biostudies-arrayexpressFanny CoulpierMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14222Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with currently no effective treatment. While half of MPNSTs arise sporadically, the others are due to the malignant progression of neurofibromas (NFs) in patients with neurofibromatosis type 1 (NF1). NFs are benign nerve sheath tumors due to bi-allelic loss of NF1, encoding a negative regulator of Ras pathway, in the Schwann cell (SC) lineage. We have conceived an Nf1-KO mouse model in which NFs spontaneously transform into MPNSTs To decipher the step-by-step evolution of the cellular composition and biological activities of tumor cells and their microenvironment during the transformation process, we performed single-cell transcriptomic profiling of pNFs, dyNFs and MPNSTs, all issued from Prss56Cre, Nf1fl/fl cohort, using 10x Chromium platform. We discovered that malignant transformation is initiated by a molecular transition of tumor SCs from glial to mesenchymal identity (GMT).biostudies-arrayexpressSequencing - The amplified cDNAs were used to generate Illumina sequencing libraries that were each sequenced on one flow cell Nextseq500 Illumina.Nucleic Acid Extraction - single-cell suspensions were labelled with DAPI (Invitrogen, #D1306) and subjected to FACS to recover viable cells. 20,000 of viable were loaded into one channel of the Chromium system using the V3 and V3.1 single-cell reagent kits (10X GenomicsLibrary Construction - Following capture and lysis, cDNAs were synthesized, then amplified by PCR for 12 cycles as per the manufacturer’s protocol (10X Genomics).Sample Collection - Mice were euthanised via isoflurane inhalation and perfused with PBS to remove circulating immune cells. Tumors were dissected and sampled for (immuno)histological and genomic analyses. Remaining tissue was minced into a slurry and digested in RPMI-1640 medium (Gibco, #42401-018) containing 0,25 mg/ml of Liberase HD (Roche, #05401089001) for 30 min at 37°C. Enzymatic digestion was halted by adding 10% FBS in RPMI (Gibcon #10270106) and tumor cell suspensions were collected by centrifugation (1200 rpmi for 5 min)and mechanical dissociation and filtering (30µm MACS SmartStratiners, Miltenyi Biotec, #130-098-458)was performed to obtain single cell suspensionOrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - BCL files were processed using 10x Genomics Cell Ranger 2 or 3. Reads were mapped onto a custom mouse transcriptome based on GRCm38 transcriptome, in which we added tdTomato sequence.Data Transformation - From the count matrices, datasets were analyzed individually using Seurat V3 package28. Cells having less than 26 UMI or less than 600 genes were filtered out. Doublets were removed using the scDblFinder tool29 and scds in hybrid mode30. Then, cells having more than 50 % of UMI related to mitochondrial genes or more than 30% related to ribosomal genes were filtered out. The UMI count matrix for remaining cells was log-normalized using the Seurat::NormalizeData function. Cell cycle annotation was made using Seurat::AddCellCycle. Single cells were annotated for cell type using the Seurat::AddModuleScore function and cell type-specific marker gene sets listedMetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 500RNA-seq of coding RNA from single cellsMus musculusFanny CoulpierfalseDeciphering cellular and molecular signatures of malignant progression in Neurofibromatosis type 1 using single-cell transcriptomic analysisMalignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with currently no effective treatment. While half of MPNSTs arise sporadically, the others are due to the malignant progression of neurofibromas (NFs) in patients with neurofibromatosis type 1 (NF1). NFs are benign nerve sheath tumors due to bi-allelic loss of NF1, encoding a negative regulator of Ras pathway, in the Schwann cell (SC) lineage. We have conceived an Nf1-KO mouse model in which NFs spontaneously transform into MPNSTs To decipher the step-by-step evolution of the cellular composition and biological activities of tumor cells and their microenvironment during the transformation process, we performed single-cell transcriptomic profiling of pNFs, dyNFs and MPNSTs, all issued from Prss56Cre, Nf1fl/fl cohort, using 10x Chromium platform. We discovered that malignant transformation is initiated by a molecular transition of tumor SCs from glial to mesenchymal identity (GMT).2025-08-26T00:00:00Z2025-08-27T00:00:53.802Z2024-07-05T13:22:45.834ZE-MTAB-14222ERP161739EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184