Synergistic anti-tumor effect of hyperthermia induced apoptosis of cancer cells and α-GalCer induced NK cells cytotoxicity in vitro.

(A) 2 µg of α-GalCer was i.p injected into 5 C57BL/6 mice and the tail vein blood was collected at 0.5, 1, 3, 6, 24, and 72 hr after treatment. The proportion variation of NK cells in peripheral blood was analyzed by flow cytometry with fluorescent CD3 and NK1.1 antibody staining. The results demonstrated that α-GalCer significantly recruited the NK cell population in peripheral blood 72 hr after treatment. (pversus other time points) (B) C57BL/6 mice were divided into 4 groups and treated by intraperitoneal hyperthermia at 43°C, 2 µg of α-GalCer, or combination. The control group mice were only opened their abdomen for 10 minutes. Intra-peritoneal cells were harvested by peritoneal lavage 3 hours after treatment. Variation on the proportions of intra-peritoneal NK cells was analyzed by flow cytometry with fluorescent CD3 and NK1.1 antibody staining. The results implied i.p. α-GalCer administration increased local NK cell proportion within a few hours. Combination of hyperthermia did not compromise this effect. (p = 0.0007, α-GalCer versus control; p = 0.0009, α-GalCer versus hyperthermia; no significant different between α-GalCer alone and combined treatment) (C) ID8 tumor-bearing C57BL/6 mice were treated with intra-peritoneal hyperthermia at 43°C for 10 min. One day after treatment, intra-peritoneal cells were lavaged and stained with Annexin V and PI. The cancer cells were gated and the apoptotic index was analyzed by flow cytometry. The proportion of apoptotic cancer cells was increased after intra-peritoneal hyperthermia. (p = 0.0084, hyperthermia versus control) (D) 2×104 of ID8-luc cells were seeded into 96-well round button plates and heated on 42°C water bath for 20 minutes. Different ratios of intra-peritoneal lavaged cells from mice i.p injected with or without α-GalCer were then added into the well as effectors cells and co-cultured overnight with heated or non-heated ID8-luc cells as target cells. Non-heated ID8-luc cells co-cultured with lavaged cells without treating α-GalCer were used as control. The cytotoxicity represented by the intensity of chemoluminence was the detected by IVIS imaging system. The results showed that co-culture of heated cancer cell with intra-peritoneal lavage cells from α-GalCer-treated mice induced highest cytotoxicity of cancer cell in vitro. (At E/T ratio = 5∶1, pversus control; p = 0.0162, heat plus α-GalCer versus heat; p = 0.0004, heat plus α-GalCer versus a-GalCer). (# p