Small RNA Delivery by Extracellular Vesicles in Cancer Signaling and Cancer Treatments

細胞外囊泡遞送小核糖核酸在癌症信號通路研究和癌症治療中的應用

Student thesis: Doctoral Thesis

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Award date21 Aug 2020

Abstract

Background: Extracellular vesicles (EVs) are natural RNA carriers that act as messengers of the intercellular communication and delivery vehicles for gene therapies. Bioactive RNAs in tumour EVs are transferred to stromal cells to influence the tumor microenvironment. On the other hand, EVs from primary cells such as red blood cells (RBCEVs) are ideal delivery vehicles due to their low toxicity, low immunogenicity and high biocompatibility. We have shown that RBCEVs can deliver therapeutic RNAs to cancer cells for effective treatments.

Methods: EVs were purified from either conditioned medium of cancer cell culture or donated blood samples using ultracentrifugation with sucrose cushion. The purified EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blot. Breast cancer cell derived EVs were electroporated with miR-125b antisense nucleotides (ASOs) or mimics for miRNA inhibition or overexpression. RBCEVs were transfected with immunomodulatory RNAs (immRNAs) for immune response activation in breast cancer cells in vitro and xenograft mouse models.

Results: i) Treatment with EVs from mouse breast cancer 4T1 cells leads to an increase in fibroblast activation in isogenic 4TO7 tumours, which is reversed by blocking miR-125b in 4T1 EVs. Uptake of EVs from human breast cancer CA1a cells significantly increases cellular levels of miR-125b and expression of multiple cancer-associated fibroblast markers in resident fibroblasts. Overexpression of miR-125b in both mouse and human fibroblasts leads to an activated phenotype similar to the knockdown of established miR-125b target mRNAs. ii) Delivery of immRNAs to 4T1 and CA1a cells using RBCEVs significantly increases cell apoptosis and up-regulates effectors of the RIG-I pathway. Treatment with immRNAs using RBCEVs suppresses 4T1 and CA1a tumour growth by activating the RIG-I cascade and increasing infiltration of T cells, dendritic cells, macrophages and neutrophils in the tumours.

Conclusion: i) miR-125b is transferred through EVs from breast cancer cells to normal fibroblasts within the tumour microenvironment and contributes to their development into cancer-associated fibroblasts. ii) immRNAs delivered by RBCEVs can be used for activation of anti-cancer immune responses. This study has shed new light into the mechanism of intercellular signaling in cancer and facilitated the development of new therapies against cancer.