Single-Cell RNA Sequencing of PBMCs Identified Junction Plakoglobin (JUP) as Stratification Biomarker for Endometriosis

Abstract

This study aimed to identify unique characteristics in the peripheral blood mononuclear cells (PBMCs) of endometriosis patients and develop a non-invasive early diagnostic tool. Using single-cell RNA sequencing (scRNA-seq), we constructed the first single-cell atlas of PBMCs from endometriosis patients based on 107,964 cells and 25,847 genes. Within CD16⁺ monocytes, we discovered JUP as a dysregulated gene. To assess its diagnostic potential, we measured peritoneal fluid (PF) and serum JUP levels in a large cohort of 199 patients including 20 women with ovarian cancer (OC). JUP was barely detectable in PF but was significantly elevated in the serum of patients with endometriosis and OC, with levels 1.33 and 2.34 times higher than controls, respectively. Additionally, JUP was found in conditioned culture media of CD14⁺/CD16⁺ monocytes aligning with our scRNA-seq data. Serum JUP levels correlated with endometriosis severity and endometrioma presence but were unaffected by dysmenorrhea, menstrual cycle, or adenomyosis. When combined with CA125 (cancer antigen 125) JUP enhanced the specificity of endometriosis diagnosis from 89.13% (CA125 measured alone) to 100%. While sensitivity remains a challenge at 19%, our results suggest that JUP's potential to enhance diagnostic accuracy warrants additional investigation. Furthermore, employing serum JUP as a stratification marker unlocked the potential to identify additional endometriosis-related genes, offering novel insights into disease pathogenesis.

Keywords: Junctional plakoglobin (JUP); adenomyosis; diagnostic biomarkers; endometriosis; peripheral blood mononuclear cells (PBMCs); single-cell RNA-sequencing (scRNA-seq).

Figure 1

Dysregulated gene expression analysis with endometriosis as endpoint. (A) Study design. Pain scores and peripheral blood were collected just before laparoscopy. PBMCs were cryopreserved until single-cell sequencing could be performed by batch. Serums were frozen before quantification of JUP, CA125 and S100A12 by ELISA. During surgery, the revised American Society for Reproductive Medicine (rASRM) score was assigned and suspect lesions were collected for confirmation of diagnosis by a trained pathologist. DGE (differential gene expression) analysis was based on endometriosis diagnosis and level of JUP in serum. (Created in https://BioRender.com, accessed on 8 November 2024) (B) Cellular composition of PBMCs based on scRNA-seq (uniform manifold approximation and projection, UMAP plot) for all samples (controls = 7; endometriosis cases = 6). (C) Dot plot showing average expression (color scale = expression intensity) and percentage of positive cells (dimension scale = proportion of positive cells) for selected cell type-specific marker genes. (D) Comparison of JUP expression profile in endometriosis cases and controls. The left panel represents the expression level of JUP in CD16⁺ monocytes (logFC 1.66; adjusted p-value = 0.004). The right panel represents the UMAP of the CD16⁺ monocytes in samples of women without endometriosis (left) and with endometriosis (right). Cells with higher expression levels are indicated by darker dots. Endo: Endometriosis group, CD4 TCM: CD4 central memory, CD4 TEM: CD4 effector memory T cells, CD8 TCM: CD8 central memory, CD8 TEM: CD8 effector memory T cells, cDC2: Type-2 conventional dendritic cells, dnT: TCRα⁺ CD4⁻ CD8⁻ double negative T cells, Eryth: erythrocytes, gdT (Yδt): Gamma-delta (γδ) T cells, HSPC: Hematopoietic Stem and Progenitor Cells, MAIT: Mucosal-Associated Invariant T cells, NK: Natural killer cells, pDC: Plasmacytoid dendritic cells, Treg: regulatory T cells.

Figure 2

JUP in endometriosis. (A) Violin plots of serum JUP level in endometriosis-free women (CTL) (median = 158.5 ng/mL), endometriosis and patients (Endo) (median = 211.4 ng/mL), patients with ovarian cancer (OC) (median = 372.3 ng/mL). CTL vs. Endo (p-value = 0.0152), CTL vs. OC (p-value < 0.0001), Endo vs. OC (p-value = 0.0027) (Mann–Whitney test). (B) Violin plots of serum JUP level in CTL (median = 158.5 ng/mL), Endo stage I–II (median = 199.5 ng/mL) and Endo stage III-IV (median = 283.4 ng/mL). Non-parametric one-way ANOVA (analysis of variance, Kruskal–Wallis test, p-value = 0.0184) followed by a non-parametric comparison (Dunn’s multiple comparisons test, CTL vs. Endo stage III-IV, p-value = 0.019). Patients with adenomyosis were included. (C) Violin plots of serum JUP level in CTL (median = 158.5 ng/mL), endometriosis patients without endometrioma (Endo WO OMA) (median = 203.1 ng/mL), and endometriosis patients with endometrioma (Endo with OMA) (median = 283.4 ng/mL). Non-parametric one-way ANOVA (Kruskal-Wallis test, p-value = 0.007) followed by a non-parametric comparison (Dunn’s multiple comparisons test, CTL vs. Endo stage III-IV, p-value = 0.006). Patients with adenomyosis were included. (D) Violin plots of serum JUP level in endometriosis-free women (CTL) (median = 165.5 ng/mL), endometriosis patients (Endo) (median = 207.4 ng/mL), endometriosis-free women with adenomyosis (Adeno) (median = 139.5 ng/mL), and endometriosis patients with adenomyosis (Endo and Adeno) (median = 255.9 ng/mL). Two-way ANOVA assessing the endometriosis and adenomyosis effects (endometriosis, p-value = 0.0151; adenomyosis, p-value = 0.1167). (E) Violin plots of serum JUP level in proliferative phase (CTL Prolif: median = 166.5 ng/mL; Endo Prolif: median = 116.8 ng/mL) and in secretory phase (CTL Secr: median = 162.2 ng/mL and Endo Secr: median = 215.5 ng/mL). Two-way ANOVA assessing the endometriosis and cycle effects (endometriosis, p-value = 0.0345; cycle, p-value = 0.2251). (F) Positive correlation of serum JUP level with BMI (Body Mass Index; Pearson’s r = 0.260, p-value = 0.003, and n = 128). (* = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, n.s. = not significant).

Figure 3

Diagnosis performance of JUP in endometriosis. (A) ROC analysis for serum JUP (vertical lines, AUC (Area Under the Curve) 0.6048, p-value = 0.0154) and serum CA125 (clear circles, AUC 0.7054, p-value = 0.0006). JUP AUC vs. CA125 AUC (p-value = 0.1476). (B) Positive correlation of JUP with CA125 in serum; Pearson’s r = 0.551; p-value 1.020 × 10⁻⁸; n = 93 (CTL: clear circles; endometriosis: black circles). (C) Box and Whiskers plots of HE4 (Human Epididymis Protein 4) quantified in serum in patients with high CA125 (>26.8 U/mL) and high JUP (>324 ng/mL). HE4 was higher in patients with ovarian cancer (median = 189.0 pM) than in patients with endometriosis (median = 26.7 pM) (p-value < 0.0001, Mann-Whitney test). (D) Positive correlation of JUP with S100A12 in serum; Pearson’s r = 0.835; p-value = 2.180 × 10⁻¹⁶; n = 59 (CTL: clear circles; endometriosis: black circles). (*** = p-value < 0.001).

Figure 4

JUP and S100A12 levels in conditioned medium of CD14⁺/CD16⁺ monocytes in culture. (A) Box and Whiskers plots of S100A12 quantified in medium at 24 h (median = 6.50 ng/mL), 72 h (median = 2.90 ng/mL), and 120 h (median = 1.00 ng/mL) of in vitro culture. One-way ANOVA (Friedman paired test) (p-value = 0.0028). and 24 h vs. 120 h (p-value = 0.0073) (Dunn’s multiple comparisons test). (B) Box and Whiskers plots of JUP quantified in medium at 24 h (median = 1.83 ng/mL), 72 h (median = 1.70 ng/mL), and 120 h (median = 0.96 ng/mL) of in vitro culture. One-way ANOVA (Friedman paired test) (p-value = 0.0054), 24 h vs. 120 h (p-value = 0.0424), and 72 h vs. 120 h (p-value = 0.0183) (Dunn’s multiple comparisons test). (C) Positive correlation of JUP with S100A12 in culture medium; Pearson’s r = 0.889; p < 0.0001; n = 18. The values collected at 24 h, 96 h and 120 h for the 6 PBMC cultures were included in this correlation plot. (* = p-value < 0.05, ** = p-value < 0.01).

Figure 5

Expression profile of endometriosis DEGs in patients with high (A–C) and low (D–F) serum JUP. The upper panels represent the expression level of the DEGs in each identified cluster; (A) RGPD2 in CD14⁺ monocytes; (B) LTB in γδt; (C) KLRC1 in γδt; (D) TMEM176A in CD14⁺ monocytes; (E) TMEM176B in CD14⁺ monocytes; and (F) TRBV2 in CD4 CTLs. The lower panels represent the UMAP of each identified cluster in samples of women without endometriosis (left) and with endometriosis (right). A color code indicates the expression level of the DEGs in each cell; cells with higher expression levels are indicated by darker dots. KLRC1: Killer cell lectin-like receptor C1, LTB: lymphotoxin beta, RGPD2: RANBP2-like and GRIP domain-containing 2, TMEM176A and B: Transmembrane proteins 176A and B, TRBV2: T Cell Receptor Beta Variable 2.