PDS-containing polymers bind C1498 cells aided by cytarabine pretreatment in vitro and activate splenocytes ex vivo. (A) Schematic of full cysteine–binding polymeric glycoadjuvant, p(Man-TLR7-PDS). (B) MFI of concentration-dependent binding of fluorescently-labeled p(PDS) or “spacer” only p(HPMA) to C1498 cells as quantified by flow cytometry. (C) C1498 cells were pretreated with varying doses of cytarabine for 4 hours before polymer incubation. MFI data of cytarabine-dependent binding of labeled p(PDS) are shown. (D) C1498 cells were pretreated with 10 μg/mL cytarabine at various time points before polymer incubation. MFI data of time-dependent binding of labeled p(PDS) are shown. C1498 cells were treated with varying doses of cytarabine for 18 hours and demonstrated dose-dependent increase in intracellular thioredoxin-1 (E) and hypoxia-inducible factor 1 alpha (F). Statistics shown are relative to untreated condition. (G) C1498 cells or (H) whole mouse splenocytes were stimulated for 8 hours with varying concentrations of p(Man-TLR7-PDS) or R848 and evaluated for TNFα secretion. All data are plotted as mean ± standard error of the mean (SEM; n = 3). All experiments were repeated with similar results. Statistical analyses were performed using ordinary 1-way analysis of variance with multiple comparisons. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; MFI, mean fluorescence intensity; ns, not significant.