Single-cell and spatial transcriptomic profiling revealed niche interactions sustaining growth of endometriotic lesions

Abstract

Endometriosis is a chronic condition with limited therapeutic options. The molecular aberrations promoting ectopic attachment and interactions with the local microenvironment sustaining lesion growth have been unclear, prohibiting development of targeted therapies. Here, we performed single-cell and spatial transcriptomic profiling of ectopic lesions and eutopic endometrium in endometriosis. We found that ectopic endometrial stromal (EnS) cells retained cyclical gene expression patterns of their eutopic counterparts while exhibiting unique gene expression that contributes to the pathogenesis of endometriosis. We identified two distinct ovarian stromal cells (OSCs) localized at different zones of the lesion, showing differential gene expression profiles associated with fibrosis and inflammation, respectively. We also identified WNT5A upregulation and aberrant activation of non-canonical WNT signaling in endometrial stromal cells that may contribute to the lesion establishment, offering novel targets for therapeutic intervention. These data will enhance our understanding of the molecular mechanisms underlying endometriosis and paves the way for developing non-hormonal treatments.

Keywords: WNT5A; endometrioma; endometriosis; menstrual cycle; ovarian stromal cells; single-cell analysis; spatial transcriptomics.

Graphical abstract

Figure 1

scRNA-seq identified ectopic EnS cells and lesion-specific stromal cells in endometriosis (A) Diagram of specimen collection for single-cell transcriptome profiling. (B and E) Uniform manifold approximation and projection (UMAP) plots of cell types identified from the 13 endometria (B) and 8 ovarian endometriomas (E). Colors indicate different cell types. See also Figures S2–S4. (C and F) Bar plots of cell compositions for the endometria (C) and endometriomas (F). Colors indicate different cell types. Error bars represent standard deviations. (D and G) Violin plots displaying the marker gene expression for each identified cell type. EnS cell, endometrial stromal cell; LEC, lymphatic endothelial cell; OSC, ovarian stromal cell. (H and I) Immunofluorescence staining of endometrium (H) and ovarian endometrioma (I). DAPI (blue) indicates the cell nucleus, Ecad (E-cadherin; green) indicates epithelial cells, TCF21 (red) indicates OSCs, and CD10 (purple) indicates EnS s. EM, endometrium; OEM, ovarian endometrioma. Scale bars represent 1 mm (upper) or 50 μm (lower).

Figure 2

The menstrual dynamics of ectopic EnS cells (A) Heatmap of the gene expression dynamics for eutopic EnS cells along the pseudotime axis of the menstrual cycle. (B and C) Dot plot of PES (B) and SES (C) for each EnS cell identified in our data along the menstrual pseudotime. PES, proliferative phase EnS score; SES, secretory phase EnS score. (D and E) Violin plots of PES (D) and SES (E) for EnS cells using the GSE111976 dataset of healthy women. Pro, proliferative phase; Sec_early, early secretory phase; Sec_early-mid, early to mid-secretory phase; Sec_mid, mid-secretory phase; Sec_late, late secretory phase. (F and G) Comparison of proliferative and secretory gene expression scores of eutopic and ecotpic EnS cells between our data and an independent endometriosis dataset (GSE213216). EM_Pro, eutopic endometrium from the proliferative phase; EM_Sec, eutopic endometrium from the secretory phase; OEM_Pro, ovarian endometriomas from the proliferative phase; OEM_Sec, ovarian endometriomas within the secretory phase. PEM_Pro and PEM_Sec denote peritoneal endometriosis within the proliferative phase and secretory phases, respectively. Student’s t test was used to compare the gene expression score of EnS cells between the proliferative phase and secretory phase. Δ = mean of PES – mean of SES. (H and I) Dot plots of representative menstrual-phase gene expression in eutopic and ectopic EnS cells for our data (H) and GSE213216. (J) Heatmap of overexpressed genes in eutopic and ectopic EnS cells across different menstrual phases. The differentially expressed genes were identified by Wilcoxon rank-sum test with log₂ fold change > 1 and adjusted p < 0.05. (K) Venn diagram displaying the number of overexpressed genes in ectopic EnS cells for the proliferative phase and secretory phase. (L) GO enrichment for the 42 overexpressed genes along the menstrual cycle. See also Figure S5.

Figure 3

Spatial profiling reveals the zonation of two subtypes of OSCs in endometriomas (A and B) UMAP plots of the two subgroups of OSCs, OSCa and OSCb, in our dataset (A) and GSE213216 (B), displaying their respective marker gene expression. (C) H&E staining of formalin-fixed, paraffin-embedded sections from a patient with ovarian endometriosis. The scale bar represents 6.5 mm. (D) Unsupervised clustering of spatial transcriptomic spots from the sections in (C). A dominant cell type is assigned to each spot following the identification of marker genes within the clusters. OSCm, spots expressing markers of both OSCa and OSCb; OUn, unclassified cells. The scale bar represents 6.5 mm. See also Figure S6. (E) Dot plot illustrating the overexpressed genes corresponding to each identified cell type from the spatial transcriptomic data. See also Figure S6. (F) Marker gene expression in the spatial transcriptomic data for EnS cells, OSCb, and OSCa. MMP11 represents ectopic EnS cells, COL10A1 and FAP mark OSCb, and TCF21 marks OSCa. Color intensity indicates gene expression levels after sctransform in Seurat. See also Figure S6. (G and H) Immunofluorescence staining of ectopic EnS cells and the two groups of OSCs in tissue sections of endometriomas. DAPI stains the cell nucleus, TCF21 marks OSCa, CD10 identifies ectopic EnS cells, and COL10A1 and FAP mark OSCb. Scale bars represent 500 μm. See also Figure S7. (I) Dot plot illustrating the overexpressed genes in three groups of OSCs derived from GSE213216, displaying the percentage of cells expressing each gene and the average expression level. (J) GO enrichment analysis of the overexpressed genes in each group of OSCs. Hypergeometric test was used to assess the gene enrichment, and false discovery rate was applied to account for multiple comparisons.

Figure 4

Spatial profiling reveals immune cell enrichment in distinct zones of endometriotic lesions (A) Marker gene expression in the spatial transcriptomic data for EnS cells, T cells, macrophages, and B cells. MMP11 represents ectopic EnS cells, while CD3D identifies T cells. CD68 and CD79A mark macrophages and B cells, respectively. Color intensity indicates gene expression levels after sctransform in Seurat. See also Figure S8. (B) Dot plot illustrating immune cell marker expression across different clusters of spots in spatial transcriptomic data. The size of the dots indicates the percentage of spots expressing the marker within each zone, and the color intensity indicates the mean expression level. Data from three samples are indicated by 1, 2, or 3 following the cluster name. These results are quantified based on the spatial transcriptomic data presented in (A) and Figures S8A and S8B. (C) Bar plots showing the percentage of CD68-positive (macrophage) and CD3D-positive spots across different zones of endometriomas. These results are linked to the dot plots in (B). Paired Student’s t test was used to compare two groups. ns, not significant. Error bars represent standard deviations. (D) Immunofluorescence staining of immune cells in different zones of endometrioma. CD10 (yellow) indicates ectopic EnS cells, CD68 (magenta) marks macrophages, CD3D (cyan) indicates T cells, TCF21 (red) marks OSCa, and FAP (orange) marks EnS cells and OSCb. The dashed lines delineate the EnS region and OSCa and OSCb regions separately. Scale bars represent 500 μm. See also Figure S8. (E) The mean fluorescence intensity of CD68 and CD3D in the EnS cell, OSCb, and OSCa regions. Paired Student’s t test was used for statistical analysis.

Figure 5

Cellular interaction analysis identified disease-specific signaling pathways associated with endometriomas (A) Heatmap illustrating the interaction strengths derived from spatial transcriptomic data of ovarian endometriomas. (B–D) Outgoing cellular interaction strengths for EnS cells (B), OSCb (C), and OSCa (D). The numbers indicate the interaction strengths between the cell types. (E) Heatmap depicting the secretory signaling pathways identified among cell types in the spatial transcriptomic data. The color represents the interaction strengths. (F) Heatmap depicting the secretory signaling pathways identified among cell types in the scRNA data of endometriomas and control ovaries. The color represents interaction strength. Green dashed lines indicate the endometriosis-specific signaling. (G) Dot plot illustrating the outgoing (x axis) and incoming signal strengths (y axis) for each cell type in endometriomas and control ovaries. Dot size represents the number of cells. (H–J) Circle plot illustrating cellular interaction strength for shared signaling pathways in endometriomas and control ovaries.

Figure 6

Ectopic EnS cells interact with OSCb through WNT5A-FZD1 signaling in endometriomas (A) Dot plot illustrating the communication probabilities for each ligand-receptor pair involved in ncWNT and BMP signaling among the cell types. The p value reflects the significance of the interactions as determined by permutation testing and is represented by the dot size, with color denoting the probability of communication. (B) Violin plots illustrating the expression levels of ncWNT signaling genes across various cell types. (C) Immunofluorescence staining reveals the interaction between ectopic EnS cells and OSCb through WNT5A-FZD1 pairings. DAPI (blue) stains cell nuclei, CD10 (white) marks ectopic EnS cells, and COL10A1 (cyan) labels OSCb cells. WNT5A (yellow), secreted by EnS cells, co-localizes with FZD1 (red) in OSCb regions. Scale bars represent 500 μm. See also Figure S9. (D and E) Dot plots illustrating WNT5A expression in EnS cells from our data (D) and the GSE213216 dataset (E). Labels denote eutopic endometrium (EM) and ovarian endometriomas (OEMs) during both proliferative (Pro) and secretory (Sec) phases as well as peritoneal endometriosis (PEM) in corresponding phases. (F) Dot plot depicting WNT5A expression in EnS cells derived from the menstrual effluent dataset GSE203191. (G and H) Immunohistochemical staining of WNT5A in ovarian endometriomas (G) and eutopic endometrium (H), accompanied by H&E sections. Blue arrows and the dashed line in (G) delineate the ectopic endometrium, and black arrows in (H) indicate WNT5A positivity. Scale bars represent 100 μm.