Fig 2: Identification, localization, and function of OX40L-mRNA protein. (A) Effect of OX40L mRNA-LNP according to RT-qPCR (B) Western blotting analysis of protein expression from the optimized and original sequences in HEK293T cells. (C) Western blotting analysis of optimized OX40L mRNA transfected into HCC cell lines. (D) Flow cytometry analysis of transfection efficiency in HEK293T cells at 24 h and 48 h. (E) Quantitative analysis of (C). (F) Immunofluorescence images of transfected HEK293T cells (scale bars: 100μm). (G) ELISA of the supernatant IL2 levels in cocultures of lymphocytes with OX40L mRNA transfected Hep3B cells. Statistical significance was calculated via t test. **P < 0.01; ***P < 0.001.

Fig 3: In vivo efficacy of intratumoral therapy with OX40L mRNA. (A) Timeline for treatment of H22 tumor-bearing mice. (B) Kaplan-Meier analysis of mouse survival after treatment with OX40L-mRNA and non-coding mRNA; log-rank test, p =0.0183. (C) Growth of subcutaneous tumors growth curves (median ± interquartile range [IQR]) of each group treated with OX40L-mRNA and the control. Two-way repeated-measures ANOVA using the Sidak multiple-comparisons test, **p≤ 0.01, ***p≤ 0.001, compared to non-coding mRNA. (D) Images of mice from the two groups at day 37. (E) Tumors; white circle, no tumor. (F) Tumor volume at day 37. Statistical significance was calculated via t test. ***p ≤ 0.001. (G) Tumor weight at day 37. Statistical significance was calculated via t test. ***p≤0.001.

Fig 4: OX40L mRNA elicited lymphocyte activation in the H22 tumor bearing mice. (A, C) T-cell activation by flow cytometry. CD4+ T cells and CD8+ T cells in the spleen of the H22 tumor-bearing BALB/c mice. (B, D) Quantitative analysis of the proportions in (A, C). Statistical significance was calculated via t test. **P ≤ 0.01, ***P ≤ 0.001. (E, F) Representative FACS profiles of CD69+ CD8+ and CD69+ CD4+ activated T cells. (G) Results of quantitative analysis of the proportions in (E, F). Statistical significance was calculated via t test. *P ≤0.05. (H) Immunohistochemistry of the mechanisms of action of OX40L mRNA in subcutaneous H22 tumors (scale bars: 100μm).

Fig 5: Organ toxicity of OX40L mRNA-LNP. Mice were euthanized at the end of treatment and their organs were removed. Heart, liver, spleen, lung, kidney and brain sections were stained with H&E to evaluate organ toxicity (scale bars: 100μm).