Large Extracellular Vesicle-Derived Latent MMP-8 and Gelatinolytically Active MMP-2 as Potential Circulating Markers...

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.

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.

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.

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.