Large Extracellular Vesicle-Derived Latent MMP-8 and Gelatinolytically Active MMP-2 as Potential Circulating Markers for Lymph Node Metastasis in Breast Cancer

Background: Lymph node metastasis (LNM) is a determinant of prognosis and in guiding chemotherapy decisions in breast cancer. Herein, we aimed to discover the protease content of the circulating large extracellular vesicles (L-EVs) as potential markers for LNM. Methods: L-EVs were isolated from the plasma of chemotherapy-naïve breast cancer patients with negative LNM (nLNM, n = 40) and positive LNM (pLNM, n = 32) patients using differential centrifugation. The isolated L-EVs were characterized by Transmission Electron Microscopy, dynamic light scattering and EV marker profiling, and their protease content was profiled using unbiased proteome profiler human protease array. Results: Protease profiling uncovered that L-EVs contained significantly elevated levels of MMP-8 (p < 0.001) and MMP-9 (p < 0.05) in pLNM versus nLNM patients. Further validation by Western blotting confirmed that latent MMP-8 was significantly increased (p < 0.01) in L-EVs from pLNM patients. Interestingly, zymography revealed that L-EVs isolated from pLNM patients contained higher levels of latent MMP-9 compared with those from nLNM patients, whereas gelatinolytically active MMP-2 was only detected in L-EVs from pLNM patients and not in those from nLNM. Online datasets revealed that higher MMP-8 and MMP-9 mRNA levels were associated with poorer overall, relapse-free, and distant metastasis-free survival. Receiver operating characteristic (ROC) plotter analyses indicated that MMP-2 and MMP-8 may serve as predictive biomarkers for response to specific chemotherapeutic regimens. Conclusions: These findings highlight the potential clinical utility of L-EV-derived MMP-2, MMP-8, and MMP-9 expressions and/or activities, as non-invasive blood-based markers associated with nodal progression and therapeutic response.