Macrophages are potent immune cells with well-established roles in the response to stress, injury, infection and inflammation. The classically activated macrophages (M1) are induced by lipopolysaccharide (LPS) and express a wide range of pro-inflammatory genes. M2 macrophages are induced by T helper type 2 cytokines such as interleukin-4 (IL4) and express high levels of anti-inflammatory and tissue repair genes. The strong association between macrophages and tumour cells as well as the high incidences of leukocyte infiltration in solid tumours have contributed to the discovery that tumour-associated macrophages (TAMs) are key to tumour progression. Here, we investigated the role of Annexin A1 (ANXA1), a well characterized immunomodulatory protein on macrophage polarization and the interaction between macrophages and breast cancer cells. Our results demonstrate that ANXA1 regulates macrophage polarization and activation. ANXA1 can act dually as an endogenous signalling molecule or as a secreted mediator which acts via its receptor, FPR2, to promote macrophage polarization. Furthermore, ANXA1 deficient mice exhibit reduced tumour growth and enhanced survival in vivo, possibly due to increased M1 macrophages within the tumor microenvironment. These results provide new insights into the molecular mechanisms of macrophage polarization with therapeutic potential to suppress breast cancer growth and metastasis.

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The RIG‐I pathway can be activated by RNA containing 5′ triphosphate, leading to type I interferon release and immune activation. Hence, RIG‐I agonists have been used to induce immune responses against cancer as potential immunotherapy. However, delivery of 5′ triphosphorylated RNA molecules as RIG‐I agonists to tumour cells in vivo is challenging due to the susceptibility of these molecules to degradation. In this study, we demonstrate the use of extracellular vesicles (EVs) from red blood cells (RBCs), which are highly amenable for RNA loading and taken up robustly by cancer cells, for RIG‐I agonist delivery. We evaluate the anti‐cancer activity of two novel RIG‐I agonists, the immunomodulatory RNA (immRNA) with a unique secondary structure for efficient RIG‐I activation, and a 5′ triphosphorylated antisense oligonucleotide with dual function of RIG‐I activation and miR‐125b inhibition (3p‐125b‐ASO). We find that RBCEV‐delivered immRNA and 3p‐125b‐ASO trigger the RIG‐I pathway, and induce cell death in both mouse and human breast cancer cells. Furthermore, we observe a significant suppression of tumour growth coupled with increased immune cell infiltration mediated by the activation of RIG‐I cascade after multiple intratumoral injections of RBCEVs loaded with immRNA or 3p‐125b‐ASO. Targeted delivery of immRNA using RBCEVs with EGFR‐binding nanobody administrated via intrapulmonary delivery facilitates the accumulation of RBCEVs in metastatic cancer cells, leading to potent tumour‐specific CD8 + T cells immune response. This contributes to prominent suppression of breast cancer metastasis in the lung. Hence, this study provides a new strategy for efficient RIG‐I agonist delivery using RBCEVs for immunotherapy against cancer and cancer metastasis.

Annexin 1 (ANXA1) is an endogenous anti-inflammatory protein implicated in cancer. ANXA1 was previously shown to be regulated by hsa-miR-196a. However, whether ANXA1 itself regulates microRNA (miR) expression is unknown. Therefore, we investigated the regulation of miR by ANXA1 in MCF7 breast cancer cells. MCF7-EV (Empty vector) and MCF7-V5 (ANXA1-V5 expressing cells) were subjected to a miR microarray. Microarray analysis revealed a number of miRNAs which were dysregulated in MCF7-V5 cells. 2 novel miRNAs (miR562 and miR26b*) were validated, cloned and functionally characterized. As ANXA1 constitutively activates NF-κB activity to modulate breast cancer metastasis, we found that miR26b* and miR562 directly targeted the canonical NF-κB pathway by targeting the 3′ UTR and inhibiting expression of Rel A (p65) and NF-κB1 (p105) respectively. MiR562 inhibited wound healing, which was reversed when ANXA1 was overexpressed. Overexpression of either miR562 or miR26b* in MCF-7 cells enhanced endothelial tube formation when cocultured with human umbilical cord endothelial cells while conversely, treatment of MCF7 cells with either anti-miR562 or anti-miR26b* inhibited endothelial tube formation after co-culture. Further analysis of miR562 revealed that miR562-transfected cell conditioned media enhances endothelial cell tube formation, indicating that miR562 increased angiogenic secreted factors from MCF-7 breast tumor cells. TNFα was increased upon overexpression of miR562, which was reversed when ANXA1 was co-transfected In conclusion, this data suggests that ANXA1-regulated miR26b* and miR562 may play a role in wound healing and tumor-induced endothelial cell tube formation by targeting NF-κB expression and point towards a potential therapeutic target for breast cancer.