Neuroblastoma-derived Extracellular Vesicles Disrupt the Integrity of Central Nervous System Barriers via Tight Junction Modulation

Background

Neuroblastoma (NB) is a pediatric malignancy that predominantly affects young children and can be associated with neurological complications even in the absence of direct central nervous system (CNS) invasion. The CNS is protected by specialized endothelial barriers whose integrity depends on tightly regulated intercellular junctions and extracellular matrix homeostasis. Here, we investigated whether extracellular vesicles (EVs) released by NB cells contribute to CNS endothelial barrier dysfunction and examined the underlying molecular mechanisms.

Methods

EVs were isolated from early- and late-differentiated murine NB cells and characterized by nanoparticle tracking analysis and transmission electron microscopy. EV protein cargo was analyzed by mass spectrometry-based proteomics. The effects of NB-derived EVs on CNS endothelial barrier function were evaluated using brain microvascular endothelial cell monolayers by measuring trans-endothelial electrical resistance and paracellular permeability. EV-associated proteolytic activity was assessed using gelatinase activity assays and zymography, and the contribution of matrix metalloproteinases (MMPs) was examined using pharmacological inhibition. Statistical analyses included two-tailed Student’s t -tests and two-way analysis of variance with appropriate corrections and multiple-comparison testing.

Results

NB-derived EVs impaired CNS endothelial barrier integrity, decreasing electrical resistance and increasing paracellular permeability. In contrast, EVs derived from non-tumor astrocytes and fibroblasts supported endothelial barrier resistance. Proteomic analysis identified proteins involved in cell junction organization and extracellular matrix remodeling within NB-derived EVs, including MMP-2 and MMP-9. Functional assays confirmed that NB-derived EVs possess gelatinase activity. Pharmacological inhibition of MMP activity substantially attenuated EV-induced barrier disruption and preserved endothelial junction integrity, supporting a major role for EV-associated proteolytic activity in the observed phenotype.

Conclusions

Our findings identify NB-derived EVs as mediators of CNS endothelial barrier dysfunction and link their barrier-disruptive effects to EV-associated metalloproteinase activity. These results reveal a potential mechanism of tumor–CNS communication and suggest that targeting EV-associated proteolytic activity may represent a strategy for preserving CNS barrier integrity in neuroblastoma.

Plain English summary

Neuroblastoma is a childhood cancer that develops from immature nerve cells. Although it rarely spreads directly to the brain, children with neuroblastoma can experience neurological complications. How neuroblastoma cells communicate with and affect the central nervous system is still not fully understood.

Cells release tiny membrane-bound particles that carry biological material and allow them to communicate with other cells. Cancer cells can use these particles to influence tissues far from the original tumor. In this study, we investigated whether particles released by neuroblastoma cells can affect the protective cellular barriers surrounding the central nervous system.

We found that particles released by neuroblastoma cells weakened the connections between the cells that form these barriers, making the barrier less effective. In contrast, particles released by non-cancerous supporting cells did not cause this damaging effect. We also found that neuroblastoma-derived particles carry active protein-degrading enzymes called matrix metalloproteinases. Blocking the activity of these enzymes substantially protected the barrier from the effects of the cancer-derived particles.

Our findings suggest that neuroblastoma cells may influence the central nervous system without directly entering it, by releasing particles that travel between cells and weaken its protective barriers. Understanding this form of communication may help explain some neurological complications associated with neuroblastoma and could eventually identify new ways to protect the central nervous system from cancer-related damage.