Fig 2: Neutralization of miR-146a-5p, together with pro-inflammatory stimulated macrophages, inhibits tumor growth. (A) RENCA cells (2 × 106 cells) were injected to the flank of BALB/c mice. After tumors became palpable, mice were i.v. injected every 7 days (black arrows), with either anti-miR-146a-5p or its negative control anti-miR-NC (0.025 mg/g BW each), alone or together with injections of RAW 264.7 (106 cells) that were stimulated in vitro with IFN? (100 U/ml) and LPS (1 µg/ml) for 24 h prior to injection. *p < 0.05, ***p < 0.001 relative to the control group, $, p < 0.05 relative to the anti-miR-146a-5p group. Representative images of tissue sections immunohistochemically stained for (B) EMMPRIN protein expression and (C) its evaluation by the h-score, and (D) inducible nitric oxide synthase (iNOS) protein expression and (E) its evaluation by the H-score (n = 6 in the miR-NC+stimulated RAW 264.7 group, and n = 5 in each of the other groups, in two biological replicates).

Fig 3: Translational inhibition of inducible nitric oxide synthase (iNOS) is reversely correlated with miR-146a expression. RENCA, CT26, and TRAMP-C2 tumor cells lines (106 cells), and RAW 264.7 macrophage-like cells (106 cells) were incubated with or without the combined stimulation of IFNγ (100 U/ml) and LPS (1 µg/ml) for 24 h. (A) A representative western blot analysis for iNOS expression and (B) a representative histogram depicting EMMPRIN expression in RENCA cells (light gray, isotype control; blue, no stimulation; hatched yellow, with the combined stimulation). (C) Accumulation of nitrites, the stable product of nitric oxide (NO), reflecting inducible nitric oxide synthase (iNOS) activity; (D) densitometric analysis of western blots for iNOS protein expression; (E) iNOS mRNA accumulation; (F) accumulation of miR-146a-5p expression; (G) mean fluorescence of EMMPRIN protein expression; (H) accumulation of EMMPRIN mRNA (n = 5–6 in each group).

Fig 4: 161-MAP active vaccination reduces EMMPRIN expression in the colon. (A) Representative images of colon sections stained for EMMPRIN and their quantification using the H-score (n = 5 per group). Scale bar is 25 µm (B) Representative image of fluorescently labeled EMMPRIN (red) and macrophages (green). Scale bar is 100 µm. (C) Determination of EMMPRIN concentrations in colon lysates (n = 9–10 per group), and in serum samples (n = 8–9 per group) by ELISA.

Fig 5: 161-MAP active vaccination reduces angiogenesis. (A) Colon sections were stained for CD31 and the vessel surface area was calculated (n = 4 per group). Scale bar is 100 µm. (B) Concentrations of VEGF and MMP-9 were determined in serum sample by ELISA, and in the colon lysates, normalized to the total protein amounts (n = 9 per group). (C) Wound scratch assay: colon lysates (25 µg of total protein) were diluted (1:4) and applied onto a confluent layer of the mouse bEND3 endothelial cells (105 cells/ 96-plate well) that was scratched with a toothpick. Images were acquired at the beginning of the experiment (T0) and at the end after 24h (T24). The migration area was calculated by subtracting the area of the wound at T24, after endothelial cell migrated and partially closed the wound, from the area of the wound at T0. An EMMPRIN specific blocking antibody (161-pAb) was added to some of the wells as indicated. (n = 9–10 for the male mice, n = 8 for the female mice). Magnification is x4.