Artemisia annua-derived extracellular vesicles reprogram breast tumor immune microenvironment via altering macrophage polarization and synergizing recruitment of T lymphocytes.

Abstract

Background: The immunosuppressive microenvironment and limited immune cell infiltration into the tumor bed contribute to the proliferation, metastasis, and invasion of breast cancer (BC) cells. Reprogramming the tumor immune microenvironment has emerged as a promising therapeutic target for BC, but remains challenging in clinical practice. Artemisia annua, a medicinal plant, has shown immune-enhancing and anti-tumor activities, although its potential therapeutic applications in BC remain underexplored.

Methods: Extracellular vesicles (EVs) were isolated from fresh Artemisia annua using gradient centrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Zetasizer Nano ZS90, ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS), and high-performance liquid chromatography (HPLC). Single-cell RNA sequencing (scRNA-seq) analysis was performed to investigate the mechanism of AEVs on tumor growth in vivo and mRNA sequencing (mRNA-seq) was employed to further explore the mechanism of AEVs-induced polarization shift from M2-like to M1-like macrophages. In vitro and in vivo assays were conducted to assess the polarization of macrophages and recruitment of T lymphocytes into the tumor bed.

Results: AEVs were successfully isolated and characterized. In vivo, AEVs inhibited tumor growth by shifting macrophage polarization from the M2-like to the M1-like phenotype, and synergistically enhancing the recruitment of CD8⁺ and CD4⁺ T cells into the tumor microenvironment. AEVs activated the NF-κB signaling pathway while inhibiting the PPARγ pathway, thereby promoting the M1-like polarization of macrophages. Polarized M1-like macrophages secreted chemokines (CCL5 and CCL3), which facilitated T lymphocyte infiltration into the tumor bed. Notably, AEVs reshaped the BC immune microenvironment without inducing systemic toxicity.

Conclusions: AEVs from Artemisia annua efficiently reprogrammed the immune microenvironment of breast cancer by inducing macrophage polarization and enhancing T lymphocyte infiltration. This study lays the foundation for using AEVs as a potential immunotherapy for BC and highlights the medicinal value of Artemisia annua in cancer treatment.