| ObjectivesRadiotherapy is a widely used and effective treatment for cancer.The abscopal effect of radiation describes radiotherapy-induced the untargeted tumor regression at sites distant to the irradiated field.Studies have shown that antitumor immune response induced by radiotherapy is the main mechanism of abscopal effect.However,such a clinically detectable abscopal effect from radiotherapy alone is rare,finding special agent to effectively trigger radiotherapy-induced abscopal effect would be important for controlling the growth of the untargeted tumor.This study investigated whether valproic acid(VPA)treatment can stimulate ionizing radiation(IR)-induced abscopal effect,and the mechanism of CD8+T cells and macrophages in this effect,to provide reliable theoretical guidance and experimental support for the application of VPA in the treatment of the untargeted tumor during radiotherapy.MethodsWe used 7,12-dimethylbenz[a]anthracene(DMBA),an environmental carcinogen,to establish a rat model with multiple breast tumors.The rats with 2-5 tumors were enrolled in this study and randomly divided into 4 groups(n=8 per group):control group,VPA group,IR group and IR+VPA group.Rats in the control group received no treatment;rats in the VPA group were intraperitoneally injected with 200mg/kg VPA,twice a day for 6 days;only one tumor was selected as the irradiated tumor of the rat(precise radiation)in the IR group and received 8Gy IR with 2Gy/day for 4 days;rats in the IR+VPA group were given 200mg/kg VPA(twice a day for 6 days)one day before IR(2Gy/day for 4 days).We monitored the volume changes of both irradiated and unirradiated tumors after treatments for 32 days.The unirradiated tumor was collected at 15 days after treatment,HE staining was performed for observing the morphological changes of tumors;the expressions of CD 8,Granzyme B,Cleaved Caspase-3,BrdU,Ki67,F4/80 and CD68 in the unirradiated tumor were analyzed by immunohistochemistry(IHC);the expressions of F4/80 and CD86 in the unirradiated tumor were analyzed by double immunofluorescence;western blot was used to detect Cleaved caspase-3 expression in the unirradiated tumor;the levels of CD86,CD163,IL-1β,IL-6,IL-12,IL-23,IL-10 and TGF-β in the unirradiated tumor were detected by real-time quantitative reverse transcriptase PCR(qRT-PCR).Results1 IR-induced abscopal effect was evoked by VPA treatmentTumor volume monitoring results showed that,compared to the control group,the tumor volume in the VPA group showed decreasing trend(P<0.05).For irradiated tumor,the tumor volume in the IR group was decreased compared to the control group(P<0.01),and the tumor volume in the IR+VPA group was further decreased compared with the IR group(P<0.01),suggesting VPA can augment the damage of IR on the irradiated tumor.Under this premise,we studied the effect of VPA on the untargeted IR tumor(ie.unirradiated tumor).For unirradiated tumor,there was no significant difference in tumor volume between the untreated control group and the IR group(P>0.05);however,compared with the IR group,the tumor volume in the IR+VPA group was significantly decreased(P<0.01).Furthermore,according to the results of HE staining,the tumor tissue in the VPA group showed a certain degree of small vacuolar necrosis;the unirradiated tumor tissue in the IR group showed similar histomorphology to the control group,exhibiting disordered arrangement of a large number of malignant cells and interstitial fibrosis;in contrast,a large area of necrosis was observed in the IR+VPA group.These results indicated that VPA can enhance the damage of IR to the irradiated tumor and trigger IR-induced abscopal effect.2 VPA treatment induced the effector CD8+T cells recruitment into the unirradiated tumorAccording to the results of IHC,the number of CD8+T cells in the tumor tissue was increased in the VPA group as compared to the control group(P<0.05),the number of CD8+ T cells in the unirradiated tumor was no difference between the IR group and the control group(P>0.05),the number of CD8+T cells in the unirradiated tumor was increased significantly in the IR+VPA group as compared to the IR group(P<0.01);in addition,the expression of Granzyme B secreted by CD8+T cells in the tumor tissue was increased slightly in the VPA group compared with the control group(P<0.05),compared with the IR group,the expression of Granzyme B in the unirradiated tumor in the IR+VPA group was significantly increased(P<0.01)Western blot results showed that the expression of Cleaved caspase-3(apoptosis-regulated protein)in the tumor tissue was increased in the VPA group compared with the control group(P<0.01),compared with the IR group,the expression of Cleaved caspase-3 in the unirradiated tumor was significantly increased in the IR+VPA group(P<0.01);in addition,the results were confirmed by immunohistochemical analysis of the number of Cleaved Caspase-3 positive cells in the unirradiated tumor.We also explored cell proliferation in the unirradiated tumor,IHC results showed that the expressions of BrdU and Ki67 in the tumor tissue in the VPA group were decreased compared with the control group(P<0.01),compared with the IR group,the expressions of BrdU and Ki67 in the unirradiated tumor were significantly decreased in the IR+VPA group(P<0.01).These findings demonstrated that VPA alone has a certain ability to stimulate antitumor immunity;importantly,combined IR+VPA treatment can induce infiltration of activated CD8+T cells into the unirradiated tumor,promoting tumor cell apoptosis and inhibiting cell proliferation3 VPA treatment promoted macrophages infiltration and polarization to the M1 phenotype in the unirradiated tumorThe IHC results showed that the number of F4/80 or CD68(macrophage markers)positive cells was increased in the VPA group compared with the control group(P<0.01),compared with the IR group,the number of F4/80 positive cells or CD68 positive cells in the unirradiated tumor was significantly increased in the IR+VPA group(P<0.01).To investigate the polarized state of the infiltrating macrophages,qRT-PCR rusults showed that CD86(M1 macrophage surface marker)mRNA expression was elevated in the VPA group as compared to the control group(P<0.01)and CD 163(M2 macrophage surface marker)mRNA was not increased(P>0.05);compared to the IR group,CD86 mRNA expression was elevated(P<0.01)in the IR+VPA group and CD 163 mRNA was decreased(P<0.05).Furthermore,double immunofluorescent staining was performed to analyze the proportion of M1 phnotype(F4/80+/CD86+)in macrophages(F4/80+),the proportion of M1 macrophages in the tumor tissue was increased in the VPA group as compared to the control group,compared with the IR group,the proportion of M1 macrophages in the unirradiated tumor was significantly increased in the IR+VPA group(P<0.01).These findings demonstrated that VPA alone has the ability to regulate the polarization of macrophages towards M1 phenotype;IR combined with VPA can induce macrophage infiltration into the unirradiated tumor and polarization into M1 phenotype,promoting the abscopal effect of radiation4 VPA treatment induced the increase of pro-inflammatory cytokines and decrease of anti-inflammatory cytokines in the unirradiated tumorAccording the results of qRT-PCR,pro-inflammatory cytokines IL-1β and IL-12 mRNA in the unirradiated tumor were increased in the IR+VPA group as compared to the IR group(P<0.05;P<0.01);there were no statistically significant differences noted with IL-6 and IL-23 mRNA between the two groups(P>0.05);anti-inflammatory cytokines IL-10 and TGF-β mRNA were decreased in the IR+VPA group compared with the IR group(P<0.01).These results indicated VPA induced the increase of macrophage-related pro-inflammatory cytokines and decrease of anti-inflammatory cytokines in the unirradiated tumor,overcoming the immunosuppression of tumor microenvironment and may enhancing antitumor immunity of CD8+T cells mediated by inflammatory factors such as IL-1β and IL-12.Conclusions1 It was first found that VPA inhibited the growth of the unirradiated tumor in rat model of multiple breast tumors induced by DMBA,suggesting VPA could trigger IR-induced abscopal effect.2 VPA could promote the infiltration of activated CD8+T cells and M1 macrophages into the unirradiated tumor;The abscopal effect of radiation triggered by VPA may be related to the enhanced antitumor immune response of CD8+T cells mediated by inflammatory factors such as IL-1β and IL-12. |
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