A Hypoxia-Inducible HIF1–GAL3ST1-Sulfatide Axis Enhances ccRCC Immune Evasion via Increased Tumor Cell–Platelet Binding

Sulfatides promote platelet binding to cancer cells. A, Wells of a 96-well plate were coated with 2 μg/mL BSA or bovine sulfatides. Washed human platelets from healthy donors were stained with calcein AM and incubated in the coated wells. Following washes, florescence was measured using a fluorescent plate reader. Results are presented as a percent change in fluorescence of control (BSA). Data were analyzed using unpaired Student t test where, ***, P < 0.001. B, 786-shSCR and 786-shGAL were coincubated with human platelets from healthy donors stained with wheat germ agglutinin (red). Actin (green) stained both cells and activated platelets, while nuclei were stained with Hoechst (blue). C, An example of flow cytometry data acquired demonstrating the emergence of a double-positive cell population. Platelets were coincubated with GFP⁺ renal cancer cells for 5 minutes. Samples were fixed, stained with PE-CD41, and analyzed using flow cytometry. D, The percentage of a GFP⁺ PE-CD41⁺ population was graphed for each platelet donor tested (n = 3).

Platelets protect GAL3ST1-sulfatide–expressing cells from NK cell–mediated destruction but not GAL3ST1-sulfatide–negative cells. NK-92 cells were stained with Calcein-Red. 786-shSCR or -shGAL3ST1 are GFP⁺. Cancer cells were incubated for 3 hours in the presence or absence of NK-92 cells ± washed healthy platelets. After incubations, cells were stained for 7-AAD and Annexin V and cell death was quantified using flow cytometry. A, An example of the gating regime applied to the experiments presented and the significant shift in AnnexinV⁺ cells upon coincubation of ccRCC cells with NK-92 cells. A total of 10,000 events were recorded for each experiment. B, Cell death in 786-shSCR and 786-shGAL3ST1 cells coincubated in the presence or absence of platelets (P) or NK-92 cells (NK). Untreated cancer cells are presented in the figure as NT. n = 5 with washed human platelets from 5 healthy donors. C, NK-mediated cell death in samples coincubated with platelets in 786-shSCR and 786-shGAL3ST1 cells. A paired t test was used to evaluate significance between the groups where n = 5 healthy donors. D, Using the same experimental setup, platelets were activated with U46619 and then coincubated with shSCR or shGAL3ST1 ± NK-92 cells. In figures B and D, a one-way ANOVA with post hoc Tukey test was applied to determine statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).