5-ALA Mediated Photodynamic Therapy Improves Antitumor Efficacy Of Immunotherapy Through Boosting Immunogenic Cell Death

Objectives:To explore whether 5-ALA-mediated photodynamic therapy(PDT)induces immunogenic cell death(ICD)in colorectal cancer cells and to study its specific molecular mechanisms,we want to developed a targeted,noninvasive,low-toxicity immunotherapy method for colorectal cancer.Methods:(1)The apoptosis of mouse MC38 or human HCT116 colorectal cancer cells induced by 5-ALA-PDT was determined by CCK-8,flow cytometry,caspase3 activity detection,hoechst staining and crystal violet staining;(2)The release of damage-associated molecular patterns(DAMPs)and the expression of autophagy-related proteins were detected by immunoblotting,flow cytometry,immunofluorescence and ELISA;(3)5-ALA-PDT-treated cancer cells were co-cultured bone marrow-derived dendritic cells(BMDCs)to detect the activation of BMDC cells;(4)To construct a mouse bilateral tumor model,and to analyze the volume and weight of bilateral tumor tissue;(5)The number and activity of T cells and DC cells in the spleen,blood and tumors of 5-ALA-PDT-treated mice were detected by flow cytometry;(6)Serum cytokine levels were detected by ELISA;(7)CCK-8 detected the decrease of cancer cell activity in the coculture of splenocytes and cancer cells of 5-ALA-PDT-treated mice.Results:(1)5-ALA-PDT reduced the viability of colorectal cancer cells,increased the activity of caspase3,and induced the apoptosis of colorectal cancer cells.(2)5-ALA-PDT induced colorectal cancer cells to release markers of immunogenic death(DAMPs),including calreticulin(CRT),HSP70,HSP90,HMGB-1,and ATP release.And 5-ALA-PDTtreated cancer cells and bone marrow-derived dendritic cells(BMDC)cocultured,BMDCs were effectively activated.(3)5-ALA-PDT decreased the expression of p-AKT and p-m TOR proteins,while 5-ALA-PDT increased the expression of autophagy-related proteins.(4)AKT activator SC79 blocked the release of 5-ALA-PDT-induced DAMPs.(5)Autophagy inhibitors Chloroquine(CQ)in combination with 5-ALA-PDT promoted more release of DAMPs and induced more cell death.(6)In mouse bilateral tumor models,5-ALA-PDT can inhibit not only primary tumors,but also distant tumors.At the same time,the primary and distal tumor growth inhibition was more pronounced in the combination of 5-ALA-PDT and CQ.(7)T cells and DC cells in the spleen,blood and tumor tissues of mice treated in the 5-ALA-PDT+CQ group were increased,and the content of cytokines(TNF-α and IFN-γ)increased in serum;(8)Splenocytes of mice in the 5-ALA-PDT and its combination therapy group specifically killed murine-derived colorectal cancer cells(not human colorectal cancer or other types of cancer cells).Conclusions : 5-ALA-PDT induces immunogenic cell death of colorectal cancer by inhibiting AKT kinase activity.Moreover,5-ALAPDT can effectively activate DC cells in vitro and can cause anti-tumor immune effects.Furthermore,5-ALA-PDT induces protective autophagy,and autophagy inhibitors combined with 5-ALA-PDT can promote the release of DAMPs and increase immunogenic cell death.Combination therapy with 5-ALA-PDT and autophagy inhibitors can synergistically activate DC and increase the number and activity of cytotoxic T cells,inducing specific antitumor immunity.