18:40 - 19:15
Room: Oregon Ballroom
Rapid Fire Abstract Session (Non-CME)
Chair/s:
Barbara Pockaj, Isaac P. Witz
Human lymphatic extracellular vesicles: early modulators of regional lymph node metastasis
Rachel Maus, James Jakub MD, Trace Christensen, Wendy Nevala MS, Svetomir Markovic MD, PhD

Background: Lymph node metastasis remains a valuable prognostic marker for many solid tumor malignancies, including melanoma. Mediating the linear progression of early lymph node metastasis, the lymphatic network enables critical communication and content exchange between the otherwise remote sites of the primary tumor and draining sentinel lymph node (SLN). Previous studies have demonstrated Th2 polarization and increased immunosuppression amenable to tumor growth and progression define SLNs in patients with melanoma. Importantly, this profile precedes clinical evidence of metastasis, suggesting the lymphatic microenvironment is initially altered independent of tumor cells. In the current study, we evaluated the role of human lymphatic-derived extracellular vesicles (EVs) isolated from lymph fluid in modulating the immune profile of regional lymph nodes.

Materials and Methods: Fresh SLN tissue and proximal afferent lymphatic channels were surgically excised from patients with primary cutaneous melanoma undergoing sentinel lymph node biopsy. Control lymph node tissue was obtained from patients undergoing prophylactic mastectomy. Lymph node immune cell profiles were comprehensively phenotyped by mass cytometry. Lymphatic fluid was perfused from the afferent channel for EV isolation, visualized by electron microscopy (EM) and quantified by nanoparticle tracking analysis. EV protein cargo was characterized by mass spectrometry and validated by EM utilizing immunogold staining.

Results: Lymphatic EVs were identified by EM to be present within the lumen of the afferent lymphatic channel (Figure 1) and isolated EVs were quantified ex vivo by nanoparticle tracking analysis. Profiling of EV cargo by mass spectrometry identified a conserved signature of 266 proteins including enrichment for known immune modulators AHNAK, Galectin-1 and Annexin A1. Co-localization of these proteins with vesicle marker CD63 on isolated lymphatic vesicles was confirmed by EM following double immuno-gold labelling.

Conclusions: To our knowledge, this pilot study provides the first examination of human lymphatic fluid as a viable bio-fluid for isolating mediators of early lymph node metastasis. Characterization of lymphatic EVs and their associated cargo is providing valuable insights regarding the mechanisms responsible for mediating the pre-metastatic immune profile of the sentinel lymph node.


Reference:
21-03
Session:
Session 21: Rapid Fire Abstract Session: Tumor Microenvironment and Cancer Progression
Presenter/s:
Rachel Maus
Presentation type:
Rapid Fire Oral Presentation
Room:
Oregon Ballroom
Chair/s:
Barbara Pockaj, Isaac P. Witz
Date:
Friday, April 21, 2017
Time:
18:50 - 18:55
Session times:
18:40 - 19:15