Human cutaneous neurofibroma matrisome revealed by single-cell RNA sequencing

Abstract

Neurofibromatosis Type I (NF1) is a neurocutaneous genetic syndrome characterized by a wide spectrum of clinical presentations, including benign peripheral nerve sheath tumor called neurofibroma. These tumors originate from the Schwann cell lineage but other cell types as well as extracellular matrix (ECM) in the neurofibroma microenvironment constitute the majority of the tumor mass. In fact, collagen accounts for up to 50% of the neurofibroma's dry weight. Although the presence of collagens in neurofibroma is indisputable, the exact repertoire of ECM genes and ECM-associated genes (i.e. the matrisome) and their functions are unknown. Here, transcriptome profiling by single-cell RNA sequencing reveals the matrisome of human cutaneous neurofibroma (cNF). We discovered that classic pro-fibrogenic collagen I myofibroblasts are rare in neurofibroma. In contrast, collagen VI, a pro-tumorigenic ECM, is abundant and mainly secreted by neurofibroma fibroblasts. This study also identified potential cell type-specific markers to further elucidate the biology of the cNF microenvironment.

Fig. 1

Single cell analysis of human cutaneous neurofibroma identifies 6 major cell types. a Uniform Manifold Approximation and Projection (UMAP) shows groupings of three human cutaneous neurofibroma cell populations totaling 3000 cells (random sampling of 1000 cells per sample). Each point represents a cell. Cells are color-coded according to cell type; 64 hematopoietic cells, 96 pericytes, 95 Schwann cells, 230 antigen-presenting cells (APCs), 619 endothelial cells, and 1896 fibroblasts were identified. b Feature plots (upper) and violin plots (lower) of genes defining different cell types in human cutaneous neurofibroma. The intensity of the purple color indicates the normalized level of gene expression

Fig. 2

Cell types within the cNF microenvironment show overlapping and unique expression of matrisome genes. a Dot plot representing the shared and common matrisome genes expressed in neurofibroma hematopoietic cells, pericytes, Schwann cells, antigen-presenting cells (APCs), endothelial cells, and fibroblasts

Fig. 3

Classic fibrogenic fibroblasts are rare in neurofibroma. a Feature plots of dipeptidyl peptidase 4 (DDP4), fibroblast-activated protein (FAP), and COL11A1 in human cutaneous neurofibroma. The intensity of the purple color indicates the normalized level of gene expression. b Characterization of activated fibroblasts in human clinical samples [keloids, normal skin (skin), cutaneous neurofibroma (cNF), normal peripheral nerve (nerve), and plexiform neurofibroma (pNF)] by immunohistochemistry [alpha smooth muscle actin (SMA), Fibroblast activation protein (FAP), and COL11A1]. c Profiling of the universal pro-fibrotic gene expression signature in human cutaneous neurofibroma (n = 5) and their normal skin margin (n = 5) by real-time PCR. Scale bar = 50 um. Arrows point fibroblasts and arrow heads point pericytes unspecific staining inherent to these fibroblasts markers

Fig. 4

Neurofibroma fibroblasts do not abundantly secrete collagen type I. a Expression of collagen type I in human clinical samples [normal skin (skin), cutaneous neurofibroma (cNF), normal peripheral nerve (nerve) and plexiform neurofibroma (pNF)] by Sirius Red staining. b Feature plots of the single cell dataset from normal skin fibroblasts (30) (blue) merged with our human neurofibroma fibroblasts (pink). c, d Feature plots from B (upper) and corresponding violin plot (bottom) of c COL1A1 and d COL1A2. The intensity of the purple color indicates the normalized level of gene expression. e Dot plot representing the differentially expressed genes between neurofibroma fibroblasts and normal skin fibroblasts. Bar = 50 um

Fig. 5

A sub population of neurofibroma fibroblasts secrete collagen type VI. a Dot plot representing the shared and common collagen genes expressed in neurofibroma hematopoietic cells, pericytes, Schwann cells, antigen-presenting cells (APCs), endothelial cells, and fibroblasts. Undetectable genes in all six clusters are omitted. b Histological characterization of cutaneous neurofibroma by H&E staining (upper), collagen I staining using Sirius Red (middle), and immunohistochemistry using anti-collagen VI antibody (bottom). c Subclustering analysis of the neurofibroma fibroblast cluster as found in Fig. 1a shows nine subclusters. d Dot plots of collagen type I (COL1A1, COL1A2) and collagen type VI (COL6A1, COL6A2, COL6A3) genes defining the distribution of expression in neurofibroma fibroblasts. The intensity of the purple color indicates the normalized level of gene expression