| Objective:To investigate the consequences of HGMB1 (high mobility group protein B1, HMGB1) releasing in vitro of hormone refractory prostate cancer cell lines after cryoablation, and its role in inducing allogeneic peripheral blood dendritic cells differentiation and maturation, preliminarily to assess its impact on cryoimmunologic reaction.Materials and Methods:Culturing hormone refractory prostate cancer cell line PC-3, HMGB1 released in the supernatant by the cells before and after cryoablation (using a freezer 1.7mm in diamete from Endocare Argon-Helium cryosurgery system) was quantitatively determined using a enzyme-linked immunosorbent assay (ELISA) kit, then HMGB1 levels in the necrosis supernatant from differdent concentration of PC-3 cell after cryoablation were confirmed with Western blot analysis. Peripheral blood from prostate cancer patients were collected, using lymphocyte separation medium (Ficoll) to get adherent mononuclear cells (PBMC), obtained immature DC (imDC) by recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin 4 (rhIL-4) stimulating in vitro. Supernatant of the necrotic PC-3 cells directly or by adding sufficient HMGB1 neutralizing antibodies were mixed with imDC and cultured under suitable culture conditions. Cell morphology was observed under inverted microscope, further its skeleton structure changes were observed under laser scanning confocal microscope. Flow cytometry (FCM)was used for mature costimulatory molecules detection such as CD80, CD86, HLA-DR. Further to observe the time-dependent and dose-dependent effect between HMGB1 and CD83, CD86 and HLA-DR expression variance of DC.Result:1. Flow cytometry:The ratio of normal, apoptosis, necrosis of PC-3 cells was respectively (95.58±1.23)%, (2.00±1.61)%, (0.56±1.09)% before cryoablation, and three states were respectively (12.22±9.53)%, (5.46±1.14) %, (82.70±11.35)% after cryoablation, the difference was significant (P <0.05).2. ELISA results:We observed HMGB1 concentrations in the cell culture supernatant of PC-3 cell examined before and after cryoablation.1±106/ml group,1±107/ml group, 1±108/ml group respectively increased from (4.59±0.25)ng/ml, (6.04±0.13)ng/ml, (7.39±0.18)ng/ml to (14.28±0.84)ng/ ml, (71.68±4.62)ng/ml, (329.64±32.89)ng/ml, the difference was significance (P<0.01). The result was also confirmed by western blot:With the increasing number of PC-3 cells, HMGB1 concentrations in the supernatant increased.3. DC when challenged with necrotic PC-3 cells supernatant rearranged their actin cytoskeleton, such as actin protein increasing, volume bulge, immunological synapse coarsening and lengthening, stress fiber formation, gelatinolytic cytoplasm thickening, toughness and strength increasing. Functional blockade of extracellular HMGB1 by mean of specific antibodies strikingly prevented the cytoskeleton modifications.4. HMGB1 in the necrosis supernatant induced the expression of mature DC surface costimulatory molecules:the cryo-challenged group, mature DC surface costimulatory molecules CD83, CD86, HLA-DR expression was significantly increased compared with the control group (P<0.01); antibody treatment group, DC surface maturation of costimulatory molecules CD83, CD86, HLA-DR expression was significantly lower than the freezing stimulation (P<0.01), but still higher than the control group (P<0.05).5. HMGB1 in the necrosis supernatant induced the expression of mature DC surface costimulatory molecules by a time-dependent effect:The expression of mature DC surface costimulatory molecules CD83, CD86 and HLA-DR in 24～72h were significantly increased (P<0.05, P<0.01), in which 48 h groups cultured DC expression of costimulatory molecules increased the most significantly (P <0.01).6. HMGB1 in the necrosis supernatant induced the expression of mature DC surface costimulatory molecules by a dose-dependent effect:Compared with the control group,1×106/ml,1×107/ml group of DC costimulatory molecules CD83, CD86, HLA-DR was significantly increased (P<0.05, P<0.01),1×108/ml group of DC surface CD83 expression was significantly increased only (P<0.05), while CD86, HLA-DR had no significant change (P> 0.05).Conclusion:Cryoablation can cause tumor cells necrosis. Necrosis tumor cells release HMGB1 as an endogenous danger signal, which can enhance the migration, and prompt the immune synapse formation by rearranging DC cytoskeleton, to facilitate their entry into lymph nodes, and trigger T cells, ultimately to initiate specific T cell immune response. At the same time, HMGB1 plays a dual regulatory role in the cryoimmunologic reaction. Lower concentration of HMGB1 as an important immune-stimulating signal, can promote the expression of mature DC costimulatory molecules, and reach peak intensity in a certain time; Higher concentrations of HMGB1 can suppress costimulatory molecule expression, and inhibite DC activation in a certain extent. |
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