Perampanel (PER), a selective AMPA receptor antagonist, has shown anti-seizure activity and has been proposed to exert anti-glioma effects. However, the molecular pathways underlying these effects in glioma remain poorly characterized. In this in vitro study, we investigated the effects of PER on microRNA (miRNA) expression in two glioblastoma cell lines (U87 and U138) and in patient-derived glioma stem cell (GSC) neurospheres. PER treatment was associated with a distinct miRNA expression profile, including the downregulation of miR-21-5p and miR-29a-3p. These miRNAs have been previously implicated in glioma cell proliferation, migration, and epithelial–mesenchymal transition (EMT)-related pathways. Functional assays showed that overexpression of miR-21-5p and miR-29a-3p increased migratory capacity and induced morphological changes consistent with a more motile phenotype. PER exposure was associated with attenuation of these phenotypic changes. In addition, PER reduced cell viability in glioblastoma cell lines and decreased neurosphere size in GSC cultures, accompanied by reduced miR-21-5p expression. These findings suggest that PER is associated with modulation of miRNAs involved in migratory and EMT-related phenotypes in glioma cells in vitro. While the data do not demonstrate direct inhibition of EMT or tumor progression, they provide preliminary insight into potential molecular pathways linked to PER exposure and support further mechanistic and in vivo studies.

Glioblastoma (GB) is the most aggressive and common malignant brain tumor, and despite current therapeutic approaches, prognosis remains poor. Given that surgical resection is frequently the sole potentially curative option, precise intraoperative tumor delineation is crucial for reducing recurrence rates and enhancing patient outcomes. In this study, we developed a novel pH-responsive imaging tool by coupling the pH-low insertion peptide (pHLIP) with fluorescein (FL) to enable targeted fluorescence-guided visualization of tumor margins. We investigated pHLIP-lipid membrane interactions using model systems, including liposomes and supported lipid bilayers (SLB), assessing peptide's pH-dependent insertion mechanism. Complementary in vitro experiments on patient-derived GB cell lines were performed to show the tracer's pH sensitivity, selective membrane targeting, and potential off-target effects. The FL-pHLIP construct showed robust, pH-dependent membrane insertion and selectivity in GB cellular models with minimal interaction under physiological conditions. These findings support FL-pHLIP as a promising candidate for fluorescence-guided surgery in GB and highlight its potential for clinical translation and for the broader development of pH-responsive diagnostic tools.

Due to the lack of effective therapeutic approach, glioblastoma (GBM) remains one of the most malignant brain tumour. By in vitro investigations on primary GBM stem cells, we highlighted one of the underlying mechanisms of drug resistance to alkylating agents, the DNA damage responses. Here, flow cytometric analysis and viability and repopulation assays were used to assess the long-term cytotoxic effect induced by the administration of a fourth-generation platinum prodrug, the (OC-6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato) platinum(IV) named Pt(IV)Ac-POA, in comparison to the most widely used Cisplatin. The immunofluorescence studies revealed changing pathways involved in the DNA damage response mechanisms in response to the two chemotherapies, suggesting in particular the role of Poly (ADP-Ribose) polymerases in the onset of resistance to Cisplatin-induced cytotoxicity. Thus, this research provides a proof of concept for how the use of a prodrug which allows the co-administration of Cisplatin and an Histone DeACetylase inhibitors, could suppress DNA repair mechanisms, suggesting a novel effective approach in GBM treatment.

Tumor-infiltrating lymphocyte (TIL)-therapy has received FDA approval for the treatment of advanced melanoma and shows potential for broader applications in solid tumors, including glioblastoma. In this study, tumor-reactive TILs (tr-TILs) are isolated and enriched for CD137 expression from cavitron ultrasonic aspirator (CUSA) emulsions of 161 adult patients diagnosed with diffuse gliomas. Tr-TILs are successfully expanded in 87 out of the 161 patients, reflecting an expansion rate of 54%. Notably, the presence of IDH1 mutation and the cumulative dose of steroids are identified as significant negative predictors of expansion efficacy. The expanded tr-TILs exhibit distinct phenotypic and molecular dysfunctional features yet show upregulated expression of progenitor/memory-like markers and polyclonal T-cell receptors. Importantly, these tr-TILs demonstrate specific antitumor reactivity against autologous tumor cells in both in vitro and in vivo xenograft models. These findings provide a compelling background for a personalized immunotherapeutic approach while tackling one of the most significant challenges in oncology.

Identification of a pathogenic variant in NF1 is diagnostic for neurofibromatosis, but is often impossible at the moment of variant detection due to many factors including allelic heterogeneity, sequence homology, and the lack of functional assays. Computational tools may aid in interpretation but are not established for NF1. Here, we optimized our random forest-based predictor RENOVO for NF1 variant interpretation. RENOVO was developed using an approach of “database archaeology”: by comparing versions of ClinVar over the years, we defined “stable” variants that maintained the same pathogenic/likely pathogenic/benign/likely benign (P/LP/B/LB) classification over time (n = 3579, the training set), and “unstable” variants that were initially classified as Variants of Unknown Significance (VUS) but were subsequently reclassified as P/LP/B/LB (n = 57, the test set). This approach allows to retrospectively measure accuracy on prediction with insufficient information, reproducing the scenario of maximal clinical utility. We further validated performance on: (i) validation set 1: 100 NF1 variants classified as VUS at the time of RENOVO development and subsequently reclassified as P/LP/B/LB in ClinVar; (ii) validation set 2: 15 de novo variants discovered in a prospective clinical cohort and subsequently reclassified per ACMG criteria. RENOVO obtained consistently high accuracy on all datasets: 98.6

Immunosuppressive myeloid cells, such as microglia and macrophages, play a key role in mediating resistance to immunotherapy in glioblastoma patients. Bulk RNA sequencing analysis revealed elevated expression of Klotho (Kl) in gliomas derived from irradiated glioma-bearing mice. Klotho, which encodes an anti-aging protein, was found to be upregulated in glioma-associated microglia/macrophages (GAMs) exhibiting an M1 pro-inflammatory phenotype. This upregulation appeared to enhance the antitumor efficacy of a combination of radiotherapy and dendritic cell (DC) immunotherapy. Furthermore, transcript levels of KL in tumor specimens and corresponding serum levels in glioblastoma patients undergoing DC immunotherapy were correlated with favorable prognostic outcomes and improved treatment responses. Given its expression in human M1-like GAMs, serum KL levels can offer valuable insights into the immune microenvironment and hold clinical significance as a peripheral biomarker. These findings highlight the pivotal role of Klotho as a prognostic biomarker for predicting responses to immunotherapy, with potential applications for monitoring tumor progression or regression through changes in serum levels.

Non-canonical tumor-specific antigens (ncTSAs) can expand the pool of targets for cancer immunotherapy, but require robust and comprehensive computational pipelines for their prediction. Here, we present NovumRNA, a fully automated Nextflow pipeline for predicting different classes of ncTSAs from patients' RNA sequencing data. We extensively benchmarked NovumRNA using publicly available and newly generated datasets, demonstrating the robustness of its analytical modules and predictions. NovumRNA analysis of RNA-seq data from colorectal cancer organoids and patients' tumor samples revealed comparable ncTSA potential for microsatellite stable and unstable tumors and candidate therapeutic targets for patients with low tumor mutational burden. Finally, our investigation of glioblastoma cell lines demonstrated increased ncTSAs burden upon indisulam treatment, and detection by NovumRNA of therapy-induced ncTSAs, which we could validate experimentally. These findings underscore the potential of NovumRNA for identifying synergistic drugs and novel therapeutic targets for immunotherapy, which could ultimately extend its benefit to a broader patient population.

Fluorescein-mediated sonodynamic therapy (FL-SDT) is an extremely promising approach for glioma treatment, resulting from the combination of low-intensity focused ultrasound (FUS) with a sonosensitizer. In the present study, we evaluated the efficacy and immunomodulation of SDT with fluorescein as the sonosensitizer in immunocompetent GL261 glioma mice for the first time. In vitro studies demonstrated that the exposure of GL261 cells to FL-SDT induced immunogenic cell death and relevant upregulation of MHC class I, CD80 and CD86 expression. In vivo studies were then performed to treat GL261 glioma-bearing mice with FL-SDT, fluorescein alone, or FUS alone. Perturbation of the glioma-associated macrophage subset within the immune microenvironment was induced by all the treatments. Notably, a relevant depletion of myeloid-derived suppressor cells (MDSCs) and concomitant robust infiltration of CD8+ T cells were observed in the SDT-FL-treated mice, resulting in a significant radiological delay in glioma progression and a consequent improvement in survival. Tumor control and improved survival were also observed in mice treated with FL alone (median survival 41.5 days, p > 0.0001 compared to untreated mice), reflecting considerable modulation of the immune microenvironment. Interestingly, a high circulating lymphocyte-to-monocyte ratio and a very low proportion of MDSCs were predictive of better survival in FL- and FL-SDT-treated mice than in untreated and FUS-treated mice, in which elevated monocyte and MDSC frequencies correlated with worse survival. The immunostimulatory potential of FL-SDT treatment and the profound modulation of most immunosuppressive components within the microenvironment encouraged the exploration of the combination of FL-SDT with immunotherapeutic strategies.

IntroductionAssessing the treatment response of glioblastoma multiforme during immunotherapy (IT) is an open issue. Treatment response assessment maps (TRAMs) might help distinguish true tumor progression (TTP) and pseudoprogression (PsP) in this setting.MethodsWe recruited 16 naïve glioblastoma patients enrolled in a phase II trial consisting of the Stupp protocol (a standardized treatment for glioblastoma involving combined radiotherapy and chemotherapy with temozolomide, followed by adjuvant temozolomide) plus IT with dendritic cells. Patients were followed up till progression or death; seven underwent a second surgery for suspected progression. Clinical, immunological, and MRI data were collected from all patients and histology in case of second surgery. Patients were classified as responders (progression-free survival, PFS > 12 months), and non-responders (PFS ≤ 12), HIGH-NK (natural killer cells, i.e., immunological responders), and LOW-NK (immunological non-responders) based on immune cell counts in peripheral blood. TRAMs differentiate contrast-enhancing lesions with different washout dynamics into hypothesized tumoral (conventionally blue-colored) vs. treatment-related (red-colored).ResultsUsing receiver operating characteristic (ROC) curves, a threshold of −0.066 in VBlue/VCE (volume of the blue portion of tumoral area/volume of contrast enhancement) variation between values obtained in the MRI performed before PsP/TTP and at TTP/PSP allowed to discriminate TTP from PsP with a sensitivity of 71.4% and a specificity of 100%. Among HIGH-NK patients, at month 6 there was a significant reduction compared to baseline and month 2 in median “blue” volumes.DiscussionIn conclusion, in our pilot study TRAMs support the discrimination between tumoral and treatment-related enhancing features in immunological responders vs. non-responders, the distinction between PsP and TTP, and might provide surrogate markers of immunological response.

Abstract BACKGROUND Despite the first classification of glioblastoma as an immunologically “cold” tumor, accumulating evidence suggests that this cancer type is susceptible to T cell infiltration, providing a glimmer of hope for the development of effective immunotherapy approaches that until now had limited success in brain tumors. To explore the contribution of biomarkers that could identify groups of patients showing durable response to immunotherapy, we examined several clinical and biological finding in 69 newly-diagnosed glioblastoma patients treated with dendritic cell (DC) immunotherapy added to standard treatment (DENDR1 NCT04801147). MATERIAL AND METHODS Patients with post-surgery volume ≤10 cc underwent leukapheresis before radiotherapy and chemotherapy with temozolomide (TMZ). Three intradermal injections of mature DC loaded with whole tumor lysate were administrated before and four after adjuvant TMZ. In all patients peripheral immune effector subsets were assessed including T and NK cells,, In all tumor samples MGMT methylation status was evaluated, and in 36 cases an immunohistochemistry (IHC) analysis was performed on tumor specimen collected before DC immunotherapy. RESULTS After a median follow-up of 23months (mo) median progression-free survival (PFS) was 12 mo (CI 95%10.34-13.6) and median overall survival (OS) was 22.6 mo (CI 95%18.7-24.5) comparing favorably with OS expected from the literature. MGMT status and expansion of NK cells positively impacted PFS and OS. Furthermore, our preliminary results suggest an association between the density and localization of specific Tumor Infiltrating Lymphocytes (TILs) and patients’ response to immunotherapy. TIL spatial patterns revealed either an immune-excluded, or an immune-infiltrated scenario with either scattered, clustered, or widespread T cell infiltration. Notably, an abundant widespread infiltration of CD8 TILs was correlated with prolonged survival of DENDR1 patients (PFS 18.0 vs. 8.9; OS 33.6 vs. 11.0; p< 0.005 compared with excluded CD8+ TIL distribution), supporting that distribution can be predictive of immunotherapy responsiveness. CONCLUSION These findings suggest that in DENDR-1 improved clinical outcomes correlate with an increase in the number of active NK cells, MGMT status, and abundant widespread infiltration of CD8+ TILs.

XLS file - 44K, List of primer pairs used for gene sequencing

Supplementary Table S2 from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

Glioblastoma (GBM) cancer stem cells (GSCs) contribute to GBM's origin, recurrence, and resistance to treatment. However, the understanding of how mRNA expression patterns of GBM subtypes are reflected at global proteome level in GSCs is limited. To characterize protein expression in GSCs, we performed in‐depth proteogenomic analysis of patient‐derived GSCs by RNA‐sequencing and mass‐spectrometry. We quantified > 10 000 proteins in two independent GSC panels and propose a GSC‐associated proteomic signature characterizing two distinct phenotypic conditions; one defined by proteins upregulated in proneural and classical GSCs (GPC‐like), and another by proteins upregulated in mesenchymal GSCs (GM‐like). The GM‐like protein set in GBM tissue was associated with necrosis, recurrence, and worse overall survival. Through proteogenomics, we discovered 252 non‐canonical peptides in the GSCs, i.e., protein sequences that are variant or derive from genome regions previously considered non‐protein‐coding, including variants of the heterogeneous ribonucleoproteins implicated in RNA splicing. In summary, GSCs express two protein sets that have an inverse association with clinical outcomes in GBM. The discovery of non‐canonical protein sequences questions existing gene models and pinpoints new protein targets for research in GBM.

<p>XLS file - 231KB, Excel file with the DEG genes from microarrays analysis.</p>

PDF file - 2.3MB, Figure S1: Histopathology of primary glioblastomas and genetic lesions of neurospheres. Figure S2: Genome-wide expression profiling and unsupervised hierarchical clustering of neurospheres (classical, mesenchymal and proneural centroids). Figure S3: Expression of MET, EGFR, and a panel of self-renewal markers in neurospheres. Figure S4: Genome-wide expression profiling and unsupervised hierarchical clustering of neurospheres (multiple signatures). Figure S5: Proliferative response, activation of tyrosine kinase receptors, and differentiation pattern in Met-neg-NS and Met-pos-NS. Figure S6: Methigh and Metneg sorting parameters. Figure S7: The Methigh subpopulation of Met-pos-NS retains clonogenic and tumorigenic cells. Figure S8: H&E and Met immunohistochemical staining of histological sections from tumors formed by BT302 neurosphere and its sorted subpopulations. Figure S9: HGF sustains the stem-like and invasive phenotype of Met-pos-NS.

XLS file - 12K, Mutations of PTEN, TP53, and NF1 genes in glioblastoma neurospheres

Glioblastoma (GBM) is known as an intractable, highly heterogeneous tumor encompassing multiple subclones, each supported by a distinct glioblastoma stem cell (GSC). The contribution of GSC genetic and transcriptional heterogeneity to tumor subclonal properties is debated. In this study, we describe the systematic derivation, propagation, and characterization of multiple distinct GSCs from single, treatment-naive GBMs (GSC families). The tumorigenic potential of each GSC better correlates with its transcriptional profile than its genetic make-up, with classical GSCs being inherently more aggressive and mesenchymal more dependent on exogenous growth factors across multiple GBMs. These GSCs can segregate and recapitulate different histopathological aspects of the same GBM, as shown in a paradigmatic tumor with two histopathologically distinct components, including a conventional GBM and a more aggressive primitive neuronal component. This study provides a resource for investigating how GSCs with distinct genetic and/or phenotypic features contribute to individual GBM heterogeneity and malignant escalation.

Supplementary Table S1 from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas