Single-cell sequencing reveals novel cellular heterogeneity in uterine leiomyomas

Abstract

Study question: What are the cellular composition and single-cell transcriptomic differences between myometrium and leiomyomas as defined by single-cell RNA sequencing?

Summary answer: We discovered cellular heterogeneity in smooth muscle cells (SMCs), fibroblast and endothelial cell populations in both myometrium and leiomyoma tissues.

What is known already: Previous studies have shown the presence of SMCs, fibroblasts, endothelial cells and immune cells in myometrium and leiomyomas. However, there is no information on the cellular heterogeneity in these tissues and the transcriptomic differences at the single-cell level between these tissues.

Study design, size, duration: We collected five leiomyoma and five myometrium samples from a total of eight patients undergoing hysterectomy. We then performed single-cell RNA sequencing to generate a cell atlas for both tissues. We utilized our single-cell sequencing data to define cell types, compare cell types by tissue type (leiomyoma versus myometrium) and determine the transcriptional changes at a single-cell resolution between leiomyomas and myometrium. Additionally, we performed MED12-variant analysis at the single-cell level to determine the genotype heterogeneity within leiomyomas.

Participants/materials, setting, methods: We collected five MED12-variant positive leiomyomas and five myometrium samples from a total of eight patients. We then performed single-cell RNA sequencing on freshly isolated single-cell preparations. Histopathological assessment confirmed the identity of the samples. Sanger sequencing was performed to confirm the presence of the MED12 variant in leiomyomas.

Main results and role of chance: Our data revealed previously unknown heterogeneity in the SMC, fibroblast cell and endothelial cell populations of myometrium and leiomyomas. We discovered the presence of two different lymphatic endothelial cell populations specific to uterine leiomyomas. We showed that both myometrium and MED12-variant leiomyomas are relatively similar in cellular composition but differ in cellular transcriptomic profiles. We found that fibroblasts influence the leiomyoma microenvironment through their interactions with endothelial cells, immune cells and SMCs. Variant analysis at the single-cell level revealed the presence of both MED12 variants as well as the wild-type MED12 allele in SMCs of leiomyomatous tissue. These results indicate genotype heterogeneity of cellular composition within leiomyomas.

Large scale data: The datasets are available in the NCBI Gene Expression Omnibus (GEO) using GSE162122.

Limitations, reasons for caution: Our study focused on MED12-variant positive leiomyomas for single-cell RNA sequencing analyses. Leiomyomas carrying other genetic rearrangements may differ in their cellular composition and transcriptomic profiles.

Wider implications for the findings: Our study provides a cellular atlas for myometrium and MED12-variant positive leiomyomas as defined by single-cell RNA sequencing. Our analysis provides significant insight into the differences between myometrium and leiomyomas at the single-cell level and reveals hitherto unknown genetic heterogeneity in multiple cell types within human leiomyomas. Our results will be important for future studies into the origin and growth of human leiomyomas.

Study funding/competing interest(s): This work was supported by funding from the National Institute of Child Health and Human Development (HD098580 and HD088629). The authors declare no competing interests.

Keywords: MED12; cellular heterogeneity; myometrium; single-cell RNA sequencing; uterine leiomyomas.

Figure 1.

Single-cell atlas of myometrium and leiomyomas. (A) Overview of the dissociation protocol for both myometrium (n = 5) and leiomyomas (n = 5). (B) Clustering of 24 605 high-quality cells from MED12-variant positive leiomyomas and myometrium. (C) Cell lineages identified by the marker gene expression. (D) Annotation of the cell clusters per sample. (E) Heatmap showing at least the top nine genes used for cluster identification. Columns denote cells; rows denote genes. SMC, smooth muscle cell; NK, natural killer cells; T, T cells; B, B cells.

Figure 2.

Heterogeneity and transcriptomic changes in smooth muscle cells (SMCs) in MED12-variant positive leiomyomas compared to the myometrium. (A) Uniform Manifold Approximation and Projection on Principal (UMAP) components showing the clusters of 4650 SMCs. (B) UMAP showing that all cell clusters are present in the myometrium and MED12-variant positive leiomyomas. (C) Heatmap of the SMC clusters. The colored bar on the top represents the cluster number. Columns denote cells; rows denote genes. (D, E) In-situ images showing the validation of the SMC clusters in MED12-variant positive leiomyomas and myometrium. Scale bar is 50 µm. (F) Volcano plots showing the transcriptomic changes in the SMC clusters of MED12-variant positive cells compared to the myometrium. (G) Gene ontology (GO) analysis of DE genes showing at least 0.5-fold change in the MED12-variant positive leiomyomas as compared to the myometrium.

Figure 3.

Intracellular heterogeneity and transcriptomic changes in fibroblast population in MED12-variant positive leiomyomas compared to the myometrium. (A) Uniform Manifold Approximation and Projection on Principal (UMAP) components showing the clusters of 1197 fibroblast cells. (B) UMAP showing that all cell clusters are present in the myometrium and MED12-variant positive leiomyomas. (C) Heatmap of the fibroblast cell clusters. The colored bar on the top represents the cluster number. Columns denote cells; rows denote genes. (D, E) In-situ images showing the validation of fibroblast populations in the myometrium and MED12-variant positive leiomyomas. Scale bar is 50 µm. (F) Volcano plots showing the transcriptomic changes in the fibroblast cell clusters of MED12-variant positive cells compared to the myometrium. (G) Gene ontology (GO) analysis of DE genes showing at least 0.5-fold change in the MED12-variant positive leiomyomas as compared to the myometrium.

Figure 4.

Lymphatic ECs are present in leiomyomas. (A) Uniform Manifold Approximation and Projection on Principal (UMAP) components showing the clusters of 10 448 endothelial cells. (B, C) UMAP showing the cluster annotation per condition: myometrium and MED12-variant positive leiomyomas. (D) Heatmap of endothelial cell clusters. The colored bar on the top represents cluster number. Columns denote cells; rows denote genes. (E, F) Immunostaining for PDPN (white arrowheads) and NUSAP1 (yellow arrowheads) showing the presence of Lymphatic EC clusters in normal myometrium (E) and MED12-variant positive leiomyomas (F). Scale bar is 100 µm.

Figure 5.

Cell–cell communication in myometrium and leiomyomas. (A) Chord diagram showing the cell–cell interactions in myometrium. (B) Chord diagram showing cell–cell communication in MED12-variant positive leiomyomas. The perimeter of the chord diagram is color-coded by cellular subtype (colored outer arcs). The inner connecting lines show the interactions between target and receiver cell types. The connecting lines are color coded consistently with cellular subtypes. The thickness of each edge (edge weight) is proportional to the interaction strength. A thicker edge line indicates a stronger signal. (C) Dot plot showing the upregulated ECM signaling pathway receptor–ligand interactions in subcellular clusters. Color indicates the communication probability. Dot size indicates the P-value. (D, E) Heatmaps show the relative importance of each cell group based on the computed four network centrality measures of ECM signaling network in (D) myometrium and (E) leiomyoma.

Figure 6.

MED12-variant positive uterine leiomyomas are not monoclonal. (A) Integrative Genome Viewer (IGV) analysis shows the presence of only wild-type MED12 allele in the myometrium (A.1) and the presence of both the variant and wild-type codon at c.131 in the MED12-variant positive leiomyomas (A.2 and A.3) in single-cell RNA sequencing data. The column med12 Bam alignment coverage shows the summary of the variant and wild-type allele detected at the c.131 location on MED12. The column MED12 alignment shows a snapshot of a small part of the data alignment track used to visualize individual MED12 aligned reads. (B) Uniform Manifold Approximation and Projection on Principal (UMAP) components of the mesenchymal cell populations in the leiomyomas. (C) UMAP showing the presence of the mutant MED12 variant (colored dots) and the wild-type MED12 (black dots) in SMCs, fibroblasts and endothelial cells.