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. 2021 Dec 2;16(12):e0259301. doi: 10.1371/journal.pone.0259301

Fig 5. Self-replicating mRNA encoding mIL12 provides tumor control and immune cell infiltration.

Fig 5

(A) Self-replicating messenger RNA (srRNA) includes machinery that allows for amplification of the transgene in the cytoplasm of transfected cells. (B) Mice inoculated with subcutaneous B16 melanoma tumors in their right flanks were treated with a single shot of polyethyleneimine-formulated-srRNA expressing mIL12 (srRNA/mIL12) on Day 7 and whole blood was collected retro-orbitally 3 days later. Serum was isolated and analyzed for IL12p70 and IFNγ (n = 4–8). Mice were inoculated subcutaneously with (C, D) B16 melanoma tumors (n = 9) or (E, F) CT26 colon carcinoma tumors in their right flanks and given weekly shots of polyethyleneimine-formulated-srRNA/mIL12 intratumorally (n = 8) (D & F, *p < 0.0001, representative data from 3 independent experiments). (G) Mice were inoculated orthotopically with 4T1 mammary carcinoma tumors in their mammary fat pads and given weekly shots of polyethyleneimine-formulated-srRNA/mIL12 either intratumorally or subcutaneously at their tail base. (H) Mice challenged with orthotopic 4T1 mammary carcinomas then treated with weekly injections of formulated srRNA/mIL12 either intratumorally or subcutaneously were sacrificed 18 days after tumor inoculation to evaluate metastatic lung nodule formation (IT, p < 0.002; SC, p < 0.0001; n = 8–10; representative data from 2 independent experiments). (I) Mice were inoculated subcutaneously with B16 melanoma tumors in their right flanks and then received weekly shots of polyethyleneimine-formulated-srRNA/mIL12. Tumors were isolated 25 days after inoculation and infiltrating immune cells were isolated and phenotyped by flow cytometry analysis (n = 5). Data points and error bars in B and H represent mean and standard error of the mean, respectively. Student’s T test was used to determine statistical significance with a 95% confidence interval. Tumor growth plots represent individual mice. Survival benefit was determined using Mantel-Cox Log-rank testing where p < 0.05 is considered significant. Numbers in each square represent the percentage stained cells of live cells found in tumor infiltrating lymphocyte population.