Childhood brain tumors instructs cranial haematopoiesis and immunotolerance

ZFTA-RELA ependymoma poses a formidable challenge due to limited treatment avenues, prompting exploration into immunotherapeutic approaches. Recent investigations have shed light on the calvaria bone marrow as a source of meningeal immune cells crucial for central nervous system (CNS) immunity. Whether or not cues from the tumour microenvironment can instruct local haematopoiesis within the skull is unknown. We used single cell RNA sequencing (snRNAseq + snATAC) to analyze the dynamic transcriptional regulation of skull, peripheral bone marrow from tumour-bearing and non-tumour bearing ZFTA-RELA fusion driven ependymoma mice. Overall design: Age-matched 6-8 week-old EPZFTA-RELA and NestinCreERT2 mice were intravenously injected with CD45-PE 3 minutes prior to schedule 1. For skulls the dura was peeled and removed with fine forceps. The skull were then cut into small pieces using sterile scissors and mechanically dissociated in FACS buffer with a pestle, followed by a filtration step through a 70-µm cell strainer. Samples were centrifuged for 5 minutes at 420 x g. For peripheral bone marrow, both tibia were flushed with 0.05% BSA PBS with 0.05% EDTA, filtered through 7- µm meshes and washed with 2% fetal bovine serum in RPMI and resuspended on 0.05% BSA PBS solution. Samples were stained with DAPI (0.2 µg/ml). Samples were centrifuged, resuspended in FACS buffer with anti-CD16/32 (FC block; Biolegend) diluted 1:50 in FACS buffer and fluorescently conjugated antibodies (anti-CD45 APC and anti-Ter 119 FITC) at 4°C, followed by for 20 minutes. Cells were sorted using the Influx Cell Sorter (BD Biosciences) or FACsAria II (BD Biosciences) into 1% BSA coated 1.5 mL Eppendorf tubes with 500 µL of DMEM