Nature 515, 563C567. 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. and in CA1a cells treated with different doses of RBCEVs made up of miR\125b ASO ((the gene encoding RIG\I) and and significantly (Physique?2c). Similarly, human TNBC MDA\MB\468 cells were highly responsive to immRNA\loaded RBCEVs as shown by substantial up\regulation of (Physique S2A). However, immRNA\loaded RBCEVs did not induce RIG\I activation in human TNBC MDA\MB\231 cells, which have very low RIG\I expression according to Biswas et?al. (2012) and our own data (Physique S2B). Open in a separate window Physique 2 RBCEVs deliver immunomodulatory RNA to activate the RIG\I pathway and induce immunogenic cell death in cancer cells. (a) The design of immRNA with 5 triphosphate (ppp). (bCd) qPCR analysis of RIG\I encoding mRNA (in mouse breast cancer 4T1 cells (b), Dutogliptin human breast cancer CA1a cells (c) and human lung cancer H358 cells (d) treated with 0.1?g/l unloaded RBCEVs, NC\RNA\loaded RBCEVs, and immRNA\loaded RBCEVs for 24?h (and but not other RIG\I\related genes in the cells (Physique S2C). We also generated mouse lung epithelial (mLE) cells using a previously established protocol (Kasinski & Slack, 2013). Surface marker EpCAM of the cells was determined by flow cytometry (Physique S1C). The uptake of immRNA\loaded RBCEVs by lung epithelial cells led to significant up\regulation of was not detectable in the cells, whereas were up\regulated significantly (Physique S2D). We further assessed the type\I\IFNs\producing activity induced by immRNA. We incubated immRNA\loaded RBCEVs with human lung epithelial carcinoma A549\Dual? and A549\Dual? RIG\I?/? cells. These two cell lines secrete the interferon regulatory factor (IRF) pathway mediated Lucia luciferase under the control of an ISG54 minimal promoter in conjunction with five IFN\stimulated response elements. As a result, the wild\type (WT) A549\Dual? cells incubated with immRNA\RBCEVs were responsive to this RIG\I activator as shown by the increased luciferase activity after 24 and 48?h (Physique?2e). Removal of the RIG\I gene from the cells resulted in complete abrogation of IFN signalling in response to immRNA (Physique?2e), confirming RIG\I as the primary immRNA sensor in the cells. To evaluate the effect of immRNA around the inflammatory Dutogliptin response, we measured the concentrations of key inflammatory mediators in 4T1\conditioned media. As expected, 4T1 cells treated with immRNA\loaded RBCEVs secreted substantially higher levels of IFN (55.95 pg/ml) and IFN (1844.33 pg/ml) (Figure?2f). Furthermore, immRNA\RBCEVs taken up by 4T1 cells also induced the secretion of proinflammatory cytokines TNF (10.91 pg/ml) and IL\6 (793.22 pg/ml) (Physique?2f). The induction of immunogenic cell death in cancer cells was subsequently quantified using Annexin V (ANXV) and Propidium Iodide (PI) staining for each EV treatment. After 72?h of incubation, immRNA\loaded RBCEVs induced apoptosis of 4T1 cells (15.9%) (Figures?2g and S2E), CA1a cells (24.6%) (Figures?2h and S2E), H358 cells (17.6%) (Figures?2i and S2E), and MDA\MB\468 cells (16.0%) (Physique S2E\F) as shown by the average percentage of ANXV+PI+ population. ImmRNA\loaded RBCEVs failed to induce cell death in RIG\I\low MDA\MB\231 cells (Physique S2E\F), indicating that RIG\I is an essential receptor in response to immRNA. MCF10A cells and mLE cells did not succumb to apoptosis after the incubation with immRNA\loaded RBCEVs (Physique S2E\F). These data suggest that immRNA\loaded RBCEVs activate the cellular RIG\I pathway, triggering type I IFN secretion and inducing cell death in cancer cells, while concurrently sparing non\malignant cells. 2.3. RBCEVs deliver bi\functional ASOs to simultaneously inhibit oncogenic miR\125b and activate the RIG\I pathway leading to cell death in cancer cells After confirming that 5 triphosphate immRNA could effectively trigger RIG\I activation, we sought to implement a combinatorial Dutogliptin approach that incorporates Rabbit Polyclonal to TBL2 ASO\mediated oncogene silencing and RIG\I\mediated immune activation simultaneously. Previous studies on miR\125b uncovered its important role in oncogenesis (B. Peng et?al., 2021). It has been reported to promote.