Fig. 2: Role of MyD88, TRIF and NO in tumor cell infiltration. | Oncogene

Fig. 2: Role of MyD88, TRIF and NO in tumor cell infiltration.

From: Intralesional TLR4 agonist treatment strengthens the organ defense against colonizing cancer cells in the brain

Fig. 2

A Organotypic brain-slice ex vivo co-culture with MCF-7 tumor cells and WT brain slices stimulated with LPS (1 µg/ml), Pam3CSK4 (1 µg/ml), recombinant IFNβ (100 ng/ml), or Poly(A:U) (1 µg/ml). Data represent the percentage degree of tumor-cell infiltration into the brain slices after 96 h (n ≥ 12; ***p < 0.001; Mann-Whitney test). B MCF-7 tumor-cell invasion after PBS (CTRL) or LPS (1 µg/ml) stimulation. Data represent the average tumor cell number migrated per high power field (HPF) after 96 hours (n ≥ 10; mean ± SD; n.s. = not significant; unpaired t-test). C Organotypic brain-slice ex vivo co-culture with MCF-7 tumor cells and brain slices from TRIF-/- mice after PBS (CTRL) or LPS (1 µg/ml) stimulation. Data represent the percentage degree of tumor-cell infiltration into the brain slices after 96 h (n ≥ 24; Mann-Whitney test). D Metabolic activity of the indicated cell types after stimulation with PBS (CTRL) or LPS (1 µg/ml) assessed by means of MTT conversion assay (n ≥ 8; mean ± SD; ***p < 0.001; one-way ANOVA followed by Sidak´s multiple comparisons test). E NO release as a function of nitrite accumulation in supernatants of WT MG after stimulation with L-NAME (1 mM), Pam3CSK4 (10 ng/ml), recombinant IFNβ (10 ng/ml), Poly(A:U) (1 µg/ml), and LPS (100 ng/ml) (n ≥ 6; mean ± SEM; ***p ≤ 0.001; unpaired t-test). F NO release as a function of nitrite accumulation in supernatants of WT, MyD88−/−, and TRIF−/− MG after LPS (100 ng/ml) stimulation. (F1) NO release is indicated as the percentage of nitrite accumulated compared to the control (CTRL; n ≥ 5; mean ± SEM; ****p < 0.0001; one-way ANOVA followed by Sidak’s multiple comparisons). (F2) Representative confocal images depicting iNos induction after LPS stimulation. G Quantitative RT-PCR analysis of Nos2 expression in wild-type (WT) or MyD88−/− MG after LPS (100 ng/ml) stimulation. Data represent the ΔCt value between the gene of interest (GOI = Nos2) and two housekeeping (HK) genes (n ≥ 4; mean; ****p < 0.0001; one-way ANOVA followed by Sidak’s multiple comparisons). H Organotypic brain-slice ex vivo co-culture with MCF-7 tumor cells and stimulated with 8-Br-cGMP (10 µM) or L-NAME (100 µM). Data represent the percentage degree of tumor-cell infiltration into the brain slices after 96 hours (n ≥ 16; **p < 0.01, ***p < 0.001; Mann-Whitney test).

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