Figure 3. α-GalCer stimulation and heat on cancer cell synergistically enhanced the phagocytosis activity of DCs both in vivo and in vitro.

(A) C57BL/6 mice were first i.p. injected with 2 µg of α-GalCer. After 18 hr, mice were sacrificed and the DCs were purified from spleens by anti-CD11c magnetic beads. For dendritic cell phagocytosis assay, ID8 cells were first stained with CFSE and heated on 42°C water bath for 20 minutes. The DCs from α-GalCer stimulated (or not-stimulated) mice were co-cultured with heated or non-heated ID8 cells for 6 hr at the ratio of 5∶1. The proportion of DCs with phagocytic activity was represented as CFSE positive in gated CD11c expressing cells analyzed by flow cytometry. The results showed that both in vitro heat treatment on cancer cells and in vivo stimulation of DC by α-GalCer could enhance the phagocytic activity of DCs (p = 0.005, heat versus control; p = 0.0037, α-GalCer versus control). The synergistic effect emerged in the combined treatment (p = 0.0002, combined treatment versus control; p = 0.0008 combined treatment versus heat; p = 0.005, combined treatment versus α-GalCer). (B) C57BL/6 mice were i.p. injected with 1×10⁶ CFSE-stained ID8 cells. On the next day, the mice were divided into four groups with treatment by i.p. hyperthermia and/or α-GalCer as previously described. One day later, intra-peritoneal cells were harvested through i.p. lavage and the proportion of CFSE-positive CD11b⁺/CD11c⁺ cells indicating phagocytic activities of DCs were analyzed by flow cytometry. It is shown that α-GalCer treatment could enhance the phagocytosis of DC in vivo (p = 0.0289, α-GalCer versus control). This effect was further enhanced by additional hyperthermia (p = 0.038, combined treatment versus α-GalCer). (# p<0.05; * p<0.01; ** p<0.001).