Figure 5.

Efficient EV‐mediated delivery of the immunomodulatory molecule mOx40L showed significant therapeutic impact in murine tumor model in vivo. a) Illustration of the proposed therapeutic short‐term and long‐term dual effect of EV‐mediated delivery of mOx40L protein and mRNA. Co‐loaded mOx40L protein is displayed immediately at the surface of targeted tumor cells, while the delivered mRNA is translated into protein for a sustained co‐stimulatory immunotherapeutic effect leading to an anti‐tumor response. Figure created using BioRender. b) Cargo composition of engineered mOx40L mRNA and protein‐loaded EVs. c) Average particle size determination by NTA of mOx40L‐PUFe mRNA EVs and control EVs with VSVg co‐expression. d) Absolute quantification of mOx40L‐PUFe mRNA molecules per 10⁶ EVs. Data presented as mean ± SD with n = 2; P‐value calculated using the two‐tailed unpaired t‐test; P = 0.05; ns (non‐significant) p > 0.05. e) Concentration of mOx40L protein in mOx40L‐PUFe mRNA and control EVs as measured by murine Ox40L sandwich ELISA. Data presented as mean ± SD with n = 2; P‐value calculated using the two‐tailed unpaired t‐test; p = 0.05; ns (non‐significant) p > 0.05. f) Injection scheme for intratumoral injections of mRNA EVs, control EVs, or suspension buffer (PBS‐HAT) at a dose of 2 ng mRNA per kg bodyweight into B16F10 melanoma‐bearing mice (n = 6 per group). After tumor engraftment, mice were injected 5 times and monitored regularly for tumor growth. Figure created using BioRender. g) Kaplan‐Meier survival analysis of mice treated with mOx40L mRNA EVs, control EVs, or buffer only with assessment of median overall survival (mOS). All curves are significantly different from each other (Log‐rank (Mantel‐Cox) test, P value 0.0003. NR – not registered. h) Tumor volumes measured regularly after the last injection. Each line represents one mouse of the respective group. Four out of six of the mRNA EV‐treated mice and one out of six of the control EV treated mice went into complete remission and lost their tumor beyond palpability for the duration of the experiment (165 days). Curve analysis (Wilcoxon Signed Rank Test): Buffer only group P value (two‐tailed) 0.0312, Control EV group P value (two‐tailed) 0.0002, mRNA EV group P value (two‐tailed) 0.0002, α = 0.05, all curves significant.