Characterization and Tracking of Melanoma and Lung Brain Metastasis-Derived Extracellular Vesicles in vitro

Abstract

Brain metastasis (BM) occurs when cancer cells from a primary tumor elsewhere in the body spread to the brain. It poses significant challenges in cancer treatment due to its aggressive nature and limited therapeutic options, with overall survival (OS) is reported to be 6 months or less. Recent research has focused on the role of intercellular communication in metastasis development, aiming to deepen our understanding of this process and develop novel therapeutic approaches. Extracellular vesicles (EVs) derived from primary tumors have emerged as key players in the formation of a pre-metastatic niche (PMN), disrupting the blood-brain barrier (BBB), and stimulating the proliferation of metastatic tumor cells.

A comparative analysis of EV isolation through differential centrifugation and size exclusion chromatography (SEC) was conducted. The findings revealed that SEC outperformed differential centrifugation in several aspects. SEC yielded up to 20 times more EVs compared to differential centrifugation based on EV protein measurements. Additionally, SEC was time-saving and required less conditioned culture medium, making it a more efficient and practical method for EV isolation. EVs derived from melanoma BM cell lines H16 and H10, as well as from the lung BM cell line LBM1, were characterized using SEC. Differential centrifugation was exclusively used for characterizing EVs derived from H16.

Labeling EVs with superparamagnetic iron oxide nanoparticles (SPIONs) facilitates their tracking using magnetic resonance imaging (MRI). By tracking SPION-labeled EVs, MRI provides a non-invasive method for researchers to visualize the distribution and behavior of the labeled EVs within the body. Electroporation was initially attempted for EV labeling but proved unsuccessful. However, successful labeling of LBM1- and H16-derived EVs was achieved through cell incubation with dextran-coated and uncoated SPIONs at concentrations ranging from 50 to 200 µg/mL prior to EV isolation. The findings indicated that uncoated SPIONs were the most optimal for labeling at high concentrations.

Lastly, agar phantoms containing SPION-labeled and unlabeled LBM1 cells were created and subjected to MRI. Different concentrations of labeled and unlabeled cells were imaged, revealing that a concentration of 4000 labeled cells/µL was excessive. Moreover, SPION-labeled EVs derived from LBM1 were effectively visualized in agar phantoms using MRI.