X-Ray Irradiation And Veliparib Induce Accelerated Senescence Of Mouse Breast Cancer Cell Line

BackgroundCarcinoma of breast now is one of the most common malignant tumor in women around the world. According to the latest statistics from International Agency for Research on Cancer (IARC), new cases of breast cancer were up to 1.7 million and accounted for 22.9% of cancer incidence of all women. The incidence of breast cancer is rising yearly and accounts for the top in female malignancy. Moreover, a gradually younger age at onset of breast cancer has been observed. Breast cancer appears to be a serious threat to the health of female. At present, the main therapeutic measures of breast cancer includes operation, chemotherapy, radiotherapy, endocrine therapy and molecule targeted treatment. With the development of comprehensive therapy and early diagnosis, the fatality rate of breast cancer has been decreased gradually. However, there were still a lot of patients undergoing recurrence and metastasis after intensive treatment. Recently, more and more studies focus on the relationship between the tumor microenvironment and host. Immunotherapy is a kind of cancer treatment which can potentially improve the prognosis of cancer patients and development of effective tumor vaccine is one of the hotspots of immunotherapy currently.Researchers found that senescent tumor cells (STC) induced by specific measure remained active to secrete different kind of immune factors and chemokines and such tumor cells avoided immune tolerance. There were more dendritic cells (DC) and macrophagocytes accumulating in vaccination site, which attracted and activated CD4+ and CD8+ cells to release more immune factors and then start the systemic immune response. STC is attracting more and more attention because of its efficient prevention of cancer.Cellular senescence, a state of irreversible cell cycle arrest, can be triggered by various stresses such as telomere shortening, DNA damage, oxidative stress, oncogene activation, tumor suppressor gene inactivation and so on. Although cancer cells is immortalized with its infinite proliferation ability, it can be accelerated to senescence by kinds of anticancer agents including ionizing radiation (IR), chemotherapeutics drugs and genetic manipulation. STC is quite different from tumor cells for its morphological and metabolic features such as permanent growth arrest, larger volume, more lysosomal granules, enhanced senescence-associated P-galactosidase (SA-β-gal). Senescent cells can influence the surrounding microenvironment by secreting a variety of growth factors, inflammatory factors and proteases, which compose senescence-associated secretory phenotype (SASP). Generally expressed by all senescent cells, SASP plays two-way adjustment role depending on different biological environment. Recent studies found that SASP produced by specific agent-induced STC can attract kinds of immune effector cells to change the tumor microenvironment into a positive immune status to promote the clearance of tumor cells.It has been reported that poly ADP-ribose polymerase (PARP) inhibitor combined with IR can induce senescence of tumor cells with persistent DNA breaks. Veliparib, one of the PARP inhibitors, can suppress the repair of DNA damage which occurred after IR and markedly accelerate senescence. A study showed that senescent melanoma cells induced by veliparib and IR attracted more macrophagocytes, lymphocytes and DCs compared with irradiated melanoma cells. STC induced DLNs to promote more IFN-γ and immune factors secreted by STC leads to infiltration of more immune effector cells. Moreover, irradiated tumor cells express enhanced MHC-I, new antigen peptide as well as danger signs(e.g. ATP, HMGB1), which contribute to more robust immune response to eliminate tumor cells. Systematic anticancer immune response was activated by these STC to prevent the development of tumor.The immunogenicity of breast cancer cell is so weak that previous vaccines often failed to promote effective immune response. We tried to combine veliparib and IR to change the irradiated breast cancer cells into active STC with stronger immune stimulation effect.Object1. To detect the expression of SA-β-gal and the percentage of senescent tumor cells to observe the aging effect of veliparib-and-X-ray irradiation-induced senescent 4T1 and determine appropriate dose and function time of X-ray irradiation.2. To compare the expression of senescence-associated secretory phenotype (SASP) and observe the lifetime of mice vaccinated with senescent 4T1 to value the prevention of senescent tumor cells against breast cancer.Methods1. Breast cancer cell line 4T1 were divided into four groups to operate:control group(4T1 were cultivated with normal DMEM), Vel group (4T1 were cultivated with DMEM containing veliperib), X-ray group (4T1 were treated with X-ray irradiation) and X-ray+Vel group (4T1 were cultivated with DMEM containing veliperib and treated with X-ray irradiation). The dose of radiation in X-ray group and X-ray+Vel group both set to 6Gy,9Gy,12Gy and linear accelerator (Varian 2100) was used to irradiate cells.2. Detect activity of SA-β-gal of each group at 2nd and 4th day respectively and compare the SA-P-gal positive rate of the four group to observe the effect of aging. Compare the SA-β-gal positive rate of the three group with different exposure dose to choose an optimal dose of irradiation. Evaluate the percentage of senescent tumor cells according to the cell volume and lysosomal granules by flow cytometry.3. Collect cells treated with X-ray+Vel at 1st,2nd,4th,6th and 8th day and cells of control group, Vel group and X-ray group at 4th day. RT-qPCR was used to detect the mRNA expression of p21 and SASP including IFN-β, TNF-α, CCL2, CCL5, CXCL9 and CXCL10. Then compare the expression of each group and observe the dynamic change of X-ray+Vel group. Cytometric Beads Array was performed to evaluate the concentration of TNF-a, IFN-Pand IFN-y of cell supernatant in each group. Collect senescent cells and non-senescent cells by flow cytometry and then examine respective secretory phenotype.4. Vacinnate Balb/c mice with cells of the four groups and then establish 4Tlbreast cancer model. Compare the life time of mice in each group.Results1.2 days after treatment, it can be observed by inverted microscope that 4T1 grew rapidly without morphologic change in control group and Vel group; 4T1 altered slightly in X-ray group; morphology of 4T1 began to change and intercellular gap was still large because of slowing proliferation in X-ray+Vel group.4T1 of X-ray+Vel group with irregular morphology, bigger cell volume and reduced nuclear plasma can be observed at 4th day.4T1 of X-ray+Vel group shed from the plate heavily and became necrotic.2. SA-β-gal staining showed that the percentage of SA-β-gal positive cells in X-ray+Vel group is the highest and it increased significantly at 4th day, while very few positive cells can be observed in control group and Vel group. SA-β-gal staining showed no significant differences in control group and Vel group (P=0.768).9Gy group showed significant increased SA-β-gal staining compared with control group (P<0.001). Significant enhanced SA-P-gal staining were observed in 9Gy+Vel group compared with control group (P<0.001) and 9G group (P<0.001). It also can be seen that the higher the dose, the higher the SA-P-gal positive rate.9Gy+Vel group had more positive cells than 6Gy+Vel group significantly (P=0.002). The percentage of SA-P-gal positive cells in 12Gy+Vel group is the highest, which showed significant increase compared with 6Gy+Vel group (P<0.001) and 9Gy+Vel group (P=0.035).3. The difference of the percentage of senescent tumor cells between Vel group and control group was not statistically significant (P=0.942). The proportion of senescent tumor cells in 9Gy group was significantly higher than the control group (P <0.001). The proportion of aging tumor cells in 9Gy+Vel group are the highest and showed significant difference compared with and the other group (P< 0.001).4.9Gy+Vel group obviously possessed the highest expression of p21, IFN-P and TNF-a among the four groups. The difference of the expression of p21 between Vel group and control group had no statistical significance (P=0.726), while difference between 9Gy group and control group was statistically significant (P=0.001); the expression of p21 in 9Gy+Vel group was significantly higher than other group (P< 0.001). There is no significant statistical difference of the expression of IFN-β between Vel group and control group (P=0.985); 9Gy group expressed more IFN-β than control group (P=0.023).The mRNA expression of IFN-P in 9Gy+Vel group was statistically higher than other group (P<0.001). The expression of TNF-a in Vel group had no statistical significance compared with control group (P=0.966), while 9Gy group expressed more TNF-a than control group (P=0.001). Expression of TNF-a in 9Gy+Vel group was higher than other group (P<0.001). After the combined treatment of X-ray and veliparib, the expression of IFN-P and TNF-a altered with the passage of time and peaked at 4th day after irradiation. The expression of chemokines including CXCL9, CXCL10, CCL2 and CCL5 in 9Gy+Vel group were the highest and significantly higher than control group (P<0.001),Vel group(P<0.001) and 9Gy group (P<0.001). The expression of CXCL9, CXCL10, CCL5 and CCL2 of the senescent cells altered with time after the treatment of drug and X-ray and peaked at 4th day.5. The secretion levels of TNF-a, IFN-βand IFN-y were detected by CBA, which were the highest in 9Gy+Vel group. The concentration of all the three cytokines in 9Gy+Vel group was significantly higher than those in the control group (P< 0.001),Vel group(P<0.001) and 9Gy group (P<0.001).6. Compared with non-senesencent cells, senescent cells expressed more p21 (P=0.017), TNF-a (P=0.001), IFN-p (P=0.001), CXCL9 (P=0.010), CXCL10 (P=0.008),CCL2 (P=0.005) and CCL5 (P=0.005)7. The mice which were inoculated with drug treated cells all formatted tumor. The percentage of tumor free mice was 22.22% in 9Gy group, while it was 77.78% in 9Gy+Vel group. The proportion of tumor bearing mice at the twentieth days after inoculation of 4T1 cells in each groups was compared and difference between the four groups was statistically significant (P=0.013).Conclusions1. Veliparib combined with X-ray irradiation can significantly accelerate the aging of mouse breast cancer cell line 4T1. Senescence doesn’t appear immediately after stress so that it needs to grasp the appropriate time to effectively use senescent tumor cell.2. SASP secreted by senescent tumor cells altered with time. SASP of cells induced by veliparib and X-ray was mainly from the large senescent tumor cells. It’s necessary to choose the senescent tumor cells in the secretion peak to act as a tumor vaccine so as to exert its effect.3. Senescent breast cancer cell 4T1 induced by veliparib and X-ray can effectively prevent tumor formation.