Characterization of E-Cadherin, SSEA-1, MSI-1, and SOX-2 Expression and Their Association with Pale Cells in Adenomyosis

Abstract

Adenomyosis (AM) is a gynecological disease characterized by the invasion of endometrial glands and stroma within the myometrium. The etiology and pathogenesis of AM remain inadequately understood. Pale cells were identified as a novel cell type characterized by the absence of desmosomal contacts and light-colored cytoplasm. These cells were observed to migrate individually through ultra-micro ruptures in the basal membrane of the endometrial glands, translocating into the stroma and then further into the myometrium. Our study aimed to explore the possible stem cell properties of these pale cells. Forty hysterectomy specimens were analyzed using immunohistochemistry and immunofluorescence to assess negative E-cadherin expression and the positive expression of stem cell markers SSEA-1, MSI-1, and SOX-2. Immunohistochemical analysis revealed the presence of pale cells and occasionally rounded, enlarged E-cadherin-negative cells predominantly in the basal endometrial epithelium. The stem cell marker SSEA-1 was significantly elevated in the basalis epithelium, as well as in the ectopic epithelium. SSEA-1 positive cells were also identified in the stroma and myometrium. Sporadic colocalization of SSEA-1+/E-cadherin- cells was confirmed through immunofluorescence. The positive staining of pale cells for SSEA-1 and MSI-1 was also confirmed at the ultrastructural level by immunoelectron microscopy. These findings indicate that pale cells may possess stem cell characteristics, particularly a positive SSEA-1 profile, warranting further in vitro investigation into their role in the pathogenesis of adenomyosis.

Keywords: E-cadherin; MSI-1; SOX-2; SSEA-1; adenomyosis; pale cell.

Figure 1

The immune expression pattern of E-cadherin, MSI-1, and SOX-2 in adenomyosis tissues. (A) E-cadherin expression in the epithelium of the non-AM group, AM group (eutopic), and in lesions (B) SOX-2 expression in the non-AM group, AM group, and lesions. (C,D) E-cadherin expression in both a lesion and the basalis region of the AM group within the same tissue section. (E,F) E-cadherin expression in the functionalis of the AM group. (G) SOX-2 expression in the basalis of the AM group. (H,I) SOX-2 expression in a lesion. (J) SOX-2 expression in the myometrium in the AM group. (K) MSI-1 expression in the epithelium of the non-AM group, AM group, and in lesions. (L,N) MSI-1 expression in the basalis of the AM group (M) MSI-1 expression in the stroma of lesions. (O) MSI-1 expression in the myometrium of the AM group. (P) MSI-1 expression in epithelium across different groups. NG = non-AM group, AG = AM group, LE = lesion epithelium, FE = functionalis epithelium, BE = basalis epithelium. Data were assessed using the Kruskal–Wallis test, and significant differences were considered at p-values of <0.05. Scale bar = 50 um.

Figure 2

SSEA-1 is upregulated in adenomyosis lesions. (A) SSEA-1 expression in the non-AM and AM groups. (B) SSEA-1 expression in the epithelium of the non-AM group, AM group (eutopic), and in lesions. (C) SSEA-1 expression according to cycle phase and groups. (D–F) SSEA-1 expression in AM group lesions. (G) SSEA-1 expression in the functionalis of the AM group. NG = non-AM group, AG = AM group, LE = lesion epithelium, FE = functionalis epithelium, BE = basalis epithelium. Data were analyzed by the Kruskal–Wallis test. Significant differences were considered at p-value < 0.05. Scale bar = 50 um.

Figure 3

Pale cell recognition by light microscopy. (A,B) PCs in eccentric, central, and luminal localizations in the non-AM group. (C,D) Eccentrically and centrally arranged PCs in the AM group. The bright cytoplasm, large roundish morphology, and enlarged cell nucleus are recognizable. (E,F) PCs centered in endometrial and adenomyotic lesions of the AM group. (G) Pale cell number per field in the non-AM group, AM group (eutopic), and lesions. (H) Pale cell number per field in different layers in each group. (I) Pale cell number per field in the secretion cycle among different layers in each group. Data were analyzed by the Kruskal–Wallis test. Significant differences were considered at p-values < 0.05. Scale bar = 50 um.

Figure 4

Immunoelectron microscopy revealed the expression of MSI-1 and SSEA-1 in pale cells. (A) Immunostaining for MSI-1 in the cytoplasm of electron-lucent cells. (B) Immunostaining for SSEA-1. Electron-dense cytoplasmic structures, as well as the cell surface, were labeled. Representative gold particles are highlighted by arrow heads. Scale bar = 500 nm.

Figure 5

Colocalization of E-cadherin-negative and SOX-2-, MSI-1-, or SSEA-1-positive cells. (A) SSEA-1 immunohistochemistry staining in the lesion. (B) E-cadherin immunohistochemistry staining in the same lesion. (C) MSI-1 immunohistochemistry staining in the lesion. (D) E-cadherin immunohistochemistry staining in the same lesion. (E,G) SOX-2 immunohistochemistry staining in the lesion. (F,H) E-cadherin immunohistochemistry staining in the same lesion. (I–K) Immunofluorescence staining of the basal epithelium in the AM group. (L–N) Immunofluorescence staining of a lesion in a stroma in the AM group. (O–Q) Immunofluorescence staining of the functionalis in the AM group. (R–T) Immunofluorescence staining of stromal basalis in the AM group. Scale bar = 50 um.