Targeting Reticulin 4 (RTN4) Within Small Extracellular Vesicles Combats Metastasis and Reinforces Immunotherapy in Triple-Negative Breast Cancer

Small extracellular vesicles (sEV) are a class of natural vesicles rich in heterogeneous cargos with the great advantage of non-invasive detection; these vesicles exhibit complex intercellular crosstalk and mediate important biological functions. However, the potential value of plasma sEV in clinical prognosis prediction of triple-negative breast cancer (TNBC) and their biological functions have not been well elucidated. In this study, we isolated sEV from non-metastatic and metastatic TNBC plasma samples. We found that the expression of reticulin 4 (RTN4) in metastatic patients was significantly higher than that in non-metastatic patients. At the same time, clinical data showed that RTN4 was associated with poor prognosis and advanced-stage TNBC patients. Subsequently, in vivo and in vitro assays showed that compared to RTN4^Low sEV, RTN4^high sEV significantly promoted tumour cell migration, invasion, epithelial-mesenchymal transition (EMT) and lung metastasis, and upregulated the expression of PD-L1 in tumour tissues and inhibited CD8⁺T cell infiltration. Regarding mechanism research, we found that RTN4 within sEV drives tumour EMT and PD-L1 expression by activating the NF-κB signalling pathway. Further, through the combined treatment experiment of anti-PD-1 and anti-RTN4, it was found that the combination of the two drugs was significantly superior to monotherapy in inhibiting tumour metastasis, EMT, and promoting CD8⁺T cell infiltration. Our results highlight the molecular mechanism of sEV protein RTN4 in tumour progression and immune system regulation, indicating that RTN4 targeting and anti-PD-1 combined therapy have clinical potential. sEV protein RTN4 is a potential new prognostic marker for non-invasive detection of TNBC and a new target for TNBC treatment.