Effect Of Melanoma Cell Surface-associated Calreticulin In The Occurrence And Development Of Tumor And The Underlying Mechanisms

Calreticulin（CRT）is a 46 KDa chaperone which can bind with Ca²⁺ions.Calreticulin is located in storage compartments associated with the endoplasmic reticulum.Full-length CRT has three domains,N domain,P domain and C domain.P domain and C domain plays an important role in the balance of Ca²⁺ions storage.The chaperone function of Calreticulin is performed by N domain,which ensures the correct folding of protein synthesis.It has been shown that the expression of CRT is closely related with tumor development,not only CRT is highly expressed in the tumor tissues but also enhanced the proliferative and migration ability of tumor cells.But CRT expressed on cell membrane boosts anti-tumor immune responses.We have constructed membrane CRT CRT/39-272 lentivirus vector,infected melanoma cells（B16）,and generated a cell line capable of stablely expressing membrane CRT/39-272.My project is based on B16-tmCRT cell line to observe how membrane CRT interacting with immuno system in tumor environment and to explore its mechanism.First of all,my project have observed the effect of proliferation、migration and adhesion of B16 cells under expressed membrane CRT in vitro.In vitro experiments,results showed that membrane CRT has no effects on proliferative migration and adhesion ability between B16-tmCRT and the control cell line B16-EGFP.We built a tumor model by inoculating C57BL/6 mice with B16-tmCRT and B16-EGFP cells to further investigating the in vivo effect of membrane CRT on tumor development.In vivo experiments,the tumor volume and incidence in B16-tmCRT-injected mice was far higher than the control group.The proportion of DC,MDSCs,NK cells and T cells in blood,spleen and tumor tissue has no difference within B16-tmCRT-injected mice and the control group.But the proportion of macrophage was higher in control group than B16-tm CRT-injected mice,pointing to the possibility that membrane CRT promotes tumor growth may be associated with macrophage.And we used clodronate liposomes which can specifically deplete macrophages in C57BL/6 mice,after depletion of macrophage we incubated C57BL/6 mice with B16-tmCRT and B16-EGFP cells.Tumor volume and incidence were significantly decreased in B16-tm CRT-injected C57BL/6 mice which depleted macrophages.We found that proportion of M2macrophages was higher in B16-tmCRT-injected C57BL/6 mice than control group.And after depletion of macrophages,there was on difference between two groups.All these evidences indicated that the reason why membrane CRT can promote tumor growth was related to M2 macrophages.To observe how macrophages interacted with B16-tmCRT in vitro,we induced C57BL/6 mice bone marrow cells into macrophages and cocultured with B16-tmCRT.We found that macrophages can phage more B16-tmCRT cells than B16-EGFP,and at the same time B16-tmCRT can enhance the phagocytic activity of macrophages.We assumed that in the differentiation of macrophages,membrane CRT may change the phenotype of macrophages.So we put B16-tmCRT cells with C57 mice bone marrow cells,and induced bone marrow cells into macrophages.The results came out that B16-tmCRT cells can polarized macrophages into M2 phenotype.In summary,membrane CRT can activate immune system and boost phagocytic activity of macrophages at early stage.But in tumor microenvironment,membrane CRT polarizes macrophages into M2 phenotype to promote tumor growth.The specific mechanisms of membrane CRT polarized macrophages into M2 remain to be further investigated,hope to find a new way to cure cancer.