Tumor-derived small extracellular vesicles loaded with functionally active miR-34a mimic can modify the anti-tumor response in 4T1 breast cancer animal model.

Abstract

Background: A highly detectable problem in Triple-negative breast cancer is reduction of tumor suppressor miRNAs. Thus, this study aimed to use tumor-derived small extracellular vesicles (tsEV) obtained from 4T1 cells as a vehicle for miR-34a-replacement therapy (tsEV-miR-34a-mimic).

Methods: We tested 4T1-tsEVs freely, loaded with miR-34a-mimic or miR-34a-inhibitor on 4T1-bearing mice. Frequency of T cells in spleen and Inguinal lymph nodes assessed by flow cytometry then the relative gene expression of target genes of miR-34a evaluated by Real-Time PCR. In addition, level of cytokine secretion considered by ELISA. Additionally, MTT and AnnexinV/PI methods were used to investigate treatments on 4T1 cell proliferation and apoptosis rate, respectively. Afterwards, histopathological evaluation is applied to determine the extent of metastasis. Ultimately, in each group, mice were followed up on to assess the effect of treatments on survival rate.

Results: Treatment with tsEV-miR-34a-mimic profoundly increased survival and reduced metastasis in 4T1-bearing mice compared to other groups. Besides, the frequency of CD8 T cells was amplified in tumor tissue and inguinal draining lymph nodes (IDLNs). CD4T cell's polarization toward regulatory T cells (Treg) was reduced. Gene expression pattern of tumor tissue showed a change from immune-suppressive to immune-activating tumor-microenvironment (TME). IL-6 and TGF-β concentrations were significantly reduced. IDLN lymphocytes performed a robust killing ability and actively proliferated in response to 4T1-lysate.

Conclusion: Totally, tsEV could be considered as a delivery carrier for miR-34a replacement therapy which also provided an anti-tumor immune response against 4T1- tumor. Thus, this platform could be considered a complementary approach to TNBC therapy.