Fig 1: 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(****).
Fig 2: Blood exchange identifies differences in clearance kinetics in healthy mice with either untreated or relapse donors.(a) Decay profiles of untreated and relapse ALL CLCs in healthy recipient mice. (b) Relapse and untreated ALL CLCs have similar kinetics as measured by equilibration time (two-tailed t test p=0.3257) (c) Decay profiles of untreated and relapse AML CLCs in healthy recipient mice. (d) Equilibration time of relapse AML CLCs significantly faster than untreated (two-tailed t test p=0.0230). (e) Schematic of E-selectin adhesion to endothelial cells and E-selectin binding assay using fluorescent E-selectin. (f) E-selectin binding capacity increases in blood and marrow at relapse in AML and ALL (two-tailed t tests: AML Blood p=0.0330, ALL Blood p=0.0443, AML Bone Marrow p=0.0008, ALL Bone Marrow p=0.0112). For decay profiles (a and c), shaded regions are represented by mean +/− standard error, and lines represent the best fit decay curve. For equilibration time analysis (b and d), each dot represents an independent donor-recipient mouse pair.
Fig 3: Interfering with E-selectin binding decreases clearance of relapse AML CLCs.(a) Overview of experiment. Recombinant E-selectin is added to the relapse donor mouse prior to blood exchange with healthy recipient. (b) Decay profiles of relapse CLCs with or without recombinant E-selectin (rEsel) treatment in healthy recipient mice (c, d) CLCs from relapse mice treated with recombinant E-selectin showed increase in both equilibration time (c, two-tailed t test p=0.021) and fraction remaining (d, two tailed t-test p=0.010). For decay profiles (b), shaded regions are represented by mean +/− standard error, and lines represent the best fit decay curve. Each dot represents an independent donor-recipient mouse pair.
Fig 4: Bone marrow microenvironment can impact clearance of CLCs.(a) Overview of experimental plan. Some mice (panels e and f) are dosed with E-selectin antibody prior to blood exchange and postscan (b) Decay profiles of RFP+ CLCs in recipients with varied tumor status. (c) Decreased clearance of CLCs in recipient mice with active disease as measured by an increase in fraction of CLCs remaining in circulation (Tukey multiple comparisons **p<0.01). (d) E-selectin expression on BMECs increases in diseased context (bars represent mean +/− standard deviation; Tukey multiple comparisons ****p<0.0001). (e) Decay profiles of RFP+ CLCs in tumor bearing recipient mice with or without E-selectin antibody treatment (f) Dosing recipient mice with E-selectin antibody allows for increased clearance of CLCs in diseased recipients as measured by decrease in fraction remaining (two-tailed t test p = 0.0254). For decay profiles (b and e), shaded regions are represented by mean +/− standard error, and lines represent the best fit decay curve. For fraction remaining analysis (c and f), each dot represents an independent donor-recipient mouse pair.
Fig 5: Physicochemical and functional characterization of sLeX‐EVs. (a) Size distribution of Nluc and mNG‐labelled EVs. (b) Cryo‐electron microscopy images of Nluc and mNG‐labelled EVs. Scale bar indicates 100 nm. (c) Detection of sLeX on Nluc‐labelled EVs captured by CD63 and CD81‐coated beads. EVs were pulled down using magnetic beads pre‐coated with capture antibodies against an EV marker and detected with sLeX antibodies using flow cytometry. (d) Elution profile of Nluc‐labelled EVs in size exclusion chromatography. Nluc in each fraction was normalized to total input. (e) Binding of Nluc‐labelled EVs to recombinant E‐selectin. Microplates were coated with E‐selectin and incubated with EVs. The amount of bound EVs was presented as fold‐change over vehicle‐coated plates. Data are shown as mean ± standard deviation. CN: CD63‐Nluc; F7CPN: FUT7 + CD63‐P19‐Nluc; F7CCN: FUT7+ CD63‐CTP‐Nluc; CM: CD63‐mNG; F7CPM: FUT7 + CD63‐P19‐mNG; F7CCM: FUT7 + CD63‐CTP‐ mNG. Two‐tailed unpaired t‐test. n.s.: non‐significant; ***p < 0.001
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