Gliomas are intrinsic brain tumors with a high degree of constitutive and acquired resistance to standard therapeutic modalities such as radiotherapy and alkylating chemotherapy. Glioma subtypes are ...recognized by characteristic mutations. Some of these characteristic mutations have shown to generate immunogenic neoepitopes suitable for targeted immunotherapy.
Using peptide-based ELISpot assays, we screened for potential recurrent glioma neoepitopes in MHC-humanized mice. Following vaccination, droplet-based single-cell T-cell receptor (TCR) sequencing from established T-cell lines was applied for neoepitope-specific TCR discovery. Efficacy of intraventricular TCR-transgenic T-cell therapy was assessed in a newly developed glioma model in MHC-humanized mice induced by CRISPR-based delivery of tumor suppressor-targeting guide RNAs.
We identify recurrent capicua transcriptional repressor (CIC) inactivating hotspot mutations at position 215 CICR215W/Q as immunogenic MHC class II (MHCII)-restricted neoepitopes. Vaccination of MHC-humanized mice resulted in the generation of robust MHCII-restricted mutation-specific T-cell responses against CICR215W/Q. Adoptive intraventricular transfer of CICR215W-specific TCR-transgenic T cells exert antitumor responses against CICR215W-expressing syngeneic gliomas.
The integration of immunocompetent MHC-humanized orthotopic glioma models in the discovery of shared immunogenic glioma neoepitopes facilitates the identification and preclinical testing of human leukocyte antigen (HLA)-restricted neoepitope-specific TCRs for locoregional TCR-transgenic T-cell adoptive therapy.
Glioblastoma is the most common malignant primary brain tumor with poor overall survival. Magnetic resonance imaging (MRI) is the main imaging modality for glioblastoma but has inherent shortcomings. ...The molecular and cellular basis of MR signals is incompletely understood. We established a ground truth-based image analysis platform to coregister MRI and light sheet microscopy (LSM) data to each other and to an anatomic reference atlas for quantification of 20 predefined anatomic subregions. Our pipeline also includes a segmentation and quantification approach for single myeloid cells in entire LSM datasets. This method was applied to three preclinical glioma models in male and female mice (GL261, U87MG, and S24), which exhibit different key features of the human glioma. Multiparametric MR data including T2-weighted sequences, diffusion tensor imaging, T2 and T2* relaxometry were acquired. Following tissue clearing, LSM focused on the analysis of tumor cell density, microvasculature, and innate immune cell infiltration. Correlated analysis revealed differences in quantitative MRI metrics between the tumor-bearing and the contralateral hemisphere. LSM identified tumor subregions that differed in their MRI characteristics, indicating tumor heterogeneity. Interestingly, MRI signatures, defined as unique combinations of different MRI parameters, differed greatly between the models. The direct correlation of MRI and LSM allows an in-depth characterization of preclinical glioma and can be used to decipher the structural, cellular, and, likely, molecular basis of tumoral MRI biomarkers. Our approach may be applied in other preclinical brain tumor or neurologic disease models, and the derived MRI signatures could ultimately inform image interpretation in a clinical setting.
We established a histologic ground truth-based approach for MR image analyses and tested this method in three preclinical glioma models exhibiting different features of glioblastoma. Coregistration of light sheet microscopy to MRI allowed for an evaluation of quantitative MRI data in histologically distinct tumor subregions. Coregistration to a mouse brain atlas enabled a regional comparison of MRI parameters with a histologically informed interpretation of the results. Our approach is transferable to other preclinical models of brain tumors and further neurologic disorders. The method can be used to decipher the structural, cellular, and molecular basis of MRI signal characteristics. Ultimately, information derived from such analyses could strengthen the neuroradiological evaluation of glioblastoma as they enhance the interpretation of MRI data.
Radiological neuro-interventions, especially endovascular stroke treatment (EST), are increasing in case numbers worldwide with increasing occupational radiation exposure. Aim of this study was to ...define the radiation exposure of neurointerventionalists (NI) during EST and to compare the accumulated dose reaching the left arm with the left temple.
This is a prospective observational study in a tertiary stroke center conducted between 11/2021 and 07/2022. Radiation exposure was measured using real time dosimetry with dosimeters being carried by the NI during EST simultaneously at the left temple and left arm. The effective dose µSV per dose area product (DAP) and potential influencing factors were compared in univariate analysis between the two dosimeter positions.
In total, 82 ESTs were analyzed with a median DAP of 6179 µGy*m2 (IQR 3271 µGy*m2–11720 µGy*m2). The accumulated dose at the left arm and left temple correlated with the DAP and fluoroscopy time of the EST (DAP and arm: p = 0.01, DAP and temple: p = 0.006). The radiation exposure (RE) showed a wide range and did not differ between the two dosimeter positions (median, IQR arm 7 µSV, IQR 3.1–16.9 µSV, min. 0.3 µSV max. 64.5 µSV) vs. head 7 µSv, IQR 3.2–17.4 µSV, min. 0.38 µSV, max. 48.6 µSV, p = 0.94). Occupational RE depends on the number of thrombectomy attempts, but not the target vessel occlusion location or the NI’s body height.
Neurointerventionalists experience a generally low but very variable radiation exposure during EST, which depends on the intervention’s fluoroscopy time and dose area product as well as thrombectomy attempts but does not differ between left temple and left arm.
Inflammatory demyelinating plaques are the pathologic hallmark of active multiple sclerosis and often precede clinical manifestations. Non-invasive early detection of active plaques would thus be ...crucial in establishing pre-symptomatic diagnosis and could lead to early preventive treatment strategies. Using murine experimental autoimmune encephalomyelitis as a model of multiple sclerosis, we demonstrate that a prototype paramagnetic myeloperoxidase (MPO) sensor can detect and confirm more, smaller, and earlier active inflammatory lesions in living mice by in vivo MRI. We show that MPO expression corresponded with areas of inflammatory cell infiltration and demyelination, and higher MPO activity as detected by MPO imaging, biochemical assays, and histopathological analyses correlated with increased clinical disease severity. Our findings present a potential new translational approach for specific non-invasive inflammatory plaque imaging. This approach could be used in longitudinal studies to identify active demyelinating plaques as well as to more accurately track disease course following treatment in clinical trials.
Diagnostic imaging criteria of multiple sclerosis (MS) include the spatial and temporal dissemination of cerebral and/or spinal cord lesions. Magnetic resonance imaging (MRI) is the method of choice ...for initial diagnosis and follow-up disease monitoring. Current guidelines for spinal MRI recommend sagittal imaging of the spinal cord and lesion confirmation on axial planes if lesions are detected. Sagittal imaging is, however, hampered by technical (e.g. partial volume effects, motion artifacts) and anatomical (e.g. scoliosis) limitations. We hypothesized that long coverage of the spinal cord by axial image acquisition has superior diagnostic performance compared to sagittal imaging and can identify otherwise undetected lesions. Our prospective clinical study included 119 MS patients. Axial MRI revealed ~2.5-fold more lesions than the sagittal angulation (axial lesion load: 4.0 ± 2.4 vs. 1.6 ± 1.2 lesions on sagittal planes,
p
< 0.001). Importantly, 20 patients (17%) with normal sagittal MRI scans had unequivocal lesions only visible on axial planes (mean lesion number on axial planes in these patients: 2.0 ± 1.3). Moreover, 45 patients (38%) showed a discrepancy of ≥3 lesions that were found additionally on axial scans (mean difference 4.4 ± 1.7). Additionally identified lesions were on average smaller in size and located more laterally within the spinal cord. No lesion on sagittal images was missed on the axial angulation. Our study demonstrates that imaging of small axial segments for lesion confirmation is insufficient in spinal imaging. We recommend implementing a long coverage axial MRI sequence for spinal imaging of MS patients.
Three-dimensional assessment of optically cleared, entire organs and organisms has recently become possible by tissue clearing and selective plane illumination microscopy (“ultramicroscopy”). ...Resulting datasets can be highly complex, encompass over a thousand images with millions of objects and data of several gigabytes per acquisition. This constitutes a major challenge for quantitative analysis. We have developed post-processing tools to quantify millions of microvessels and their distribution in three-dimensional datasets from ultramicroscopy and demonstrate the capabilities of our pipeline within entire mouse brains and embryos. Using our developed acquisition, segmentation, and analysis platform, we quantify physiological vascular networks in development and the healthy brain. We compare various geometric vessel parameters (e.g. vessel density, radius, tortuosity) in the embryonic spinal cord and brain as well as in different brain regions (basal ganglia, corpus callosum, cortex). White matter tract structures (corpus callosum, spinal cord) showed lower microvascular branch densities and longer vessel branch length compared to grey matter (cortex, basal ganglia). Furthermore, we assess tumor neoangiogenesis in a mouse glioma model to compare tumor core and tumor border. The developed methodology allows rapid quantification of three-dimensional datasets by semi-automated segmentation of fluorescently labeled objects with conventional computer hardware. Our approach can aid preclinical investigations and paves the way towards “quantitative ultramicroscopy”.
Specific biological properties of those circulating cancer cells that are the origin of brain metastases (BM) are not well understood. Here, single circulating breast cancer cells were fate-tracked ...during all steps of the brain metastatic cascade in mice after intracardial injection over weeks. A novel
two-photon microscopy methodology was developed that allowed to determine the specific cellular and molecular features of breast cancer cells that homed in the brain, extravasated, and successfully established a brain macrometastasis. Those BM-initiating breast cancer cells (BMIC) were mainly originating from a slow-cycling subpopulation that included only 16% to 20% of all circulating cancer cells. BMICs showed enrichment of various markers of cellular stemness. As a proof of principle for the principal usefulness of this approach, expression profiling of BMICs versus non-BMICs was performed, which revealed upregulation of NDRG1 in the slow-cycling BMIC subpopulation in one BM model. Here, BM development was completely suppressed when NDRG1 expression was downregulated. In accordance, in primary human breast cancer, NDRG1 expression was heterogeneous, and high NDRG1 expression was associated with shorter metastasis-free survival. In conclusion, our data identify temporary slow-cycling breast cancer cells as the dominant source of brain and other metastases and demonstrates that this can lead to better understanding of BMIC-relevant pathways, including potential new approaches to prevent BM in patients. IMPLICATIONS: Cancer cells responsible for successful brain metastasis outgrowth are slow cycling and harbor stemness features. The molecular characteristics of these metastasis-initiating cells can be studied using intravital microscopy technology.
Glioblastoma is the most frequent, primary brain tumor that is characterized by a highly immunosuppressive tumor microenvironment (TME). The TME plays a key role for tumor biology and the ...effectiveness of immunotherapies. Composition of the TME correlates with overall survival and governs therapy response. Non invasive assessment of the TME has been notoriously difficult.
We have designed an
imaging approach to non invasively visualize innate immune cell dynamics in the TME in a mouse glioma model by correlated MRI and multiphoton microscopy (MR-MPM) using a bimodal, fluorescently labeled iron oxide nanoparticle (NP). The introduction of Teflon cranial windows instead of conventional Titanium rings dramatically reduced susceptibility artifacts on MRI and allowed longitudinal MR-MPM imaging for innate immune cell tracking in the same animal.
We visualized tumor associated macrophage and microglia (TAM) dynamics in the TME and dissect the single steps of NP uptake by blood-born monocytes that give rise to tumor-associated macrophages. Next to peripheral NP-loading, we identified a second route of direct nanoparticle uptake via the disrupted blood-brain barrier to directly label tissue resident TAMs.
Our approach allows innate immune cell tracking by MRI and multiphoton microscopy in the same animal to longitudinally investigate innate immune cell dynamics in the TME.
Neoangiogenesis is a pivotal therapeutic target in glioblastoma. Tumor monitoring requires imaging methods to assess treatment effects and disease progression. Until now mapping of the tumor ...vasculature has been difficult. We have developed a combined magnetic resonance and optical toolkit to study neoangiogenesis in glioma models. We use in vivo magnetic resonance imaging (MRI) and correlative ultramicroscopy (UM) of ex vivo cleared whole brains to track neovascularization. T2* imaging allows the identification of single vessels in glioma development and the quantification of neovessels over time. Pharmacological VEGF inhibition leads to partial vascular normalization with decreased vessel caliber, density, and permeability. To further resolve the tumor microvasculature, we performed correlated UM of fluorescently labeled microvessels in cleared brains. UM resolved typical features of neoangiogenesis and tumor cell invasion with a spatial resolution of ~5 µm. MR-UM can be used as a platform for three-dimensional mapping and high-resolution quantification of tumor angiogenesis.
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