Interrogation of the microenvironmental landscape in spinal ependymomas reveals dual functions of tumor-associated macrophages

Spinal ependymomas are the most common spinal cord tumors in adults, but their intratumoral cellular heterogeneity has been less studied, and how spinal microglia are involved in tumor progression is still unknown. Here, our single-cell RNA-sequencing analyses of three spinal ependymoma subtypes dissect the microenvironmental landscape of spinal ependymomas and reveal tumor-associated macrophage (TAM) subsets with distinct functional phenotypes. CCL2+ TAMs are related to the immune response and exhibit a high capacity for apoptosis, while CD44+ TAMs are associated with tumor angiogenesis. By combining these results with those of single-cell ATAC-sequencing data analysis, we reveal that TEAD1 and EGR3 play roles in regulating the functional diversity of TAMs. We further identify diverse characteristics of both malignant cells and TAMs that might underlie the different malignant degrees of each subtype. Finally, assessment of cell-cell interactions reveal that stromal cells act as extracellular factors that mediate TAM diversity. Overall, our results reveal dual functions of TAMs in tumor progression, providing valuable insights for TAM-targeting immunotherapy.

For scRNA-seq processing,We filtered cells using scRNA-seq analysis standard procedure, with less than 2000 UMI count or 500 detected genes, as well as cells with more than 20% mitochondrial gene count. To remove potential doublets, we also removed cells with UMI count above 70,000 and number of detected genes above 10,000.we removed potential doublets predicted by Scrublet. For scATAC-seq processing, cell by feature matrix with window size of 2.5kb was generated as described previously by first reading fragment file into R. These criteria were pre-established and generally used for scRNA-seq data and scATAC-seq data processing.
For multi-omics data, we used more than 2 biological samples for each type. For IF images, images represent the results from 3 experiments. All replications were consistent for results.
The samples were allocated into each experimental groups based on the tumor clinical pathology.The final nuclei /cell suspension was homogeneous with no artificial preference.
The investigators were blinded to group allocation during data collection and analysis.
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March 2021
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Policy information about studies involving human research participants All antibodies used in this study were obtained from commercial source, and validated according to manufacturers' instruction.
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Human tumor tissues of 17 treatment-naïve patients diagnosed with different subtypes of spinal ependymoma, including subependymoma (SE, 3 patients), ependymoma (EPN, 9 patients), and anaplastic ependymoma (AEP, 5 patients). All the patients are Asian. No restriction of ages or genders were used.
Patients who were diagnosed spinal endymoma were recruited. Since the cases were very rare, especially for surgery removal, all the samples collected were used for experiments.

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March 2021

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Patients were enrolled in this study after approved by the Ethics Committee of Beijing Tiantan Hospital, Capital Medical University. All patients provided written informed consent for sample collection and data analyses.
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