3.1 The injury of mammary duct epithelial cells is a common pathologic feature of PCM and GM
Fig. 1. shows the typical pathological characteristics of PCM, GM, and the normal breast tissues. Normal mammary gland tissues are composed of independently existing mammary lobules. The mammary ducts in the lobules are arranged neatly, without inflammatory cell infiltration and damage to the duct epithelial cells (Fig.1-A1). Ductal epithelial cells of the breast were placed within a double layer of tubes. The inner cells were glandular epithelial cells, and the outer ones were myoepithelial cells (Fig. 1-A2). Extensive inflammatory cells around the dilated duct are a typical histopathological feature of PCM, and a high percentage of plasma cells have diagnostic values (Fig.1-B1). Inflammatory infiltration caused injury and apoptosis of mammary duct epithelial cells, which showed typical nuclear pyknosis, nuclear fragmentation, and nucleolysis, the reddish phagosome found in PCM. Usually, it engulfs apoptotic cell fragments (Fig.1-B2). Typical pathological features of GM (Fig.1-C1) are granulomatous structures and lipid vacuoles. In the high background, the remaining ductal epithelial breast cells showed classic signs of apoptosis, as seen in PCM (Fig.1-C2). Inflammatory cell infiltration and mammary duct epithelial cell injury were elevated in the PCM and GM groups than the control group (*P＜0.01)(Fig.1-D). However, the inflammatory cell infiltration and mammary duct epithelial cell injury of the GM group were apparent compared with the PCM group (*P＜0.01)(Fig.1-D).
3.3 Damage of mammary ductal epithelial cells under transmission electron microscopy
Fig. 2. shows the ultrastructural changes of PCM, GM, and the normal breast tissues. Within normal mammary ducts, the epithelial cells were closely arranged without infiltration of the inflammatory cells. The capillaries were observed between fibrous tissues, and different white blood cells were observed in the blood vessels (Fig.2-A1). The ultrastructure of PCM showed that the epithelial cells of mammary ducts were disordered and depicted different morphological changes, accompanied by many inflammatory cells (Fig.2-A2). The ultrastructure of GM was very similar to PCM (Fig.2-A3). The ultrastructure of normal mammary duct epithelial cells was intact, and the intercellular links were well developed, along with gap links and tight junctions. The intracellular staining was homogeneous, and the cell and nuclear membranes were intact without damage. There were poorly developed microvilli on the surface of the cell membrane (Fig.2 B1-B2). PCM and GM ductal epithelial cells had similar configuration changes, including connections with the microvilli on the cell membrane surface. There was a noticeable increase in cell differences, lysosomes, the nucleus, irregular shapes, increased heterochromatin, and chromatin edge set. Some cells with prominent nucleoli and nuclear pulp ratio also increased, along with abnormal chromatin phenomenon (Fig.2 C1-C2,Fig.2 D1-D2). Mammary ductal epithelial cells within the PCM and GM tissues showed typical damage and apoptosis, including cell and nuclear membrane deterioration, chromatin shrinkage, and fragmentation (Fig.2 E1-E2). In addition, a large number of exosomes were found in the PCM and GM tissues but rarely within normal breast tissues (Fig.2-F2 and Fig.2-F3); however, no exosomes were found in normal breast tissue (Fig.2-F1).
3.3 The expression of MAC was significantly increased in PCM and GM
Figure 3 shows the increased expression of MAC and its related molecules in PCM and GM tissues.We observed the high expression of MAC in PCM, GM and NC groups cells with tissue immunoelectron microscopy (Fig.3 A1-A3). The more the black particles, the higher was the positive charge. The black particles indicated that MAC was located in the cytoplasm and membrane. As shown in the figure, the expression of MAC in PCM and GM groups was significantly increased than in the NC group (Fig.3-B) (*P＜0.05). Representative micrographs of immunohistochemical staining are illustrated in Fig.3-C MAC immunoreactivities were detected in epithelial cells, plasma cells, macrophages, lymphocytes, and neutrophils. In addition, immunohistochemical staining scores were analyzed using ANOVA or Welch’s ANOVA test followed by a posthoc test. The results showed that MAC immunoreactivities were significantly elevated in the PCM and GM than in the NC groups (Fig.3-D) (both *P＜0.05). However, there was no statistical significance between the PCM and GM groups (Fig.3-D) (*P＞0.05). C5b and C5b-9n (MAC) expression in the supernatants of peripheral blood in the PCM, GM, and NC group was detected using ELISA. Compared with the NC group, C5b and C5b-9n(MAC) expression in the PCM and GM groups was significantly increased (Fig.3-E) (*P＜0.05). The mRNA levels of MAC in PCM and GM groups were significantly increased than in the NC group (*P＜0.001). Representative Western blot data are illustrated in Fig. 3G, with quantitation and comparison by ANOVA or Welch’s ANOVA test followed by the posthoc test in Fig.3-H. Specifically, Western blot results indicated that MAC was significantly expressed in most PCM and GM samples. In addition, the protein contents of MAC were no observable statistical difference between the PCM and GM groups (*P＞0.05).
3.4 The concentration of exosome increased significantly in PCM and GM
The transmission electron microscopy analysis showed that the exosomes isolated from PCM, GM, and the normal breast tissues were morphologically homogeneous. They ranged from 30 to 150 nm in size, with a standard round or cup-shaped appearance (Fig.4A1-A3). The particle size distribution and concentration of PCM/Exo, GM/Exo, and NC/Exo were detected using the Nano-FCM (N30E). The exosome concentration was 4.68 × 1010 particles/mL in PCM/Exo, 3.99 × 1010 particles/mL in GM/Exo, and 1.07 × 1010 particles/mL in NC/Exo (Fig.4-B). There was a statistically significant difference in the concentration of exosomes (Fig.4 -C) (*P＜0.05) . The average particle size of exosomes was 70.05 nm, 75.26 nm, and 76.27 nm in PCM/Exo, GM/Exo, and NC/Exo (Fig.4-B), respectively, and there was no statistically significant difference in the particle size (*P＞0.05).
3.5 Elevated MAC amounts in exosomes
Overrepresented MAC in PCM/Exo, GM/Exo and NC/Exo were confirmed using immune electron microscopy (Fig.4 D1-D3). There was a statistically significant difference in the PCM/Exo and GM/Exo compared with NC/Exo (Fig.4-E)(*P＜0.05). The levels of MAC in exosomes were further detected by Western blotting (Fig.4-F). Furthermore, the results indicated that the protein level of MAC was significantly upregulated in PCM/Exo and GM/Exo than in NC/Exo (Fig.4-G, Both* P＜0.05). These results suggest that exosomes derived from the tissues of PCM and GM contain concentrated MAC, which could be related to the MAC removal process.
Furthermore, we assessed the levels of exosomal MAC in breast tissue by flow cytometry analysis to explore the predictive value of MAC for investigating the injury to breast ductal epithelial cells in PCM and GM patients. The results showed that exosomal MAC in the breast tissue of PCM and GM was significantly higher than in the NC group (Fig.4-H and Fig.4-I, Both *P＜0.05).
3.6 P-selectin, E-selectin, and ICAM-1 were highly expressed in PCM and GM
Representative micrographs of immunohistochemical staining are illustrated in Fig.5-A. P-selectin, E-selectin, and ICAM-1 are mainly found in the nucleus and membrane of the epithelial cells, macrophages, neutrophils, and lymphocytes. Compared with the PCM and GM group, the immunoreactivities of P-selectin, E-selectin, and ICAM-1 were weaker and detected only in epithelial cells. Immunohistochemical staining scores were analyzed by ANOVA or Welch’s ANOVA test followed by the posthoc test (Fig.5-B). The results showed that the immunoreactivities of P-selectin, E-selectin, and ICAM-1 were significantly elevated in macrophages, neutrophils, and lymphocytes in the PCM and GM group compared with NC cases (both P＜0.0001). There were no statistical differences in P-selectin, E-selectin, and ICAM-1 between the PCM and GM groups (p＞0.05).
Furthermore, we assessed the mRNA levels of P-selectin, E-selectin, and ICAM-1 in breast tissue by Q-PCR. The mRNA levels of P-selectin, E-selectin, and ICAM-1 in PCM and GM groups were significantly increased compared with the NC group (*P＜0.001)(Fig.5-C).