Valproic Acid Intervenes The Abscopal Effect Of Normal Tissues Induced By Radiation In Situ Breast Tumors By Regulating The Polarization Of Macrophages

ObjectivesBreast cancer is very common and its morbidity and mortality are major threats to women’s health.As an effective treatment for cancer,about 40%of cancer cases have been cured by RT.Ionizing radiation(IR)can damage the tissues through both direct targeted and indirect non-targeted effects.The non-targeted effect,or abscopal effect(RAE),includes the killing of metastatic tumors and the damage to normal tissues.Our project focuses on the RAE in normal tissues,and mechanisms of the occurrence and intervention of RAE are still unclear,which are the research hotspot in this field.Valproic acid(VPA)is a kind of histone deacetylase inhibitors(HDACis),which is often used in antiepileptic treatments.It has been confirmed that VPA has anti-tumor effects.Previous studies of our group showed that based on the animal model of primary mammary tumors,a rat model of IR-induced RAE was established.And we found 200 mg/kg VPA not only played a radiosensitizing effect on tumors,but also alleviated the loss of weight and the decrease of organ index(OI)of normal organs in rats which induced by IR,suggesting that VPA can be used as an ideal adjuvant for RT and alleviates the toxic and side effects of RT on normal tissues.But mechanisms of its protective effects remain unclear.It has been reported that inflammatory and immune responses may be involved in the development of RAE.Macrophages are important immune cells,and the polarization of Ml or M2 phenotype determines the occurrence or regression of inflammation in vivo.It has been confirmed that macrophages participate in the occurrence of IR-induced RAE in normal tissues by secreting ROS and various cytokines to mediate inflammatory responses,potentially suggesting that the protective effect of VPA may be related to macrophages-mediated inflammatory immune responses.Therefore,our research will further study whether VPA can alleviate the toxic and side effects of IR in normal tissues by regulating the polarization phenotype of macrophages and changing its immune functions,so as to reduce inflammatory responses in normal tissues.The aim of our research is to enrich the mechanism of pathogenesis and intervention of RAE,and to provide the experimental evidence and theoretical guidance for reducing RAE damages in normal tissues of cancer patients in clinical practice.MethodsPrimary breast tumors were induced in 50-day-old female SD rats by one-time intragastric administration of 20 mg DMBA.A large number of malignant cell proliferation and disordered duct arrangement were observed in the breast mass by HE staining.On the basis of the successful establishment of the model,rats were randomly divided into four groups:Control,VPA,IR and IR+VPA.And then we established the animal model of RAE.In order to simulate the RT model commonly used in clinical practice,the situ tumor in rats was treated with fractional irradiation in low doses(2 Gy each time,once a day for four consecutive days)and the dose rate was 2.25 Gy/min.200 mg/kg VPA(i.p.)was applied throughout irradiation(twice a day for 6 consecutive days).The experiment was observed for 32 days after treatments.In spleen,small intestine,lung,liver and brain tissues,we used IHC to observe the expression of cell proliferation markers Brdu and Ki67.IHC to detect the expression of macrophages surface markers F4/80,CD68 and myeloid derived cell marker CD11b.IHC to observe the expression of M1-phenotype macrophages surface marker CD80.Western blotting(WB)was used to detect the expression of Arg-1 protein,a functional marker of M2-phenotype macrophages.RT-qPCR was used to detect the mRNA expression of M1-phenotype macrophages surface marker CD86,M2-phenotype macrophages surface marker CD163,related pro-inflammatory factors(IFN-γ,IL-12)and anti-inflammatory factors(TGF-β,IL-10).IHC to observe the expression of CD8+T lymphocytes and Granzyme-B.The dose of VPA was ultimately selected as 200 mg/kg according to its safe concentration in blood and toxicity to rats which was equivalent to the clinical dose for epilepsy treatments.Thus,the VPA concentration in the cell model was calculated to be 0.5 mM.Experimental designs to investigate the protective effect of VPA against RAE damages in normal tissues at the cellular level:(1)To explore the effect of VPA on the polarization of macrophages induced by irradiated MCF-7 cell medium:MCF-7 cells were irradiated with a single dose of 8 Gy and treated with VPA at a concentration of 0.5 mM.The medium was directly applied to RAW264.7,and the expression of related inflammatory factors was detected by RT-qPCR.(2)To explore the role of VPA in protecting normal mammary epithelial cells(MCF-10A)by regulating the polarization of macrophages:Firstly,the dynamic process model of RAE at the global level was established.MCF-7 was irradiated with 8 Gy for a single dose and treated with 0.5 mM VPA.MCF-10A was treated with this medium and then cultured with normal medium.Thus,conditional medium(VPA/IR-TCM)treated with VPA and IR from MCF-10A was obtained.RAW264.7 was cultured with VPA/IR-TCM,and then MCF-10A was treated with the macrophage lysate.After treated for 48 h,the proliferation ability of MCF-10A cells was detected by CCK-8 assay,and the expression of macrophages-related inflammatory factors was detected by RT-qPCR.(3)To explore the effect of VPA on the repolarization of M1-phenotype macrophages:RAW264.7 directly cultured in tumor cell medium was cultured with VPA/IR-TCM,and the expression of macrophages-related inflammatory factors was detected by RT-qPCR.All experimental data were represented as mean±standard deviation(X±S)and analyzed through one-way ANOVA by Student-Newman-Keuls multiple comparison test on SPSS software.P value is expressed as:*represents P<0.05;or**represents P<0.01,the difference is considered statistically significant.Results1.In the established animal model of IR-induced RAE,VPA can regulate the polarization of macrophages towards M2 phenotype to protect normal tissues against RAE damages(1)VPA promotes the cell proliferation and alleviates the RAE damage in non-targeted normal tissuesBased on our previous study,we observed VPA rescued the IR-induced decrease of BrdU positive cells in spleen,small intestine,lung and brain tissues.VPA attenuated IR-induced decreases of Ki67 positive cells in spleen,lung and liver tissues.(2)VPA reduces the infiltration of medullary derived macrophages in non-targeted normal tissuesVPA significantly reduced the IR-induced increases of F4/80 positive cells in non-targeted normal organs.VPA significantly decreased the IR-induced increases of CD68 positive cells in spleen,small intestine,liver and brain tissues.And compared with IR group,VPA decreased the infiltration of CD11b positive cells in non-targeted normal organs.(3)VPA regulates the polarization of macrophages towards M2 phenotype in non-targeted normal tissuesThe result showed that VPA significantly reduced the IR-induced increases of CD80 positive cells in each non-targeted normal tissue.WB showed that VPA rescued the IR-induced decreases of Arg-1 expression in small intestine and lung tissues.RT-qPCR showed that compared with IR group,VPA decreased the mRNA expression of CD86,pro-inflammatory cytokines IFN-y and IL-12.But VPA increased the expression level of CD163,CD163/CD86,anti-inflammatory factors TGF-β and IL-10 in spleen.In small intestine,compared with IR group,VPA decreased the mRNA expression of CD86,IFN-γ and IL-12.On the contrary,VPA elevated the expression level of CD163,CD163/CD86,anti-inflammatory factors TGF-β and IL-10.In lung,VPA decreased the mRNA expression of IFN-y compared with IR group.But the mRNA expression of CD 163,CD163/CD86,TGF-βand IL-10 were increased.In liver,compared with IR group,VPA decreased the mRNA expression of CD86,IFN-y and IL-12.On the contrary,VPA elevated the expression level of CD163/CD86.In brain,VPA decreased the mRNA expression of CD86,IFN-y and IL-12 compared with IR group.The expression level of CD163/CD86 and anti-inflammatory factor TGF-β were increased oppositely.(4)VPA reduces the infiltration of activated CD8-T lymphocytes in non-targeted normal tissuesIn non-targeted normal organs,VPA significantly reduced the number of CD8+T lymphocytes and Granzyme-B positive cells,thus alleviating the inflammatory response in non-targeted normal tissues.2.In the cellular level,VPA regulates the polarization of macrophages towards M2 phenotype and promotes the proliferation of non-irradiated normal mammary epithelial cells(1)VPA can regulate the polarization of macrophages towards M1 phenotype induced by irradiated MCF-7 cell mediumIn the co-culture model of macrophages directly treated by irradiated tumor cell medium,RT-qPCR showed that compared with IR group,VPA decreased the expression level of macrophages M2 phenotype surface markers CD 163,CD209,CD209/CD86 and anti-inflammatory factor IL-10,stimulating the polarization of macrophages towards M1 phenotype,and played a pro-inflammatory role.It was suggested that there were other cellular function models on exerting protective effects of VPA by regulating the polarization of macrophages.(2)VPA can protect normal cells(MCF-10A)by regulating the polarization of macrophagesIn the dynamic process model of RAE at the global level,RT-qPCR showed that compared with IR group,VPA increased the expression level of M2-phenotype macrophage surface markers CD163,CD209,CD209/CD86 and anti-inflammatory factor IL-10.VPA decreased the expression level of IFN-y oppositely.After MCF-10A cells were treated with the macrophage lysate,it was found that VPA restored the IR-induced decrease of the proliferation ability of MCF-10A cells,suggesting that VPA can protect normal cells by regulating the polarization of macrophages towards M2 phenotype.(3)VPA can reverse the polarization of Ml-phenotype macrophages into M2 phenotypeThe M1-phenotype macrophage RAW264.7 induced by irradiated MCF-7 cell medium was cultured with VPA/IR-TCM.RT-qPCR showed that compared with IR group,VPA elevated the expression level of CD209/CD86 and anti-inflammatory factor IL-10.The expression level of pro-inflammatory cytokines IFN-γ and IL-12 was decreased oppositely,stimulating the polarization of macrophages towards M2 phenotype.Conclusion1.In the rat model of IR-induced RAE,VPA can regulate the polarization of macrophages towards M2 phenotype and mediate the anti-inflammatory responses to alleviate the RAE damage in non-targeted normal tissues.2.In the cellular model of IR-induced RAE,it is further demonstrated that VPA can exert a radiological protective effect on normal cells by regulating the polarization of macrophages towards M2 phenotype,promoting the secretion of anti-inflammatory cytokines and decreasing the expression level of pro-inflammatory cytokines.