O25 - Profiling Extracellular Vesicles from Breast Cancer Cells Reveals Subtype-Specific Differences in Size Distribution and Functional Modulation of Lymphatic Endothelial Cells

Introduction: : Breast cancer remains a global health crisis, ranking as the second leading cause of cancer-related deaths. In approximately 90% of metastatic cases, breast cancer spreads through the lymphatic system, which plays a crucial role in maintaining fluid balance and immune surveillance. Cancer cells access the lymphatic system via lymphatic vessels in breast tissue, ultimately traveling to lymph nodes and distant metastatic sites.
O-linked glycosylation—the attachment of sugars to serine or threonine residues on membrane-associated proteins—facilitates cancer cell interactions with each other and with the extracellular matrix. Altered glycosylation patterns, including expression of tumor-associated antigens such as T, Thomsen-Friedenreich, (Galβ1-3GalNAc) and Tn, Thomsen-nouveau, (GalNAc), are commonly observed in cancers and contribute to disease progression. Aberrant O-linked glycosylation in cancer is associated with increased production of extracellular vesicles, particularly microvesicles (MVs), which promote long-range cellular communication and support metastatic processes.
In the lymphatic system, podoplanin, a heavily O-glycosylated sialomucin expressed on lymphatic endothelial cells, plays a key role in vessel integrity and tumor cell interaction. However, the relationship between microvesicle exposure and glycosylation changes in lymphatic endothelial cells, including podoplanin modulation, remains poorly defined. Understanding how tumor-derived vesicles and their glycan content influence lymphatic function and glycoprotein regulation is essential for identifying mechanisms that drive metastasis and for developing subtype-specific therapeutic strategies.

Materials and

Methods: : Four cell lines were used in this study: two triple-negative breast cancer lines (MDA-MB-231 and MDA-MB-468), one estrogen receptor–positive line (T47D), and one non-cancerous breast epithelial control line (MCF10A). Cells were seeded at a density of 10,000 cells/cm² and cultured under standard conditions (37°C, 5% CO₂). After 42 hours of incubation to allow for attachment and growth, cultures were rinsed with Dulbecco's phosphate-buffered saline (DPBS) and switched to serum-free media. After 6 hours in serum-free conditions, conditioned media containing microvesicles was collected. To isolate MVs, the media was first centrifuged twice at 600 × g for 5 minutes to remove cellular debris, then filtered through a 0.45 µm filter to eliminate larger particles. Nanoparticle Tracking Analysis (NTA) was performed using a NanoSight NS300 to assess the size distribution and concentration of the extracellular vesicles. Simultaneously, cells were detached and counted to calculate MV production rates.
To assess surface O-linked glycosylation, we performed live-cell lectin staining followed by flow cytometry. Cells were incubated with Vicia villosa agglutinin (VVA), Peanut agglutinin (PNA), and Sambucus nigra agglutinin (SNA) to detect the Tn antigen, T antigen, and sialylated O-glycans, respectively. Lectin binding was quantified to compare glycan expression patterns across breast cancer subtypes and the non-cancerous control. For downstream experiments, human lymphatic endothelial cells were cultured to confluence and then treated with either control media or MV-enriched media for 24 to 72 hours. Following exposure, cells were analyzed by immunohistochemistry to assess podoplanin, glycosylation, sialylation, morphology, and markers of proliferation and adhesion.

Results, Conclusions, and Discussions:: Nanoparticle tracking analysis (NTA) revealed significant extracellular vesicle (EV) size distributions across the breast cancer subtypes and the non-cancerous control indicating that EV size profiles are influenced by cell type. These subtype-specific EV patterns suggest differences in vesicle biogenesis or secretion mechanisms, potentially reflecting the distinct behaviors of each cell line.
Lectin-based glycan profiling further revealed that the triple-negative cell lines expressed high levels of the T antigen, but minimal Tn antigen expression. In contrast, the estrogen receptor–positive T47D cells showed elevated levels of the Tn antigen, indicating a distinct glycosylation signature between subtypes. These differences may contribute to the variation in EV output and structure, as O-linked glycosylation has been shown to influence vesicle biogenesis and membrane composition.
The findings are consistent with prior research showing that EVs from malignant cells differ in size and abundance compared to those from non-cancerous cells. One key driver of these differences is aberrant O-linked glycosylation, which has been linked to both increased vesicle shedding and enhanced cancer cell invasiveness. Thus, glycosylation-dependent regulation of EV production may be an important contributor to the observed size distribution differences.
Together, these findings indicate that both breast cancer subtype and glycosylation profile are associated with differences in EV production and characteristics. Triple-negative cell lines showed high expression of the T antigen and produced smaller, more uniform vesicles, while the estrogen receptor–positive T47D line exhibited greater Tn antigen expression and released a higher concentration of more heterogeneous vesicles. These results support a link between surface glycan expression and EV output, highlighting the potential value of glycosylation profiling in understanding subtype-specific EV behavior.
Current work is focused on investigating how exposure to MV-enriched media over 24–72 modulate facets of lymphatic endothelial cell behavior and function. We are examining alterations in podoplanin expression as an indicator of lymphatic marker shifts, as well as changes in surface glycosylation and sialylation. Functionally, we are assessing effects on proliferative capacity and adhesive behavior. Together, these studies aim to define how breast cancer–derived MVs modulate facets of lymphatic endothelial cell behavior and function.

Acknowledgements and/or References (Optional):: This work was supported by the National Institutes of Health (NIH) and the National Cancer Institute (NCI) under Award Number NIH-NCI P30CA012197