Glioblastoma cells increase expression of notch signaling and synaptic genes within infiltrated brain tissue.

Glioblastoma remains one of the deadliest brain malignancies. First-line therapy consists of maximal surgical tumor resection, accompanied by chemotherapy and radiotherapy. Malignant cells escape surgical resection by migrating into the surrounding healthy brain tissue, where they give rise to the recurrent tumor. Based on gene expression, tumor cores can be subtyped into mesenchymal, proneural, and classical tumors, each being associated with differences in genetic alterations and cellular composition. In contrast, the adjacent brain parenchyma where infiltrating malignant cells escape surgical resection is less characterized in patients. Using spatial transcriptomics (n = 11), we show that malignant cells within proneural or mesenchymal tumor cores display spatially organized differences in gene expression, although such differences decrease within the infiltrated brain tissue. Malignant cells residing in infiltrated brain tissue have increased expression of genes related to neurodevelopmental pathways and glial cell differentiation. Our findings provide an updated view of the spatial landscape of glioblastomas and further our understanding of the malignant cells that infiltrate the healthy brain, providing new avenues for the targeted therapy of these cells after surgical resection.

GDNF/GFRA1 signaling contributes to chemo- and radioresistance in glioblastoma.

Glioblastoma is the most common primary brain tumor in adults, characterized by an inherent aggressivity and resistance to treatment leading to poor prognoses. While some resistance mechanisms have been elucidated, a deeper understanding of these mechanisms is needed to increase therapeutic efficacy. In this study we first discovered glial-cell derived neurotrophic factor (GDNF) to be upregulated in patient-derived glioblastoma spheroid cultures after chemotherapeutic temozolomide treatment, through RNA-Seq experiments. Therefore, we investigated the role of the GDNF/GDNF receptor alpha 1 (GFRA1) signaling pathway as a resistance mechanism to chemotherapy with temozolomide and lomustine, as well as irradiation using patient-derived glioblastoma spheroid cultures. With qPCR experiments we showed a consistent upregulation of GDNF and its primary receptor GFRA1 following all three lines of treatment. Moreover, CRISPR/Cas9 knock-outs of GDNF in two patient-derived models sensitized these cells to chemotherapy treatment, but not radiotherapy. The increased sensitivity was completely reversed by the addition of exogeneous GDNF, confirming the key role of this factor in chemoresistance. Finally, a CRISPR KO of GFRA1 demonstrated a similar increased sensitivity to temozolomide and lomustine treatment, as well as radiotherapy. Together, our findings support the role of the GDNF/GFRA1 signaling pathway in glioblastoma chemo and radioresistance.

Nivolumab Reaches Brain Lesions in Patients with Recurrent Glioblastoma and Induces T-cell Activity and Upregulation of Checkpoint Pathways.

Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis. Although immunotherapy is being explored as a potential treatment option for patients with GBM, it is unclear whether systemic immunotherapy can reach and modify the tumor microenvironment in the brain. We evaluated immune characteristics in patients receiving the anti-PD-1 immune checkpoint inhibitor nivolumab 1 week prior to surgery, compared with control patients receiving salvage resection without prior nivolumab treatment. We observed saturating levels of nivolumab bound to intratumorally and tissue-resident T cells in the brain, implicating saturating levels of nivolumab reaching brain tumors. Following nivolumab treatment, significant changes in T-cell activation and proliferation were observed in the tumor-resident T-cell population, and peripheral T cells upregulated chemokine receptors related to brain homing. A strong nivolumab-driven upregulation in compensatory checkpoint inhibition molecules, i.e., TIGIT, LAG-3, TIM-3, and CTLA-4, was observed, potentially counteracting the treatment effect. Finally, tumor-reactive tumor-infiltrating lymphocytes (TIL) were found in a subset of nivolumab-treated patients with prolonged survival, and neoantigen-reactive T cells were identified in both TILs and blood. This indicates a systemic response toward GBM in a subset of patients, which was further boosted by nivolumab, with T-cell responses toward tumor-derived neoantigens. Our study demonstrates that nivolumab does reach the GBM tumor lesion and enhances antitumor T-cell responses both intratumorally and systemically. However, various anti-inflammatory mechanisms mitigate the clinical efficacy of the anti-PD-1 treatment.

Homeostatic iron regulatory protein drives glioblastoma growth via tumor cell-intrinsic and sex-specific responses.

Background: Glioblastoma (GBM) displays alterations in iron that drive proliferation and tumor growth. Iron regulation is complex and involves many regulatory mechanisms, including the homeostatic iron regulator (HFE) gene, which encodes the homeostatic iron regulatory protein. While HFE is upregulated in GBM and correlates with poor survival outcomes, the function of HFE in GBM remains unclear. Methods: We interrogated the impact of cell-intrinsic Hfe expression on proliferation and survival of intracranially implanted animals through genetic gain- and loss-of-function approaches in syngeneic mouse glioma models, along with in vivo immune assessments. We also determined the expression of iron-associated genes and their relationship to survival in GBM using public data sets and used transcriptional profiling to identify differentially expressed pathways in control compared to Hfe-knockdown cells. Results: Overexpression of Hfe accelerated GBM proliferation and reduced animal survival, whereas suppression of Hfe induced apoptotic cell death and extended survival, which was more pronounced in females and associated with attenuation of natural killer cells and CD8+ T cell activity. Analysis of iron gene signatures in Hfe-knockdown cells revealed alterations in the expression of several iron-associated genes, suggesting global disruption of intracellular iron homeostasis. Further analysis of differentially expressed pathways revealed oxidative stress as the top pathway upregulated following Hfe loss. Hfe knockdown indeed resulted in enhanced 55Fe uptake and generation of reactive oxygen species. Conclusions: These findings reveal an essential function for HFE in GBM cell growth and survival, as well as a sex-specific interaction with the immune response.

Preclinical evaluation of [58mCo]Co-DOTA-PSMA-617 for Auger electron therapy of prostate cancer.

Prostate-specific membrane antigen (PSMA), highly expressed in prostate cancer, is a promising target for radionuclide therapy. Auger electron-emitting radionuclides are well suited for targeted radionuclide therapy if they can be delivered close to the DNA of the targeted cells. This preclinical study evaluated the theranostic pair [55/58mCo]Co-DOTA-PSMA-617 for PET imaging and Auger electron therapy of prostate cancer. [58mCo]Co-DOTA-PSMA-617 was successfully prepared with > 99% radiochemical yield and purity. In vitro, uptake and subcellular distribution assays in PSMA-positive prostate cancer cells showed PSMA-specific uptake with high cell-associated activity in the nucleus. Incubation with [58mCo]Co-DOTA-PSMA-617 reduced cell viability and clonogenic survival in a significant dose-dependent manner (p < 0.05). Biodistribution of xenografted mice showed high specific tumor uptake of the cobalt-labeled PSMA ligand for all time points with rapid clearance from normal tissues, which PET imaging confirmed. In vivo, therapy with [58mCo]Co-DOTA-PSMA-617 in tumor-bearing mice demonstrated significantly increased median survival for treated mice compared to control animals (p = 0.0014). In conclusion, [55/58mCo]Co-DOTA-PSMA-617 displayed excellent in vitro and in vivo properties, offering significant survival benefits in mice with no observed toxicities.

Meningioma animal models: a systematic review and meta-analysis.

BACKGROUND: Animal models are widely used to study pathological processes and drug (side) effects in a controlled environment. There is a wide variety of methods available for establishing animal models depending on the research question. Commonly used methods in tumor research include xenografting cells (established/commercially available or primary patient-derived) or whole tumor pieces either orthotopically or heterotopically and the more recent genetically engineered models-each type with their own advantages and disadvantages. The current systematic review aimed to investigate the meningioma model types used, perform a meta-analysis on tumor take rate (TTR), and perform critical appraisal of the included studies. The study also aimed to assess reproducibility, reliability, means of validation and verification of models, alongside pros and cons and uses of the model types. METHODS: We searched Medline, Embase, and Web of Science for all in vivo meningioma models. The primary outcome was tumor take rate. Meta-analysis was performed on tumor take rate followed by subgroup analyses on the number of cells and duration of incubation. The validity of the tumor models was assessed qualitatively. We performed critical appraisal of the methodological quality and quality of reporting for all included studies. RESULTS: We included 114 unique records (78 using established cell line models (ECLM), 21 using primary patient-derived tumor models (PTM), 10 using genetically engineered models (GEM), and 11 using uncategorized models). TTRs for ECLM were 94% (95% CI 92-96) for orthotopic and 95% (93-96) for heterotopic. PTM showed lower TTRs [orthotopic 53% (33-72) and heterotopic 82% (73-89)] and finally GEM revealed a TTR of 34% (26-43). CONCLUSION: This systematic review shows high consistent TTRs in established cell line models and varying TTRs in primary patient-derived models and genetically engineered models. However, we identified several issues regarding the quality of reporting and the methodological approach that reduce the validity, transparency, and reproducibility of studies and suggest a high risk of publication bias. Finally, each tumor model type has specific roles in research based on their advantages (and disadvantages). SYSTEMATIC REVIEW REGISTRATION: PROSPERO-ID CRD42022308833.

D-2-hydroxyglutarate regulates human brain vascular endothelial cell proliferation and barrier function.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) genes result in excessive production of (D)-2-hydroxyglutarate (D-2HG) which intrinsically modifies tumor cell epigenetics and impacts surrounding noncancerous cells through nonepigenetic pathways. However, whether D-2HG has a paracrine effect on endothelial cells in the tumor microenvironment needs further clarification. We quantified microvessel density by immunohistochemistry using tissue sections from 60 high-grade astrocytic gliomas with or without IDH mutation. Microvessel density was found to be reduced in tumors carrying an IDH mutation. Ex vivo experiments showed that D-2HG inhibited endothelial cell migration, wound healing, and tube formation by suppressing cell proliferation but not viability, possibly through reduced activation of the mTOR/STAT3 pathway. Further, D-2HG reduced fluorescent dextran permeability and decreased paracellular T-cell transendothelial migration by augmenting expression of junctional proteins thereby collectively increasing endothelial barrier function. These results indicate that D-2HG may influence the tumor vascular microenvironment by reducing the intratumoral vasculature density and by inhibiting the transport of metabolites and extravasation of circulating cells into the astrocytoma microenvironment. These observations provide a rationale for combining IDH inhibition with antitumor immunological/angiogenic approaches and suggest a molecular basis for resistance to antiangiogenic drugs in patients whose tumors express a mutant IDH allele.

Loss of cancer cell-derived ADAM15 alters the tumor microenvironment in colorectal tumors.

Tumor progression and response to treatment are highly affected by interactions between cancer cells and the tumor microenvironment (TME). Many of the soluble factors and signaling receptors involved in this crosstalk are shed by a disintegrin and metalloproteinases (ADAMs). Upregulation of ADAM15 has been linked to worse survival in cancer patients and a tumor-promoting function both in vitro and in murine cancer models. Although ADAM15 has been involved in cell-cell and cell-extracellular matrix interactions, its role in the crosstalk between cancer cells and the TME in vivo remains unexplored. Therefore, we aimed to understand how ADAM15 regulates the cell composition of the TME and how it affects tumor progression. Here, we showed an upregulation of ADAM15 in tumor tissues from rectal cancer patients. Subcutaneous injection of wildtype and ADAM15-knockout CT26 colon cancer cells in syngeneic mice confirmed the protumorigenic role of ADAM15. Profiling of tumors revealed higher immune cell infiltration and cancer cell apoptosis in the ADAM15-deficient tumors. Specifically, loss of ADAM15 led to a reduced number of granulocytes and higher infiltration of antigen-presenting cells, including dendritic cells and macrophages, as well as more T cells. Using in vitro assays, we confirmed the regulatory effect of ADAM15 on macrophage migration and identified ADAM15-derived CYR61 as a potential molecular mediator of this effect. Based on these findings, we speculate that targeting ADAM15 could increase the infiltration of immune cells in colorectal tumors, which is a prerequisite for effective immunotherapy.

Molecular diagnostic tools for the World Health Organization (WHO) 2021 classification of gliomas, glioneuronal and neuronal tumors; an EANO guideline.

In the 5th edition of the WHO CNS tumor classification (CNS5, 2021), multiple molecular characteristics became essential diagnostic criteria for many additional CNS tumor types. For those tumors, an integrated, "histomolecular" diagnosis is required. A variety of approaches exists for determining the status of the underlying molecular markers. The present guideline focuses on the methods that can be used for assessment of the currently most informative diagnostic and prognostic molecular markers for the diagnosis of gliomas, glioneuronal and neuronal tumors. The main characteristics of the molecular methods are systematically discussed, followed by recommendations and information on available evidence levels for diagnostic measures. The recommendations cover DNA and RNA next-generation-sequencing, methylome profiling, and select assays for single/limited target analyses, including immunohistochemistry. Additionally, because of its importance as a predictive marker in IDH-wildtype glioblastomas, tools for the analysis of MGMT promoter methylation status are covered. A structured overview of the different assays with their characteristics, especially their advantages and limitations, is provided, and requirements for input material and reporting of results are clarified. General aspects of molecular diagnostic testing regarding clinical relevance, accessibility, cost, implementation, regulatory, and ethical aspects are discussed as well. Finally, we provide an outlook on new developments in the landscape of molecular testing technologies in neuro-oncology.

A variability in response of osteoclasts to zoledronic acid is mediated by smoking-associated modification in the DNA methylome.

BACKGROUND: Clinical trials have shown zoledronic acid as a potent bisphosphonate in preventing bone loss, but with varying potency between patients. Human osteoclasts ex vivo reportedly displayed a variable sensitivity to zoledronic acid > 200-fold, determined by the half-maximal inhibitory concentration (IC50), with cigarette smoking as one of the reported contributors to this variation. To reveal the molecular basis of the smoking-mediated variation on treatment sensitivity, we performed a DNA methylome profiling on whole blood cells from 34 healthy female blood donors. Multiple regression models were fitted to associate DNA methylation with ex vivo determined IC50 values, smoking, and their interaction adjusting for age and cell compositions. RESULTS: We identified 59 CpGs displaying genome-wide significance (p < 1e-08) with a false discovery rate (FDR) < 0.05 for the smoking-dependent association with IC50. Among them, 3 CpGs have p < 1e-08 and FDR < 2e-03. By comparing with genome-wide association studies, 15 significant CpGs were locally enriched (within < 50,000 bp) by SNPs associated with bone and body size measures. Furthermore, through a replication analysis using data from a published multi-omics association study on bone mineral density (BMD), we could validate that 29 out of the 59 CpGs were in close vicinity of genomic sites significantly associated with BMD. Gene Ontology (GO) analysis on genes linked to the 59 CpGs displaying smoking-dependent association with IC50, detected 18 significant GO terms including cation:cation antiporter activity, extracellular matrix conferring tensile strength, ligand-gated ion channel activity, etc. CONCLUSIONS: Our results suggest that smoking mediates individual sensitivity to zoledronic acid treatment through epigenetic regulation. Our novel findings could have important clinical implications since DNA methylation analysis may enable personalized zoledronic acid treatment.