Purpose: Targeting immunosuppressive and pro-tumorigenic glioblastoma (GBM)-associated macrophages and microglial cells (GAM) has great potential to improve patient outcomes. Colony-stimulating factor-1 receptor (CSF1R) has emerged as a promising target for reprograming anti-inflammatory M2-like GAMs. However, treatment data on patient-derived, tumor-educated GAMs and their influence on the adaptive immunity are lacking. Experimental Design: CD11b+-GAMs freshly isolated from patient tumors were treated with CSF1R-targeting drugs PLX3397, BLZ945, and GW2580. Phenotypical changes upon treatment were assessed using RNA sequencing, flow cytometry, and cytokine quantification. Functional analyses included inducible nitric oxide synthase activity, phagocytosis, transmigration, and autologous tumor cell killing assays. Antitumor effects and changes in GAM activation were confirmed in a complex patient-derived 3D tumor organoid model serving as a tumor avatar. Results: The most effective reprogramming of GAMs was observed upon GW2580 treatment, which led to the downregulation of M2-related markers, IL6, IL10, ERK1/2, and MAPK signaling pathways, while M1-like markers, gene set enrichment indicating activated MHC-II presentation, phagocytosis, and T-cell killing were substantially increased. Moreover, treatment of patient-derived GBM organoids with GW2580 confirmed successful reprogramming, resulting in impaired tumor cell proliferation. In line with its failure in clinical trials, PLX3397 was ineffective in our analysis. Conclusions: This comparative analysis of CSF1R-targeting drugs on patient-derived GAMs and human GBM avatars identified GW2580 as the most powerful inhibitor with the ability to polarize immunosuppressive GAMs to a proinflammatory phenotype, supporting antitumor T-cell responses while also exerting a direct antitumor effect. These data indicate that GW2580 could be an important pillar in future therapies for GBM.

IDH-wildtype glioblastoma (GBM) represents the most common malignant form of brain tumor and is still incurable despite comprehensive therapeutic efforts. Due to tumor location and patient condition, open surgical resection of recurrent GBM is not always feasible. In these cases, frame-based stereotactic biopsies represent a less invasive technique to obtain tissue samples for diagnostics. However, whether this material would also be sufficient to prepare tumor organoids (TOs) and perform drug screenings has not been addressed so far. In this study, we present our highly optimized workflow for generating standardized patient-derived GBM TOs from single-cell suspensions using limited biopsy-derived material. We highlight crucial steps within the procedure, such as reliable cell counting, viable cell recovery, enzymatic digestion, and the requirement of an extracellular matrix as a scaffold. Furthermore, we showcase the potential of personalized drug testing as a promising application of GBM TOs. In conclusion, we successfully developed a robust workflow that effectively utilizes the limited material derived from stereotactic biopsies to reproducibly form standardized TOs. Moreover, we demonstrate that biopsy-derived TOs represent a valuable tool for testing drug vulnerabilities in a personalized setting, which might be especially useful in the case of non-resectable GBM.

Breast cancer brain metastases (BCBM) are a severe condition with high demand for improved personalized treatment, but a comprehensive understanding of BCBM immune-microenvironment heterogeneity and susceptibility to immunotherapy is lacking. Here, we multimodally profile the immune niche in a clinically well-annotated cohort of 156 BCBM applying tissue cytometry, bulk and single nuclei RNA-sequencing, flow cytometry, and spatial transcriptomics, complemented by functional studies in patient-derived models. Integrative analyses reveal two immune landscapes predicting prolonged patient survival and that are not deducible from paired primary tumors: 1) BCBM with a high proportion of CD8⁺ tissue-resident-like memory T cells as major players of tumor immune control. 2) BCBM containing tertiary lymphoid structures. Surrogate signatures of these landscapes are prognostic in independent BCBM and primary breast cancer cohorts, are associated with fewer metastases, and predict immunotherapy response. Our work provides critical insights into anti-tumor immunity in BCBM and identifies novel biomarkers with translational relevance.

Managing aggressive meningiomas remains challenging because of limited treatment options besides surgical tumor removal and radiotherapy. To increase the repertoire of promising therapies for aggressive meningiomas, we established a multistep drug screening workflow, focusing on targetable genes obtained from transcriptome data of highly aggressive grade 3 meningiomas. In vitro screening of 107 targeted drugs identified nine effective inhibitors. To study these drugs in a more natural environment, we established a standardized patient-derived tumor organoid (TO) model preserving accurately the original tissue’s genotype and phenotype. Individual drug responses were assessed in TOs from 60 meningioma cases characterized at the molecular level. In particular, the US Food and Drug Administration–approved epigenetic drug panobinostat demonstrated high antimeningioma efficacy in 70% of TOs, mediated through histone deacetylase 1 and 2 (HDAC1/2) inhibition. In addition, treatment in an orthotopic in vivo model revealed improved survival. In search of the molecular mechanism underlying a potentially intrinsic panobinostat resistance, we identified up-regulation of the HDAC8–transforming growth factor–β (TGFβ)–epithelial-to-mesenchymal transition (EMT) axis in the TO model, whereas subsequent HDAC8 depletion increased the sensitivity to panobinostat. These data highlight the utility of personalized drug screenings on TOs to identify suitable drug targets and inhibitors for more effective treatment of clinically aggressive meningiomas and to help advance our understanding of counteracting resistance mechanisms.

Purpose:Targeting immunosuppressive and pro-tumorigenic glioblastoma (GBM)-associated macrophages and microglial cells (GAM) has great potential to improve patient outcomes. Colony-stimulating factor-1 receptor (CSF1R) has emerged as a promising target for reprograming anti-inflammatory M2-like GAMs. However, treatment data on patient-derived, tumor-educated GAMs and their influence on the adaptive immunity are lacking.Experimental Design:CD11b+-GAMs freshly isolated from patient tumors were treated with CSF1R-targeting drugs PLX3397, BLZ945, and GW2580. Phenotypical changes upon treatment were assessed using RNA sequencing, flow cytometry, and cytokine quantification. Functional analyses included inducible nitric oxide synthase activity, phagocytosis, transmigration, and autologous tumor cell killing assays. Antitumor effects and changes in GAM activation were confirmed in a complex patient-derived 3D tumor organoid model serving as a tumor avatar.Results:The most effective reprogramming of GAMs was observed upon GW2580 treatment, which led to the downregulation of M2-related markers, IL6, IL10, ERK1/2, and MAPK signaling pathways, while M1-like markers, gene set enrichment indicating activated MHC-II presentation, phagocytosis, and T-cell killing were substantially increased. Moreover, treatment of patient-derived GBM organoids with GW2580 confirmed successful reprogramming, resulting in impaired tumor cell proliferation. In line with its failure in clinical trials, PLX3397 was ineffective in our analysis.Conclusions:This comparative analysis of CSF1R-targeting drugs on patient-derived GAMs and human GBM avatars identified GW2580 as the most powerful inhibitor with the ability to polarize immunosuppressive GAMs to a proinflammatory phenotype, supporting antitumor T-cell responses while also exerting a direct antitumor effect. These data indicate that GW2580 could be an important pillar in future therapies for GBM.

Purpose:Targeting immunosuppressive and pro-tumorigenic glioblastoma (GBM)-associated macrophages and microglial cells (GAM) has great potential to improve patient outcomes. Colony-stimulating factor-1 receptor (CSF1R) has emerged as a promising target for reprograming anti-inflammatory M2-like GAMs. However, treatment data on patient-derived, tumor-educated GAMs and their influence on the adaptive immunity are lacking.Experimental Design:CD11b+-GAMs freshly isolated from patient tumors were treated with CSF1R-targeting drugs PLX3397, BLZ945, and GW2580. Phenotypical changes upon treatment were assessed using RNA sequencing, flow cytometry, and cytokine quantification. Functional analyses included inducible nitric oxide synthase activity, phagocytosis, transmigration, and autologous tumor cell killing assays. Antitumor effects and changes in GAM activation were confirmed in a complex patient-derived 3D tumor organoid model serving as a tumor avatar.Results:The most effective reprogramming of GAMs was observed upon GW2580 treatment, which led to the downregulation of M2-related markers, IL6, IL10, ERK1/2, and MAPK signaling pathways, while M1-like markers, gene set enrichment indicating activated MHC-II presentation, phagocytosis, and T-cell killing were substantially increased. Moreover, treatment of patient-derived GBM organoids with GW2580 confirmed successful reprogramming, resulting in impaired tumor cell proliferation. In line with its failure in clinical trials, PLX3397 was ineffective in our analysis.Conclusions:This comparative analysis of CSF1R-targeting drugs on patient-derived GAMs and human GBM avatars identified GW2580 as the most powerful inhibitor with the ability to polarize immunosuppressive GAMs to a proinflammatory phenotype, supporting antitumor T-cell responses while also exerting a direct antitumor effect. These data indicate that GW2580 could be an important pillar in future therapies for GBM.