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

• Published in: Acta neuropathologica communications 2023-09, Vol.11 (1), p.1-158, Article 158
• Main Authors: Amani, Vladimir, Riemondy, Kent A, Fu, Rui, Griesinger, Andrea M, Grimaldo, Enrique, De Sousa, Graziella Ribeiro, Gilani, Ahmed, Hemenway, Molly, Foreman, Nicholas K, Donson, Andrew M, Willard, Nicholas
• Format: Article
• Language: English
• Subjects: Airway management; Cell growth; Data analysis; Gene expression; Genomics; Hyperplasia; Immunohistochemistry; Kinases; Leukemia; Ligands; Morbidity; Patients; Proteins; Tumors
• ISSN: 2051-5960; 2051-5960
• DOI: 10.1186/s40478-023-01639-1

Plexiform neurofibroma (PN) is a leading cause of morbidity in children with the genetic condition Neurofibromatosis Type 1 (NF1), often disfiguring or threatening vital structures. During formation of PN, a complex tumor microenvironment (TME) develops, with recruitment of neoplastic and non-neoplastic cell types being critical for growth and progression. 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. To bypass this barrier, we performed single-nuclei RNA-sequencing (snRNA-seq) on 8 frozen PN samples, and integrated this with spatial transcriptomics (ST) in 4 PN samples and immunohistochemistry to provide morphological context to transcriptomic data. SnRNA-seq analysis definitively charted the heterogeneous cellular subpopulations in the PN TME, with the predominant fraction being fibroblast subtypes. PN showed 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 and correlated with histological features. Schwann cell/fibroblast interactions were identified by receptor/ligand interaction analysis demonstrating a high probability of Neurexin 1/Neuroligin 1 (NRXN1/NLGN1) receptor-ligand cross-talk predicted between fibroblasts and non-myelinated Schwann cells (NM-SC) and subtypes, respectively. We observed aberrant expression of NRXN1 and NLGN1 in our PN snRNA-seq data compared to a normal mouse sciatic nerve single-cell RNA-seq dataset. This pathway has never been described in PN 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 TME and 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.