The Research Of Cu-Cy Combined With X-ray Treatment Melanoma And Its Induced Anti-tumor Immunity

Background and Objective:As a promising approach to cancer treatment,photodynamic therapy(PDT)has been used for cancer treatment for many years.When the photosensitizer is irradiated with excitation light,PDT can kill cancer cells by generating reactive oxygen species(ROS).However,the application of PDT is very limited due to the inability of the excitation light to penetrate deep tissue.Here,we use copper-cysteamine complex nanoparticles(Cu-Cy)that can be easily activated by X-rays and can penetrate directly into the skin.Treatment with a combination of Cu-Cy and X-rays promotes apoptosis and/or necrosis on B16 cells,which may depend on the STAT3 signaling pathway.In addition,when B16 tumor-bearing mice were treated with Cu-Cy and X-rays,significant tumor ablation was found to be effective.Strikingly,combination therapy increases the number of white blood cells in the spleen and tumors,including dendritic cells(DC),CD4~+T and CD8~+T cells,natural killer(NK)cells,and,on the other hand,M2 macrophages.The number has dropped strongly.It is worth noting that the neglected drug toxicity of Cu-Cy nanoparticles was found in vivo and in vitro.These findings indicate that Cu-Cy-mediated PDT enhances tumor killing activity and promotes the formation of an anti-tumor microenvironment.MethodIn vitro experiments were performed by fluorescence confocal microscopy to observe the time and intracellul distribution of Cu-Cy into B16 cells.The toxic side effects of on B16 cells were determined by CCK8 cytotoxicity assay.The toxic side effects of Cu-Cy on B16 cells after 2.5 Gy X-ray irradiation were also determined.B16 cells were analyzed and recorded from the morphological.After a different treatment,the shape of cells changed.At the same time,flow cytometry and fluorescence confocal microscopy were used to analyze and observe the production of intracellular ROS by PDT after B16 cells.Flow cytometry was used to detect the apoptosis of B16 cells after different treatments.The apoptosis-related signaling pathway was detected and verified by western blot.In addition,we analyzed the changes of cytokines such as TGF-?after B16 cells received PDT by RT-PCR.We then conducted a related in vivo study by first establishing a tumor model by subcutaneous injection of mouse B16melanoma cells.When the tumor volume reached 300 mm~3,intraperitoneal injection of PBS and Cu-Cy were performed,and it was divided into X-ray irradiation group and non-irradiation group.After 6 h,the tumor-bearing mice were exposed to 5 Gy X-rays,and treated every other day for a total of three times.Changes in tumor and body weight of the mice were continuously recorded during the course.At the end of all treatments,the tumor,heart,liver,spleen,lung and kidney of the mice were taken.The ratio of immune cells in the spleen and tumor was detected by flow cytometry.At the same time,we used hematoxylin-eosin staining(H&E)was used to detect the toxic side effects of Cu-Cy on the main organs of the body.H&E-staining was also used to analyze the changes of tumor tissue after Cu-Cy combined with X-ray treatment.ResultIn vitro experiments showed that was co-cultured with B16 cells for 2,4,and 6 h,and it was found that the amount of entering cells increased significantly after 6 hours of culture,and Cu-Cy was concentrated in the nucleus.CCK8 cytotoxicity experiments showed that Cu-Cy itself had no obvious cytotoxicity to B16 cells,but after X-ray irradiation,could significantly kill tumor cells.At the same time,we found that the amount of intracellular ROS increased significantly after treatment of cells by Cu-Cy and X-ray.Moreover,flow cytometry analysis showed that X-ray irradiation further promoted apoptosis of B16 cells.Through western experiments,we found that the expression of cytochrome C increased after PDT.At the same time,the expression of p-STAT3 and Bcl2 was decreased,and the expression of Bax was significantly increased,indicating that Cu-Cy can promote apoptosis.Through in vitro experiments,we found that tumor growth was significantly inhibited after intratumoral injection of and X-ray irradiation,but the spleen of mouse body weight was not affected by PDT.We analyzed the proportion of relevant immune cells in the spleen and tumor of mice and found that CD4~+T cells and CD8~+T cells in the spleen were increased in the Cu-Cy+X group compared with the PBS,Cu-Cy and PBS+X-ray groups.At the same time,we found that in the Cu-Cy+X group,the proportion of DC cells and NK cells increased in addition to the ratio of CD4~+T cells and CD8~+T cells.Moreover,the M2type is a decrease in the proportion of cells.Finally,H&E staining results showed that combined with Cu-Cy+X-ray treatment did not cause pathological side effects on the heart,liver,spleen,lung and kidney of the wire harness,but it significantly promoted the death of tumor cells.ConclusionThe above results indicate that the novel photosensitizer Cu-Cy used in this study can be easily introduced into cells.It can be activated by low doses of X-rays and produces ROS,which has obvious effects of killing tumor cells,and can further promote cell apoptosis and affect the expression of intracellular related cytokines.In addition,in vitro studies have shown that Cu-Cy can significantly grow B16melanoma after X-ray irradiation,and induce anti-tumor immunity in vivo.In addition,our in vitro and in vivo results show that Cu-Cy has no obvious toxic side effects on cells and mouse body.