Adenomyosis: single-cell transcriptomic analysis reveals a paracrine mesenchymal-epithelial interaction involving the WNT/SFRP pathway

Abstract

Objective: To assess the cellular and molecular landscape of adenomyosis.

Design: Single-cell analysis of genome-wide messenger RNA (mRNA) expression (single-cell RNA sequencing) of matched tissues of endometrium, adenomyosis, and myometrium using relatively large numbers of viable cells.

Setting: Not applicable.

Patient(s): Patients (n = 3, age range 40-44 years) undergoing hysterectomy for diffuse adenomyosis.

Main outcome measure(s): Definition of the molecular landscape of matched adenomyotic, endometrial and myometrial tissues from the same uterus using single-cell RNA sequencing and comparison of distinct cell types in these tissues to identify disease-specific cell populations, abnormal gene expression and pathway activation, and mesenchymal-epithelial interactions.

Result(s): The largest cell population in the endometrium was composed of closely clustered fibroblast groups, which comprise 36% of all cells and seem to originate from pericyte progenitors differentiating to estrogen/progesterone receptor-expressing endometrial stromal- cells. In contrast, the entire fibroblast population in adenomyosis comprised a larger (50%) portion of all cells and was not linked to any pericyte progenitors. Adenomyotic fibroblasts eventually differentiate into extracellular matrix protein-expressing fibroblasts and smooth muscle cells. Hierarchical clustering of mRNA expression revealed a unique adenomyotic fibroblast population that clustered transcriptomically with endometrial fibroblasts, suggestive of an endometrial stromal cell population serving as progenitors of adenomyosis. Four other adenomyotic fibroblast clusters with disease-specific transcriptomes were distinct from those of endometrial or myometrial fibroblasts. The mRNA levels of the natural WNT inhibitors, named, secreted frizzled-related proteins 1, 2, and 4, were higher in these 4 adenomyotic fibroblast clusters than in endometrial fibroblast clusters. Moreover, we found that multiple WNTs, which originate from fibroblasts and target ciliated and unciliated epithelial cells and endothelial cells, constitute a critical paracrine signaling network in adenomyotic tissue. Compared with endometrial tissue, unciliated and ciliated epithelial cells in adenomyosis comprised a significantly smaller portion of this tissue and exhibited molecular evidence of progesterone resistance and diminished regulation of estrogen signaling.

Conclusion(s): We found a high degree of heterogeneity in fibroblast-like cells in the adenomyotic uterus. The WNT signaling involving differential expression of secreted frizzled-related proteins, which act as decoy receptors for WNTs, in adenomyotic fibroblasts may have a key role in the pathophysiology of this disease.

Keywords: Adenomyosis; SFRP; endometriosis; endometrium; fibroblast; scRNA-seq.

FIGURE 1

Single-cell transcriptomic analysis of endometrium. (A) Experimental design of the study. (B) UMAP presentation of endometrial cell clusters. Although the fibroblast-like population contained the highest numbers of cells, there also are prominent groups of T cells, NK cells, endothelial cells and unciliated and ciliated epithelial cells. (C) Dot plot of identified markers of each cell type. (D) Pie chart showing percentages of identified cell types. (E) Bar graph showing the number of cells from each patient (n = 3) in each cluster of integrated data. Each patient sample contributed data to each cluster. (F) Heatmap of highly expressed genes compared between progenitor cells, fibroblast-like cells, SMCs, and matrix Gla protein (MGP)-expressing fibroblasts. Ciliated epithelial cells highly expressed cilia-associated genes such as TPPP3, PIFO, and RSPH1. (G) Feature plots showing the distribution of key messenger RNAs s such as estrogen and progesterone receptors and select ECM-associated genes in fibroblast-like cells. (H) Pseudotime trajectory that was started from pericytes. It appeared that the pericytes might differentiate to the fibroblast-like group, including E0/E1/E11/E13, a process terminated in ciliated epithelial cells, which suggested mesenchymal–epithelial transition. (I) Heatmap of the PROGENy-predicted pathway activity scores of selected clusters (ciliated epithelial cells, pericytes, fibroblast-like cells, VSMCs). Interestingly, estrogenic and WNT pathways were upregulated in most fibroblast-like cells and downregulated in ciliated epithelial cells. UMAP, uniform manifold approximation and projection; SMCs, smooth muscle cells; VSMCs, vascular smooth muscle cells.

FIGURE 2

Single-cell transcriptomic analysis of adenomyosis tissue. (A) UMAP presentation of adenomyotic cell clusters. There is a larger and more heterogeneous group of fibroblast-like cells than endometrial fibroblasts (see Fig. 1). In contrast to endometrial tissue, there is no readily recognizable connection between the pericyte progenitors and fibroblast-like cells. (B) Fibroblasts comprised a much larger portion (50%) of adenomyosis, compared with endometrial fibroblast population (36%, see Fig. 1). In contrast, epithelial cells made up only 3% of adenomyosis versus 14% of endometrial tissue (see Fig. 1). Macrophage (MQ)-monocyte clusters seem to compose the most prominent immune cell population in adenomyosis. (C) Bar graph showing the number of cells from each patient (n = 3) in clusters of integrated data. (D) Dot plot of identified markers of each cell type. (E) Heatmap of highly expressed genes in progenitor cells, fibroblast-like cells, SMCs, and MGP-expressing fibroblasts. MGP-fibroblast-like cluster (A4) prominently expresses ECM-related genes and SFRPs. SMC cluster (A2) expresses SMC markers such as ACTA2. (F) Feature plots showing the distribution of expression of ESR1 and PGR, which concentrated in fibroblast-like cells. (G) Pseudotime trajectory of fibroblast-like cells suggesting that fibroblast-like cells might originate from cluster A0, differentiate through A1/A6/A12/A5, and terminate in 2 endpoints, A2 SMCs and A4 MGP-fibroblast-like cells. (H) Heatmap of the PROGENy-predicted pathway activity scores of selected cell clusters (fibroblast-like cells, MGP-expressing fibroblasts, and SMCs). Estrogen and WNT pathways are coactivated in A1/A5/A12, consistent with high ESR1 expression in these cluster (see Fig 2F). MGP, matrix gla protein; Cil epi, ciliated epithelial cells; CMP, common myeloid progenitor; fibro-like, fibroblast-like; Lymph endothelia, lymphatic endothelial cells; NK, natural killer; PC, Progenitor-like cells; VSMC, vascular SMCs; Uncil epi, unciliated epithelial cells.

FIGURE 3

Comparative analysis of differential gene expression in fibroblast populations across the 3 tissue types. (A) Heatmap of highly expressed genes in fibroblast-like cell populations in adenomyosis versus endometrium. The adenomyotic fibroblasts population A0 clustered together with the endometrial fibroblast types, suggesting that it may be of endometrial origin. (B) Heatmap of highly expressed genes in fibroblast-like cell populations in adenomyosis versus myometrium. The adenomyotic fibroblast-like population A4 clustered together with the myometrial fibroblast populations, suggesting that A4 might originate from myometrial tissue. (C) Violin plots displaying statistically significant differential expression of SFRPs (SFRP1, SFRP4, SFRP2) between the fibroblast-like clusters of adenomyosis (A), endometrium (E), and myometrium (M). (D) Violin plots displaying statistically significant differential gene expression of SFRPs (SFRP1, SFRP4, SFRP2) between selected fibroblast-like clusters in adenomyosis.

FIGURE 4

Analysis of epithelial cell profiles in adenomyosis and endometrium. (A) Molecular characterization of epithelial cell types in adenomyosis. Scatter plot showing average log fold change in gene expression between ciliated epithelial cells and unciliated epithelial cells in adenomyotic tissue. Expression of only several select genes, such as PAEP, defined the specific transcriptional profile of unciliated epithelial cells. (B) Heatmap of highly expressed genes in unciliated epithelial cells in adenomyosis (A) versus endometrium (E). Expression of PAEP, a well-defined progesterone-responsive differentiation marker, is significantly higher in endometrial unciliated cells. (C) Heatmap of highly expressed genes in ciliated epithelial cells in adenomyosis (A) versus endometrium (E). Secretoglobin 2A1 (SCGB2A1) is prominently expressed in ciliated endometrial cells. (D) Over-representation analysis comparing the single unciliated epithelial cell cluster in adenomyosis (A13) compared with the 2 unciliated epithelial clusters in endometrium (E19 and E2). The canonical WNT pathway was uniquely activated in the adenomyotic ciliated epithelial cell cluster. (E) Inferred communication network of the entire WNT signaling pathway in adenomyosis. The general signaling direction is such that WNT ligands originate primarily from the fibroblast-like cells (A0, A1, A5 and A12), whereas the primary WNT-responsive cell types are the epithelial and endothelial cells. A13 unciliated epithelial cell cluster is unique in that it shows an autocrine signaling pattern; moreover, A13 epithelial cells also send signals to ciliated epithelial (A18) and endothelial (A10) cells. (F) Expression of WNT signaling pathway genes in each cell cluster in adenomyosis. The general pattern of expression is such that the WNT ligands are highly expressed in fibroblast-like cells, whereas their receptors or coreceptors are prominently expressed in epithelial and endothelial cells.