| BackgroundBreast cancer is a kind of malignancy derives from mammary gland epithelial tissue.According to the epidemiological study,incidence of breast cancer increased in recent years.Breast cancer has become an important public health problem with a priority incidence of malignant tumors in women.Moreover,it has reported that a tendency was found to the young.Therapetics against breast carcinoma is guided by neoplasm staging and clinicopathologic information that mainly reflects molecular features.Multidisciplinary combination approach with surgery,chemotherapy,targeted therapy,radiotherapy and hormonal therapy has been proved more successful than conventional single treatment method.However,patients with triple-negative breast carcinoma has always correlated with poor prognosis because of a lack of effective approach.Therefore,it is a urgency to explore novel therapy strategy to deal with the puzzle.Immunotherapy was regarded as the major scientific breakthrough in 2013.Vaccination is one of the therapeutics to stimulate endogenous immune response against tumors.It has been reported to be associated with favorable outcomes for patients with triple-negative breast carcinoma.However,because of the high metabolic rate or low expression level of cytokines and activation of the negative immune regulation mechanism,tumor vaccines,such as peptides,tumor-associated antigens protein and inactivated tumor cells,usually cause an insufficient efficacy in clinical.Thus,reseachers has focused on developing new tumor vaccines to conquer those barriers.Many researches have discovered a change in cytokines expression by tumor cells receiving radiotherapy or chemotherapy.Futher study has revealed the essence.Cellular senescence occured in tumor cells triggered by therapy stresses.Senescence tumor cells are quite different from tumor cells for their morphological and metabolic features such as permanent growth arrest,larger volume,more lysosomal granules,enhanced senescence-associated-?-galactosidase,a variety of growth factors,inflammatory factors secretion.Senescence cells not only persisted a large amount of tumor antigens as others,but also maintained high local cytokines concentration.In a word,senescence cells possessed the potential of working as a novel vaccine against tumor.Microenvironment surrounding tumor cells would change following treatments.Meanwhile,effects of therapy would be cut off due to the activation of immunosuppressive mechanism.PD-1/PD-L1 pathway is one of the major immunosuppressive mechanisms.PD-1/PD-L1 signaling plays an important role in manitaining the immune balance of organism in condition.As for tumor cells,it is a mechanism to prevent from elimination by immune cells.Obviously,the phenomena meant that PD-1/PD-L1 signaling would certainly cripple efficiency of vaccine against tumors because of microenvironment shift.In other words,an optimization of blockage could recover efficacy of tumor vaccines.sPD-1 is what has been detected in blood of patients with autoimmune diseases and associated with intense inflammation by interacted with PD-L1.A possible approach for reversing the vaccination failure was the combination usage of sPD-1 and senescence tumor cells.The combination therapy not only solved the problem the vaccined faced,but also reduce the financial burden and side effects of anti-PD-1/PD-L1 antibody.Object1.To detect the percentage of PD-L1+4T1 cells after treated with radiation or different doses of IFN-? and ensure that whether treatments would lead to immune escape.2.To developed a sPD1-expressing 4T1 cell subset denoted as 4T1/sPD-1 cells.4T1/NC cells were set as a control.And to find out whether lentivirus infection had impacts on cell varients characteristics.3.To investigate the senescence status of 4T1,4T 1/NC,4T1/sPD-1 cells after induced by radiation with Veliparib by SA-?-gal staining and p21 expression test.Meanwhile,to compared the expression of senescence-associated secretory phenotype of senescence and non-senescence cells.4.To observe the tumor onset of mice caused by senescent 4T1,4T1/NC or 4T1/sPD-1 cells injection and analyze the activation of DCs and T cells.5.To value the prevention and therapeutic effect of senescent 4T1,4T1/NC and 4T1/sPD-1 cells as a vaccine against breast cancer.Methods and Materials1.4T1 cells were underwent 10Gy irradiation and mantained for 3 to 5 days or cultured in the presence of IFN-? for 24h in different doses range from 0ng/ml,5ng/ml,10ng/ml,20ng/ml to 30ng/ml before harvested and PD-L1 detection by flow cytometry.2.4T1 Cells were infected with lentiviral particles carrying sPD-1 vector or negative control mock sequence.72h post infection,both 4T1/NC and 4T1/sPD-1 cell subsets were collected for sPD-1 examination by microscope,qRT-PCR,western blotting and flow cytometry.3.EdU assay,transwell assay and wound healing assay were used to measure cell subsets proliferation,invasion and migration ability.4.Tumor cell subsets were exposured to radiation and incubated in medium containing Veliparib for 5 days.Cells were harvested for senescence test by SA-?-gal staining and senescence marker p21 expression examination by western blotting.5.Supernate collected from 4T1,4T1/NC and 4T1/sPD-1 cells were collected for IL-10,IL-12P40,IL-12P70,IL-17,IFN-?,TNF-?,TGF-? and sPD-1 measurements.6.To determine whether sPD-1 could enhance the safety of the vaccine,mice were injected with senescent or non-senescent tumor cells.Tumor occurrence as well as immune cells activation were analysed in mice.7.To determine the effciacy of senescence 4T1,4T1/NC and 4T1/sPD-1 cells,mice were vaccinated with those tumor cells before/after 4T1 cells inoculated.Tumor occurrence and survival were analysed in mice.Results1.LIFN-? and radiation stimulation up-regulated PD-L1 expression of 4T1 TNBC cells.Both radiation and IFN-? treatments significantly up-regulated the expression of PD-L1.After irradiated,the proportions of PD-L1+ cells in the groups were(3.93±0.,9)%,(5.14±0.19)%and(9.25±0.34)%,respectively(P<0.001).With the stimulated of IFN-?,PD-L1+ variant of 4T1 cells increased in a dosage-dependent manner from(2.61 ±0.23)%to(19.3± 1.59)%,(48.51±1.14)%,(63.79±0.92)%and(86.69±1.04)%(P<0.001).2.4T1 cells were re-engineered to secrete biological activity sPD-1 through lentivirus-mediated delivery.4T1 Cells were infected with lentiviral particles carrying sPD-1 vector or negative control mock sequence.72 h post infection,both 4T1/NC and 4T1/sPD-1 cell subsets presented in a strong fluorescence intensity under microscope.The consistent expression of sPD-1 at RNA and protein levels were confirmed in 4T1/sPD-1 cells but not in cells of control group by qRT-PCR and western blotting.Adding supernate collected from 4T1/sPD-1 cells culture medium could significantly increased the population of PD-1+4T1 cells which pre-treated with IFN-?.All results demostrated the successful cell genetically modified via lentivirus and sPD-1 secreted by 4T1/sPD-1 possessd biological activity.3.Lentivirus-mediated sPD-1 gene transfer had no impacts on cell varients characteristics in vitro.In EdU assay,the typical pitures insinuated similar vitality among 4T1,4T1/NC and 4T1/sPD-I cells.The percentage of EdU positive cells were(50.33±2.52)%,(51.33±3.21)%and(50.67±2.52)%,respectively(P>0.05).In Transwell assay,4T1 cells showed undifferentiated performance in invasion as compared to 4T1/NC and 4T1/sPD-1 cells.Migration index for 4T1 was(905.3±26.27)vs(903.7±23.03)of 4T1/NC and vs(897.7±13.05)of 4T1/sPD-1 cells(P>0.05).For repairing a wound,4T1 showed a mean%of repair of(12.3 3±0.58)%vs(12.33±1.15)%of 4T1/NC vs(12.67±1.53)of 4T1/sPD-1 cells after 12h and(49.67±2.08)%vs(50.67±1.53)%vs(51.7±1.53)%after 24h(P>0.05).4.Senescence tumor cells induced by radiation and Veliparib revealed potential vaccinal character.Flattened cell morphology with enhanced SA-?-ga1 staining and increased p21 expression of 4T1,4T1/NC and 4T1/sPD-1 cells indicated that tumor cells actually shifted toward senescence.As expected,senescence tumor cells expressed higher levels of immunostimulatory cytokines including IFN-?,TNF-?,IL-17,IL-12P70,which correlated with boosting anti-tumor immune responds.Secretion of mmunosuppressive cytokines such as TGF-?,IL-12P40 were no significant changes except IL-10.Only senescence and non-senescence 4T1/sPD-1 cells secreted sPD-1.5.sPD-1 expression enhanced the safety of STCV.When challenged with non-senescent cells,we observd that mice inoculated with non-senescent 4T1/sPD-1 cells had smaller tumor volumns and longer survival time compared with PBS,4T1 and 4T1/NC inoculation groups.Mice received STCV/sPD-1 alone or before/after 4T1 implantation.As a result,STCV/sPD1-treated mice showed no tumor occurrence.When injection STCV/4T1 or STCV/NC alone,mice were seen to bear tumor but eventually free to tumor.A similar tumorigenesis rate was obtained in mice with STCV/4T1 or STCV/NC treatment before/after 4T1 implantation.STCV/sPD-1 stimulated DCs to maturity and led to fewer dysfunction PD-1+T cells.6.sPD-1 expression ehanced the efficacy of STCV.In a prevention tumor model,we discoverd that STCV could induce intense antitumor immunity which resulted in a higher tumor-free rate,longer survival time and lower tumor volumes.The percentage of tumor-free mice and survival rates even rised to 100%in STCV/sPD-1 group.In therapeutic setting,tumor onset and growth was suppressed for a period of time in STCV/sPD-1 treated tumor-bearing mice.ConclusionsSenescence tumor cells vaccine has anti-tumor effect against breast cancer in mice,and sPD-1 over-expression could enhance this effect of the vaccine. |
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