This international consensus statement synthesizes key findings on the complex roles of microglia and macrophages (tumor-associated microglia/macrophages or TAMs) in glioma progression and therapeutic resistance. Recent advances have highlighted the cellular, spatial, and temporal heterogeneity of TAMs, their functional plasticity, and the intricate interactions between TAMs, glioma stem cells, and the neuronal microenvironment, challenging the M1/M2 classification paradigm for TAMs in gliomas and other misconceptions. The statement emphasizes that glioma cells manipulate TAMs to suppress anti-tumor functions, while microglia-mediated modulation of neuron-glioma cell interactions promotes tumor progression. Furthermore, glioblastoma-derived extracellular vesicles (EVs) reprogram microglia to support tumor progression, offering novel therapeutic targets. To advance research and develop more effective treatments, the statement advocates for precision therapies targeting specific TAM subsets or functions, the use of bioengineered EVs as a therapeutic approach, and a shift away from simplistic terminology like “M1/M2” and “neuroinflammation”. Ultimately, this new understanding can support innovative strategies to modulate the tumor microenvironment, turning immunosuppression into immunostimulation and improving outcomes for patients with glioblastoma and other types of gliomas.

Human surgery and autopsy specimens are routinely stored as formalin-fixed paraffin-embedded (FFPE) tissue blocks for decades, creating vast archives of healthy and diseased tissues. While tissue clearing and whole-mount microscopy enable 3D analysis, FFPE human tissue blocks are often unsuitable for clearing and immunolabeling due to their large size and extensive cross-linking. Here, we introduce "archival" DISCO (aDISCO), a clearing method designed to overcome these challenges. aDISCO achieves effective clearing and immunolabeling of large samples stored for 15 years or more. We applied aDISCO to human brain, spinal cord, peripheral nerve, skin, muscle, heart, kidney, liver, spleen, colon, and lung, using a broad range of antibodies. Combining aDISCO with deep learning-based analysis to study focal cortical dysplasia (FCD), we found disrupted cortical layering with both focal and global neuronal density variations, features likely to be overlooked by conventional histology. In summary, aDISCO delivers datasets suitable for deep learning-based processing, enabling the detection of subtle and sparse pathologies in large archival human tissue specimens.

Abstract Background Targeting the Ras/MAPK pathway is driving a paradigm shift in pediatric low-grade glioma (pLGG) treatment. Despite their slow growth, tumors often recur, and many patients undergo multiple and prolonged lines of treatment. Most treatment decisions are based on the analyses of the primary tissue, and the mechanisms driving tumor progression remain unclear. Methods We analyzed the molecular profiles of 130 tumor samples from 109 patients with pLGG [ 17 patients with matched samples]. Using imaging mass cytometry (IMC), we compared single-cell phenotypes and the spatial cytoarchitecture of primary and matched recurrent pLGGs. Results BRAF alterations were observed in 53% of patients, with rare BRAF fusion variants, and PTPN11 and PIK3CA alterations enriched in patients with progressive disease. In matched primary/recurrent samples, new genetic alterations were detected in 14/21 samples at progression, particularly in patients who received non-surgical treatments, suggesting the emergence or selection of neoplastic subpopulations during chemo/radiotherapy. Using 70 unique protein markers we profiled 782934 cells using IMC to capture the tumor, immune, and stromal compartments from 53 pLGG samples. We observed a complex tumor microenvironment with substantial inter- and intra-patient heterogeneity, including up to 30% non-neoplastic cells. GFAP,Vimentin+ cells were significantly more abundant in recurrent samples, particularly within reactive regions, and exhibited significant cellular interactions with GFAP,S100B+ cells and tumor-associated macrophages/microglia. Conclusion Molecular characterization of progressive pLGGs can reveal additional alterations with potential treatment implications, underscoring the importance of re-biopsy in patients with progressive disease. Spatial analyses of tumor cell phenotypes are critical for understanding the variability between primary and recurrent disease, potentially offering clinically actionable insights into treatment failure in pLGG.

Pediatric cancers are thought to arise from dysregulation of developmental programs, otherwise tightly regulated in time and space. Infant-type hemispheric gliomas (IHGs) arise in early childhood, driven by characteristic ALK/ROS1/MET/NTRK receptor tyrosine kinase (RTK) gene fusions. We dissected the cellular hierarchies of 24 fusion-positive gliomas, spanning infants through adolescents, using single-cell and single-nucleus RNA/ATAC-seq, and spatial transcriptomics. We identified five cancer cell states, with radial glia-like cells at the apex of a neoplastic hierarchy resembling neuronal- and glial-like trajectories. Neuronal-like cells were enriched in most IHGs but diminished in ROS1-fused IHGs and older patients. Integration of chromatin profiling revealed FOS/JUN-driven oncogenic programs and high inferred plasticity across all cancer cell populations. Myeloid cells, the most abundant non-neoplastic population, comprised distinct subgroups, suggesting context-dependent functions. Despite lacking high-order structure, spatial transcriptomics revealed discrete cellular niches within IHGs. Collectively, our findings elucidate the cellular states and developmental programs underlying IHGs and RTK-fused gliomas in older patients, opening new avenues for research and therapy innovation.

Brain metastases (BM) require a multidisciplinary treatment approach combining surgery, radiotherapy, and systemic therapy. While current guidelines recommend the analysis of established cancer driver genes in BM tissue, little is known about its real-life implementation and relevance on oncological treatment strategies. This retrospective multicenter study includes patients with BM from melanoma, lung and breast cancer operated between 2010 and 2022. Clinical records were evaluated for BM molecular analyses of predefined cancer drivers and their discordance to extracranial tumor sites, that is, ALK, BRAF, EGFR, KRAS, NTRK for lung, HER2, estrogen/progesterone receptor, BRCA1/2 for breast cancer and BRAF, KIT, NRAS among others for melanoma. Adjustment of systemic therapies based on BM molecular profiles was analyzed. Among 1431 BMs screened for availability of molecular analysis, molecular profiling was performed at least partially in 723 BMs (51%). In cases with matched extracranial tumor samples (n = 276), discordant alterations were found in 18% of lung, 4.7% of melanoma and 45% of breast cancer BMs. Molecular BM analyses informed systemic therapy adjustments in 13%-27% of patients depending on the primary tumor. Overall survival was significantly better in patients who underwent BM profiling, especially when operated in recensst years (median 19.3 vs. 9.9 months; p < 0.0001) and in patients with breast cancer who had a change in systemic therapies upon BM profiling (p = 0.0085). In conclusion, molecular profiling of BM allows selecting available treatment options for a substantial subset of patients. This study underscores the need for more systematic molecular testing in BM patients to guide systemic treatment decisions.

Background:A significant proportion of meningiomas are resistant to current treatments. Somatostatin receptor 2 (SSTR2) is highly and consistently expressed in most meningiomas, providing a promising target for localized chimeric antigen receptor (CAR)-T cell therapy. Short-lived small-molecule CAR adapters can potentially prevent CAR-T cell exhaustion in solid tumors by alternating between active and resting states. Methods:We developed the CAR adapter peptide Octofluo, which combines fluorescein-5-isothiocyanate (FITC) with a high-avidity SSTR2 antagonist. After determining its biodistribution, killing efficiency of CAR-T cells plus Octofluo against human meningioma was evaluated in vitro and ex vivo. Therapeutic capacity was assessed in vivo against xenograft and syngeneic genetically engineered mouse meningioma models. Results:We herein demonstrate rapid tissue diffusion and transient tumor persistence for only a few hours after intravenous administration of Octofluo, making a suitable switch for on-demand CAR-T cell activation. Nanomolar concentrations of Octofluo effectively directed FITC-specific CAR-T cells against SSTR2-expressing meningioma cells. Combined intratumoral CAR-T cell delivery and intravenous octofluo infusion at periodic intervals showed limited efficacy in an immunocompromised xenograft model but cured most mice with an intact immune system harboring highly aggressive, genetically induced higher grade meningiomas. Cures were accompanied by CAR-T cell expansion and an endogenous T cell response, suggesting a role for the host immune system in tumor elimination. Ex vivo lysis of patient-derived meningioma cells was observed. Conclusions:The combination of systemic octofluo administration and locally applied CAR-T cells is a promising strategy for future clinical development for patients with refractory meningiomas.

During human surgeries and autopsies, specimens are regularly sampled and stored as formalin-fixed paraffin-embedded (FFPE) tissue blocks. Diagnoses are rendered by microscopical examination of two-dimensional sections. Good clinical practice requires that samples be retained for decades, thus giving rise to enormous archives of healthy and diseased human tissues. Tissue-clearing technologies and whole-mount microscopy would enable 3D analyses, but whole FFPE tissue blocks are often unsuitable for clearing due to their size, their extensive covalent cross-linking, and their embedding in solid wax. Here, we present ‘archival DISCO’ (aDISCO), a versatile and robust clearing method for whole-mount archival FFPE human tissue blocks. aDISCO enabled complete clearing and consistent antibody staining of samples stored for at least 15 years. We show that aDISCO can be applied to human brain, spinal cord, peripheral nerve, skin, muscle, heart, kidney, liver, spleen, colon, and lung, and is compatible with a wide range of commonly used antibodies. We applied aDISCO to the 3D study of focal cortical dysplasia (FCD), a neurodevelopmental disease associated with epilepsy. We performed deep-learning-based segmentation and object detection to identify both focal and global neuronal density variations in FCD and to precisely quantify the disruption of cortical layering, features that are likely to be overlooked by conventional histology. When combined with selective-plane illumination microscopy, aDISCO delivers natively digital data suitable for deep-learning-based processing, thus enabling the detection of subtle and sparse pathologies in large archival human tissue specimens. ### Competing Interest Statement The authors have declared no competing interest. Filling-the-Gap grant, University of Zurich EMPIRIS and Lazarus grants, University Hospital Zurich Foundation Investment Fund, University of Zurich URPP Adaptive Brain Circuits in Development and Learning (AdaBD)?, University of Zurich

Inclusion body myositis (IBM) is a progressive muscle disorder characterized by inflammation and degeneration with altered proteostasis. To better understand the interrelationship between these two features, we aimed to establish a novel preclinical mouse model. First, we used quantitative PCR, in situ hybridization and immunohistochemistry to determine the expression of pro-inflammatory chemokines and cytokines including lymphotoxin (LT)-signalling pathway components in human skeletal muscle tissue diagnosed with myositis. Based on these results, we generated a mouse model that we analysed at the histological, ultrastructural, transcriptional, biochemical and behavioural level. Lastly, we subjected this model to anti-inflammatory treatments. After confirming and extending previous data on activation of LT-signalling in human myositis, we generated distinct transgenic mouse lines co-expressing LTα and -β in skeletal muscle fibres. Transgenic mice displayed chronic myositis accompanied by dysregulated proteostasis, including an altered autophagolysosomal pathway that initially showed signs of activation and later exhaustion and decreased flux. To enhance the latter, we genetically impaired autophagy in skeletal muscle cells. Autophagy impairment alone induced a pro-inflammatory transcriptional state, but no obvious cellular inflammation. However, the combination of LT-driven myositis with autophagy impairment induced the full spectrum of characteristic molecular and pathological features of IBM in skeletal muscle, including protein inclusions with typical ultrastructural morphology and mild mitochondrial pathology. Our attempts to treat the pathology by subjecting these mice to corticosteroids or anti-Thy1.2 antibodies mirrored recent treatment failures in humans, i.e. none of these treatments resulted in significant clinical improvement of motor performance or the transcriptional profile of muscle pathology. In summary, these data provide evidence that inflammation and autophagy disruption play a synergistic role in the development of IBM-like muscular pathology. Furthermore, once established, IBM-like pathology in these mice, as in human IBM patients, cannot be reverted or prevented from progression by conventional means of immunosuppression. We expect that this novel mouse model will help to identify future treatment modalities for IBM.

Abstract Effective treatment options for meningioma patients beyond surgical resection and radiotherapy are limited. This study presents preclinical and translational evidence supporting the efficacy of an adaptor CAR-T cell system targeting Somatostatin Receptor (SSTR) 2 in otherwise untreatable meningiomas. Tissue microarray analysis revealed that SSTR2 expression was generally heterogeneous but high or intermediate in most World Health Organization (WHO) grade 2 (87.8%) and grade 3 (87.5%) meningiomas. The fluorescein-linked, SSTR2 antagonist peptide octofluo was used in conjunction with adaptor FITC (AdFITC) CAR-T cells to treat experimental meningiomas. In vitro, 10 nM octofluo induced tumor cell killing within 72 hours at effector-target cell ratios (E:T) as low as 1:100. This was observed in two human meningioma cell lines, Ben-Men-1 (CAR-T cells vs. untransduced T-cells [UTT]: 47.9% vs. 4.7%, P < 0.001) and IOMM-Lee (CAR-T cells vs. UTT: 40.2% vs. 4.9%, P < 0.001). In vivo, the AdFITC CAR-T cell system inhibited meningioma growth (P = 0.002) and prolonged survival (P = 0.003) in the IOMM-Lee orthotopic model compared to PBS. In a genetically engineered murine meningioma model, the AdFITC CAR-T cell system not only restricted tumor growth (P = 0.0018) and improved survival (P < 0.0001), but cured a substantial proportion of meningioma bearing mice. This treatment also induced the expansion of both CD8+ CAR-T cells (P = 0.0041) and a host T-cell response. Preliminary ex vivo killing assays, involving co-culture of the AdFITC CAR-T cell system with patient-derived meningioma tissue, showed specific lysis rates of 15-20% (ET 1:1) within 12 hours. Targeting SSTR2 with the AdFITC CAR-T cell system emerges as a promising therapeutic approach for meningioma, an early phase clinical trial is warranted.

Introduction MRI is the imaging modality of choice for assessing patients with encephalopathy. In this context, we discuss a novel biomarker, the “split ADC sign,” where the cerebral cortex demonstrates restricted diffusion (high DWI signal and low ADC) and the underlying white matter demonstrates facilitated diffusion (high or low DWI signal and high ADC). We hypothesize that this sign can be used as a biomarker to suggest either acute encephalitis onset or to raise the possibility of an autoimmune etiology. Materials and Methods A full-text radiological information system search of radiological reports was performed for all entities known to produce restricted diffusion in the cortex excluding stroke between January 2012 and June 2022. Initial MRI studies performed upon onset of clinical symptoms were screened for the split ADC sign. Results 25 subjects were encountered with a positive split ADC sign (15 female; median age = 57 years, range 18–82). Diagnosis included six herpes simplex encephalitis, three peri-ictal MRI changes, eight PRES, two MELAS, and six autoimmune (3 anti-GABA A R, two seronegative, and one anti-Ma2/Ta). Subjects were imaged at a mean 1.8 days after the onset of symptoms (range 0–8). Discussion We present a novel visual MRI biomarker, the split ADC sign, and highlight its potential usefulness in subjects with encephalopathy to suggest acute disease onset or to raise the possibility of an autoimmune etiology when location-based criteria are applied. When positive, the sign was present on the initial MRI and can therefore be used to help focus further clinical and laboratory workup.

Dynamic perviousness is a novel imaging biomarker, with clot density measurements at multiple timepoints to allow longer contrast to thrombus interaction. We investigated the correlations between dynamic perviousness and clot composition in the setting of acute ischemic stroke. Thirty-nine patients with large vessel occlusion (LVO) undergoing mechanical thrombectomy (MT) were analyzed. Patients received a three-phase CT imaging pre-thrombectomy and histopathological analysis of retrieved clots. Clot densities for every phase and change in densities between phases were calculated, leading to four patterns of dynamic perviousness: no contrast uptake, early contrast uptake with and without washout and late uptake. Clots were categorized into three groups based on dominant histologic composition: red blood cell (RBC)-rich, fibrin/platelet-rich and mixed. Clot composition was correlated with dynamic perviousness using the Kruskal–Wallis test and Pearson’s correlation analysis. The dynamic perviousness categories showed a significant difference between fibrin-rich clots when compared to RBC-rich plus mixed groups. The uptake without washout category had significantly fewer fibrin clots compared to the uptake with washout (p = 0.036), and nearly significantly fewer fibrin clots when compared to the no uptake category (p = 0.057). Contrast uptake with different patterns of contrast washout showed significant differences of the likelihood for fibrin-rich clots.

Background Advancements in metastatic breast cancer (BC) treatment have enhanced overall survival (OS), leading to increased rates of brain metastases (BM). This study analyzes the association between microsurgical tumor reduction and OS in patients with BCBM, considering tumor molecular subtypes and perioperative treatment approaches.Methods Retrospective analysis of surgically treated patients with BCBM from two tertiary brain tumor Swiss centers. The association of extent of resection (EOR), gross-total resection (GTR) achievement, and postoperative residual tumor volume (RV) with OS and intracranial progression-free survival (IC-PFS) was evaluated using Cox proportional hazard model.Results 101 patients were included in the final analysis, most patients (38%) exhibited HER2-/HR + BC molecular subtype, followed by HER2 + /HR + (25%), HER2-/HR- (21%), and HER2 + /HR- subtypes (13%). The majority received postoperative systemic treatment (75%) and radiotherapy (84%). Median OS and intracranial PFS were 22 and 8 months, respectively. The mean pre-surgery intracranial tumor volume was 26 cm3, reduced to 3 cm3 post-surgery. EOR, GTR achievement and RV were not significantly associated with OS or IC-PFS, but higher EOR and lower RV correlated with extended OS in patients without extracranial metastases. HER2-positive tumor status was associated with longer OS, extracranial metastases at BM diagnosis and symptomatic lesions with shorter OS and IC-PFS.Conclusions Our study found that BC molecular subtypes, extracranial disease status, and BM-related symptoms were associated with OS in surgically treated patients with BCBM. Additionally, while extensive resection to minimize residual tumor volume did not significantly affect OS across the entire cohort, it appeared beneficial for patients without extracranial metastases.

Background The incidence of brain cancer and neurodegenerative diseases is increasing with a demographic shift towards aging populations. Biological parallels have been observed between glioblastoma and Alzheimer's disease (AD), which converge on accelerated brain aging. Here, we aimed to map the cooccurrence of AD neuropathological change (ADNC) in the tumor-adjacent cortex of patients with glioblastoma. Methods Immunohistochemical screening of AD markers amyloid beta (Abeta), amyloid precursor protein (APP), and hyperphosphorylated tau (pTau) was conducted in 420 tumor samples of 205 patients. For each cortex area, we quantified ADNC, neurons, tumor cells, and microglia. Results Fifty-two percent of patients (N = 106/205) showed ADNC (Abeta and pTau, Abeta or pTau) in the tumor-adjacent cortex, with histological patterns widely consistent with AD. ADNC was positively correlated with patient age and varied spatially according to Thal phases and Braak stages. It decreased with increasing tumor cell infiltration (P < .0001) and was independent of frequent expression of APP in neuronal cell bodies (N = 182/205) and in tumor necrosis-related axonal spheroids (N = 195/205; P = .46). Microglia response was most present in tumor cell infiltration plus ADNC, being further modulated by patient age and sex. ADNC did not impact patient survival in the present cohort. Conclusions Our findings highlight the frequent presence of ADNC in the glioblastoma vicinity, which was linked to patient age and tumor location. The cooccurrence of AD and glioblastoma seemed stochastic without clear spatial relation. ADNC did not impact patient survival in our cohort.

Extracellularly released molecular inflammasome assemblies -ASC specks- cross-seed Aβ amyloid in Alzheimer’s disease. Here we show that ASC governs the extent of inflammation-induced amyloid A (AA) amyloidosis, a systemic disease caused by the aggregation and peripheral deposition of the acute-phase reactant serum amyloid A (SAA) in chronic inflammatory conditions. Using super-resolution microscopy, we found that ASC colocalized tightly with SAA in human AA amyloidosis. Recombinant ASC specks accelerated SAA fibril formation and mass spectrometry after limited proteolysis showed that ASC interacts with SAA via its pyrin domain (PYD). In a murine model of inflammatory AA amyloidosis, splenic amyloid load was conspicuously decreased in Pycard−/− mice which lack ASC. Treatment with anti-ASCPYD antibodies decreased amyloid loads in wild-type mice suffering from AA amyloidosis. The prevalence of natural anti-ASC IgG (−logEC50 ≥ 2) in 19,334 hospital patients was <0.01%, suggesting that anti-ASC antibody treatment modalities would not be confounded by natural autoimmunity. These findings expand the role played by ASC and IL-1 independent inflammasome employments to extraneural proteinopathies and suggest that anti-ASC immunotherapy may contribute to resolving such diseases.

Gliomas are the most common primary central nervous system (CNS) tumors and a major cause of cancer-related mortality in children (age <15 years), adolescents and young adults (AYA, ages 15–39 years), and adults (age >39 years). Molecular pathology has helped enhance the characterization of these tumors, revealing a heterogeneous and ever more complex group of malignancies. Recent molecular analyses have led to an increased appreciation of common genomic alterations prevalent across all ages. The 2021 World Health Organization (WHO) CNS tumor classification, 5th edition (WHO CNS5) brings forward a nomenclature distinguishing “pediatric-type” and “adult-type” gliomas. The spectrum of gliomas in AYA comprises both “pediatric-like” and “adult-like” tumor entities but remains ill-defined. With fragmentation of clinical management between pediatric and adult centers, AYAs face challenges related to gaps in medical care, lower rates of enrollment in clinical trials and additional psychosocial and economic challenges. This calls for a rethinking of diagnostic and therapeutic approaches, to improve access to appropriate testing and potentially beneficial treatments to patients of all ages.