FIGURE 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). (b–d) qPCR analysis of RIG‐I encoding mRNA (Ddx58) and its downstream effectors relative to Gapdh in mouse breast cancer 4T1 cells (b), human breast cancer CA1a cells (c) and human lung cancer H358 cells (d) treated with 0.1 μg/μl unloaded RBCEVs, NC‐RNA‐loaded RBCEVs, and immRNA‐loaded RBCEVs for 24 h (n = 4, RNA loaded using REG1). (e) Average luciferase activity in A549‐Dual™ and A549‐Dual™ RIG‐I^−/− cells treated with PBS, 0.05 μg/μl unloaded RBCEVs, NC‐RNA‐loaded RBCEVs and immRNA‐loaded RBCEVs for 24 and 48 h (n = 5–9). (f) Multiplex immunoassay analysis of cytokines in the conditioned media of 4T1 cells treated with 0.1 μg/μl unloaded RBCEVs, NC‐RNA‐loaded RBCEVs and immRNA‐loaded RBCEVs for 48 h (n = 3). (g–i) Flow cytometry analysis showing the average percentage of ANXV⁺PI⁺ population in 4T1 cells (g), CA1a cells (h) and H358 cells (i) after a treatment with 0.1 μg/μl unloaded RBCEVs, NC‐RNA‐loaded RBCEVs and immRNA‐loaded RBCEVs for 72 h (n = 4). All bar graphs represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 determined by Student's two‐tailed t‐test