Neoplasia

Glioblastoma multiforme (GBM) is one of the most aggressive and therapy-resistant brain tumors prevalent in both adults and children. Despite extensive research to understand GBM pathology, it remains unclear how neural cells in the human brain interact with GBM cells to support their brain propagation and therapy resistance and whether GBM cells exert any influence on the properties of human neural cells. In this study, we co-culture human stem cell-derived subpallial telencephalic organoids with patient-derived proneural or mesenchymal GBM spheroids to investigate their reciprocal interactions. We show that both proneural and mesenchymal GBM spheroids readily fuse and propagate with human organoids, forming organoid-GBM chimeras, without the need for exogenous growth factors. GBM cells within the chimeras adapt by modulating gene expression profiles consistent with diminished proliferation, heightened hypoxia, increased angiogenesis, and proneural-to-mesenchymal transition in proneural GBM. Both proneural or mesenchymal GBMs also exert an impact on the properties of neural cells in the chimeras, leading to the suppression of neuronal genes and an upregulation expression of genes associated with hypoxia and angiogenesis. Collectively, this study identifies specific genes and molecular pathways that can be altered in GBM and neural cells by reciprocal interactions in a human developing brain-like environment for an increased understanding of GBM pathology and future therapy development.

Gliomas are primary brain tumors that develop from glial cells within the central nervous system and are among the deadliest human cancers. Glioblastoma (GBM) is the most malignant form of glioma. NLRX1 is an innate immune pattern recognition receptor that exhibits tumor-suppressive and tumor-promoting effects that may be cancer or cell-type, context-dependent, aided by differences in the microenvironment. Here, we report that NLRX1 is differentially expressed in microglia, astrocytes, GBM cell lines, and glioma patient tissues. siRNA-mediated silencing of Nlrx1 decreases the ability of the GBM cell line, LN-229, to proliferate and migrate. Nlrx1-/- GBM cells exhibit attenuated ability to generate 3D spheroids and enhanced capability to form tunneling nanotubes. Moreover, Nlrx1-/- GBM cells show decreased expression of autophagy markers, suggesting that NLRX1 plays a role in maintaining autophagy in GBM. In summary, our findings indicate that NLRX1 may modulate GBM pathophysiology by regulating GBM cell proliferation, migration, and metabolism. We believe our understanding of NLRX1 in GBM pathophysiology paves the potential development of GBM-targeting therapeutics that may delay disease progression and/or improve survival.

Glioblastoma (GBM) is the most common primary tumor of the central nervous system. One major challenge in GBM treatment is the resistance to chemotherapy and radiotherapy observed in subpopulations of cancer cells, including GBM stem-like cells (GSCs). These cells hold the ability to self-renew or differentiate following treatment, participating in tumor recurrence. The gap junction protein connexin43 (Cx43) has complex roles in oncogenesis and we have previously demonstrated an association between Cx43 and GBM chemotherapy resistance. Here, we report, for the first time, increased direct interaction between non-junctional Cx43 with microtubules in the cytoplasm of GSCs. We hypothesize that non-junctional Cx43/microtubule complexing is critical for GSC maintenance and survival and sought to specifically disrupt this interaction while maintaining other Cx43 functions, such as gap junction formation. Using a Cx43 mimetic peptide of the carboxyl terminal tubulin-binding domain of Cx43 (JM2), we successfully ablated Cx43 interaction with microtubules in GSCs. Importantly, administration of JM2 significantly decreased GSC survival in vitro, and limited GSC-derived tumor growth in vivo. Together, these results identify JM2 as a novel peptide drug to ablate GSCs in GBM treatment.

One of the main glioblastoma (GB) features is the diffuse migration of the tumor cells within the surrounding brain parenchyma, rendering almost impossible the complete tumor resection and irradiation, leading to inexorable lethal relapse of the disease. In the past years, we demonstrated that IRE1 (hereafter IRE1), one of the Endoplasmic Reticulum (ER) stress sensors, plays a key role in GB biology by impacting on immune infiltration, angiogenesis and tumor cell migration/invasion, all these features being linked to an alteration of protein secretion. In the present study, we investigated if and how IRE1 could regulate the functionality of the secretory machinery in GB cells and identified GOLIM4, a Golgi-associated molecule whose expression is regulated downstream of IRE1 through the transcription of XBP1s. Interestingly, GOLIM4 silencing led to decreased surface expression of multiple molecules including MHC class I molecules, growth factor receptors (PDGFRA and IL13RA2) and proteins involved in cell-cell adhesion (CD44, CD54, NCAM1), adhesion to matrix (ITGB1) or cell migration (CD90) without alteration of their encoding transcripts expression levels. Moreover, GOLIM4 silencing phenotypically affected GB cell-cell adhesion and cell migration in multiple models. Overall, we have described a novel IRE1/XBP1s/GOLIM4 operon that controls the secretion of specific proteins and impacts the tumor aggressiveness. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/619629v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@511636org.highwire.dtl.DTLVardef@18971f6org.highwire.dtl.DTLVardef@98ff81org.highwire.dtl.DTLVardef@ae7189_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bone morphogenetic protein 4 (BMP4) has emerged as a potential glioblastoma therapy due to its anti- proliferative effect via SOX2 downregulation and differentiation promotion. However, BMP4 responses vary across and within tumors. Our previous data indicate that BMP4 induces transition to a mesenchymal-like cell state. Mesenchymal transition is associated with therapy-resistance and tumor recurrence, as is senescence in cancer. In this study, we investigated BMP4s potential to induce senescence in primary glioblastoma cells, including proneural- and mesenchymal-like clones derived from the same tumor. BMP4 treatment induced senescence-associated genes and phenotypic changes such as cell enlargement, senescence- associated-{beta}-gal expression, lamin B1 downregulation, and elevated p21 levels. The most robust senescence induction was observed in the mesenchymal-like clone, compared to its proneural counterpart. Notably, mesenchymal-like cells displayed high basal levels of p21 and other senescence- associated markers, suggesting a convergence of mesenchymal and senescent traits. p21 knockout abolished BMP4-induced senescence, maintaining proliferation and cell size despite SOX2 downregulation. Additionally, senolytic treatment effectively eliminated senescent cells through apoptosis, thereby favoring survival of cells retaining normal p21 levels. Our findings demonstrate BMP4s ability to induce p21-dependent senescence in glioblastoma, particularly in therapy-resistant mesenchymal-like cells. These insights provide potential therapeutic strategies targeting senescence pathways in this challenging disease.

Nucleotide-binding domain leucine-rich repeat-containing receptors (NLR) are cytosolic pattern recognition receptors that regulate inflammation by sensing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). NLRP12 is a cytosolic protein with inflammation-promoting and inflammation-attenuating properties. NLRP12 exhibits tumor-suppressive or tumor-promoting effects that may be cancer, cell-type, context-dependent, aided by differences in the microenvironment contributing to the pathophysiology of hepatocellular carcinoma, prostate cancer, colitis-associated cancer, and glioblastoma (GBM). GBM is a grade IV malignant brain tumor with poor patient survival and high tumor recurrence due to a heterogeneous cell population and angiogenesis. Our previous research reported NLRP12 as a potential prognostic marker for GBM with high expression in GBM patient tissues. In the present study, we investigated the role of NLRP12-mediated signalling between GBM and tumor-adjacent astrocytes, using a comprehensive panel of experimental models, including LN-229 GBM and SVG astrocyte cell lines, cell line-derived spheroids, patient-derived primary glioma cells, and patient-derived glioma organoids. We report that NLRP12 deficiency reduces cell proliferation and viability in the LN-229 cells, but increases these parameters in SVG cells. Furthermore, NLRP12-deficient LN-229 cells display attenuated ability to form three-dimensional spheroids, indicating a role of NLRP12 in anchorage-independent growth, which is considered a hallmark of tumorigenicity. Analysis of patient-derived glioma tissue and patient-derived GBM organoids revealed differential expression of NLRP12. Our findings suggest that NLRP12 modulates GBM cell proliferation, viability, and anchorage-independent growth in a cell-context dependent manner. The cell-type-specific roles of NLRP12 may underlie its complex contribution to GBM pathophysiology with implications for therapy and prognosis. SummaryNLRP12, a cytosolic regulator of inflammation, exhibits context-dependent roles in cancer. Its deficiency reduces glioblastoma cell proliferation, viability, and spheroid formation, while enhancing these properties in astrocytes, indicating a cell-type-dependent role for NLRP12 in GBM progression.

Prolyl-4-hydroxylase subunit 2 (P4HA2), as a member of collagen modification enzymes, is induced under hypoxic conditions with essential roles in the collagen maturation, deposition as well as the remodeling of extracellular matrix(ECM). Mounting evidence has suggested that deregulation of P4HA2 is common in cancer. However, the expression pattern and molecular mechanisms of P4HA2 in glioma remain unknown. Here, we demonstrate that P4HA2 is overexpressed in glioma and inversely correlates with patient survival. Knockdown of P4HA2 inhibits proliferation, migration, invasion, and epithelial-to-mesenchymal transition (EMT)-like phenotype of glioma cells in vitro and suppressed tumor xenograft growth in vivo. Mechanistically, bioinformatics analysis shows that ECM-receptor interaction and PI3K/AKT pathway are the most enriched pathways of the co-expressed genes with P4HA2. Furthermore, P4HA2 mRNA was positively correlated with mRNA expressions of a series of collagen genes, but not mRNA of PI3K or AKT1/2. Conversely, both the protein expressions of collagens and phosphorylated PI3K/AKT could be downregulated either by silencing of P4HA2 expression or inhibition of its prolyl hydroxylase. Moreover, the inhibitory effects on the migration, invasion and the EMT-related molecules by P4HA2 knockdown can be recapitulated by the Akt phosphorylation activator. Taken together, our findings for the first time reveal an oncogenic role of P4HA2 in the glioma malignancy. By regulating the expression of fibrillar collagens and the downstream PI3K/AKT signaling pathway, it may serve as a potential anti-cancer target for the treatment of glioma. HighlightsP4HA2 is overexpressed and correlated with poor prognosis in glioma. P4HA2 depletion inhibits glioma proliferation, migration, invasion and EMT-like phenotype in vitro and tumorigenesis in vivo. P4HA2 depletion attenuates the PI3K/AKT signaling pathway in a collagen-dependent manner.

Glioblastoma multiforme (GBM) is an aggressive brain cancer with a very poor prognosis. It has been shown that GBM stem cells within a GBM tumour have increased resistance to standard therapies, so new approaches are needed to increase the range of treatment options available. Here we use two GBM stem cell lines, representing the classical/pro-neural and mesenchymal GBM subtypes, to investigate the effects of three different EZH2 inhibitors on GBM stem cell survival and gene expression: EPZ6438, GSK343 and UNC1999. EZH2 is the catalytic component of the PRC2 chromatin repressor complex, which represses transcription through methylation of histone H3 at lysine 27. Both cell lines showed significantly reduced colony formation after 48-hour exposure to the inhibitors, indicating they were sensitive to all three EZH2 inhibitors. RNA-seq analysis revealed that all three EZH2 inhibitors led to increased expression of genes related to neurogenesis and/or neuronal structure in both GBM stem cell lines. Chromatin immunoprecipitation (ChIP-Seq) was used to identify potential direct targets of the histone methylation activity of EZH2 that might be driving the increase in neuronal gene expression. Three genes were identified as candidate regulatory targets common to both cell lines: MAFB, ZIC2 and ZNF423. These transcription factors all have known roles in regulating neurogenesis, brain development and/or neuronal function. Through analysis of three different EZH2 inhibitors and two GBM stem cell lines, this study demonstrates a common underlying mechanism for how inhibition of EZH2 activity reduces GBM stem cell proliferation and survival.

Current therapies offer only a short relief for patients with pediatric glioblastoma (PED-GBM). Therefore, expanding treatment options for this fatal disease is of utmost importance. We found that PED-GBM cell lines, originated from diffuse intrinsic pontine glioma expressing H3K27M mutation (DIPG), or from hemispheric glioma expressing H3G34R mutation, are sensitive to combinations of histone deacetylase and PARP-1 inhibitors (vorinostat with either olaparib or veliparib). These combinations led to an enhanced decrease in their survival, and to increased phosphorylation of eIF2. Experiments with the S51D phosphomimetic variant of eIF2 and with brain-penetrating inhibitors of phosphorylated eIF2 (p-eIF2) dephospohrylation, salubrinal and raphin1, showed that increased eIF2 phosphorylation diminished PED-GBM cell survival and sensitized them to PARP-1 inhibitors as well as to ionizing irradiation, which is the main treatment modality in these patients. PED-GBM cells were also remarkably more sensitive to combination of vorinostat and PARP-1 inhibitors and to salubrinal and raphin1 than normal human astrocytes and fibroblasts. Importantly, although the overall effect of increased eIF2 phosphorylation was a reduced survival of PED-GBM cells, it also increased the cellular level of MTH1, an enzyme that protects treated cells against the incorporation of oxidized nucleotides into nucleic acids, resulting in an enhanced decrease in cell survival in response to the combination of salubrinal and MTH1 inhibitor, TH588. Our results indicate that combinations of the FDA approved drugs, vorinostat and either veliparib or olaparib, could potentially be included in PED-GBM treatment protocols and that the effect of salubrinal and raphin1 on PED-GBM survival warrants further evaluation.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially due to subventricular zone (SVZ) contact. Despite this, crosstalk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. Additionally, GBM brain tumor initiating cells (BTICs) increase expression of CTSB upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Finally, we show LV-proximal CTSB upregulation in patients, showing the relevance of this crosstalk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM. HighlightsO_LIPeriventricular GBM is more malignant and disrupts neurogenesis in a rodent model. C_LIO_LICell-specific proteomics elucidates tumor-promoting crosstalk between GBM and NPCs. C_LIO_LINPCs induce upregulated CTSB expression in GBM, promoting tumor progression. C_LIO_LIGBM stalls neurogenesis and promotes NPC senescence via CTSB. C_LI

BackgroundGlioblastoma multiforme (GBM) is characterized by an unfavorable prognosis for patients affected. During standard-of-care chemotherapy using temozolomide (TMZ), tumors acquire resistance thereby causing tumor recurrence. Thus, deciphering essential molecular pathways causing TMZ resistance are of high therapeutic relevance. MethodsMass spectrometry based proteomics were used to study the GBM proteome. Immunohistochemistry staining of human GBM tissue for either calpain-1 or -2 was performed to locate expression of proteases. In vitro cell based assays were used to measure cell viability and survival of primary patient-derived GBM cells and established GBM cell lines after TMZ +/- calpain inhibitor administration. shRNA expression knockdowns of either calpain-1 or calpain-2 were generated to study TMZ sensitivity of the specific subunits. The Comet assay and {gamma}H2AX signal measurements were performed in order to assess the DNA damage amount and recognition. Finally, quantitative real-time PCR of target proteins was applied to differentiate between transcriptional and post-translational regulation. ResultsCalcium-dependent calpain proteases, in particular calpain-2, are more abundant in glioblastoma compared to normal brain and increased in patient-matched initial and recurrent glioblastomas. On the cellular level, pharmacological calpain inhibition increased the sensitivities of primary glioblastoma cells towards TMZ. A genetic knockdown of calpain-2 in U251 cells led to increased caspase-3 cleavage and sensitivity to neocarzinostatin, which rapidly induces DNA strand breakage. We hypothesize that calpain-2 causes desensitization of tumor cells against TMZ by preventing strong DNA damage and subsequent apoptosis via post-translational TP53 inhibition. Indeed, proteomic comparison of U251 control vs. U251 calpain-2 knockdown cells highlights perturbed levels of numerous proteins involved in DNA damage response and downstream pathways affecting TP53 and NF-{kappa}B signaling. TP53 showed increased protein abundance, but no transcriptional regulation. ConclusionTMZ-induced cell death in the presence of calpain-2 expression appears to favor DNA repair and promote cell survival. We conclude from our experiments that calpain-2 expression represents a proteomic mode that is associated with higher resistance via "priming" GBM cells to TMZ chemotherapy. Thus, calpain-2 could serve as a prognostic factor for GBM outcome.

BackgroundColorectal cancer (CRC) is frequently associated with metastasis, resulting in high mortality rates. Platelets are known to play a crucial role in the metastatic cascade influencing tumor microenvironment remodeling, promoting cell transformation, facilitating metastatic niche formation, and shielding circulating tumor cells from immune surveillance. However, platelet proteomic alterations during tumor cell-induced platelet aggregation (TCIPA) remain largely unexplored. This study aims to characterize the proteomic profile of TCIPA in CRC using an in vitro model that recapitulates key aspects of CRC metastasis. MethodsTCIPA was assessed via light transmission aggregometry using an in vitro model incorporating paired primary and metastatic cell cultures. Stable Isotope Labeling with Amino Acids in Cell culture (SILAC) allowed for the discrimination of healthy platelet and tumor cell proteomes prior to and following TCIPA. Data-independent acquisition mass spectrometry was employed to analyze intra- and extracellular tumor and platelet proteomes. Comparative proteomic profiling was performed using a range of bioinformatic analyses, including clustering, differential expression, and Gene Set Enrichment Analyses (GSEA). ResultsComparison of the baseline proteome profiles of the CRC cell lines SW480 and SW620 identified 263 significant differentially expressed proteins (FDR [≤] 0.05, log2FC [≥] 1). The GSEA demonstrated enrichment of the epithelial-mesenchymal transition (FDR: 5.617 x 10-5) gene set in SW480 cells. While SW480 exhibited rapid TCIPA, SW620 did not consistently interact with healthy platelets. Following TCIPA, 34 tumor proteins showed differential expression compared to their naive status (without platelet-exposure). Notably, 17 of these proteins were significantly associated with CRC progression, particularly in the promotion of EMT, metastasis, tumor cell survival, proliferation, and metabolic reprogramming. ConclusionsThis study successfully characterized the proteomic profiles of platelets, platelet secretomes, and colorectal tumor cells following TCIPA-induced activation. The findings highlight the significant role of several tumor proteins and their metabolic effects in colorectal cancer progression, particularly with regard to metastasis.

Glioblastoma (GBM) are highly aggressive tumors treated mainly with surgery, radiotherapy, and chemotherapy. Innovative multimodal therapies are needed, targeting the immune system, tumor metabolism, and cell signaling. Our research focuses on the role of the actin cytoskeleton and Rho GTPases in modulating DNA damage repair and therapeutic sensitivity in GBM cells. We developed GBM sublines resistant to temozolomide (TMZ) and cisplatin (CP), and assessed actin stress fiber organization, Rho pathway activity, and resistance phenotype. TMZ-resistant clones exhibited increased Rho pathway activity, elevated p53 and DNA double-strand break (DSB) repair pathways, but reduced MMR protein levels. Importantly, Rho GTPase inhibition restored TMZ-resistant clones sensitivity to TMZ and CP, counteracting chemoresistance. While both drugs reduced DNA repair capacity in normal GBM cells--exacerbated by Rho inhibition--TMZ-resistant clones with overactivated Rho pathways did not show this effect. This response was p53-wild-type dependent, as p53-mutant GBM cells were unresponsive to Rho inhibition. However, p53-mutant cells treated with PRIMA-1 showed restored sensitivity to chemotherapeutics with Rho inhibition. Furthermore, modulation of the actin cytoskeleton and Rho GTPases affected sensitivity and viability in GBM spheroid models exposed to chemotherapy. In summary, Rho pathway activity and actin cytoskeleton dynamics are critical for both the development and reversal of chemoresistance in GBM tumors. STATEMENT OF SIGNIFICANCEChemoresistance in glioblastomas modulates the Rho GTPases pathway and actin cytoskeleton, while negatively affecting DNA repair. Downmodulating the actin circuitry in resistant GBMs sensitizes them to TMZ and CP drugs.

Glioblastoma (GBM) is an aggressive primary malignant brain tumor in adults with a median patient survival of 12-18 months post-diagnosis. The PI3K/Akt and Wnt/{beta}-catenin signaling pathways promote GBM cell growth, survival, invasiveness and therapeutic resistance. We hypothesize that inhibiting Akt and {beta}-catenin, which are central regulatory proteins of the PI3K/Akt and Wnt/{beta}-catenin pathway, will suppress GBM growth and progression. Our in vitro studies demonstrate that MK-2206, a pan-Akt inhibitor, effectively reduced cell viability, induced apoptosis, and inhibited {beta}-catenin activity; consistently outperforming iCRT3, a {beta}-catenin-TCF interaction inhibitor, in CT-2A mouse glioma cells, and N08-30 human glioma stem cells. Luciferase-expressing CT-2A cells were then intracranially implanted in C57BL/6J mice followed by MK-2206 treatment, and we observed a reduction in phosphorylated Akt and GSK-3{beta} levels, consistent with disruption of the Akt and Wnt/{beta}-catenin signaling axis causing tumor suppression. In summary, MK-2206 outperformed iCRT3 efficacy in vitro, and suppressed GBM progression, in vivo. These findings suggest that Akt inhibition via MK-2206 may offer a promising therapeutic strategy for treating GBM characterized by dysregulation of PI3K/Akt or Wnt/{beta}-catenin pathways.

Glioblastoma is the most common malignant brain tumor in adults. Cellular plasticity and the poorly differentiated features result in a fast relapse of the tumors following treatment. Moreover, the immunosuppressive microenvironment proved to be a major obstacle to immunotherapeutic approaches. Branched-chain amino acid transaminase 1 (BCAT1) is a metabolic enzyme that converts branched-chain amino acids into branched-chain keto acids, depleting cellular -ketoglutarate and producing glutamate. BCAT1 is expressed in and drives the growth of glioblastoma and other cancers. Here we show that low-BCAT1 expression correlates with differentiated glioblastoma subtypes and its knockout (KO) results in a differentiated phenotype in human and mouse glioblastoma cells. Consistent with these observations, Bcat1-KO mouse glioblastoma cells were highly susceptible to serum-induced differentiation in vitro. The transition to a differentiated cell state was linked to the increased activity of TET demethylases and the hypomethylation and activation of neuronal differentiation genes. Orthotopic tumor injection into immunocompetent mice demonstrated that the brain microenvironment is sufficient to induce differentiation of Bcat1-KO tumors in vivo. In addition, the knockout of Bcat1 attenuated immunosuppression, allowing for an extensive infiltration of CD8+ cytotoxic T-cells and complete abrogation of tumor growth. Additional analysis in immunodeficient hosts revealed that both Bcat1-KO-induced differentiation and immunomodulation contribute to the long-term suppression of tumor growth. In summary, our study demonstrates that BCAT1 promotes glioblastoma growth by blocking tumor cell differentiation and sustaining an immunosuppressive microenvironment. These findings suggest novel modes limiting glioblastoma phenotypic plasticity and therapeutic failure through targeting BCAT1. Importance of the studyHigh expression of BCAT1 occurs in many tumor entities and is related to aggressiveness, proliferation and invasion of tumor cells. In this study, we show that its expression is crucial for the continuous growth of glioblastoma cells by preventing their differentiation. Furthermore, we show that the expression of BCAT1 modulates the tumor immune microenvironment, suppressing the CD8 T-cell response. BCAT1 knockout causes glioblastoma cell differentiation and a persistent CD8 T-cell response, which is sufficient to abrogate tumor growth and prolong survival in in vivo immunocompetent and immunodeficient models, respectively. Our findings consolidate BCAT1 as a major player in glioblastoma and highlight its importance as a potential future target of research in this and other tumor entities. Key PointsO_LIBCAT1 expression maintains poorly differentiated features of glioblastoma cells and provides resistance to differentiation. C_LIO_LIExpression of BCAT1 in glioblastoma cells contributes to the immunosuppressive features of the tumor. C_LI

BackgroundInfiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo- and radio-therapy. A deeper understanding of the mechanisms invoked by GMB to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this. MethodsCRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterised EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived extracellular matrix (ECM) to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice. ResultsGBM cells expressing a p53 mutant (p53273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion of PODXL opposes infiltration of GBM in vivo. ConclusionsThis work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue. KEY POINTSThe p53R273H oncogene encourages GBM cells to release EVs containing podocalyxin. Podocalyxin-containing EVs from GBM increase hyaluronic acid production by astrocytes. Hyaluronic acid production by astrocytes drives GBM migration. IMPORTANCE OF THE STUDYThe infiltrative behaviour of glioblastoma (GBM) leads to widespread dissemination of cancer cells throughout the brain. Thus, even following successful resection of the primary tumour these disseminated cells inevitably contribute to post-surgical relapse. In this study, we have discovered a new mechanism through which GBM can release small extracellular vesicles (EVs) to reprogramme extracellular matrix (ECM) production by astrocytes in a way that supports increased invasive behaviour of the GBM cells. Moreover, we have discovered several key components of the pathway which contribute to this EV-mediated GBM-glial cell communication. Principal amongst these, we show that a particular mutant of the p53 tumour suppressor, p53273H drives the release of EVs which foster the deposition of pro-invasive ECM by astrocytes. This study provides mechanistic insight into why brain tumours expressing p53273H are associated with particularly poor patient survival and highlights the possibility of deploying agents which target astrocyte ECM deposition to reduce the morbidity of p53273H- expressing GBM.

Invasion of high-grade glioma (HGG) cells through the brain and spinal cord is a leading cause of cancer death in children. Despite advances in treatment, survivors often suffer from lifelong adverse effects of the current toxic therapies used for management. This study investigated the influence of nutritional ketosis on the therapeutic action of mebendazole (MBZ) and devimistat (CPI-613) against the highly invasive VM-M3 and non-invasive CT-2A glioblastoma cells grown orthotopically in juvenile syngeneic mice. Additionally, both drugs were tested in the human pediatric GBM cell line SF-188. DON (6-Diazo-5-oxo-L-norleucine) was used as a positive drug control for glutamine targeting. Cerebral implantation of the VM-M3 cells, which are mesenchymal origin, invaded throughout the brain and the spinal column similar to that seen in children with HGG. Neither the CT-2A nor the VM-NM1 glioblastoma stem cell tumors showed distal invasion in syngeneic juvenile mouse brains. The maximum therapeutic benefit of MBZ and CPI-613 on tumor invasion, growth, and mouse survival occurred only when the drugs were administered together with a ketogenic diet (KD). MBZ treatment inhibited both the glutaminolysis and the glycolysis pathways in VM-M3 cells grown either in vivo or in vitro. Both MBZ and CPI-613 significantly reduced the in vitro growth and viability of the SF-188 cells. Moreover, drug administration together with the KD allowed for lower dosing thus minimizing toxicity while improving overall survival of the mice. This preclinical study in two different HGGs, grown in syngeneic juvenile mice, highlights the potential importance of diet/drug therapeutic strategies for managing childhood brain cancer.

Gliomas account for ~80% of primary malignant brain tumors. Many CNS WHO grade 2-3 and some grade 4 gliomas harbor mutant isocitrate dehydrogenase 1 (mIDH1), which causes a gain of function mutation (IDH1 R132H) leading to the production of 2-hydroxyglutarate (2HG). Mutant IDH1-induced 2HG, through epigenetic reprogramming elicits an immune-permissive tumor microenvironment (TME). An immunosuppressive mechanism in the glioma TME involves adenosine production via the ectoenzyme CD73. This study investigates mIDH1s influence on CD73 expression and adenosine levels. We demonstrate that mIDH1 glioma cells exhibit reduced CD73 expression, driven by DNA hypermethylation, leading to reduced adenosine levels. Since wtIDH1 gliomas have high CD73 expression, we evaluated CD73 blockade as an immunotherapy target. We show that CD73 inhibition used as monotherapy, did not improve survival in wtIDH1 glioma-bearing mice. However, when combined with immune-stimulatory Ad-TK (adenoviral vectors encoding herpes simplex virus thymidine kinase) and Ad-Flt3L (adenoviral vectors encoding FMS-like tyrosine kinase 3 ligand) gene therapy, CD73 blockade significantly enhanced therapeutic efficacy and increased anti-glioma effector T cell activity. These findings reveal that CD73 inhibition used in combination with immune stimulatory Ad-TK/Ad-Flt3L gene therapy may be an effective treatment for wtIDH1 gliomas, which could be readily translated to the clinical arena.

MYCN is a major driver for neuroblastoma (NB) and the tyrosine hydroxylase (TH)-MYCN transgenic mouse model is extensively used for preclinical NB studies. However, spatio-temporal NB progression in the TH-MYCN model has not been studied, and questions remain about the value of implanted models as a surrogate for transgenic mice. In this work, we used magnetic resonance imaging (MRI) to study tumor progression and nanoparticle contrast-enhanced computed tomography (n-CECT) to assess tumor vascular architecture in TH-MYCN transgenic mice (2-7 weeks of age) and TH-MYCN+/+-derived orthotopic allograft and syngeneic mice (2-5 weeks post-tumor implantation). Tumors in TH-MYCN transgenic mice became evident in the abdominal paraspinal region at week 5. A delayed thoracic paraspinal mass became evident at week 6 and most mice succumbed by week 7. In allograft and syngeneic mice, single mass tumor growth was restricted to the peritoneal cavity. N-CECT revealed a predominantly microvascular network in TH-MYCN tumors while implanted tumors exhibited heterogeneous and tortuous vessels. N-CECT quantitative analysis demonstrated high vascularity (tumor fractional blood volume ~ 0.12) in all models. Multi-modal imaging of TH-MYCN transgenic and implanted models revealed differences in growth patterns and vascular architecture that should be considered in designing preclinical studies.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood and is stratified into four molecular groups - WNT, SHH, Group 3 and Group 4. Group 3 MB patients exhibit the poorest prognosis, with a 5-year overall survival of <60%, followed by Group 4 MB patients. Apart from MYC amplification in a subset of Group 3 MBs, the molecular pathomechanisms driving aggressiveness of these tumors remain incompletely characterized. The gene encoding the mTOR substrate and mRNA translation inhibitor eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) represents a possible MYC target gene whose corresponding protein, 4EBP1, was shown to be more active in Group 3 versus Group 4 MBs. However, the prognostic role of 4EBP1 in MB and the mechanisms supporting 4EBP1 overexpression in Group 3 MB are still elusive. We analyzed EIF4EBP1 mRNA expression in publicly available data sets and found an upregulation in MB as compared to non-neoblastic brain. EIF4EBP1 mRNA expression levels were higher in Group 3 compared to Group 4 MBs. EIF4EBP1 mRNA expression was correlated with MYC expression, most prominently in Group 3 MBs. Survival analyses highlighted that high EIF4EBP1 mRNA expression was associated with reduced overall and event-free survival across all MB patients and in Group 3/Group 4 MB patients. Immunohistochemical evaluation of 4EBP1 protein expression in MB tissues confirmed that high levels of 4EBP1 are associated with poor outcome. Functional analyses revealed that MYC directly regulates EIF4EBP1 promoter activity, providing a mechanism for increased EIF4EBP1 mRNA levels in Group 3 MBs. Finally, we observed that 4EBP1 may support colony formation of in vitro cultured MB cells. Our data highlight that transcriptional upregulation of EIF4EBP1 by MYC promotes in vitro tumorigenicity of MB cells and associates with shorter survival of MB patients.