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Fig. 2 | Cancer Cell International

Fig. 2

From: AGI-134: a fully synthetic α-Gal glycolipid that converts tumors into in situ autologous vaccines, induces anti-tumor immunity and is synergistic with an anti-PD-1 antibody in mouse melanoma models

Fig. 2

AGI-134-treated cells are phagocytosed by antigen-presenting cells and antigen cross-presented. a CFSE-labeled A549 cells were treated with PBS or 500 μg/mL AGI-134 and then incubated with or without normal human serum (NHS) to opsonize them with anti-Gal and complement. Subsequently, human macrophages were added at a A549 to macrophage ratio of 3:1. Subsequently, the co-cultures were stained with an anti-CD11 antibody and analyzed by flow cytometry. CFSE (for A549 cells) vs. CD11b (for macrophages) dot plots are shown for the various conditions. Double-positive events were assumed to be macrophages with associated (adherent or phagocytosed) A549 cells. In the bar graphs, the results of three independent experiments, specifically the average percentages of double positive events + SD are shown (*p < 0.05; **p < 0.005; ns, not significant; one-way ANOVA). b CHO-K1 cells were treated with 1 mg/ml AGI-134 and then with or without 50% NHS. Cell killing was determined by DAPI-staining of a cell aliquot. The range gates in the histogram plots quantified dead cells. The remaining CHO-K1 cells were stained with CellVue Claret dye and incubated with GFP-expressing MutuDC cells at 1:1 ratios. Samples were removed from the co-culture after 30–120 min, and analyzed by flow cytometry. The CellVue Claret dye geometric mean fluorescence intensities (gMFIs) were normalized as described in the methods and then plotted against time. c CHO-K1 cells were transduced to express OVA tagged with the fluorophore mCherry. The histogram shows an overlay for the mCherry signal for CHO-K1 parental cells (open curve) and CHO-OVA cells (closed curve). After treatment with vehicle or 1 mg/ml AGI-134, the CHO-OVA cells were incubated with 50% NHS before co-culture with wild-type or DNGR-1 KO MutuDCs at the indicated range of dead CHO-OVA:MutuDC cell ratios. After 4 h, OT-1 CD8+ T cells were added to the co-culture and incubated overnight. OT-1 T cell activation was quantified by IFN-γ ELISA of the co-culture supernatants

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