| Objective: Hepatocellular carcinoma(HCC)is the most common primary liver cancer and is ranked as the sixth most common tumor and the third leading cause of cancer-related death in the world.Surgical resection is the main treatment of primary or metastatic liver cancer.However,due to the difficultly anatomical location of the lesion,multiple lesions,insufficient liver function reservation,extrahepatic metastases or other comorbidities,many patients are not suitable for liver resection.Radiofrequency ablation(RFA)for tumor is the most common alternative treatment.Radiofrequency ablation leads to coagulative necrosis in situ by heating and releases tumor-associated antigens.Tumor-associated antigens can induce specific immune response to the tumor.The systemic anti-tumor effect can inhibit the metastatic tumors and circulatory tumor cells.However,the immune effect of RFA-induced tumor-specific T cells is limited.The tumor growth rate resumes and even accelerates after a short-time inhibition.The anti-tumor immune response activated by radiofrequency ablation can only temporarily inhibit the growth of distant tumors,which is not enough to prevent tumor recurrence.However,the mechanism of the immune status change of distant tumors after RFA is not clear.The aim of our study is to evaluate the different immune status of distant HCC after RFA by contrast-enhanced ultrasound(CEUS)and to explore the mechanism of the weakened anti-tumor immune induced by RFA in distant tumors,which can provide appropriate timing and other treatments combined with RFA.Methods: 1.To select the time points of different immune status according to the right tumor growth curve after RFA,24 mice with bilateral axillary subcutaneous H22 hepatocellular carcinoma were randomly divided into two groups(n = 12 in each group).The left tumors of mice in the RFA group were ablated,while the left tumors of the NonRFA group were not treated.The growth of the right tumor was monitored in both groups after RFA.To plot the growth curve of tumor volume,the right tumor growth of both groups was measured by ultrasound volumetry each day after RFA.Then,according to the tumor growth curve,two time points of tumor growth inhibition and restoration were selected to study,respectively.2.Contrast-enhanced ultrasound was to evaluate the immune status of distant tumors after RFA: Next,another 24 mouse models were randomized into RFA(12 mice with the left tumor ablated)and Non-RFA groups(12 mice with bilateral non-ablated tumors).CEUS and histochemistry of the right tumors were analyzed at the time points mentioned above.To demonstrate the immune status of the right tumors,the number of CD8 + T cells in the tumor was analyzed by CD8 immunohistochemical staining after RFA.The apoptosis in the right tumors was analyzed by TUNEL staining to demonstrate the effect of RFAinduced immune response on tumor cells.Microvessel density(MVD)in tumors was analyzed by CD31 immunohistochemical staining.CD31+PAS dual staining was used to analyze angiogenesis mimicry density(VMD)in the right tumors after RFA.The parameters of time-intensity curve(TIC)of CEUS was analyzed at different immune status.And the relationship between the parameters of TIC of CEUS and VMD was evaluated.3.Distant HCC immune status was inhibited via IL-6/STAT3/PD-L1 pathway after RFA:Another 24 mouse models were subjected to pathological histology analysis in the right tumors of the two groups at the two time points mentioned above.And H22 cells was stimulated with IL-6 and BP-1-102(p-STAT3 inhibitor).The mouse models were also grouped according to the time points of the tumor growth curve.The cell was divided into four groups: H22 group,H22 + IL-6 group,H22 + BP-1-102 group,and H22 + IL-6 + BP-1-102 group.The number of CD8 + T cells in the tumor was analyzed by CD8 immunohistochemical staining to evaluate the immune status in the right tumor after RFA.The expression of PD-L1 in the tumor was analyzed by PD-L1 immunohistochemical staining.The apoptosis of the right tumor was analyzed by TUNEL staining.The changes of IL-6 in mouse serum of each group of was accessed by ELISA.The mRNA level of PDL1 was analyzed by real-time PCR both in vivo and in vitro.The changes of PD-L1,pSTAT3 protein expression levels in tumors of each group and the four cell groups were all analyzed by Western blot to verify the role of the RFA/IL-6/STAT3 signaling pathway to regulate PD-L1.The proliferation of the four groups was accessed by CCK-8 in vitro.Results: 1.Compared with the Non-RFA group,on day 3 after RFA,the right tumor growth of the RFA group was slightly slowed(P =0.038).After a short-time suppression,the right tumor growth of the RFA group was restored on day 6,which was same as that in the NonRFA group(P=0.665).Therefore,according to the tumor growth curve,day 3 and 6 were selected as the time points to study.2.CD8+ T cells: In the control group,there was no difference in the number of CD8 + T cells infiltrated in the right tumors on day 3 and day 6.However,the CD8 + T cells in the right tumor were significantly increased in day 3 RFA group compared with that in day 3Non-RFA group.There was no difference in the number of CD8 + T cells infiltrated in the tumor in day 6 RFA group compared with that in day 6 Non-RFA group.In the RFA group,CD8 + T cells in the right tumor were significantly higher on day 3 than that on day 6(P<0.001).CEUS: The right tumors of each group were hyper-enhanced in CEUS.All tumors showed “fast in” and “slow out” on the TIC.Compared with the non-RFA group,the parameters of TIC are decreased on day 3 and increased on day 6 after RFA in the RFA group,though there is no difference on each time point.The parameters of TIC showed that PI,TTP,AWI,and AWO increased significantly in the day 6 RFA group vs.the day 3RFA group(P < 0.001,P = 0.017,P = 0.005,P = 0.002,respectively).But there was no difference in the slope and MTT between day 3 RFA group and day 6 RFA group.(P =0.595,P = 0.563,respectively).TUNEL: On day 3,apoptosis of the right tumors in the RFA group was increased compared with that of the non-RFA group(P < 0.001).However,apoptosis of the right tumors was significantly decreased on day 6 compared with that on day 3 in the RFA group(P < 0.001).There were no significant differences in apoptosis between RFA and Non-RFA group on day 6(P = 0.773).The results revealed that RFAinduced immune response can lead to apoptosis in distant tumors and the amount of apoptosis is related to the immune status.VMD: No difference in VMD of the right tumors was observed between the non-RFA and the RFA group on day 3(P = 0.389)and day 6(P= 0.155),respectively.In the RFA group,the VMD of the right tumors was higher on day6 compared with that on day 3(P = 0.003).There was no difference in MVD between day3 and day 6 in the RFA group(P = 0.632).Correlation between the TIC parameters of CEUS and VM: There is a positive correlation between TIC parameters(PI,TTP,AWI and AWO)and VMD in the distant right tumor,which is statistically significant(PI: r = 0.75,P <0.001;TTP: r = 0.56,P = 0.005;AWI: r = 0.77,P <0.001;AWO: r = 0.48,P <0.001,respectively).3.PD-L1: In Non-RFA group,there was no significant difference between PD-L1 on day3 and day 6.The PD-L1 in the right tumor of day 3 RFA group was slightly lower than that of day 3 Non-RFA group.The PD-L1 in the tumor was slightly elevated on the 6th day compared with the control group.And there was no difference between RFA group and Non-RFA group both on day 3 and day 6(P <0.05).The mRNA levels of PD-L1 detected by PCR in the right tumor of the RFA group was increased on day 6 compared with that on day 3.The expression of PD-L1 and p-STAT3 protein detected by Western blot in the right tumor of the RFA group was also increased on day 6 compared with that on day 3.The mRNA levels of PD-L1 detected by PCR in H22 cells stimulated by IL-6 was higher than that of other groups in vitro.The expression of PD-L1 protein in IL-6 + H22 cells was also significantly higher than that in H22,H22 + BP-1-102 and H22 + IL-6 + BP-1-102 group.The proliferation detected by CCK-8 of IL-6 + H22 cells was higher than the other three groups.Conclusions: 1.RFA can induce tumor-specific immunity within the distant HCC.The growth of distant tumors is related to the infiltrated T cells in the tumor.2.Immune response induced by RFA can cause apoptosis in distant HCC.The number of VM formed by tumor cells can also be reduced.Therefore,the number of VM indirectly reflects the immune status of tumors after RFA.CEUS is a noninvasive assessment of intratumoral VM.These results indicate that CEUS can be used as a noninvasive imaging method to monitor the immune response of distant tumors after RFA.3.Increased PD-L1 in tumors can suppress RFA-activated tumor-specific immune responses.Our results indicated that tumor-specific immune responses can be suppressed through the IL-6/STAT3/PD-L1 signaling pathway after RFA. |
PDF Full Text Request