2023. 623(7986): p. 283–291. Capobianco, D.L., et al., Human neural stem cells derived from fetal human brain communicate with each other and rescue ischemic neuronal cells through tunneling nanotubes . Cell Death Dis, 2024. 15(8): p. 639. Pustchi, S.E., et al., Astrocytes Decreased the Sensitivity of Glioblastoma Cells to Temozolomide and Bay 11-7082. Int J Mol Sci, 2020. 21(19). Baldwin, J.G., et al., Intercellular nanotube-mediated mitochondrial transfer enhances T cell metabolic fitness and antitumor efficacy. Cell, 2024. 187(23): p. 6614–6630.e21. Valdebenito, S., et al., Tunneling Nanotubes Mediate Adaptation of Glioblastoma Cells to Temozolomide and Ionizing Radiation Treatment . iScience, 2020. 23(9). Rath, B.H., et al., Astrocytes enhance the invasion potential of glioblastoma stem-like cells . PLoS One, 2013. 8(1): p. e54752. Zhang, H., et al., Novel insights into astrocyte-mediated signaling of proliferation, invasion and tumor immune microenvironment in glioblastoma . Biomedicine & Pharmacotherapy, 2020. 126: p. 110086. Sisakht, A.K., et al., Cellular Conversations in Glioblastoma Progression, Diagnosis and Treatment . Cellular and Molecular Neurobiology, 2022. Liu, R., et al., PI3K/AKT pathway as a key link modulates the multidrug resistance of cancers . Cell Death & Disease, 2020. 11(9): p. 797. Li, X., et al., PI3K/Akt/mTOR signaling pathway and targeted therapy for glioblastoma . Oncotarget, 2016. 7(22): p. 33440–50. Lin, X., et al., ROS/mtROS promotes TNTs formation via the PI3K/AKT/mTOR pathway to protect against mitochondrial damages in glial cells induced by engineered nanomaterials . Part Fibre Toxicol, 2024. 21(1): p. 1. Laux, T., et al., GAP43, MARCKS, and CAP23 modulate PI(4,5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism . J Cell Biol, 2000. 149(7): p. 1455–72. Du, L., et al., Research progress on the role of PTEN deletion or mutation in the immune microenvironment of glioblastoma . Front Oncol, 2024. 14: p. 1409519. Luongo, F., et al., PTEN Tumor-Suppressor: The Dam of Stemness in Cancer . Cancers (Basel), 2019. 11(8). Verhaak, R.G., et al., Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1 . Cancer Cell, 2010. 17(1): p. 98–110. Hamed, A.A., et al., Gliomagenesis mimics an injury response orchestrated by neural crest-like cells . Nature, 2025. Tarasiuk, O. and A. Scuteri, Role of Tunneling Nanotubes in the Nervous System . Int J Mol Sci, 2022. 23(20). Dong, L., et al., Smurf1 Suppression Enhances Temozolomide Chemosensitivity in Glioblastoma by Facilitating PTEN Nuclear Translocation . Cells, 2022. 11(20). Jarboe, J.S., et al., MARCKS regulates growth and radiation sensitivity and is a novel prognostic factor for glioma . Clin Cancer Res, 2012. 18(11): p. 3030–41. Fong, L.W.R., D.C. Yang, and C.H. Chen, Myristoylated alanine-rich C kinase substrate (MARCKS): a multirole signaling protein in cancers . Cancer Metastasis Rev, 2017. 36(4): p. 737–747. Eustace, N.J., et al., A cell-penetrating MARCKS mimetic selectively triggers cytolytic death in glioblastoma . Oncogene, 2020. 39(46): p. 6961–6974. Eustace, N.J., et al., Myristoylated alanine-rich C-kinase substrate effector domain phosphorylation regulates the growth and radiation sensitization of glioblastoma . Int J Oncol, 2019. 54(6): p. 2039–2053. El Amri, M., U. Fitzgerald, and G. Schlosser, MARCKS and MARCKS-like proteins in development and regeneration . Journal of Biomedical Science, 2018. 25(1): p. 43. Brudvig, J.J., et al., MARCKS regulates neuritogenesis and interacts with a CDC42 signaling network . Scientific Reports, 2018. 8(1): p. 13278. Hartl, M. and R. Schneider, A Unique Family of Neuronal Signaling Proteins Implicated in Oncogenesis and Tumor Suppression . Front Oncol, 2019. 9: p. 289. Iioka, H., N. Ueno, and N. Kinoshita, Essential role of MARCKS in cortical actin dynamics during gastrulation movements . J Cell Biol, 2004. 164(2): p. 169–74. Rohrbach, T.D., et al., Targeting the effector domain of the myristoylated alanine rich C-kinase substrate enhances lung cancer radiation sensitivity . Int J Oncol, 2015. 46(3): p. 1079–88. Cooke, M., et al., Gi/o GPCRs drive the formation of actin-rich tunneling nanotubes in cancer cells via a Gβγ/PKCα/FARP1/Cdc42 axis . J Biol Chem, 2023. 299(8): p. 104983. Rohrbach, T.D., et al., MARCKS phosphorylation is modulated by a peptide mimetic of MARCKS effector domain leading to increased radiation sensitivity in lung cancer cell lines . Oncol Lett, 2017. 13(3): p. 1216–1222. Kitange, G.J., et al., Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts . Neuro Oncol, 2009. 11(3): p. 281–91. Zhao, Z., et al., Chinese Glioma Genome Atlas (CGGA): A Comprehensive Resource with Functional Genomic Data from Chinese Glioma Patients . Genomics, Proteomics & Bioinformatics, 2021. 19(1): p. 1–12. Bowman, R.L., et al., GlioVis data portal for visualization and analysis of brain tumor expression datasets . Neuro-Oncology, 2016. 19(1): p. 139–141. Ahmad, T., et al., Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy . The EMBO Journal, 2014. 33(9): p. 994–1010. Gousset, K., et al., Myo10 is a key regulator of TNT formation in neuronal cells . Journal of Cell Science, 2013. 126(19): p. 4424–4435. Dubois, F., et al., Investigating Tunneling Nanotubes in Cancer Cells: Guidelines for Structural and Functional Studies through Cell Imaging. BioMed Research International, 2020. 2020: p. 16. Han, X. and X. Wang, Opportunities and Challenges in Tunneling Nanotubes Research: How Far from Clinical Application? Int J Mol Sci, 2021. 22(5). Halász, H., et al., Cooperation of Various Cytoskeletal Components Orchestrates Intercellular Spread of Mitochondria between B-Lymphoma Cells through Tunnelling Nanotubes . Cells, 2024. 13(7): p. 607. Osswald, M., et al., Tunneling nanotube-like structures in brain tumors . Cancer Rep (Hoboken). 2019;2(6):e1181. doi: 10.1002/cnr2.1181 . eCollection 2019 Dec. Patel, A.P., et al., Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma . Science, 2014. 344(6190): p. 1396–401. Tyner, J.W., et al., Understanding Drug Sensitivity and Tackling Resistance in Cancer . Cancer Research, 2022. 82(8): p. 1448–1460. Anderson, J.C., et al., Kinomic exploration of temozolomide and radiation resistance in Glioblastoma multiforme xenolines . Radiother Oncol, 2014. 111(3): p. 468–74. Stackhouse, C.T., et al., An in vivo model of glioblastoma radiation resistance identifies long noncoding RNAs and targetable kinases . JCI Insight, 2022. 7(16). Luo, C., et al., Tumor treating fields for high-grade gliomas . Biomed Pharmacother, 2020. 127: p. 110193. Jo, H.J., et al., FLASH Radiotherapy: A FLASHing Idea to Preserve Neurocognitive Function . Brain Tumor Res Treat, 2023. 11(4): p. 223–231. Nassour-Caswell, L.C., et al., Abstract 1273: Glioblastoma brain tumor-initiating cells are protected from hypoxia when co-cultured with normal human astrocytes revealing a potential role for mitochondrial transfer via tunneling nanotubes . Cancer Research, 2023. 83(7_Supplement): p. 1273–1273. Willey, C.D., et al., Patient-Derived Xenografts as a Model System for Radiation Research . Semin Radiat Oncol, 2015. 25(4): p. 273–80. Thayanithy, V., et al., A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication . Cell Communication and Signaling, 2017. 15(1): p. 46. Brown, K.C., et al., An Experimental Protocol for the Boyden Chamber Invasion Assay With Absorbance Readout . Bio Protoc, 2024. 14(15): p. e5040. Additional Declarations (Not answered) Supplementary Files SupplementalMethods02.17.2026.docx Supplemental Methods FigS1.png Figure S2: Proposed MARCKS-related TNT mechanism. MARCKS is typically bound to the PIP 2 at the cell membrane. Upon phosphorylation by PKC, MARCKS is released from PIP 2 and TNT protrusions result from the dissociation. MARCKS may also be crosslinking necessary actin at the cell membrane in preparation for TNT protrusions. Made in Biorender.com. FigS2.png Figure S1: Homotypic TNT Dynamics in JX14. A) Confocal imaging of JX14 cells under different PKC-targeting drug conditions. B) Average TNT-connected cells (VEH vs PMA, P=0.4327; VEH vs EZ, P=0.0293, VEH vs PMA +CytoB, P=0.0031). C) Average TNTs per cell (VEH vs PMA, P=0.1260; VEH vs EZ, P=0.5694, VEH vs PMA +CytoB, P=0.2219). D) Average TNT length (VEH vs PMA, P=0.0285; VEH vs EZ, P=0.0849, VEH vs PMA +CytoB, P=0.0072). E) % JX14 with JX14-derived mitochondria quantified longitudinally every 30min over 18h. Cite Share Download PDF Status: Under Revision Version 2 posted Editorial decision: revise 20 Mar, 2026 Review # 1 received at journal 09 Mar, 2026 Reviewer # 1 agreed at journal 21 Feb, 2026 Reviewers invited by journal 20 Feb, 2026 Submission checks completed at journal 18 Feb, 2026 Editor assigned by journal 17 Feb, 2026 First submitted to journal 17 Feb, 2026 You are reading this latest preprint version Show more versions 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-6479274","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2025-04-29 17:47:10","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 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TMZ resistance in JX14.\u003c/strong\u003eViability of TMZ-treated JX14 cells and NHAs 5 days post-treatment. Cells were tested in homogeneous (monoculture) and heterogeneous (co-culture) conditions separated by a transwell insert. JX14 cells indirectly cultured with NHAs exhibited a two-fold increase in viability compared to those in homogenous culture (P\u0026lt;0.0001). NHAs exhibited no difference in viability in either condition (P=0.1157).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/ea12d4fdf4463fada46310dc.png"},{"id":103621201,"identity":"481b172b-a35e-408e-accc-383a9a4e1e6c","added_by":"auto","created_at":"2026-02-27 18:19:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6564305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMARCKS localizes in GBM-Astrocyte TNTs.\u003c/strong\u003e A) Confocal imaging of GBM PDX-derived JX14 and NHAs incubated for 18h probed with MARCKS, phalloidin, and TNFAIP2. Left panel shows merged view of NHAs and JX14 indicated with a 20µm scale bar. Image set on right show individual fluorescent channels with indicated antibody targets and 2µm scale bars. ROI shows close view of tri-color merge of single TNT (white box, 2µm inset). B) Correlation of target proteins shown through line intensity profile plot (red line across TNTs on grayscale inset). MARCKS vs F-actin: R\u003csup\u003e2\u003c/sup\u003e=0.6795, P\u0026lt;0.0001; MARCKS vs TNFAIP2: R\u003csup\u003e2\u003c/sup\u003e=0.4639, P\u0026lt;0.0001; TNFAIP2 vs F-actin: R\u003csup\u003e2\u003c/sup\u003e=0.8268, P\u0026lt;0.0001. C) Correlation metrics of target proteins and F-actin in single TNT ROI. MARCKS vs F-actin: R\u003csup\u003e2\u003c/sup\u003e=0.6393, P\u0026lt;0.0001; MARCKS vs TNFAIP2: R\u003csup\u003e2\u003c/sup\u003e=0.6381, P\u0026lt;0.0001; TNFAIP2 vs F-actin: R\u003csup\u003e2\u003c/sup\u003e=0.8034, P\u0026lt;0.0001. D) Imaris-rendered 3D depictions and layers. Various views of NHA (Labeled with CellTrackerBlue) and JX14 (labeled with MitoTrackerRed) probed for anti-MARCKS (magenta) and F-actin (green) in 3D-rendered wide view (D1, D3, D7-D9), TNT zoom (D4 and D5), and TNT-ROI zoom (D2 and D6).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/209ad997ccd6ab8a53bba80b.png"},{"id":103621204,"identity":"6868edd7-5161-49bc-82c0-a10ef688ea26","added_by":"auto","created_at":"2026-02-27 18:19:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5058817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMARCKS Effector Domain Peptide Alters TNT mechanics and functionality. \u003c/strong\u003eA) Confocal imaging of JX14 and NHAs when treated with 3µM MED2 for 4h. ROIs show distorted TNT morphology (white box, white arrows) and TNT-like fragments shed into the extracellular space (orange box, orange arrows), 20µm scale bar. B) Number of cells connected by TNTs decreases under MED2 conditions (P=0.0241). C) Average TNTs per cell in quantified between NHAs and JX14 was decreased under MED2 conditions compared to VEH (P=0.0064) D) Average length of TNTs between JX14 and NHAs exhibited a decrease compared to control (P=0.0220). E) % NHAs with JX14-derived mitochondria quantified longitudinally every 30min over 18h (2h peak: P=0.0247). F) Mean fluorescent intensity of NHA mitochondrial confocal imaging at 3.5h (P\u0026gt;0.2641). G) Live cell kinetic confocal imaging depicting TNT formation, extension, and retraction between JX14 (magenta, white arrows) and NHA (green, yellow arrows), 20µm scale bar. H) Live cell kinetic confocal imaging close-up of MED-treated JX14 TNT formation and extension (white box) and TNT extension attempt (yellow box). TNT-like extension tethered to JX14 appears to fail extension and pinch off into the extracellular space (yellow box), 1µm scale bar.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/733081c760ee795ba921e0e4.png"},{"id":104399576,"identity":"ad7482de-1cd3-4772-af59-cca4741ec559","added_by":"auto","created_at":"2026-03-11 12:06:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5639075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePKC Stimulation Increases TNTs Between JX14 and NHAs. \u003c/strong\u003eA) Confocal imaging of NHAs and JX14 directly co-cultured under different treatment conditions for 4h (1µM PMA, 3µM EZ, and 1µM PMA + 1µM CytoB), 20µm scale bar. White arrows indicate TNT-like structures. B) Average number of cells connected by TNTs (VEH vs PMA, P=0.1928; VEH vs EZ, P\u0026lt;0.0001; VEH vs PMA +CytoB, P=0.0005). C) Average TNTs quantified per cell (VEH vs PMA, P = 0.5208; VEH vs EZ, P = 0.1244; VEH vs PMA +CytoB, P =0.0021). D) Average length of TNTs normalized to total cell number (VEH vs PMA, P=0.0003; VEH vs EZ, P=0.3593; VEH vs PMA +CytoB, P=0.0708). E) % NHAs with JX14-derived mitochondria quantified longitudinally every 30min over 18h (2h Peak: VEH vs PMA: P=0.0129; VEH vs EZ, P=0.0955; VEH vs PMA+CytoB: P=0.0549). F) Mean fluorescent intensity of NHA mitochondria confocal images at 3.5h (VEH vs PMA: P\u0026gt;0.5986; VEH vs EZ: P\u0026gt;0.6352; VEH vs PMA+CytoB: P\u0026gt;0.7856).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/1f1b1dd2cab69a1f3ced04aa.png"},{"id":103621208,"identity":"3a8e5ea7-8764-49df-b077-c3d55f86ee88","added_by":"auto","created_at":"2026-02-27 18:19:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8142544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eU87 MARCKS ED Mutants Exhibit Differential TNT morphology.\u003c/strong\u003e A-C) U87 MARCKS ED mutants following 72h induction, indicated by V5-positive cells A) 3D-rendered IMARIS confocal images of U87-WT depicting several views of TNTs as a whole (A1) indicated with a 10µm scale bar or in magnified ROI’s depicting numerous TNTs in U87-WT (A1-3). Bottom panel shows individual fluorescent channels with indicated antibody targets indicated with a 20µm scale bar. B) 3D-rendered IMARIS confocal images of U87-PP showing different views of TNTs as a whole (B1) indicated with a 10µm scale bar or as magnified ROI’s showing U87-PP’s ability to form TNTs (B1-3). Bottom panel shows individual fluorophore channels with antibody targets indicated with a 20µm scale bar. C) 3D-rendered IMARIS confocal images of U87-NP depicting several views of TNTs as a whole (C1) indicated with a 10µm scale bar or in magnified ROI’s (C1-3). Bottom panel shows individual fluorophore channels with indicated antibody targets indicated with a 20µm scale bar. D-F) Negative stain TEM of U87-WT (D), U87-PP (E), and U87-NP (F) indicated with a 200nm scale bar. U87-NP exhibits clubbed tips (white arrow) while filaments maintain integrity in U87-WT and U87-PP. G-I) Average TNT-connected cells (WT vs PP: P=0.5307; WT vs NP: P\u0026lt;0.0001) (G), Average TNTs per cell (WT vs PP: P=0.1366; WT vs NP: P=0.0089) (H), and average TNT length (WT vs PP: P=0.0.4217; WT vs NP: P=0.0744) (I).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/38fb7c463a23370c3d4ef4cb.png"},{"id":104398699,"identity":"e1faf01d-f203-4c15-b1cd-f249b677c2b2","added_by":"auto","created_at":"2026-03-11 12:03:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4182296,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNHA co-cultures with U87 MARCKS mutants closely mimic pharmacologically stimulated phoshpho-events with JX14. \u003c/strong\u003eA) Direct co-culture of NHAs with induced U87-WT, -NP, or –PP stained with phalloidin and anti-V5 showing NHAs form direct extended connections with U87-WT and U87-PP. Co-culture with U87-NP resulted in a decrease in cells connected by TNTs and more cells connected by thick filopodial-like connections, 20µm scale bar. B) Average TNT-connected cells across conditions (WT vs PP: P=0.0296; WT vs NP: P\u0026lt;0.0001) C) Average number of TNTs per cell (WT vs PP: P0.3217; WT vs NP: P=0.0261). D) Average TNT length (WT vs PP: P=0.8249; WT vs NP: P=0.8302) E) Representative images of NHAs stained with CellTrackerBlue co-cultured with MARCKS ED mutants stained with MitoTracker Red and both for phalloidin and V5. F) % NHAs with U87-derived mitochondria quantified longitudinally every 30min over 18h (NHA-WT vs NHA-PP: P=0.0031; NHA-WT vs NHA-NP: P\u0026lt;0.0319). G) Mean fluorescent intensity of NHA confocal imaging at 3.5h (NHA-WT vs NHA-PP: P\u0026lt;0.0107; NHA-WT vs NHA-NP: P\u0026lt;0.0031). H-J) % NHAs with U87-derived mitochondria under the influence of PKC targeting drugs quantified longitudinally. H) 3h peak NHA (U87-WT): VEH vs PMA: P=0.0457; VEH vs EZ: P=0.0028. I) 3h peak NHA (U87-PP): VEH vs PMA: P=0.0728; VEH vs EZ: P=0.0010. J) 3h peak NHA (U87-NP): VEH vs PMA: P=0.0083; VEH vs EZ: P\u0026gt;0.4111.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/951e40869f67c53425d2aa14.png"},{"id":104399546,"identity":"292e929d-5e7b-4f49-9521-4141da855c58","added_by":"auto","created_at":"2026-03-11 12:06:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2212247,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMARCKS Positively Correlates with TNT-related Protein-Encoding Genes in Primary Classical GBM\u003c/strong\u003e. Pearson’s Correlation between various proteins. Obtained from CGGA GlioVis public patient data with parameters set to primary classical GBM. MARCKS vs. MyosinX (R\u003csup\u003e2\u003c/sup\u003e=0.741); MARCKS vs. ACTB (R\u003csup\u003e2\u003c/sup\u003e=0.699); MARCKS vs. ACTG1 (R\u003csup\u003e2\u003c/sup\u003e=0.746); MARCKS vs. RHOT1 (R\u003csup\u003e2\u003c/sup\u003e=0.740); P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/52562076d7f636450ed80c09.png"},{"id":104407747,"identity":"404bf248-29cd-40e8-ae44-10f84bc1fcd4","added_by":"auto","created_at":"2026-03-11 12:39:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":37059045,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/40b002b4-9dff-4681-88f6-bc34d81eadd5.pdf"},{"id":103621200,"identity":"2a0932b6-6604-4d48-a819-87c9685d2d87","added_by":"auto","created_at":"2026-02-27 18:19:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18689,"visible":true,"origin":"","legend":"Supplemental Methods","description":"","filename":"SupplementalMethods02.17.2026.docx","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/6504365ae880498699b85c4f.docx"},{"id":103621206,"identity":"0555d69f-3b86-41a6-bed7-26d4c4bd9934","added_by":"auto","created_at":"2026-02-27 18:19:10","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5009751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2:\u003c/strong\u003e \u003cstrong\u003eProposed MARCKS-related TNT mechanism\u003c/strong\u003e. MARCKS is typically bound to the PIP\u003csub\u003e2\u003c/sub\u003e at the cell membrane. Upon phosphorylation by PKC, MARCKS is released from PIP\u003csub\u003e2\u003c/sub\u003e and TNT protrusions result from the dissociation. MARCKS may also be crosslinking necessary actin at the cell membrane in preparation for TNT protrusions. Made in Biorender.com.\u003c/p\u003e","description":"","filename":"FigS1.png","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/7a9d2d473de8c9e3ece914b0.png"},{"id":103621202,"identity":"f6ec5f15-7cad-47bf-8468-9ffc448b3d8c","added_by":"auto","created_at":"2026-02-27 18:19:10","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1017297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1: Homotypic TNT Dynamics in JX14. \u003c/strong\u003eA) Confocal imaging of JX14 cells under different PKC-targeting drug conditions. B) Average TNT-connected cells (VEH vs PMA, P=0.4327; VEH vs EZ, P=0.0293, VEH vs PMA +CytoB, P=0.0031). C) Average TNTs per cell (VEH vs PMA, P=0.1260; VEH vs EZ, P=0.5694, VEH vs PMA +CytoB, P=0.2219). D) Average TNT length (VEH vs PMA, P=0.0285; VEH vs EZ, P=0.0849, VEH vs PMA +CytoB, P=0.0072). E) % JX14 with JX14-derived mitochondria quantified longitudinally every 30min over 18h.\u003c/p\u003e","description":"","filename":"FigS2.png","url":"https://assets-eu.researchsquare.com/files/rs-6479274/v2/c086f4aac69048cbe4771f3d.png"}],"financialInterests":"(Not answered)","formattedTitle":"MARCKS as a Target for Pathological Tunneling Nanotubes in Glioblastoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe development of therapeutic resistance in glioblastoma (GBM) is a significant obstacle to improve patient outcomes and increase survival times. GBM forms interconnected networks comprised of heterogeneous populations of tumor and non-tumor cells, which in turn, establishes a resilient tumor microenvironment (TME) that promotes the development of treatment resistance and diffusion from the origin site [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Rapid communication and organelle exchange amongst intertwined cell populations mediates metabolic rescue of tumor cells, infiltration of normal cell regions, and acquired resistance over time [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, targeting these structures through novel mediators remains to be explored.\u003c/p\u003e \u003cp\u003eSeveral forms of communication ensue, including but not limited to paracrine signaling via vesicles, tumor microtubes (TMs) predominantly found extending from tumor cells and tunneling nanotubes (TNTs), which are a universal transport highway between multiple cell types [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Tunneling nanotubes were first identified in rat pheochromocytoma PC12 cells in the early 2000\u0026rsquo;s from the Rustom, et al. group and were shown to also be an in vivo phenomenon shortly after [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Since their discovery, TNTs have been identified in cellular communication and treatment resistance across several diseases, including GBM [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. While these membrane-extending structures originate from normal physiological events such as embryonic development, their prevalence decreases with age. In adults, TNTs are more associated with metabolic challenges, where TNTs can help mediate metabolic rescue [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. When cells become metabolically stressed, such as a neuron during an ischemic stroke or when oxygen capacity is exceeded due to rapid proliferation of GBM, TNTs can mediate the transfer of mitochondria from healthy cells to damaged ones [\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Astrocytes are ideal targets for rescue due to their abundance and engagement in normal brain processes. The TNT-mediated exchange of mitochondria between astrocytes and neurons is intrinsic and is considered a useful stress-related TNT event [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. GBM cells on the other hand are suspected of self-preserving by plundering mitochondria from astrocytes that have become tumor-supportive [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. GBM cells have been documented to distribute mitochondria with mutated mitochondrial DNA (mtDNA) to surrounding NHAs to resist chemotherapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This accounts for one of the ways astrocytes become adapted to TME by reprogramming into tumor-associated astrocytes (TAA) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Several groups have shown TAA\u0026rsquo;s can decrease GBM sensitivity to temozolomide (TMZ) and radiotherapy, enhance their invasive capacity, and recruit other cells to the TME in a TNT-mediated manner [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Though \u0026ldquo;normal\u0026rdquo; stressful TNT events are mediated by tightly controlled signaling, GBM harbors aberrant signaling mechanisms that manipulate these normal processes leading to pathological TNTs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, our understanding of key mediators of TNTs warrants further investigation in GBM.\u003c/p\u003e \u003cp\u003eTNTs are rich in filamentous actin (F-actin), therefore actin-modulating dynamics are crucial to consider in addition to known, associated pathways. One pathway of interest is the PI3K-AKT-mTOR pathway, a known driver of TNTs and one of the most dysregulated and overactive pathways in GBM [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This pathway is activated by the phosphorylation and conversion of phosphatidylinositol 4,5-bisphosphate (PIP\u003csub\u003e2\u003c/sub\u003e) to phosphatidylinositol 3,4,5-trisphosphate (PIP\u003csub\u003e3\u003c/sub\u003e), by phosphoinositide 3-kinase (PI3K) which activates downstream mediators such as protein kinase B (AKT) and mammalian target of rapamycin (mTOR), resulting in increased TNT formation, proliferation, migration, and resistance [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The pathway is negatively regulated by the tumor suppressor, phosphatase and tensin homolog (PTEN), however, 40% of GBM cases present a loss of PTEN function [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, PTEN deletion has been shown to elicit an injury-like response and promote self-renewal, both of which involve TNT utilization [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Moreover, PTEN loss is associated with increased TMZ resistance in addition to unmethylated MGMT promoter status [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the absence of PTEN, PIP\u003csub\u003e2\u003c/sub\u003e substrate availability largely becomes reliant on the protein Myristoylated Alanine-Rich C Kinase Substrate (MARCKS), yet MARCKS signaling has never been investigated in the context of TNT dynamics [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMARCKS is implicated in GBM treatment resistance, stemness, migration, and other tumor progression factors [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. MARCKS is known for its regulation of polarized signaling networks and the cytoskeleton through actin modulation, much of which is regulated through its electrostatically charged effector domain (ED) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan additionalcitationids=\"CR40 CR41 CR42 CR43\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. MARCKS cycles between binding PIP\u003csub\u003e2\u003c/sub\u003e at the membrane and translocating to the cytosol upon phosphorylation by protein kinase C (PKC), Rho-associated kinase (ROCK), or by binding to other substrates such as calmodulin or phosphatidylserine [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Following these modifications, MARCKS releases PIP\u003csub\u003e2\u003c/sub\u003e, leaving PIP\u003csub\u003e2\u003c/sub\u003e available for PI3k [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Interestingly, we have previously published that U87 lines modified to overexpress a pseudo-phosphorylated MARCKS ED exhibit increased resistance to radiation compared to those that overexpress a non-phosphorylated MARCKS ED [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Moreover, PKC is highly influential in MARCKS ED phosphorylation and has been implicated in adrenocorticoid carcinoma cell TNT formation, but its role in GBM-TNTs has not been reported [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur group has previously investigated the cytotoxic benefits of utilizing a peptide derived from the MARCKS ED, termed MED2 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition to inducing cytotoxicity, MED2 induced the retraction of the cytoplasmic extensions in GBM patient-derived xenograft (PDX) brain tumor-initiating cells (BTICs). Because TNTs are products of the cell membrane, we hypothesized that MARCKS is influencing TNTs as well through its ED. Moreover, due to the strong linkage between MARCKS phosphorylation events and the reciprocal effect on the PI3K-AKT-mTOR pathway in the absence of PTEN, we aimed to determine if MARCKS phosphorylation and upstream PKC activity may be regulating aberrant TNT function.\u003c/p\u003e \u003cp\u003eSince tumor cells alone typically favor the use of tumor microtubes, we utilized a TNT- promoting co-culture model of GBM cells (BTICs or U87s) and normal human astrocytes (NHAs). Importantly, both GBM models possess null PTEN status, favoring MARCKS regulation of the downstream pathway. PKC stimulation results in increased TNT structures and the transfer of mitochondria from GBM cells to NHAs, while PKC inhibition and MED2 administration demonstrated contrasting results. We then investigated co-cultures of astrocytes and lentivirus-modified, doxycycline-inducible, mutated-MARCKS ED U87 models, validated as previously described [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Immortalized PTEN-null U87 cells that contain inducible differential mutations that overexpress endogenous MARCKS ED as wild-type (U87-WT), pseudo-phosphorylated (U87-PP), or non-phosphorylated (U87-NP) upon induction revealed distinct alterations in TNTs, phenocopying what we observed with PKC manipulation drugs. These data reveal a new role for PKC and MARCKS in TNT dynamics between NHAs and GBM cells.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAstrocytes increase TMZ resistance in JX14\u003c/h2\u003e \u003cp\u003eJX14 cells naturally possess an epigenetically silenced unmethylated MGMT promoter and are PTEN-null. Previous studies have reported that although JX14 has unmethylated MGMT promoter, it is partially sensitive to TMZ [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. We investigated if JX14 co-cultured with NHAs exhibited increased resistance to TMZ. We began culturing JX14 and NHAs as monocultures and indirect co-cultures designed to favor TNT-mediated events to assess for cytotoxicity after 5 days. NHA monocultures and co-cultures were both sensitive to TMZ when cultured alone, resulting in 15% viability (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:0.0366)\\:\\)\u003c/span\u003e\u003c/span\u003eand 18.5% viability (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:0.0109)\\:\\:\\)\u003c/span\u003e\u003c/span\u003erespectively (P\u0026thinsp;=\u0026thinsp;0.1157) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Monocultured JX14 resulted in 18.5% viability (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:0.0078)\\:\\)\u003c/span\u003e\u003c/span\u003efollowing TMZ treatment, however, JX14 demonstrated a survival advantage when exposed to NHAs, with increased viability of 37.9% (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:0.0376)\\:\\)\u003c/span\u003e\u003c/span\u003e(P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Our next goal was to determine if our protein of interest was present in TNT structures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMARCKS is present in functional TNTs between JX14 and NHAs\u003c/h3\u003e\n\u003cp\u003eTo determine if MARCKS is involved in TNT dynamics, we performed immunofluorescence on NHAs co-cultured with JX14 cells for 18h at a low cell density. We identified MARCKS in heterotypic TNT-like structures that stained positive for F-actin and the TNT marker, TNFα-induced protein 2 (TNFAIP2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Next, we sought to determine the degree of overlap of these proteins by plotting the line intensity profile across the numerous TNT structures present. Pearson\u0026rsquo;s correlation revealed positive correlations amongst all proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To increase specificity, we performed the same analysis on a single TNT-ROI, for which we observed similar positive correlation trends across all proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To determine if the TNTs were functional, we investigated if mitochondria were present in heterotypic TNTs by staining JX14 with a MitoTracker Red and NHAs differentially with a cell tracker then incubating the two cell types for 3.5h at a higher cell density. Using high-resolution confocal microscopy, we identified mitochondria within a clearly defined TNT structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, subpanel D2). Using 3D-reconstruction and processing in Imaris, we visualized MARCKS co-localizing with mitochondria in the TNT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, subpanel D6). A MitoTracker map overlayed with anti-MARCKS and phalloidin staining suggests a close relationship between TNT transport and MARCKS expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, subpanel D7-9).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMARCKS effector domain peptide alters TNT formation and functionality\u003c/h3\u003e\n\u003cp\u003eOur aforementioned MED2, generated by conjugating the MARCKS ED to a cell-penetrating HIV-TAT sequence, induced the retraction of cellular extensions in BTIC xenoline, XD456, when adhered to Geltrex [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Due to the differential PTEN transcription profiles of XD456 (PTEN-WT) and JX14 (PTEN-null), we aimed to determine if this same result occurred in JX14. We observed retraction of cellular membrane extensions in JX14 infected with mCherry lentivirus upon exposure to 3\u0026micro;M MED2 for 1.5h in 30-minute increments (data not shown). Next, we aimed to determine how MED2 affected the integrity and function of heterotypic TNTs between JX14 and NHAs. We noted a decrease in TNTs and abnormal structures in any TNT-like structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, gray box/arrows). TNT-like fragments were released into the extracellular space (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, orange box/arrows). MED2-treated cells exhibited decreased average TNT-connected cells, TNTs per cell, and TNT length compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D). To investigate functionality, mitochondrial transfer in direct contact was quantified based on MitoTracker Red accumulation in acceptor cells, which is dependent on membrane potential. MED2 treatment reduced NHAs uptake of JX14-derived mitochondria and NHAs\u0026rsquo; mitochondrial intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). Next, we aimed to determine the effects of MED2 on transient TNTs in real-time using live cell confocal imaging. We observed that TNTs readily form between JX14 and NHAs under normal conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Extension of the TNT, contact with the opposing cell, and retraction into the originating cell occurs over a short period of approximately 6 minutes, with each event occurring 3 minutes apart (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, white and orange arrows). Upon addition of MED2, the time to form, extend, and retract a TNT increased to approximately 12 minutes, indicating MED2 might slow the TNT formation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, white box). It also appeared that the TNT attempting to form failed to maintain canonical structure and linearity upon extension. A nearby TNT (tethered to the membrane) was pinched off (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, yellow box), and we suspect this may account for the TNT-like fragments we observed in the fixed sample noted earlier (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, yellow box/arrows). These results strongly suggest that MARCKS ED influences heterotypic TNTs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePKC stimulation increases TNTs between NHAs and JX14\u003c/h3\u003e\n\u003cp\u003eSince MARCKS phosphorylation is largely regulated by PKC, we investigated the effect of PKC stimulation on TNTs. Cells were treated with drugs for a total of 3.5h before fixation. Phorbol 12-myristate 13-acetate (PMA) was utilized to stimulate PKC, while Enzastaurin (EZ) inhibited PKC. We observed increased heterotypic TNT-like structures positive for anti-MARCKS between JX14 and NHAs under vehicle conditions and upon induction of PKC activation, which promotes MARCKS phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). Contrastingly, we observed abnormal directionality of TNT-like structures when treated with EZ, resulting in decreased TNT-connected cells, TNTs per cell, and average TNT length compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). Interestingly, we could rescue the TNT phenotype observed during PMA treatment with dual administration with the TNT-inhibitor, Cytochalasin B (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). Kinetic measurements of mitochondrial uptake by NHAs over 18h were obtained every 30 minutes, during which we observed the peak of transfer around 2h. PMA-treated cells exhibited a significant increase in mitochondria uptake compared to all other conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). MED2 resulted in the least amount of mitochondria uptake, followed closely by EZ and PMA\u0026thinsp;+\u0026thinsp;CB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). We also quantified mitochondrial intensity in NHAs at 3.5h showing similar trends (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results support PKC activity to be a key element in TNT formation. JX14 was also assessed in monoculture under these conditions, however, JX14 exhibited less homotypic TNT-related alterations, suggesting the potential primary utilization of TMs over TNTs (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eU87 MARCKS Effector Domain mutants have altered TNT phenotypes\u003c/h3\u003e\n\u003cp\u003eTo further investigate MARCKS ED function in TNT formation in JX14 and NHAs, we utilized U87-MARCKS ED mutant cells under PDX (serum-free) conditions to promote TNT dynamics. We first sought to characterize homotypic TNTs in the modified MARCKS ED U87s (full validation of mutations detailed previously) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Anti-V5 was probed to ensure sufficient mutation induction and visualization of mutated MARCKS localization. WT conditions demonstrate MARCKS overexpression, in which phosphorylation is not altered. U87-WT exhibited a plethora of TNT-like structures, connecting most if not all cells present (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). When pseudophosphorylated MARCKS (U87-PP) was induced, multiple TNT-like structures maintaining structure and appearance were observed, phenocopying PMA conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Contrastingly, non-phosphorylatable MARCKS (U87-NP) induction exhibited cellular aggregation by adhering membranes, with TNT numbers greatly reduced and exhibiting compromised directionality and stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Next, we used negative stain transmission electron microscopy (TEM) on each cell line to investigate TNT morphology. U87-WT exhibited long branched TNT-like filaments, while U87-PP filaments appeared shorter, though still maintaining integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). Strikingly, U87-NP exhibited TNT-like filaments with clubbed tips (white arrow) or deformed structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, white arrow). In line with these results, we quantified fewer average TNTs per cell, TNT-connected cells, and TNT length in U87-NP compared to both U87-PP and U87-WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-I). These results phenocopy the morphological and functional aspects observed with PKC activation and inhibition, supporting a potential role for MARCKS ED in TNT dynamics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNHA co-cultures with U87 MARCKS mutants phenocopy pharmacological findings\u003c/h2\u003e \u003cp\u003eNext, we aimed to characterize heterotypic TNT dynamics between U87 MARCKS ED mutants co-cultured with NHAs. Co-cultures were maintained in the same conditions as JX14 and NHAs. We noted increased TNT-like structures and TNT-connected cells in the -WT and \u0026ndash;PP, but not in the \u0026ndash;NP co-culture group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). Interestingly, average TNT length was not significantly different in all groups, suggesting astrocytes may partially rescue this phenotype in \u0026ndash;NP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Next, TNT functionality was assessed by examining mitochondrial transfer from U87 cells to NHAs. NHAs received the most mitochondria from U87-WT cells, followed by U87-PP, and least from U87-NP, possibly indicating endogenous MARCKS activity contributes to increased transfer (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F). We then stimulated PKC in all co-cultures to detect any further changes in mitochondrial dynamics. PMA-treatment increased mitochondrial transfer to NHAs compared to vehicle and EZ-treatment conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-I). This suggests PKC activity may promote MARCKS phosphorylation in NHAs, increasing NHA-derived TNTs which may also function to uptake mitochondria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMARCKS co-expression in public database\u003c/h3\u003e\n\u003cp\u003eTo further support the relevance of MARCKS in TNT regulation in GBM, we investigated public patient data available on GlioVis using the Chinese Glioma Genome Atlas dataset [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. For relevance purposes, we set the parameters to include only CGGA data for primary classical GBM since JX14 is a primary, classically derived GBM xenoline [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. We determined the correlation between MARCKS and other genes that are implicated in TNT formation and TNT-mediated mitochondrial trafficking: MyosinX (motor), ACTB and ACTG1 (actin-binding), and RHOT1 (mitochondria trafficking) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. MARCKS exhibited strong positive correlations with all these protein-encoding genes, suggesting some potentially overlapping signaling dynamics that could aid in TNT regulation (MARCKS vs. MyosinX (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.741); vs. ACTB (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.699); vs. ACTG1 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.746); vs. RHOT1 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.740); P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eThe current study provides evidence supporting an important role for MARCKS in heterotypic TNT regulation between GBM cells and NHAs. PKC stimulation, well-known to phosphorylate MARCKS ED, appears to be critical for proper TNT formation and function. Lentivirus overexpression of WT and pseudo-phosphorylated MARCKS in U87s supported these findings by demonstrating increased TNT formation and function compared to non-phosphorylatable MARCKS ED. In addition, we show strong positive correlations between MARCKS and TNT-related protein-encoding genes in classical, primary GBM patient data, obtained from the Chinese Glioma Genome Atlas (CGGA) using GlioVis [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePKC and downstream regulation of TNTs have been minimally investigated previously and are not well explored in the context of the brain or GBM, especially in relation to MARCKS [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Importantly, we demonstrate MARCKS\u0026rsquo; localization in TNTs, and manipulation of PKC or MARCKS ED phospho-mutant expression alters TNT phenotypes. Interestingly, we were able to rescue increases in PMA-treated TNTs when dual-treating with the TNT-inhibitor, Cytochalasin B. We observed increased TNT-connected cells and mitochondrial transfer to NHAs when stimulating PKC with PMA, overexpressing WT or pseudo-phosphorylated MARCKS.\u003c/p\u003e \u003cp\u003eUsing fixed and live cell confocal imaging, we were able to determine that TNTs form readily between our stem-like GBM model, JX14, and NHAs, however, the timing of this process is significantly delayed when treated with MED2. MED2-treated TNTs presented with altered morphology and functionality, further supporting the role of MARCKS ED in TNT dynamics. Decreased TNT dynamics following EZ treatment or dual PMA and Cytochalasin B treatment were also observed. Based on MARCKS known role in f-actin bundling, we suspect membrane-bound\u003c/p\u003e \u003cp\u003eThere are several limitations in the present study. The usage of pharmacological agents over genetic or lentivirus modification in our most physiologically relevant GBM model, JX14, can be perceived as a weakness. However, we were able to support our findings using U87-MARCKS ED mutants that possess the same null PTEN status as the JX14 model, making both models suitable for MARCKS exploration studies. Additionally, by using PKC-targeting drugs, we were able to discern a role for PKC upstream of MARCKS. Another drawback is the current state of the TNT field, which lacks a single biomarker for TNTs, resulting in TNT assessments being largely based on three-dimensional (3D) morphology, proper staining, and organelle-passaging abilities [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Much debate still exists regarding the semantics of membrane-derived tubes. \u0026ldquo;Thin\u0026rdquo; and \u0026ldquo;thick\u0026rdquo; TNTs versus TMs have reported similarities and differences, further complicating the discovery of a single biomarker. Recent studies in B-Lymphoma cells have indicated that \u0026ldquo;thick\u0026rdquo; TNTs contain microtubules which raises debate over what constitutes a TNT compared to a TM, or if they are transient products of each other [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Nonetheless, the current state of the field considers them to be distinct entities and because TNTs are prevalent in several diseases, proper terminology could be cell-type or disease-specific [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Branching off this, it is important to acknowledge that our co-culture system does not represent the complexity of the heterogeneous TME, therefore, TNT-related signaling pathways may differ amongst various non-tumor cell populations when in contact with tumor cells. Though limiting, it was necessary to focus only on tumor cell outcome in the current study, therefore it will be important to determine the reciprocal effect on NHA-derived mito-transfer and NHAs that have received GBM-derived mitochondria from cells with altered MARCKS phosphorylation in the future. Additional tumor models can also be examined to determine whether variations in MARCKS levels and its endogenous phosphorylation influence TNTs. Indeed, prior studies have identified MARCKS as a potential gene biomarker for GBM \u0026ldquo;stemness\u0026rdquo; using-single cell cultures from GBM patients [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Due to clonal evolution of stem-like brain tumor-initiating cells, there is a possibility that clinical therapies (e.g. radiation and temozolomide) will lead to clonal selection such that the originally active TNT-related pathway may change to other alternative pathways during the selection process [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Future studies using our acquired therapeutic resistance models could help identify these potential alterations [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite these limitations, we anticipate that MARCKS-related therapies, such as MED2, could be successfully combined with other cytotoxic therapies for GBM treatment. In the current study, JX14 exposed to NHAs had increased viability following TMZ treatment compared to monoculture, therefore we are eager to investigate if this survival advantage is diminished upon dual treatment with MED2. We have previously shown MED2 is not cytotoxic to NHAs unless a considerably high concentration is administered, however, GBM xenolines are sensitive at low to moderate doses [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. We postulate that coupling MED2 with therapies that are suggested to spare normal cells (i.e. tumor treating fields, FLASH radiation) will result in increased normal cell protection and tumoral cytotoxicity due to the reduction in TNT-mediated cellular interactions between GBM cells and NHAs [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings suggest MARCKS expression can increase TNTs, but ED phosphorylation status and PKC activation seem imperative for proper formation and functionality (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). We believe the field of cellular communication and GBM will greatly benefit from these insights and other groups will be able to build upon our findings in the future. Importantly, elucidating the role of MARCKS ED and upstream PKC in GBM-NHA TNT regulation may reveal a new potential target or combinatorial therapy for overcoming treatment resistance in GBM. To the best of our knowledge, this is the first study investigating MARCKS' role in TNT regulation between NHAs and GBM cells (i.e. BTICs or U87s). We anticipate our work will contribute to advancing the existing TNT and cellular communication field in GBM and other brain-related diseases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture Conditions\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003ePatient Derived Xenograft (PDX) media\u003c/h2\u003e \u003cp\u003eDulbecco's Modified Eagle Medium (DMEM)/F12 50/50 (Corning, Cat. #10-090-CV), B27 supplement (50X) Gibco, Cat.#17504044), 1% penicillin-streptomycin (Corning, Cat.#30-001-CI), 1% sodium pyruvate (Corning Cat.#5000CI), EGF (20ng/mL), FGF (20ng/mL).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSerum-Free Astrocyte (SFA) media\u003c/h2\u003e \u003cp\u003eNeurobasal medium (Gibco, Cat. #12349015) and Dulbecco's Modified Eagle Medium (DMEM)/F12, 50/50 (Corning, Cat. #10-090-CV) (1:1 v/v) supplemented with 1% Penicillin-streptomycin (Corning, Cat.#30-001-CI), 1 mM sodium pyruvate (Corning, Cat.#5000CI), bovine serum albumin (100mg/L) (Sigma Cat.# A9418-5G), N2 supplement (100X) (Gibco, Cat.#17502048), N-Acetyl Cysteine (2.5 mg/L) (Thermofisher, Cat. #160280250), Human EGF-basic (20ng/mL) (Gibco,, Cat. #PHG0311), and Human FGF (20ng/mL) (Gibco, Cat. #PHG0261).\u003c/p\u003e \u003cp\u003eModel validation [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell Lines\u003c/h2\u003e \u003cp\u003eJX14P (also known as \u0026ldquo;GBM14\u0026rdquo; or \u0026ldquo;14\u0026rdquo;) was purchased from Mayo Clinic and maintained in the UAB Brain Tumor Model Core. JX14P was kept in PDX media and re-passaged in the flank of athymic nude mice after passage 10 in vitro. PDX Generation described previously[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Their use was approved by the UAB IRB (IRB-300002910). Normal human astrocytes (NHA) were purchased from Lonza (cat. #CC-2565) and kept in SFA media.\u003c/p\u003e \u003cp\u003eU87 MARCKS mutants were established as previously described [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In long-term culture, U87 cells were maintained in a 1:1 mixture of PDX media and DMEM/F12 50/50 (Corning, Cat. #10-090-CV) supplemented with 10% fetal bovine serum (FBS) (Sigma, Cat. #F0926). U87 cells were placed in complete PDX media 3 days prior to induction. 300 000 cells were plated in a 60mm petri dish and induced with 2 \u0026micro;g/ml of doxycycline in PDX media for 72h. For experiments, cells were maintained in only PDX media +/- doxycycline to promote stem-like properties and TNT formation. U87 MARCKS mutant and normal human astrocyte co-cultures were maintained in a 1:1 medium of PDX +/- doxycycline and SFA for experimental purposes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDrug Experiments\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVendor\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemozolomide (TMZ)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSandoz, Basel, Switzerland, cat. # 0781-2694-44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhorbol-Myristate Acetate (PMA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSigma Aldrich, Cat #P8139\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnzastaurin (EZ)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSelleckchem, Cat. #S1055\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMARCKS Effector Domain Peptide (MED2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEstablished previously [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCytochalasinB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSigma Aldrich, Cat # 250233\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry (Nikon A1R-HD25 Ti2)\u003c/h2\u003e \u003cp\u003e200 000 cells were seeded in monoculture or in co-culture at 1:1 ratio onto Geltrex-coated (Gibco, Cat. #A1413302) 12mm coverslips (Neuvitro, Cat. #NC0706236). Cells were fixed with a 1:1 ratio of 4% PFA and 0.05% glutaraldehyde for 15min. Cells were quenched for 10min with 100mM NH4Cl and then permeabilized with 0.01% triton-X for 3min.\u003c/p\u003e \u003cp\u003e \u003cem\u003eProtocol adapted from: In\u0026eacute;s S\u0026aacute;enz-De-Santa-Mar\u0026iacute;a, J Michael Henderson, Anna Pepe, Chiara Zurzolo. Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking. Current Protocols, 2023, 3 (11), pp.e939. ff10.1002/cpz1.939ff. ffpasteur-04315571f\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCells were stained with anti-MARCKS (Abcam, Cat. #1446Y), anti-TNFAIP2 (Invitrogen, Cat. #PA5-34708 or sc-28318), anti-V5 (Invitrogen, Cat. #A01805-100), Phalloidin (Invitrogen, Cat. # A12379 or Cat. #R415), according to the manufacturer protocol. Cells were incubated with secondary antibodies conjugated to AF-488 (Thermofisher, Cat # A32731) or AF-647 (Thermofisher, Cat # A32733) respectively. Images were obtained on a Nikon A1 Confocal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIndirect Co-culture Viability Assay\u003c/h2\u003e \u003cp\u003e500 000 GBM cells (JX14 or U87) were seeded onto Geltrex-coated (Gibco, Cat. #A1413302) 6 well plates. A 1.0 \u0026micro;m Transparent PET Membrane (Corning, Cat. #353102) was placed in the well and 500 000 NHAs were seeded onto the insert. Samples were then incubated for 5 days then individually assessed for viability using CellTiter-Glo 2.0 (Promega Cat: G9241).\u003c/p\u003e \u003cp\u003e \u003cem\u003eProtocol adapted from\u003c/em\u003e [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]: \u003cem\u003eThayanithy, V., O\u0026rsquo;Hare, P., Wong, P. et al. A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication. Cell Commun Signal 15, 46 (2017).\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12964-017-0201-2\u003c/span\u003e\u003cspan address=\"10.1186/s12964-017-0201-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMitochondria Transfer Assay\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003eDirect contact, Cytation5\u003c/h2\u003e \u003cp\u003e5 000 NHAs were stained with 5 \u0026micro;g/ml cell tracker blue according to the manufacturer protocol and designated \u0026ldquo;Acceptor cells\u0026rdquo;. 5 000 JX14 or induced U87 cells were separately stained with 500nM MitoTrackerRed according to the manufacturer protocol and were designated \u0026ldquo;Donor cells\u0026rdquo;. All groups were plated in a black-walled 96 well plate and were imaged immediately after plating on the Cytation5 very 30min for 18h. Double positive (acceptor cells containing mito-tracker) were quantified using the Gen5 software. Values were normalized by dividing the later timepoints by time 0h. Statistical analysis was conducted in GraphPad Prism as a two-way ANOVA with repeated measures and multiple comparisons.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDirect contact, Nikon A1R\u003c/h2\u003e \u003cp\u003e200 000 GBM cells (JX14 or U87) and NHAs per well were seeded at a 1:1 ratio and stained as described above. Cells were fixed as described in the immunocytochemistry section. Z-stacked images were acquired with a Nikon A1R confocal microscope. MitoTracker Red accumulation in at least 40 acceptor cells (NHA) was quantified in Bitplane Imaris 10.1. (details in supplemental methods).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTNT Quantification\u003c/h2\u003e \u003cp\u003e200 000 cells were seeded in monoculture or in co-culture at 1:1 ratio as described above. Cells were stained with appropriate markers described above. Homotypic and heterotypic TNTs and TNT-connected cells were identified using ICY Bioimage software (version 2.4.3.0.) TNT manual plug-in\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://icy.bioimageanalysis.org/\u003c/span\u003e\u003cspan address=\"https://icy.bioimageanalysis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Average length was quantified using FIJI (version 2.14.0). At least 100 cells were assessed per condition.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eCytation5 Cell Imaging Multimode Reader\u003c/h2\u003e \u003cp\u003eSamples were imaged on an Agilent Cytation5 fluorescence widefield microscope, equipped with xenon flash lamp (Agilent), using an Olympus 4x/0.13 NA air lens, 4x Phase/0.1 NA air lens, 20x/20x Phase 0.45 NA air lens. EGFP was detected using a 469/35nm EX (Agilent), 525/39nm EM (Agilent), and 497nm dichroic mirror (Agilent). Texas Red was detected using a 586/15nm EX (Agilent), 647/57nm EM (Agilent), and 605nm dichroic mirror. The samples were acquired with a Sony IMX 264 CMOS WFOV camera (1992 x 1992 pixels), pixel size: 3.45\u0026micro;m.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eConfocal Microscopy Image Acquisition\u003c/h2\u003e \u003cp\u003eSamples were acquired on a Nikon Ti2 inverted fluorescence microscope with a tandem galvano and Nikon A1R-HD25 resonance scanner. Images were acquired with 1024x1024 or 2048x2048 Nyquist pixel dimensions using an Apo 60x/1.4 NA gamma oil DIC wd 140 objective. EGFP was detected using a 488nm laser, TexasRed was detected using a 567nm laser, DAPI was detected using a 405nm laser, and far red (Cy5) was detected using a 637nm laser. Nis Elements 5.0 imaging software was used to acquire Z-stack images.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eLive Cell Confocal (Nikon A1R-HD25 Ti2)\u003c/h2\u003e \u003cp\u003eNHAs were stained with 1 \u0026micro;M CellMask\u0026trade; Orange Actin Tracking stain based on the manufacturer\u0026rsquo;s protocol (Thermofisher Cat. #A57244). 200,000 cells were seeded at a 1:1 concentration (100,000 of each cell type) onto geltrex-coated 35mm coverglass petri dishes (Mattek Cat. # P35G-1.5-14-C) and allowed to settle for 2h. Cells were imaged at 60x/1.4NA on Nikon A1R-HD25 Ti2 eclipse inverted microscope. During imaging, cells were housed in a live cell temperature- (37C) and gas-controlled (5% CO2) Tokai Hit incubation stage chamber to mimic incubator conditions. Nis Elements 5.0 imaging software was used to acquire Z-stack images at 1024x1024 pixels every 3 minutes for 1.5h. Timelapses were then 3D-rendered in IMARIS or NIS Nikon Elements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eNegative Stain Transmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eSamples were induced for 72h in PDX media then collected for to assess morphological changes present in mutant TNTs with negative stain TEM. 300 mesh carbon film, copper EM grids (EMS) were glow-discharged using a PELCO easiGlow 9000 system (Ted Pella). 4.5 mL of sample was applied to each grid and incubated for 1 min. Excess liquid was blotted away on blotting paper. One drop of 2% uranyl acetate (UA) solution (EMS) was applied to the grid followed by blotting excess. This step was repeated three times in total. The grids were then allowed to dry for 5 minutes prior to storage. Negative stain TEM images were collected on a 120 kV Technai T12 transmission electron microscope (TEM) (FEI) with a 4k CCD camera and total dose of 20\u0026ndash;30 e-/\u0026Aring;2.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict Of Interest Statement\u003c/h2\u003e \u003cp\u003eThe authors indicate no direct conflicts of interest for the manuscript but for full disclosure: CDW has received funding from AACR-Novocure, Varian Medical Systems, and OMS Foundation and has been a consultant and/or received honorarium from EMD Serono, LifeNet Health, and Guidepoint Global.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding Statement\u003c/h2\u003e \u003cp\u003eResearch reported in this publication was supported by The National Cancer Institute/National Institutes of Health (U01CA223976 to CDW, 3U01 CA223976-03S1 to CDW); National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award number T32NS121721 (to TLS); O\u0026rsquo;Neal Invests Young Supporters Board Predoctoral and Postdoctoral NextGen Scholars Award (to LCN-C, Predoctoral, and MK, Postdoctoral); the National Cancer Institute Cancer Center Support Grant P30 CA013148.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe UAB High-Resolution Imaging Facility was used to acquire all high-resolution images. We would like to give special thanks to Dr. Robert Grabski and Shawn Williams for their attentive help with protocol optimization for high-resolution imaging and IMARIS analysis. We would like to acknowledge Dr. Leti Beltran for collecting the TEM images at the University of Virginia.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eAll data related to the manuscript are available in the main, or supplementary figures. Data can be made available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFine, H.A., \u003cem\u003eGlioblastoma: Not Just Another Cancer\u003c/em\u003e. Cancer Discov, 2024. 14(4): p. 648\u0026ndash;652.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeker-Polat, F., et al., \u003cem\u003eTumor Cell Infiltration into the Brain in Glioblastoma: From Mechanisms to Clinical Perspectives\u003c/em\u003e. Cancers (Basel), 2022. 14(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValdebenito, S., et al., \u003cem\u003eTunneling nanotubes, TNT, communicate glioblastoma with surrounding non-tumor astrocytes to adapt them to hypoxic and metabolic tumor conditions\u003c/em\u003e. Scientific Reports, 2021. 11(1): p. 14556.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVenkataramani, V., et al., \u003cem\u003eDisconnecting multicellular networks in brain tumours\u003c/em\u003e. Nature Reviews Cancer, 2022. 22(8): p. 481\u0026ndash;491.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeCordova, S., et al., \u003cem\u003eMolecular Heterogeneity and Immunosuppressive Microenvironment in Glioblastoma\u003c/em\u003e. Frontiers in Immunology, 2020. 11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinto, G., et al., \u003cem\u003ePatient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids\u003c/em\u003e. Biochemical Journal, 2021. 478(1): p. 21\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin, Y., et al., \u003cem\u003eThe Functions, Methods, and Mobility of Mitochondrial Transfer Between Cells\u003c/em\u003e. Front Oncol, 2021. 11: p. 672781.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoehlecke, C. and M.H.H. Schmidt, \u003cem\u003eTunneling Nanotubes and Tumor Microtubes in Cancer\u003c/em\u003e. Cancers, 2020. 12(4): p. 857.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLou, E., et al., \u003cem\u003eTunneling Nanotubes Provide a Unique Conduit for Intercellular Transfer of Cellular Contents in Human Malignant Pleural Mesothelioma\u003c/em\u003e. PLoS ONE, 2012. 7(3): p. e33093.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLou, E., \u003cem\u003eA Ticket to Ride: The Implications of Direct Intercellular Communication via Tunneling Nanotubes in Peritoneal and Other Invasive Malignancies\u003c/em\u003e. Frontiers in Oncology, 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChinnery, H.R., E. Pearlman, and P.G. McMenamin, \u003cem\u003eCutting edge: Membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea\u003c/em\u003e. J Immunol, 2008. 180(9): p. 5779\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRustom, A., et al., \u003cem\u003eNanotubular highways for intercellular organelle transport\u003c/em\u003e. Science, 2004. 303(5660): p. 1007\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsswald, M., et al., \u003cem\u003eBrain tumour cells interconnect to a functional and resistant network\u003c/em\u003e. Nature, 2015. 528(7580): p. 93\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbounit, S., E. Delage, and C. Zurzolo, \u003cem\u003eIdentification and Characterization of Tunneling Nanotubes for Intercellular Trafficking.\u003c/em\u003e Current Protocols in Cell Biology, 2015. 67(1): p. 12.10.1\u0026ndash;12.10.21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, S., M.G. Kazanietz, and M. Cooke, \u003cem\u003eRho GTPases and the emerging role of tunneling nanotubes in physiology and disease\u003c/em\u003e. American Journal of Physiology-Cell Physiology, 2020. 319(5): p. C877-C884.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayakawa, K., et al., \u003cem\u003eTransfer of mitochondria from astrocytes to neurons after stroke\u003c/em\u003e. Nature, 2016. 535(7613): p. 551\u0026ndash;555.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyineni, J., et al., \u003cem\u003eProspective Approach to Deciphering the Impact of Intercellular Mitochondrial Transfer from Human Neural Stem Cells and Brain Tumor-Initiating Cells to Neighboring Astrocytes\u003c/em\u003e. Cells, 2024. 13(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, C., et al., \u003cem\u003eTunneling nanotubes: The transport highway for astrocyte-neuron communication in the central nervous system\u003c/em\u003e. Brain Res Bull, 2024. 209: p. 110921.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan, F., et al., \u003cem\u003eMitochondrial transfer in tunneling nanotubes\u0026mdash;a new target for cancer therapy\u003c/em\u003e. Journal of Experimental \u0026amp; Clinical Cancer Research, 2024. 43(1): p. 147.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorcherding, N. and J.R. Brestoff, \u003cem\u003eThe power and potential of mitochondria transfer\u003c/em\u003e. Nature, 2023. 623(7986): p. 283\u0026ndash;291.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCapobianco, D.L., et al., \u003cem\u003eHuman neural stem cells derived from fetal human brain communicate with each other and rescue ischemic neuronal cells through tunneling nanotubes\u003c/em\u003e. Cell Death Dis, 2024. 15(8): p. 639.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePustchi, S.E., et al., \u003cem\u003eAstrocytes Decreased the Sensitivity of Glioblastoma Cells to Temozolomide and Bay 11-7082.\u003c/em\u003e Int J Mol Sci, 2020. 21(19).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaldwin, J.G., et al., \u003cem\u003eIntercellular nanotube-mediated mitochondrial transfer enhances T\u0026nbsp;cell metabolic fitness and antitumor efficacy.\u003c/em\u003e Cell, 2024. 187(23): p. 6614\u0026ndash;6630.e21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValdebenito, S., et al., \u003cem\u003eTunneling Nanotubes Mediate Adaptation of Glioblastoma Cells to Temozolomide and Ionizing Radiation Treatment\u003c/em\u003e. iScience, 2020. 23(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRath, B.H., et al., \u003cem\u003eAstrocytes enhance the invasion potential of glioblastoma stem-like cells\u003c/em\u003e. PLoS One, 2013. 8(1): p. e54752.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, H., et al., \u003cem\u003eNovel insights into astrocyte-mediated signaling of proliferation, invasion and tumor immune microenvironment in glioblastoma\u003c/em\u003e. Biomedicine \u0026amp; Pharmacotherapy, 2020. 126: p. 110086.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSisakht, A.K., et al., \u003cem\u003eCellular Conversations in Glioblastoma Progression, Diagnosis and Treatment\u003c/em\u003e. Cellular and Molecular Neurobiology, 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, R., et al., \u003cem\u003ePI3K/AKT pathway as a key link modulates the multidrug resistance of cancers\u003c/em\u003e. Cell Death \u0026amp; Disease, 2020. 11(9): p. 797.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, X., et al., \u003cem\u003ePI3K/Akt/mTOR signaling pathway and targeted therapy for glioblastoma\u003c/em\u003e. Oncotarget, 2016. 7(22): p. 33440\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin, X., et al., \u003cem\u003eROS/mtROS promotes TNTs formation via the PI3K/AKT/mTOR pathway to protect against mitochondrial damages in glial cells induced by engineered nanomaterials\u003c/em\u003e. Part Fibre Toxicol, 2024. 21(1): p. 1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaux, T., et al., \u003cem\u003eGAP43, MARCKS, and CAP23 modulate PI(4,5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism\u003c/em\u003e. J Cell Biol, 2000. 149(7): p. 1455\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu, L., et al., \u003cem\u003eResearch progress on the role of PTEN deletion or mutation in the immune microenvironment of glioblastoma\u003c/em\u003e. Front Oncol, 2024. 14: p. 1409519.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuongo, F., et al., \u003cem\u003ePTEN Tumor-Suppressor: The Dam of Stemness in Cancer\u003c/em\u003e. Cancers (Basel), 2019. 11(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerhaak, R.G., et al., \u003cem\u003eIntegrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1\u003c/em\u003e. Cancer Cell, 2010. 17(1): p. 98\u0026ndash;110.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamed, A.A., et al., \u003cem\u003eGliomagenesis mimics an injury response orchestrated by neural crest-like cells\u003c/em\u003e. Nature, 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTarasiuk, O. and A. Scuteri, \u003cem\u003eRole of Tunneling Nanotubes in the Nervous System\u003c/em\u003e. Int J Mol Sci, 2022. 23(20).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong, L., et al., \u003cem\u003eSmurf1 Suppression Enhances Temozolomide Chemosensitivity in Glioblastoma by Facilitating PTEN Nuclear Translocation\u003c/em\u003e. Cells, 2022. 11(20).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJarboe, J.S., et al., \u003cem\u003eMARCKS regulates growth and radiation sensitivity and is a novel prognostic factor for glioma\u003c/em\u003e. Clin Cancer Res, 2012. 18(11): p. 3030\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFong, L.W.R., D.C. Yang, and C.H. Chen, \u003cem\u003eMyristoylated alanine-rich C kinase substrate (MARCKS): a multirole signaling protein in cancers\u003c/em\u003e. Cancer Metastasis Rev, 2017. 36(4): p. 737\u0026ndash;747.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEustace, N.J., et al., \u003cem\u003eA cell-penetrating MARCKS mimetic selectively triggers cytolytic death in glioblastoma\u003c/em\u003e. Oncogene, 2020. 39(46): p. 6961\u0026ndash;6974.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEustace, N.J., et al., \u003cem\u003eMyristoylated alanine-rich C-kinase substrate effector domain phosphorylation regulates the growth and radiation sensitization of glioblastoma\u003c/em\u003e. Int J Oncol, 2019. 54(6): p. 2039\u0026ndash;2053.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl Amri, M., U. Fitzgerald, and G. Schlosser, \u003cem\u003eMARCKS and MARCKS-like proteins in development and regeneration\u003c/em\u003e. Journal of Biomedical Science, 2018. 25(1): p. 43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrudvig, J.J., et al., \u003cem\u003eMARCKS regulates neuritogenesis and interacts with a CDC42 signaling network\u003c/em\u003e. Scientific Reports, 2018. 8(1): p. 13278.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHartl, M. and R. Schneider, \u003cem\u003eA Unique Family of Neuronal Signaling Proteins Implicated in Oncogenesis and Tumor Suppression\u003c/em\u003e. Front Oncol, 2019. 9: p. 289.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIioka, H., N. Ueno, and N. Kinoshita, \u003cem\u003eEssential role of MARCKS in cortical actin dynamics during gastrulation movements\u003c/em\u003e. J Cell Biol, 2004. 164(2): p. 169\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRohrbach, T.D., et al., \u003cem\u003eTargeting the effector domain of the myristoylated alanine rich C-kinase substrate enhances lung cancer radiation sensitivity\u003c/em\u003e. Int J Oncol, 2015. 46(3): p. 1079\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCooke, M., et al., \u003cem\u003eGi/o GPCRs drive the formation of actin-rich tunneling nanotubes in cancer cells via a Gβγ/PKCα/FARP1/Cdc42 axis\u003c/em\u003e. J Biol Chem, 2023. 299(8): p. 104983.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRohrbach, T.D., et al., \u003cem\u003eMARCKS phosphorylation is modulated by a peptide mimetic of MARCKS effector domain leading to increased radiation sensitivity in lung cancer cell lines\u003c/em\u003e. Oncol Lett, 2017. 13(3): p. 1216\u0026ndash;1222.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKitange, G.J., et al., \u003cem\u003eInduction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts\u003c/em\u003e. Neuro Oncol, 2009. 11(3): p. 281\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, Z., et al., \u003cem\u003eChinese Glioma Genome Atlas (CGGA): A Comprehensive Resource with Functional Genomic Data from Chinese Glioma Patients\u003c/em\u003e. Genomics, Proteomics \u0026amp; Bioinformatics, 2021. 19(1): p. 1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBowman, R.L., et al., \u003cem\u003eGlioVis data portal for visualization and analysis of brain tumor expression datasets\u003c/em\u003e. Neuro-Oncology, 2016. 19(1): p. 139\u0026ndash;141.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad, T., et al., \u003cem\u003eMiro1 regulates intercellular mitochondrial transport \u0026amp; enhances mesenchymal stem cell rescue efficacy\u003c/em\u003e. The EMBO Journal, 2014. 33(9): p. 994\u0026ndash;1010.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGousset, K., et al., \u003cem\u003eMyo10 is a key regulator of TNT formation in neuronal cells\u003c/em\u003e. Journal of Cell Science, 2013. 126(19): p. 4424\u0026ndash;4435.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDubois, F., et al., \u003cem\u003eInvestigating Tunneling Nanotubes in Cancer Cells: Guidelines for Structural and Functional Studies through Cell Imaging.\u003c/em\u003e BioMed Research International, 2020. 2020: p. 16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan, X. and X. Wang, \u003cem\u003eOpportunities and Challenges in Tunneling Nanotubes Research: How Far from Clinical Application?\u003c/em\u003e Int J Mol Sci, 2021. 22(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHal\u0026aacute;sz, H., et al., \u003cem\u003eCooperation of Various Cytoskeletal Components Orchestrates Intercellular Spread of Mitochondria between B-Lymphoma Cells through Tunnelling Nanotubes\u003c/em\u003e. Cells, 2024. 13(7): p. 607.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsswald, M., et al., \u003cem\u003eTunneling nanotube-like structures in brain tumors\u003c/em\u003e. Cancer Rep (Hoboken). 2019;2(6):e1181. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/cnr2.1181\u003c/span\u003e\u003cspan address=\"10.1002/cnr2.1181\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. eCollection 2019 Dec.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel, A.P., et al., \u003cem\u003eSingle-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma\u003c/em\u003e. Science, 2014. 344(6190): p. 1396\u0026ndash;401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTyner, J.W., et al., \u003cem\u003eUnderstanding Drug Sensitivity and Tackling Resistance in Cancer\u003c/em\u003e. Cancer Research, 2022. 82(8): p. 1448\u0026ndash;1460.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson, J.C., et al., \u003cem\u003eKinomic exploration of temozolomide and radiation resistance in Glioblastoma multiforme xenolines\u003c/em\u003e. Radiother Oncol, 2014. 111(3): p. 468\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStackhouse, C.T., et al., \u003cem\u003eAn in vivo model of glioblastoma radiation resistance identifies long noncoding RNAs and targetable kinases\u003c/em\u003e. JCI Insight, 2022. 7(16).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo, C., et al., \u003cem\u003eTumor treating fields for high-grade gliomas\u003c/em\u003e. Biomed Pharmacother, 2020. 127: p. 110193.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJo, H.J., et al., \u003cem\u003eFLASH Radiotherapy: A FLASHing Idea to Preserve Neurocognitive Function\u003c/em\u003e. Brain Tumor Res Treat, 2023. 11(4): p. 223\u0026ndash;231.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNassour-Caswell, L.C., et al., \u003cem\u003eAbstract 1273: Glioblastoma brain tumor-initiating cells are protected from hypoxia when co-cultured with normal human astrocytes revealing a potential role for mitochondrial transfer via tunneling nanotubes\u003c/em\u003e. Cancer Research, 2023. 83(7_Supplement): p. 1273\u0026ndash;1273.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilley, C.D., et al., \u003cem\u003ePatient-Derived Xenografts as a Model System for Radiation Research\u003c/em\u003e. Semin Radiat Oncol, 2015. 25(4): p. 273\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThayanithy, V., et al., \u003cem\u003eA transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication\u003c/em\u003e. Cell Communication and Signaling, 2017. 15(1): p. 46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown, K.C., et al., \u003cem\u003eAn Experimental Protocol for the Boyden Chamber Invasion Assay With Absorbance Readout\u003c/em\u003e. Bio Protoc, 2024. 14(15): p. e5040.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6479274/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6479274/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDuring glioblastoma (GBM) progression, therapeutic resistance is influenced by a heterogeneous network of tumor- and tumor-promoting cells in the tumor microenvironment. Biological attacks against tumor cells (i.e. chemoradiotherapy) induce tumoral defense mechanisms bolstered by sophisticated communication mechanisms and aberrant signaling pathways. Tunneling nanotubes (TNTs) have been well documented to mediate this process by aiding the metabolic rescue of tumor cells or facilitating the recruitment and reprogramming of normal cells to become tumor-supportive. GBM brain tumor-initiating cells (BTIC) target normal human astrocytes (NHA) using TNTs, therefore investigating this interaction and the potential mediators involved is critical. Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) has never been investigated as a potential regulator of TNTs despite several overlapping signaling pathways. In the present study, we demonstrate a role for the MARCKS effector domain (ED) and PKC activation in the formation and functionality of TNTs between GBM BTICs and NHAs. We employ a cell-penetrable peptide derived from MARCKS effector domain (MED2), PKC-targeting drugs, and an inducible MARCKS ED U87 model to elucidate a potential role for MARCKS and PKC in TNT regulation between GBM cells (i.e. BTICs or U87s) and NHAs.\u003c/p\u003e","manuscriptTitle":"MARCKS as a Target for Pathological Tunneling Nanotubes in Glioblastoma","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2026-02-27 18:19:05","doi":"10.21203/rs.3.rs-6479274/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-20T16:47:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-09T23:03:00+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-21T12:59:54+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-02-20T23:11:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-18T12:41:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-17T16:53:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2026-02-17T16:53:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c866c84b-9dad-4e27-9cf9-a8f91c6e4ad7","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63554573,"name":"Biological sciences/Cancer/CNS cancer"},{"id":63554574,"name":"Biological sciences/Cancer/Cancer microenvironment"}],"tags":[],"updatedAt":"2026-03-20T16:52:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 18:19:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-6479274","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6479274","identity":"rs-6479274","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}