Integration of single-nuclei RNA-sequencing and spatial transcriptomics defines the complex microenvironment of NF1-associated plexiform neurofibromas

Due to the cohesive cellularity of PN, single-cell RNA-sequencing is difficult and may result in a loss of detection of critical cellular subpopulations, therefor single-nuclei RNA-sequencing (snRNA-seq) was applied retrospectively to 8 frozen PN, a large enough sample cohort required to adequately describe the disease TME. Additionally, 4 frozen PN samples were OCT embedded and spatial transcriptomics (ST) was run, adding morphological context to the transcriptomic data generated. Our snRNA-seq analysis definitively charted the heterogeneous cellular subpopulations in the PN TME, with the predominant fraction being fibroblast-like cells. PN have a remarkable amount of inter-sample homogeneity regarding cellular subpopulation proportions despite being resected from a variety of anatomical locations. ST analysis identified distinct cellular subpopulations which were annotated using snRNA-seq data that correlated with histological features. Schwann cell/fibroblast interactions were further characterized by receptor/ligand interaction analysis (CellChat) that was applied to snRNA-seq data. A high probability of Neurexin 1/Neuroligin 1 (NRXN1/NRGLN1) receptor-ligand crosstalk was predicted between non-myelinated Schwann cells (NM_SC) and fibroblast subpopulations, respectively. We observed aberrant expression of NRXN1 and NRGLN1 in our snRNA-seq data versus normal mouse sciatic nerve. This pathway has never been described in PN but has been observed in other NF1-associated tumors and may indicate a clear and direct communication pathway between putative NM_SC cells of origin and surrounding fibroblasts, potentially driving disease progression. SnRNA-seq integrated with spatial transcriptomics advances our understanding of the complex cellular heterogeneity of PN. These data identify potential novel communication pathways that may drive disease progression, a finding that could provide translational therapy options for patients with these devastating tumors of childhood and early adulthood.