Fig 1: Restoration of miR-200c in EO771 alters expression of MHC class I SNV-derived neoantigens and putative SNV-derived peptides upregulated in mesenchymal-like EO771 cells elicit superior cytotoxic responses against epithelial-like EO771-miR-200c+ cells. (A) Overview of study design vaccinating mice with pooled MHC class I peptides followed by ex vivo T cell assays. (B) Quantification of putative peptides resulting from somatic variants per H-2Db/H-2Kb allele with strong binding affinity predictions (<500 nM) is shown. The gray bar fraction corresponds to the number of peptides with significant change in expression ± miR-200c. (C) Flow cytometry analysis depicting CD8+ T cell proliferation in response to stimulation with individual MHC class I SNV-derived peptides. The gray peaks represent the dilution of cell trace violet (CTV) stain as CD8+ T cells proliferate in response to specific peptide. The black peak represents CTV stain in non-proliferating CD8+ T cells treated with negative control (no peptide). (D) Quantification of percent cytotoxicity and (E) IFNγ measurements from splenocytes of mice vaccinated with pooled peptides expressed more in the mesenchymal conditions and challenged with EO771-B7.1 ± miR-200c target cells ex vivo at the respective Effector: Target cell ratio (E:T). A two-way ANOVA was performed using Sidak’s multiple comparisons and found significant differences in cytotoxicity against EO771-B7.1-miR-200c target cells at both E:T ratios (p < 0.0001). The numerical p values listed in the figure legend correlate with the asterisks in the figure.
Fig 2: Following miR-200c restoration, enhanced cytotoxicity against EO771 target cells is associated with MHC class I expression. (A) Overview of study design. (B) Quantification of percent cytotoxicity and (C) IFNγ measurements from splenocytes of mice against EO771-B7.1 ± miR-200c target cells following vaccination with EO771 WCV ± miR-200c. Cells were co-cultured at E:T ratio of 20:1. Target cells are shown in the figure legend. A two-way ANOVA was performed using Sidak’s multiple comparisons test, finding a significant increase in cytotoxicity against EO771-B7.1-miR-200c+ target cells in both the EO771 WCV (p = 0.001) and EO771-miR-200c WCV (p = 0.0012) groups. Data are representative of three independent experiments and are shown as mean ± SD. (D) Quantification of geometric mean of Zeb1 (n = 5) and (E) MHC class I (n = 5) on EO771 cells transfected with miR-200c for 72 h by flow cytometric analysis is shown. An unpaired t test shows a significant difference in Zeb1 expression (p < 0.0001) and MHC class I expression (p < 0.024) following miR-200c transfection. Data are representative of three independent experiments and are shown as mean ±SD. (F) Tumor growth curve of EO771 (mesenchymal-like) cells in mice treated with adjuvant only (n = 20), EO771 WCV (mesenchymal-like) (n = 16) or EO771-miR-200c treated WCV (epithelial-like) (n = 20). Error bars depict mean with SEM. A two-way ANOVA was performed using Tukey’s multiple comparisons test and significant protection was provided by the EO771 WCV (p < 0.0001) and EO771-miR-200c WCV (p < 0.0001). (G) Survival curve of the same mice as in f; log rank (Mantel-Cox) test = 0.0035. (H) Representative histograms of change in Zeb1 expression in EO771 tumors at day 50 in mice that received adjuvant, EO771 WCV, or EO771-miR-200c vaccine relative to baseline Zeb1 expression in EO771 cells ± miR-200c. The numerical p values listed in the figure legend correlate with the asterisks in the figure.
Supplier Page from Sino Biological, Inc. for Mouse CD80/B7-1 Gene ORF cDNA clone in cloning vector