Therapeutic Effect And Immunological Mechanism Of A Novel In Situ Photoimmunotherapy On Cutaneous Melanoma In Mice

BackgroundSkin melanoma is the greatest harmful of the human skin tumors,but the traditional treatment had poor efficacy,and high recurrence rate.In situ immunotherapy is a new method for tumor therapy.The pro-clinical treatment of melanoma is effective and the recurrence rate is low.Previous studies have shown that the therapy increases the expression of heat shock protein by generating photothermal effects,which in turn activates cellular immunity is the main anti-cancer mechanism.During the course heat treatment,the temperature inside the tumor tissue was gradient,that is,the photothermal gradient.In this study,we mainly studied the efficacy and immunological mechanism of ln situ immunotherapy in the treatment of cutaneous melanoma,and tried to study the temperature gradient and DAMPs expression in different temperature gradients during the photothermal therapy,to provide theoretical basis for further laser immunotherapy.MethodsB16F10 mouse melanoma cells were inoculated into C57BL/6 mice subcutaneously to construct a skin melanoma model.Forty-eight tumor-bearing mice were randomly divided into four groups:control group,imiquimod group,laser group and in situ photoimmunopherapy group,12 mice in each group were treated as follows:control group,had no treatment;the laser group were treated with only 808 nm laser irradiation,0.75 W/cm~2 for 10 mins;the imiquimod group was treated with imiquimod twice a day for 7 days.In situ immunotherapy group,imiquimod coated 2 times a day for 3 days,then treated with 808 nm laser for 10 mins,0.75 W/cm~2,after the irradiation coated imiquimod 2 times for 3 days.Observed and recorded the size of mouse tumor and survival every 3 day.The histopathological changes were observed at the first day and 30 days after the end of light exposure.After 7 days of irradiation,CD4~+,CD8~+T cell infiltration was detected by immunohistochemistry in the tumor tissues of each group.Flow cytometry to detect the activation of DC cells at 48h after the end of irradiation.CD3~+,CD4~+,CD8~+T cells in drainage related lymph nodes tissues and spleen were detected by flow cytometry.The changes of temperature in different regions in tumor tissues were different,and the tumor surface temperature was measured by thermal imaging,thermocouples measured the changes in tumor tissue temperature.The expression of DAMPs(HSP70,HSP90,HMGB1,CRT)in the temperature region was studied after 24 hours of incubation with different temperature(37℃,40℃,45℃,50℃,55℃,60℃)and the different temperteure combined with imiquimod water bath B16F10 melanoma cells.ResultsIn situ photoimmunotherapy could significantly inhibit the growth of the tumor and prolong the survival rate,by plotting the changes of the tumor volume and the survival time of mice.The pathological results of each group were 7 days after the end of the treatment,in situ photoimmunotherapy group was complete cured,the cells were sacrificed and the cells were cleared in the laser group.Bilateral interval inoculation of tumor cells,only the treatment of side of the tumor,found that laser immunotherapy on the untreated side of the tumor also inhibited.The activation of DC cells was detected by flow cytometry in the drainage-related lymph nodes of at 48h after the end of the irradiation.It was found that the DC cells were activated in situ photoimmunotherapy group,CD4~+,CD8~+T cells were detected by flow cytometry,but the immunological cells in each group had no statistically different.In vitro,Western blot was used to detect the expression of HSP70,HSP90 HMGB1 and CRT in B16F10 cells treated with different concentrations of imiquimid.HSP70,HSP90 HMGB1 and CRT were obtained by the gray scale ratio ofβ-actin 40℃,high expression of HSP70 at 55℃,HSP90,HMGB1 and CRT appeared at the second peak at 50℃.The protein expression of HSP70,HSP90 and HMGB1 at 40℃ and 45℃,45℃ and 45℃ protein were significantly increased after treatment with different temperature.The Elisa showed that the release peak of HSP70,HSP90 and HMGB1 was consistent with the supernatant of different temperature treatment and different temperature combined with imiquimod,and the release peak at 50℃,45℃,50℃ and CRT was 45℃,combined with imiquimod at different temperatures is 50℃;and the different temperature combined with imiquimod DAMPs release than the temperature alone.ConclusionIn situ photoimmunotherapy can significantly inhibit the growth of mouse melanoma and prolong the survival rate.The immunological mechanism of laser immunotherapy to kill tumor cells is to stimulate the activation and maturation of DC cells,to extract antigen to T cells and further kill tumor cells.The expression of DAMPs in different temperature regions was mainly range 40-55℃.