The Mechanism Of Ceramide Analog Induced Cell Death In Tumour-associated Immunosuppressive Cell

Cancer immunotherapy is the use of immunology principles and methods, to inspire and enhance immune response, so as to kill tumor cells and inhibit tumor growth. Nowadays, it is much more progress in immunotherapy methods against cancer, such as monoclonal antibody therapy, cancer vaccine therapy and immune cell therapy. In the journal of Science, cancer immunotherapy is the top one of the 2013 scientific Breakthrough of the Year. Although the body has a variety of antitumor immune response, however, tumor can still occur, develop, and grow in the body. These factors which produce by tumor cells can induce the body to form immunosuppressive cells including immature dendritic cells (iDC), tumor-associated macrophage (TAM), myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg). Bioactive sphingolipid (SPL) metabolites, specifically ceramides (Cer), sphingosine (Sph), and sphingosine 1-phosphate (SIP), are increasingly recognized for their important roles as signaling molecules involved in regulation of survival, proliferation, and cell death. During the decades, it is found that ceramide analogs are potentially an effective approach to overcome tumor. However, the role of these analogs in tumor immunosuppressive cell are still unclear. From in vivo and in vitro, we study the influence of cell activity about the ceramide analogues treated tumor immune suppressive cells, and a relatively well-developed signal pathway has been established, which elucidate the complex role of ceramide analogues in tumor immune escape on experimental basis, and theoretical support a new possibly view for anti-tumor drug research and development which targets tumor immunosuppressive cells.The main results of this study are as follows:1. Increased the percentage of CD11b+Gr1+MDSCs in tumor-bearing mice promote tumor initiation and progression. Sarcoma CMS4-met tumor cells were injected into BALB/c mice s.c. Spleen, BM and blood were collected one month later from tumor-bearing mice (TB) and stained for CD11b+Gr1+.Tumor-free mice (TF) were used as control. The results are that CD11b+Gr14MDSCs were dramatically increased in the tumor-bearing mice compared with tumor-free mice, which indicate the promotion of CD11b+Grl+MDSCs in tumor initiation and progression.2. Ceramide analog LCL521 decreased the percentage of CD11b+Gr1+MDSCs and suppressed CMS4-met isograft growth in vivo. Sarcoma CMS4-met tumor cells were injected into BALB/c mice s.c. Thirty days later, tumor-bearing mice were treated with ceramide analog LCL521 at a dose of 75 mg/kg body weight every two days for twice. Mice were then sacrificed. Spleens, blood, BM were collected and stained with CD11b-, Grl-specific mAbs. The conclusion is that LCL521 decreased the percentage of CDllb+Gr1+MDSCs in spleen and tumor in vivo. CMS4-met cells were injected to the BALB/c s.c.when the size of tumor is around 60-70mm3, Tumor-bearing mice were then treated either with Solvent(5%ethamol,15% cremphor,80% PBS) or LCL521 (75 mg/kg body weight)for 6 times. Measured the tumor size everyday, we found that LCL521 suppressed CMS4-met isograft growth in vivo.3. J774 and RAW cell lines can be used as CD11b+Gr1+MDSCs and macrophage in vitro model, respectively. J774 and RAW were stained with fluorescent dye-conjugated mAbs as indicated and analyzed by flow cytometry. IgG isotype controls were used as negative control for the gating.The results of Flow cytometry indicated that J774 cells were mostly CD11b+Gr1+ and Raw cells were CD11b+but mostly Gr1". Therefore J774 and RAW were respectively used as an in vitro cell model for MDSCs and macrophages, which were used to study the inhibition of ceramide analogues to tumor associated immunosuppressive cells and its molecular mechanism.4.Ceramide analog LCL521 induced J774 and RAW cell death through an apoptosis-,necroptosis-,autophagy-independent pathway.LCL521 triggered J774, RAW cell death on dose-dependent. To illuminate the death pathway, we used inhibitors of caspase, REP1, RIP3 and autophagy. They did not block LCL521-induced J774 and RAW cell death. So the conclusion was that LCL521 induced J774, RAW cell death through an apoptosis-,necroptosis-,autophagy-independent pathway.5. Target of ceramide analog LCL521 in the J774 and RAW cells located in lysosome and mitochondria. Using the methods of ultra-high speed density gradient centrifugation and cell mitochondria isolation kit, we successfully separated the lysosome and mitochondria, then got the two organelles lysate. Analyzed by LC-MS/MS, We found LCL521 located mitochondria and lysosome. Meanwhile, we also found that content of sphingosine in lysosome and mitochondria were significantly reduced.6. Ceramide analog LCL521 induced mitochondrial Ca2+ uptake, and generation of ROS. Using the indicators of Ca2+ and ROS, flow cytometry demonstrated that treatment of cells with LCL521 dramatically increased in mitochondrial Ca2+ and the cellular ROS levels. Then using extracellular chelator and intracellular inhibitors of Ca2+, we found that the Ca2+ of J774 was from extracellular, and RAW was from intracellular. The level of Ca2+ was blocked by chelator or inhibitor, which directly reduced cellular ROS, and then resulting in the decrease of LCL521-induced J774 and RAW cell death.7. Ceramide analog LCL521 induced J774 and RAW cell death through a lysosomal cathepsin-dependent mechanism. Using alone or combined the inhibitors of cathepsin B and D to analyze cell viability of LCL521-treated J774 and RAW cell, we found neither inhibitor could block cell death induced by LCL521, except combining the two inhibitors together. The result indicated the ceramide analog LCL521 induced cell death based on cathepsin B and D signaling pathways.In summary, our studies confirm that ceramide analog LCL521 can effectively induce tumor immunosuppressive cell death, and illuminates the molecular mechanism of LCL521 in tumor immunosuppressive cells. For the first time, we reveal that the ceramide analog LCL521 can be used as an inhibitor of tumor immunosuppressive cells, and it reveal the potential of Ceramide analog in the inhibition of tumor immunosuppressive cells and provides a new train of thought for immunotherapy which aims tumor-induced immunosuppressive cells.