Fig 1: EcN mi-IL2 slows down the growth of CT26 tumors. CB6F1 mice harboring CT26 tumors on both flanks were treated with PBS or 1 × 108 CFU of bacteria intravenously. Mice were sacrificed when tumors reached 2000 mm3. (A) Study design. Figure made using biorender under the agreement Number YB25KBCY7U. (B) CT26 tumor volumes of all mice treated with PBS (n = 12), EcN Ctrl (n = 6) and EcN mi-IL-2 (n = 8). Mean tumor volume with SEM is shown. Significance determined with ANOVA and post-hoc analysis of comparison between groups using Tukey’s Honest Significant Difference test: (ns) p > 0.05, *p = 0.05. (C) Longitudinal analysis of growth rate of tumors using a linear regression model. The 95% confidence interval of the coefficient estimates are shown. The interpretation of the model output is the change in rate of growth of tumors when compared to PBS group. (D) IL-2 levels in colonized tumors with mean and SEM shown.
Fig 2: Optimization of signal peptide and solubility tag leads to higher cytokine production and activity in the supernatant. (A) Different signal peptides were cloned upstream of the IL-2-His6 gene. Bacterial supernatants were collected and IL-2 levels in the supernatant were measured using an IL-2 ELISA kit (indicated in red). Activity of IL-2 in the supernatants was measured using the CTLL-2 activity assay. RFUs normalized to non-expressing bacteria are shown (indicated in blue). (B) The InfB solubility tag was cloned to the N or C-terminus of the cytokine. Supernatants were collected and IL-2 signal was measured by ELISA (indicated in red). Activity of IL-2 in the supernatants was measured using a CTLL-2 activity assay. RFUs normalized to non-expressing bacteria are shown (indicated in blue). Values from three biological replicates with standard deviations are shown. *The LamB strain with C-terminal InfB tag was chosen for further experiments. RFU relative fluorescent unit, RBS ribosome binding site, Sig peptide signal peptide, His6 hexahistidine tag.
Fig 3: Gene expression associated with immune cell-mediated cytotoxicity is induced by the supernatant from mi-IL2. (A) Cytotoxicity was measured from co-cultures exposed to purified IL-2 or supernatant from mi-IL2 using Cytotox Green and the Incucyte imaging system. Three spheroids were analyzed for each condition. Supernatant from non-expressing bacteria were used as negative control (Neg.Ctrl). (B) Representative images of spheroid integrity after 3 days of co-culture. (C) RT-qPCR analysis of IL-2 response genes from tumor spheroid and PBMC co-culture. Gene expression was normalized to GAPDH expression. Mean values of relative gene expression and S.E.M. from three independent experiments are shown. qPCR primer sequences are listed in Table 3. (D) IFN? levels from the tumor spheroid and PBMC co-cultures. Protein levels were measured by ELISA. Mean values of protein levels and S.E.M. from three independent experiments are shown. Significance determined with ANOVA and post-hoc analysis of comparison between groups using Tukey’s Honest Significant Difference test: (ns) p > 0.05, *p = 0.05, **p = 0.01, ***p = 0.001.
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