Fig 2: scIL-12 and DRIL18 mRNA induce changes in cell-surface glycosylation and generate E-selectin ligands important for efficacy on distant non-injected tumors(A) Heatmap of the Z-scored log2counts/million (CPM) expression of differentially expressed genes with the indicated mRNAs involved in protein glycosylation showing a prominent enhancement of Gcnt3. “BP GLYCOSILATION” term appears as enriched in the GO analysis (adjusted P value = 0.02180856; q-value = 0.01409151).(B) Fluorescent-lectin-binding assays represented as a matrix heatmap for binding to Pmel-1 cells transfected with the indicated mRNAs 48 h prior to the assay. Binding is represented as the fold change over non-electroporated counterparts.(C) Grouping of the lectin-binding assays, according to their primary glycan specificity and the monosaccharide to which they bind. Complex structures indicate N-glycan-linked structures (PHA-L and PHA-E binding).(D) Immunostaining and flow cytometry analyses of Pmel-1 cells electroporated with the indicated mRNA 48 h prior to the assay to assess the percentage of cells stained with the 1B11 mAb that detects CD43 decorated with core 2 O-glycans.(E) Flow cytometry for quantification of a(1,3) fucosylated structures by AAL binding and evaluation of fucosylated glycoepitopes in O-glycans by inhibition with benzyl-a-GalNAc.(F) Comparative E-selectin adhesion assays of IL-12/DRIL18-electroporated cells precultured for 48 h in comparison with the other indicated mRNAs similarly transduced cultured Pmel-1 cells. Comparative results of adhesion in 15-min assays are provided. When indicated, the O-glycosylation inhibitor benzyl-a-GalNAc was added during the 48 h preculture.(G) Representative images of the endpoint of the adhesion assay with IL-12/DRIL18 cells are in green, while the other transduced and untransduced cells are in red.(H) Shear stress adhesion assays under flow of the indicated mRNA-electroporated Pmel-1 cultures on MS1 mouse endothelium cells preactivated with TNF-a (see also Video S1). The number of Pmel-1 cells rolling or arrested on the endothelium are provided.(I) Similar experiments as in (H) but performed on recombinant E-selectin attached to the bottom of the chambers.(J) Length of the tracks of rolling Pmel-1 cells on recombinant E-selectin and endothelial cells in recorded fluorescence microscopy time-lapse videos.(K) Treatment experiments as in Figure 1H were undertaken on B16-OVA bilateral tumor-bearing mice treated with the indicated mRNA-electroporated pmel-1 cells. In the conditions pointed out, benzyl-a-GalNAc was added during 2-h culture before in vivo transfer to inhibit the O-glycan elongation.(L) Flow cytometry quantification of CD90.1+ pmel-1 T cells in the contralateral tumor in experiments in which IL-12/DRIL18 mRNA-electroporated pmel-1 cells were injected into the other contralateral tumor. When indicated, mice were given neutralizing anti-E-selectin mAb.Experiments are representative of at least two repetitions, and one-way ANOVA (D–F and L), two-way ANOVA (K), and Mann–Whitney U (J) tests were used for statistical comparisons. Biological duplicates were performed in experiments (A)–(H). For antitumor efficacy experiments (K and L), we randomly assigned six mice per group. Data are represented as mean ± SD. Con A, Concanavalin A; DBA, Dolichos biflorus lectin; GSL-I, Griffonia simplicifolia lectin I; PHA-E, Phaseolus vulgaris erythroagglutinin; PHA-L, Phaseolus vulgaris leucoagglutinin; PNA, peanut agglutinin; SBA, soybean agglutinin; UEA-I, Ulex europaeus agglutinin I; WGA, wheat germ agglutinin.The significance is represented with asterisks (*) according to the following values: p<0.05 (*), p<0.01(**), p<0.001(***) and p<0.0001(****).