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.

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.

Personalized drug screening aims to improve the survival of glioblastoma (GBM) patients by identifying effective patient-individual drugs. Therefore, we conducted an automated high-throughput drug screening (aHTS) on standardized patient-derived glioblastoma tumor organoids (TOs). Robot-assisted aHTS was performed on TOs from 11 GBM patients. TOs fully compacted after two days, and the size remained stable over 10 days. TOs proliferated over time, and immunofluorescence stainings (GFAP, Tenascin C) confirmed tissue-like architecture. Anti-glioma effects with the lowest drug concentrations were achieved for proteasome inhibitors (carfilzomib, bortezomib, ixazomib), and HDAC inhibitors (panobinostat, romidepsin). Plasma C-max-based drug levels (C-max/IC50 > 1) used as a surrogate were only achieved for the three proteasome inhibitors and the HDACi romidepsin. The impact of their drug targets PSMB5 and HDAC1/2 on the growth of GBM cells was successfully validated by RNAi experiments. We established an aHTS platform for GBM TOs, and identified proteasome and HDAC inhibitors as promising drugs for the treatment of GBMs.

Meningiomas (MGMs) are the most common primary intracranial tumors in adults with a substantial subset exhibiting aggressive clinical behavior. Immunotherapy represents a potential alternative treatment option, even though MGMs have traditionally been considered “immunologically cold” tumors. This study explored less characterized immune cell subsets —B cells, natural killer (NK) cells, and granulocytes— and their associations with major immune cell populations as well as their prognostic implications. For this purpose, we performed tissue cytometry analysis in a clinically well-annotated multi-center cohort of 97 newly diagnosed MGMs encompassing all WHO grades (1, 2, 3) and DNA methylation classes (benign, intermediate, malignant). Resulting infiltration data were integrated with previously published data on tumor-associated macrophages (TAMs) and tumor-infiltrating T lymphocytes (TILs) to identify MGM immune ecotypes. Overall, infiltration rates of B cells, NK cells, neutrophils, and eosinophils showed lower frequencies and varied widely across tumors. Notably, we observed significantly lower numbers of B cells in MGM with losses in chromosomal arms 10q and 22q, while lower number of T cells were found in patients with a loss of chromosomal arm 1p. In addition, NK cells and eosinophils were enriched in grade 1 and benign tumors, whereas neutrophils predominated in malignant cases. Despite their relatively low abundance, elevated neutrophil frequencies turned out to be an independent of prognostic factor for poor survival. Importantly, subsequent integration of TAM and TIL data derived from the same patient cohort unraveled five distinct immune ecotypes, each displaying characteristic immune cell infiltration patterns and differential survival outcomes. Altogether, this study provides an expanded overview of various rare immune cell subtypes in MGM and demonstrates their integration into different prognostic immune ecotypes, enabling better stratification in future clinical studies.

IntroductionTumor-associated macrophages (TAMs) belong to the most frequent immune cells in the tumor microenvironment of head and neck squamous cell carcinomas (HNSCC). They can undergo an anti- or pro-tumoral polarization, the latter often referred to as M2-like activation. Because existing data are either not consistent, not well-connected to clinicopathological parameters or lack a detailed spatial distribution, we aimed to get more insight into the relevance of this immune cell population and their activation status.MethodsThis study analyzed the spatial distribution and prognostic value of CD68+TAMs and CD68+CD163+M2-like TAMs in different tumor compartments and tumor-distant stromal areas, in 85 treatment-naïve HNSCC. Various clinicopathological features were considered, such as major tumor sites, stage, T-stage, nodal status and p16-status. TAM and M2-like TAM densities were analyzed using multicolor immunofluorescence stainings followed by an objective tissue cytometry-based quantification at the single-cell level in whole tissue sections and subsequent uni- and multivariate survival analyses.ResultsWhereas we observed higher TAM and M2-like TAM densities in p16-negative HNSCC specimens, densities of M2-like TAMs were highest in the tumor-near stroma of advanced nodal-positive p16-negative HNSCC compared to tumor cell nests and tumor-distant stroma, particularly in younger and male patients and patients with hypopharynx carcinomas. Moreover, higher infiltration of M2-like TAMs turned out to be an independent prognostic factor of poorer survival even exceeding the impact of the p16-status and the tumor site.DiscussionIn summary, our data provide a strong rationale to target M2-like TAMs to improve success of immune-modulatory treatments and survival of patients suffering from p16-negative HNSCC.

Objectives: The lymph node yield of a curative neck dissection for advanced head and neck squamous cell carcinoma (HNSCC) is an important factor in improving patient outcomes. Achieving adequate resection while minimising surgical risk is important. This retrospective study investigated the role of the lymph node yield for the survival of patients with HNSCC. Methods: A total of 234 patients with advanced HNSCC who were treated at a German university hospital between 1997 and 2018 were analysed. The analysis included patient data, tumour-specific characteristics, and the extent of neck dissection performed. Statistical analysis was performed using multivariate Cox proportional hazards models, supplemented by Kaplan-Meier analyses. Results: The median age was 60.0 years (range: 30-85 years, interquartile range: 12.3 years). The follow-up period covered up to 25 years. According to the Union for International Cancer Control, n = 64 patients had UICC-stage III, and 170 patients had UICC-stage IV. The above-average lymph node yield was superior to average, but especially to below-average lymph node yields in bilateral (2.9-fold), ipsilateral (2.6-fold), and contralateral (10.7-fold) neck dissection. In particular, contralateral neck dissection was found to correlate with a significantly better prognosis in terms of overall survival when a higher number of lymph nodes were removed. Conclusions: The study suggests that a thorough and careful neck dissection involving the removal of a greater number of lymph nodes and including the contralateral side could significantly improve the survival for patients with advanced HNSCC.

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.

There are currently no systemic treatment options available for patients with recurrent or refractory meningiomas. This study aims to address this gap by developing a comprehensive pharmacological atlas of FDA-approved oncology drugs using a large cohort of patient-derived meningioma organoids. We constructed a pharmacological atlas of meningiomas by generating 8,950 dose-response curves from 179 FDA-approved oncology drugs tested on over 60,000 tumor organoids derived from 50 meningioma patients. The cohort included 37 grade 1, 11 grade 2, and 2 grade 3 meningiomas, with 11 of these being recurrent tumors. Of the drugs tested, 21% (n=39/179) demonstrated sensitivity (median IC50 < 30 µM), with significant enrichment for topoisomerase, RNA/protein synthesis, proteasome, and HDAC inhibitors. The top five drugs with the lowest IC50 values were romidepsin, dactinomycin, carfilzomib, plicamycin, and omacetaxine, with median values of 10, 31, 145, 303, and 400 nM, respectively. Hierarchical clustering of IC50 data revealed four distinct drug response clusters characterized by sensitivity to tyrosine kinase inhibitors, sensitivity to classical chemotherapeutics, overall resistance, and heterogeneous drug sensitivity. Additionally, an ongoing multiomics analysis, including RNA and DNA-panel sequencing and methylation profiling, is being conducted to correlate genetic alterations with drug responses and drug clusters. Considering the ratio of peak serum concentration (Cmax) to IC50 as a potential predictor of treatment efficacy (>1), the HDAC inhibitors belinostat (74.5), romidepsin (69.7), along with the proteasome inhibitor carfilzomib (34.6), and the anthracyclines epirubicin (2.6), and doxorubicin (1.8), and the PI3K inhibitor idelalisib (1.6) are the most promising drug candidates for further evaluation. This study offers the first comprehensive insight into the pharmacological landscape of meningiomas, providing a crucial foundation for future clinical trials aimed at developing systemic treatments for aggressive meningiomas.

The development of breast cancer brain metastases (BCBM) is among the most critical predictive factors for patient survival. However, understanding the drug sensitivity of BCBM cells to various drugs, particularly in terms of how effectively they eliminate specific tumor cell clones responsible for the disease, remains limited. Acquiring this knowledge is crucial for improving drug selection and personalizing treatment strategies for individual patients. For this purpose, we generated novel patient-derived BCBM cell lines from three individuals with triple-negative breast cancer and three with HER2-enriched breast cancer. Whole genome and transcriptome analyses verified their resemblance to the original tumor tissues. We performed high-throughput drug screening to evaluate responses to over 250 drugs, testing more than 10 dosage points within our framework HeiDePEx (Heidelberg Drug Screening Platform for Experimental Neurosurgery). The findings were further explored in relation to patient-matched multi-omics data generated as part of the CATCH study, a metastatic breast cancer precision oncology program, which integrates drug screening results, clinical data, and molecular information. Overall, the newly established BCBM cell lines exhibited drug responses that closely reflected the individual patient and breast cancer subtype. Furthermore, they enhanced the clinically assessed multi-omics data by providing extensive insights into functional cellular vulnerabilities across a wide range of targeted anticancer drugs. Taken together, these findings emphasize the critical importance of personalized therapeutic strategies for BCBM patients and demonstrate the value of integrating functional drug screening with multi-omics data. Ultimately, the newly developed and characterized BCBM cell lines serve as a valuable resource for future research.

Tumor-associated macrophages (TAMs) represent the main immune cell population in various brain malignancies, but there is rare knowledge on the functional and, in particular, the prognostic role of TAMs in the meningioma (MGM) microenvironment. Here, we investigated TAM frequencies, activation state, survival-associated changes, and their association with tumor-infiltrating T lymphocytes (TILs) in two independent study samples comprising altogether 680 MGMs. To this end, we performed tissue cytometry analyses, quantified tissue cytokine levels, and integrated previously published TIL infiltration and microarray datasets in the discovery cohort comprising n = 195 clinically well-annotated cases. This was complemented by a DNA methylation-based deconvolution approach to predict TAM and TIL infiltration rates using immune cell-specific CpG sites as well as survival associations in an independent validation cohort of n = 485 MGMs. Our findings revealed substantial but heterogeneous TAM infiltration in newly diagnosed MGMs, with increased numbers of pro-tumoral TAMs in clinically aggressive tumors. Additional cytokine and transcriptome analyses corroborated the presence of an immunosuppressive niche in TAM-enriched MGMs. Importantly, a high frequency of pro-tumoral TAMs was associated with poor patient outcome, and high TAM infiltration was further identified as an independent prognostic factor for inferior survival, counteracting the beneficial prognostic effect of TILs. Moreover, methylation-based deconvolution analyses confirmed the opposing prognostic roles of TAMs and TILs in the validation cohort. Altogether, higher numbers of TAMs appear to be a hallmark of clinically aggressive behavior in newly diagnosed and recurrent MGMs. Unlike TILs, immunosuppressive TAMs seem to play a dominant role in the immunological landscape of MGMs with a significant negative impact on patient outcome, highlighting pro-tumoral TAMs to be an attractive treatment target in MGMs. Furthermore, our deconvolution approach presents a pipeline to computationally determine TAM and TIL infiltrates in the MGM microenvironment, which might be highly valuable for patient stratification for future immunotherapeutic treatments.

Aim: Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) is thought to involve both reduced drug uptake and altered molecular responses. However, the relative contribution of these mechanisms remains unclear. Methods: Two HNSCC cell lines with differing sensitivity (HNO97 and HNO41) were analyzed using cytotoxicity assays, atomic absorption spectroscopy-based quantification of intracellular cisplatin, caspase 3/7 assays, Western blotting, polymerase chain reaction (PCR)-based transcriptomic analysis of DNA damage response and cell cycle arrest pathways, and RNA-seq data from The Cancer Genome Atlas (TCGA) to characterize the resistance phenotype. Results: HNO97 (IC50 = 440 µM) was 7.6-fold more resistant to cisplatin than HNO41 (IC50 = 57.8 µM; P = 0.0286). After quantifying intracellular uptake (pg Pt/µg protein) and normalizing cytotoxicity to intracellular drug levels, HNO97 (IC50 = 778.9 pg Pt/µg protein) remained 5-fold more resistant than HNO41 (IC50 = 153.5 pg Pt/µg protein), indicating only a partial reduction in resistance (33% decrease, from 7.6-fold to 5-fold; P = 0.0286). At cisplatin concentrations yielding comparable intracellular exposure (HNO97: 440 µM; HNO41: 196 µM; both ≈ 725 pg Pt/µg protein), caspase 3/7 activation and induction of CDKN1A, GADD45A, GADD45G, and PPP1R15A were weaker in HNO97 than in HNO41. Notably, baseline expression of these genes was significantly higher in HNO97. In the TCGA cohort, multivariate analysis showed that high FANCD2 expression was associated with unfavorable recurrence-free survival in platinum-treated patients (hazard ratio = 4.0; P = 0.011), but not in those who did not receive platinum chemotherapy. Conclusion: Cisplatin resistance in HNSCC appears to be driven primarily by molecular mechanisms involving DNA damage response and cell cycle arrest pathways, rather than poor drug uptake.

Efficacy of the standard cytotoxic drugs against head and neck squamous cell carcinoma (HNSCC) is limited, underlining the potential relevance of multidrug resistance (MDR), mediated by drug transporters and drug-metabolising enzymes. While the major regulator of these proteins, the pregnane-X-receptor, is of minor relevance for HNSCC, little is known about the aryl hydrocarbon receptor (AhR) signaling, its transcriptional effect on MDR genes and phenotypic MDR upon activation. Using established HNSCC cell lines, AhR reporter gene assays, quantitative reverse transcription polymerase chain reaction, and proliferation assays, this study demonstrates that AhR and its major cofactors (heat shock protein 90, AhR nuclear translocator, and AhR-interacting protein 1) are expressed and that AhR is active, and druggable. The potent AhR ligand, 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) increased AhR activity in these cells up to 5.4-fold and strongly induced mRNA expression of cytochrome P450 (CYP) 1A1 (up to 224-fold) and CYP1B1 (up to 20-fold), while breast cancer resistance protein (ABCG2) was hardly enhanced (up to 2.2-fold). The endogenous ligand of AhR kynurenine (2-4-fold) and its "activated" condensation product (2-250-fold) also enhanced these genes' expression levels. However, AhR activation and target gene induction were not accompanied by relevant alterations of the antiproliferative effects of docetaxel, paclitaxel, cisplatin, carboplatin, or 5-fluorouracil. Together, this data shows that AhR signaling is in fact active in HNSCC, but its therapeutic role in HNSCC is unlikely related to induction of MDR genes. In contrast, the immune system-regulating effects of kynurenine-mediated AhR activation is likely of higher relevance and thus needs further evaluation.

Brain metastases are a severe complication for lung adenocarcinoma patients. Immunotherapies build a promising treatment option, but there is still a limited understanding of the patient-individual immune cell composition within these brain metastases and its prognostic and predictive implications. We studied a clinically annotated cohort of n = 76 neurosurgically resected lung adenocarcinoma brain metastases for their infiltration by various immune cells, including T cells, B cells, plasmablasts, macrophages, and granulocytes, applying multiplexed imaging of whole slides and cell quantification by tissue cytometry. In addition, broad clinical data, matched bulk transcriptomes, and serum cytokine levels of patients were assessed for integrative analyses with the tissue immune cell quantities. Our analyses revealed the grouping of brain metastases into four cell-type integrative immune ecotypes, each characterized by a unique immune cell composition and infiltration rate. The immune ecotypes were associated with a distinct overall survival, independent of clinical prognostic variables in a multivariate model. Interestingly, a subgroup of brain metastases enriched for B cells, often spatially arranged with T cells and other specialized cell types in tertiary lymphoid structures, was especially linked to a favorable prognosis. Integrative analyses of bulk transcriptome data further enabled the identification of related gene expression signatures. Our current analyses assess the suitability of serum cytokines as surrogate markers for the brain metastases immune ecotypes, evaluating patient-matched samples. Together, our analyses advance the knowledge of lung adenocarcinoma immune landscapes in the brain metastatic niche and suggest characteristics that could translate to clinically applicable biomarkers in the future.

Patients with head and neck squamous cell carcinoma (HNSCC) suffer from severe morbidity and mortality. Immunotherapy represents a novel promising treatment option. Therefore, a better understanding of the immune niche is needed. This study focuses on the spatial distribution and prognostic value of different T cell subtypes in 84 HNSCC specimens as well as chemokine and cytokine levels associated with spatial T cell infiltration. Density of T helper (TH), cytotoxic (CTL), and regulatory T cells (Treg) was quantified by multicolor tissue cytometry on a single cell level in whole tissue sections, discriminating between T cells located in epithelial tumor cell nests or tumor stroma, respectively. In addition, quantitative levels of 27 immune-related factors were assessed. Survival analysis of patients with p16-negative HNSCC revealed higher stromal Treg densities to be an independent prognostic factor for better progression-free and overall survival. Furthermore, high levels of CXCL10, IL-9, and CCL4 were associated with significantly higher numbers of T cells, especially for CTL with direct contact to tumor cells, whereas for VEGF the opposite effect was observed in the tumor stroma. In conclusion, Treg cell infiltration as well as distinct cytokine levels could serve as new immune biomarkers in p16-negative HNSCC to predict survival and the spatial distribution of T cells.

Tumor organoids are important tools for cancer research, but current models have drawbacks that limit their applications for predicting response to therapy. Here, we developed a fast, efficient, and complex culture system (IPTO, individualized patient tumor organoid) that accurately recapitulates the cellular and molecular pathology of human brain tumors. Patient-derived tumor explants were cultured in induced pluripotent stem cell (iPSC)-derived cerebral organoids, thus enabling culture of a wide range of human tumors in the central nervous system (CNS), including adult, pediatric, and metastatic brain cancers. Histopathological, genomic, epigenomic, and single-cell RNA sequencing (scRNA-seq) analyses demonstrated that the IPTO model recapitulates cellular heterogeneity and molecular features of original tumors. Crucially, we showed that the IPTO model predicts patient-specific drug responses, including resistance mechanisms, in a prospective patient cohort. Collectively, the IPTO model represents a major breakthrough in preclinical modeling of human cancers, which provides a path toward personalized cancer therapy.

Abstract BACKGROUND Meningiomas represent the most common primary brain malignancies in adults with a subset of tumors exhibiting aggressive clinical behavior. Immunotherapy might present a new treatment strategy but is highly dependent on the immunological composition of the tumor microenvironment. Our previous data have shown that tumor-associated macrophages (TAMs) make up the main immune cell population in meningiomas with a significant negative impact on patient outcome. In this study, we investigated whether TAMs from meningioma tissue could be reprogrammed to an immunologically active and tumoricidal phenotype. MATERIAL AND METHODS For this purpose, CD11b+ sorted macrophages derived from tumor samples from > 40 patients including a substantial number of clinically aggressive meningiomas were treated with small molecule inhibitors targeting the colony-stimulating factor-1 receptor (CSF-1R). In a first analysis, the direct treatment response of CD11b+ patient-derived macrophages has been investigated by various techniques including flow cytometry and bulk RNA-sequencing of treated TAMs and further analysis of the macrophage-conditioned media after treatment. In addition, we studied the influence of CSF-1R-targeted macrophage treatment on the phenotype and functional activity of T cells to assess a potential indirect treatment response in the tumor microenvironment. RESULTS Our data revealed that CSF-1R-targeted treatment of CD11b+ TAMs induced significant changes in the protein and gene expression of macrophage polarization markers towards a more immunologically active state and a significantly higher metabolic nitric oxide production as another sign of immunological activation. Subsequent analysis of indirect effects on T cells showed not only a significantly increased expression of the T cell activation marker CD69+, but also a significantly increased tumor cell killing by autologous T cells after macrophage-targeted treatment. CONCLUSION Together these data suggest both a direct and indirect CSF-1R-targeted macrophage treatment response in the local tumor microenvironment and give first promising results on the efficacy of macrophage-targeted immunotherapy in human meningiomas.

Tumor-associated macrophages (TAMs) represent the main immune cell population in various brain malignancies. To elucidate their biological impact in the tumor microenvironment (TME) of meningiomas (MGMs), we assessed TAM numbers, activation state, malignancy- and survival-associated changes, as well as their association with tumor-infiltrating T lymphocytes (TILs). TAM infiltration was analyzed in a multicenter cohort of 195 clinically well-annotated cases (follow-up >5 years, n=120 newly-diagnosed and n=75 recurrent MGMs) enriched for higher-grade MGMs. TAMs and M2-TAMs were quantified by tissue cytometry on whole-tumor sections. Further, we assessed levels of 27 cyto- and chemokines in a subset of tissues (n=46 cases), and re-analyzed our previously published T cell infiltration (n=94 cases) and expanded microarray (n=97 cases) datasets. Newly-diagnosed MGMs showed a substantial but highly heterogeneous TAM infiltration that was four times higher than for TILs. Anti-inflammatory M2-TAMs were increased in higher WHO grade tumors and in recurrent MGMs. Importantly, high M2-TAM infiltration was associated with poor progression-free survival independent of other prognostic confounders and even mitigated the beneficial prognostic effect of TIL infiltration. Additional cytokine, gene expression and pathway analyses corroborated the presence of an immunosuppressive niche in M2-TAM-enriched MGMs. Altogether, higher numbers of TAMs and M2-TAMs appear to be a hallmark of clinically aggressive behavior in newly-diagnosed and recurrent MGMs. Unlike TILs, immunosuppressive TAMs seem to play a dominant negative role in the immunological landscape of MGMs, highlighting M2-TAMs to be an attractive treatment target for immunotherapeutic approaches. ### Competing Interest Statement The authors have declared no competing interest.

Abstract To date, there are no effective systemic treatment options for recurrent glioblastoma (GBM). Furthermore, often surgical resection is not possible and tumors have acquired an increased chemo- or radioresistance. Patient-derived tumor organoids (PDTOs) provide a new exciting tool to test individual treatment responses. Aim of this study was to assess the feasibility of a personalized PDTO-based drug testing even from rare material such as stereotactic biopsies. PDTOs were prepared based on single cell suspensions from tumor material obtained either from open GBM resections (reference data set, n = 37) or from stereotactic biopsies (n = 8). Drug response curves (6-9 doses) were performed for 9 drugs including commonly used drugs such as temozolomide, lomustine, etoposide and temsirolimus. Viability was measured using CellTiterGlo (Promega) to assess half-maximal inhibitory concentrations (IC50). Medical records were reviewed for survival and molecular information such as the MGMT methylation status. Drug testing on PDTOs from open resections allowed to adjust the test range for each drug. Additionally, a significant association between increased temozolomide sensitivity of PDTOs and both, MGMT methylation and increased survival was observed. However, in 59% of the cases, no sensitivity to any of the drugs was seen, and only 17% were highly sensitive to more than one drug. Subsequent drug testing on PDTOs from biopsies of recurrent GBM revealed sensitivity in 3/8 cases against one (n=2) or two (n=1) of the drugs. However, sensitivity against drugs varied substantially in a patient-individual manner. We successfully developed a workflow to test drug sensitivity on PDTO even from low starting material obtained from stereotactic biopsies. Association of clinical outcome and MGMT methylation with temozolomide sensitivity corroborated the strength of this approach. Furthermore, patient-individual treatment responses strongly suggest for a future personalized drug testing.