Exosomes Exist in Nipple Discharge of Breast Cancer
Exosomes are nanoscale extracellular vesicles, typically ranging between 50 and 100 nm in diameter, that are secreted by various cell types into the extracellular matrix. These vesicles are enriched with proteins, lipids, and nucleic acids, enabling them to mediate intercellular communication and participate in diverse physiological and pathological processes. Prior studies have established the presence of exosomes in biological fluids such as blood, saliva, and urine. However, the existence of exosomes in nipple discharge—a fluid of clinical relevance in breast cancer diagnosis—remained unexplored. This study provides the first conclusive evidence that exosomes are present in nipple discharge from breast cancer patients and in colostrum from postpartum women, employing a combination of advanced isolation and characterization techniques.
The research utilized ultracentrifugation to isolate exosomes from nipple discharge samples obtained from two breast cancer patients and colostrum from two postpartum women. Key experimental methods included transmission electron microscopy (TEM) for morphological validation, Western blot analysis for protein marker identification, and nanoparticle tracking analysis (NTA) for determining particle size and concentration. Ethical approval was obtained from the Institutional Review Board of Qilu Hospital of Shandong University, and informed consent was secured from all participants.
Clinical Characteristics of Nipple Discharge Cases
Two nipple discharge samples were analyzed. The first sample (“nipple discharge 1”) was collected from a 43-year-old woman diagnosed with low-grade intraductal papillary carcinoma in the left breast. Preoperative galactography revealed no overt tumor signs, but cytological examination identified ductal epithelial cells with mild atypia. Tumor marker analysis of the discharge fluid showed elevated carcinoembryonic antigen (CEA) and carbohydrate antigen-153 (CA-153) levels at 4,775.50 ng/mL and 4,259.00 U/mL, respectively. The second sample (“nipple discharge 2”) was obtained from a 34-year-old woman diagnosed with invasive ductal carcinoma accompanied by high-grade intraductal carcinoma components. CEA and CA-153 levels in this sample were even higher, measuring 6,900.00 ng/mL and 5,940.00 U/mL. Colostrum samples from two postpartum women (“colostrum 1” and “colostrum 2,” aged 28 and 35) served as comparative controls.
Morphological and Biochemical Identification of Exosomes
TEM imaging revealed that vesicles isolated from both nipple discharge and colostrum exhibited the classic exosomal morphology: spherical structures with a cup-shaped concavity, averaging approximately 100 nm in diameter (Figure 1A). Western blot analysis further confirmed the presence of exosome-specific membrane proteins CD9 and TSG101 in both nipple discharge-derived exosomes and breast cancer cell lysates (MDA-MB-231 and MCF-7). CD81, another exosomal surface marker, was also detected in these samples. Critically, nuclear markers TFIIB and lamin A/C were absent in the exosomal fractions, confirming the purity of the isolates and the exclusion of cellular debris (Figure 1B). These results aligned with observations from cell lysates, where nuclear markers were present alongside exosomal proteins, while β-actin served as a loading control for lysate quantification.
Quantification and Size Distribution of Exosomes
NTA was employed to measure the size and concentration of exosomes. The median diameter of vesicles isolated from nipple discharge was 107.8 nm, with a concentration of 4.4 × 10¹⁰ particles/mL. In contrast, colostrum-derived exosomes were larger (median diameter: 141.9 nm) and less concentrated (2.4 × 10¹⁰ particles/mL) (Figure 1C). These findings not only validated the presence of exosomes in nipple discharge but also highlighted distinct biophysical properties between exosomes from pathological (nipple discharge) and physiological (colostrum) sources.
Technical Considerations and Classification Challenges
The study addressed ongoing debates in extracellular vesicle classification, particularly regarding size criteria. While exosomes are traditionally defined as vesicles between 50–100 nm, discrepancies exist across studies due to variations in isolation methods and measurement techniques. For instance, prior research on rat milk exosomes reported sizes exceeding 200 nm, attributed to potential exosome aggregation. In this study, the larger median diameter of colostrum-derived exosomes (141.9 nm) compared to nipple discharge exosomes (107.8 nm) underscores the influence of biological source and methodological factors on vesicle size. Despite these variations, the consistent detection of exosomal protein markers (CD9, CD81, TSG101) and the absence of nuclear contaminants strongly support the identification of these vesicles as exosomes.
Implications for Breast Cancer Diagnosis and Research
The identification of exosomes in nipple discharge opens new avenues for non-invasive breast cancer diagnostics. Exosomes carry molecular cargo reflective of their cell of origin, including tumor-specific proteins, miRNAs, and mRNAs. The elevated tumor marker levels (CEA and CA-153) in nipple discharge samples further suggest that exosomal content could serve as a reservoir of biomarkers for early cancer detection or monitoring. Additionally, comparing exosomes from malignant nipple discharge and benign breast conditions may yield insights into disease-specific molecular signatures.
Limitations and Future Directions
The study’s primary limitation is its small sample size, with only two nipple discharge and two colostrum samples analyzed. Future investigations should expand participant cohorts to include patients with benign breast tumors, enabling direct comparisons of exosomal properties across disease states. Furthermore, functional studies are needed to explore the biological roles of nipple discharge exosomes—such as their potential involvement in tumor progression or immune modulation—and to evaluate their utility in liquid biopsy platforms.
Conclusion
This study conclusively demonstrates the presence of exosomes in nipple discharge from breast cancer patients and colostrum from postpartum women. The integration of TEM, Western blot, and NTA methodologies provided robust validation of exosomal characteristics, including morphology, protein markers, and size distribution. The findings emphasize the ubiquity of exosomes across diverse biological fluids and underscore their potential as diagnostic and therapeutic tools in oncology. Future research should focus on elucidating the clinical relevance of nipple discharge exosomes and refining isolation protocols to enhance their applicability in personalized medicine.
https://doi.org/10.1097/CM9.0000000000001043
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