Study Of MR Guided And Monitored Precise Ablation Of HCC And The Regulation Of Antitumor Immunity By Different Ablation Techniques

BackgroundHepatocellular carcinoma is one of the most common malignancies.In recent years,image-guided ablation therapy has become a conventional treatment method for patients with postoperative recurrence of early hepatocellular carcinoma and HCC,but tumor recurrence after ablation has become an urgent problem in clinical practice.With the proposal of precision medicine and the advent of the era of tumor immunotherapy,precise ablation combined with immunotherapy has become a hot research topic.The combination treatment will have great prospects in improving the immune efficacy and reducing tumor recurrence.On the one hand,precision ablation treatment can not only effectively improve the overall success rate of tumor ablation,but also can make tumor immunogenic cell death,release tumor antigen,stimulate local and systemic immune response,make part of the"cold tumor"(immune desert,immune rejection tumor)to "hot tumor"(immune inflammatory tumor),thus change tumor immune suppression microenvironment,on the other hand,immunotherapy can enlarge or extend the immunomodulatory effect of ablation.Therefore,precision ablation combined with immunotherapy may bring hope for solving the problem of recurrence after HCC ablation.Currently,US and CT are the most commonly used guidance methods for ablation of HCC.But,because of its good soft tissue resolution,multiple parameters and any orientation imaging ability,MR can show lesions from different angles,also has a unique vascular flow empty effect,unenhance can clearly show peripheral vascular distribution of lesions,but also has the typical MRI performance in the process of ablation,which make it to accurate guidance and monitoring.RFA,MWA and CRYO is the most commonly used technology for ablation of liver cancer.MWA is one of the main treatment methods in recent years,it has higher tumor ablation temperature,larger ablation range and faster ablation time,at the same time less affected by the heat sink effect around the lesion,so ithad achieved good results in the clinical treatment of liver cancer.Therefore,we believe that the MR-guided and monitored HCC microwave ablation treatment can achieve the purpose of accurate ablation,which can improve the overall ablation efficiency of the treatment,but few relevant studies have reported on it.With the advent of tumor immunotherapy era,the ablation and immunity of liver cancer have become a research hotspot.Many studies had shown,not only RFA、MWA and CRYO ablation therapy can directly destroy the tumor cells,but also they can induce antitumor immune response,It can even produce a distant effect,However,in different ways of tumor necrosis due to different ablation techniques,The various damage-related molecular patterns(DAMP)released by tumor cells after immunogenic cell death may also differ,Therefore,the strength of the anti-tumor immune response produced by the body and the corresponding immune changes may be different,however,there are few comparative studies in this area,and the studies that had been reported are also controversy.Over the past decade,immunotherapies of targeting various immune-checkpoint inhibitory receptors have provided ample evidence for clear clinical therapeutic effects with a wide variety of solid tumors.However,the objective response rate of single agent is only 20%-30%.In order to make up for the deficiency of monotherapy,many researchers are constantly exploring new immunotherapy targets and the optimal methods of combination therapy.Among them,T cell immune receptor(TIGIT)with T cell immunoglobulin and ITIM domains is a promising new target for immunotherapy,which mainly restricted express on the subsets of activated T cells and natural killer(NK)cells,and interacts with CD155 to induce immunosuppression.In the research field of immunotherapy for liver cancer,TIGIT has become a hot research topic.A new study found that gene knockdown or antibody blocking of TIGIT enhanced NK cell killing and enhanced CD8+T cells tumor activity.However,whether the different ablation methods of liver cancer can lead to the changes in TIGIT,and whether the change has an association with immune cells and PD-L1,which has not been previously reported,which may provide a valuable research point for the current anti-tumor immune combination therapy based on the immune checkpoint.To sum up,improving the accuracy of guidance and monitoring with ablation of HCC,exploring different ablation methods lead to the change of immune response and the influence of new immunotherapy targets,and making the ablation and immunotherapy more effective combination to reduce postoperative tumor recurrence probability,which make clinical ablation combined immunotherapy of liver cancer to get more detailed and effective theory and clinical basis.The paper consists of three parts,the abstract are as follows:Part 1:Clinical application of multimodal MR-guided and monitored precision microwave ablation for HCCPurposeTo explore the feasibility,safety and effectiveness of precision microwave ablation with MRI multimodal guidance and monitoring ofMethodsCollect patients receiving MR-guided and monitoring MWA ablation therapy in our hospital from 2017.01 to 2019.05,of these,45 patients met the enrollment criteria,(men 34,women 11;mean age 58.2±8.5 years,range:40-72 years old),Contains 56 liver lesions(10 primary hepatocellular carcinoma,46 recurrent hepatocellular carcinoma;38 are located in the right lobe,15 are located in the left lobe,3 are located in the caudate leaves).Lesions were divided into 3 groups according to their diameter:17 lesions in<1.0cm group,19 lesions in 1.02.0cm group,and 20 lesions in>2.0cm group.The mean diameter of the liver lesion was 1.7 ±,0.9 cm(range 0.5-4.6 cm).T2WI-TSE,Thrive,DWI and T1WI-FFE are guided and monitored.Technical success rate,technical success rate,local tumor progression rate,surgical duration,and complications were evaluated after surgery.Technical success is defined as a precise targeting of the tumor and a complete ablation of the tumor tissue,and a single technical effectiveness rate is defined as a complete tumor ablation effect achieved within 3 months after the initial MWA ablation treatment.Local tumor progression was defined as recurrence of the tumor surrounding the ablation area more than 3 months after MWA ablation.Follow-up time to assess response to treatment ranged from 12 to 30 months(median of 23 months).ResultThe technical success rate of HCC microwave ablation was 100%,and the single technical response rate was 92.8%(52/56).Ten of these lesions cannot be clearly displayed on US images due to the influence of cirrhosis and US sound window(6<1cm diameter,41-2c m diameter),however,the lesions can be clearly visualized by MR and complete ablation were achieved.6 lesions with>3cm diameter are not completely ablated by WMA treatment under US or CT guidance,but completely inactivated under the precise guidance and monitoring of MR multiple sequence and any orientation imaging.During the follow-up procedure,only three lesions revealed local tumor progression,with a time interval of 7,9,and 12 months,respectively.The local progression rate was just 5.3%(3/56),LTP of<1.0cm,1.0-2.0cm and>2.0cm was 0/17(0%),0/18(0%)and 3/20(15%)respectively.The mean duration of each tumour treatment during the interventional process was 66 minutes(range 40-156 minutes).There was no significant difference in the technical success rate,the technical response rate,and the rate of local tumor progression,respectively(P>0.05).There were no serious intraoperative complications,only three patients underwent pleural effusion drainage after ablation,and four patients developed perihepatic bleeding of self-limited small amounts without additional treatment.ConclusionMR guidance and monitoring of HCC microwave ablation treatment can achieve accurate control of all aspects of the ablation,and effectively improve the complete ablation rate of tumors.Part 2:Comparative study of antitumor immunity induced by radiofrequency microwave and cryoablationPurposeTo compare the immune changes of MWA,RFA and CRYO,and clarify the difference in immune response and the time node of immune change after ablation with the C57 mouse bilateral subcutaneous tumor-forming HCC model.Methods6-8week-old C57BL/6 male mice were used,and hepa1-6 HCC cells were expanded at 5× 107/ml concentration of was injected into the bilateral axilla,200ul on each side,tumor size was measured three times per week,and tumor volume was calculated using the formula(AxB 2)× 0.4,where A was the largest diameter and B was the shortest diameter.Tumor bearing mice were divided into four groups,including control group(n=9),microwave ablation group(n=15),radiofrequency ablation group(n=15),cryoablation group(n=15),and a blank group(n=3).The mice of control group were dissected from the spleen tissue and the tumor tissue before surgery,and three mice in the ablation groups were performed under ultrasound guidance.Mice in the three ablation groups underwent ultrasound-guided ablation of one axillary tumor.3 mice in the three ablation groups were sacrificed at 1,2,3 and 4 weeks after operation,respectively,and the spleen tissue and the tumor tissue of the unablated side were dissected.After treatment of the obtained tissues:1.To test the proportion of CD8+T cells(the main anti-tumor immune cells with adaptive immune response),NK cells(the most important antitumor immune cells with innate immune response),and Treg cells(the main immunosuppressive cells in the body)by flow cytometry,contrast the differences in the change of immune cell proportions after the three ablation procedures,and the regulation of immune change;2 To detect PD-L1 protein expression in tumor tissues by Western Blot,the differences in the changes of PD-L1 protein expression after the three ablation procedures was observed.Result1.The results of mouse HCC molding show that the second week after hepa1-6 cell implantation is the largest time point for their natural growth volume,and the maximum diameter range from 8mm to 15mm.2.The proportion of immune cells showed an increase in the TIL and spleen with CD8+T cells and NK cells after the three ablation procedures compared with preoperative surgery(P<0.05).The pattern of immune changing showed continuous increasing in the first three weeks after ablation,entered a flat phase in week 3-4,and the fourth week showed a downward trend;the proportion of Treg cells decreased compared with before surgery(P<0.05).Treg in CRYO ablation group decreased in the first three weeks and increased in the fourth week,But,the proportion of Treg in RFA and MWA ablation groups was continuously down-regulated.3.Change of immune cells showed as to:(1)Compared with RFA and MWA,CRYO caused the most significant i ncrease in CD8+Tcells,while RFA was better than MWA.The comparison data is as follows(spleen tissue CRYO37.45%vsRFA32.8%,P=0.029;CRYO 37.45%vsMWA30.15%,P=0.029;MWA30.15%vsRFA32.8%,P=0.114),(tumor t issue,CRYO34.65%vsRFA31.92%,P=0.149;CRYO34.65%vsMWA24.87%,P=0.029;MWA24.87%vsRFA31.92%,P=0.043).(2)There was no significant difference in the proportion of Treg cells after the three ablation methods(P>0.05),however,CRYO led to the proportion of Treg cells with the most obvious downward trend.(3)There was no significant difference in the proportion of NK cells after the three ablation methods(P>0.05),But compared to the CRYO,In TIL,RFA and MWA led to a higher trend in the proportion of cells.4.The results of PD-L1 protein expression in tumor tissues showed that:(1)The three ablation procedures could lead to the downregulation of PD-L1 protein expression comparing with the preoperative comparison(P<0.05),While patterns of their immune change are the same,the first three weeks showed continuous downregulation after ablation,an inflection point occurs at the 3-4 weeks,the fourth week shown the trend of upregulation.(2)the comparison between groups,no significant difference in PD-L1 protein expression changes after the three ablation methods(P>0.05).However,the downregulation trend of PD-L1 protein expression caused by CRYO was more obvious than that of RFA and MWA.ConclusionIn the HCC mouse model,the immune changes after radiofrequency ablation,microwave ablation and cryoablation are consistent,and the time node of immune changes occurs in 3-4 weeks.Among them,the adaptive immune response stimulated by cryoablation is the strongest,and the adaptive immune response stimulated by radiofrequency ablation is better than that of microwave ablation.Part 3:Studies on the regulation of TIGIT/CD155 signaling pathway by radiofrequency,microwave and cryoablationPurposeTo further explore the effect of three ablation methods on TIGIT/CD155,a novel immunotherapeutic target.MethodsDescribed in the second part,after successful moding and ablation therapy,3 mice were sacrificed at postoperative 1,2,3 and 4 weeks in each ablation group,untreated side tumors and spleen tissue were also dissected,after selecting some tissues for treatment:1.Test the proportion change of TIGIT+T cells in tissues by flow cytometry;2.To test expression of TIGIT,CD 155,and PD-L1 proteins in tumor tissues by western blot,the trend of TIGIT change after different ablation was observed,and the correlation between TIGIT change and the expression of immune cells and PD-L1 was analyzed.Result1.The change of the TIGIT+T ratio in the spleen tissues and the unt reated tumor tissues after three ablation was decreased comparing with the preoperative TIGIT+T ratio(P<0.05).Compared with RFA and MWA,cr yoablation downregulate the ratio of TIGIT+T most significantly,but there is no significant difference between radiofrequency ablation and microwave ablation.The comparison data is as follows(spleen tissue,CRYO9.25vsRFA 17.45,P=0.083;CRYO9.25vsMWA16.5,P=0.043;RFA17.45vsMWA16.5,P=1.00),(TIL,CRYO 9.52vsRFA23.55,P=0.021;CRYO9.52vsMWA15.48,P=0.343;RFA23.55vsMWA15.48,P=0.200).2.The results of the correlation between TIGIT+T cells and immune cell changes showed that with the downregulation of TIGIT+Tcells,CD8+T cells and NK cells were gradually upregulated after ablation,while the expression trend of Treg cells was basically consistent with that of TIGIT+Tcells,and both showed continuous downregulation.3.The results of correlation between the expression of immune checkpoint protein showed that the changes of TIGIT/CD155 signaling protein and PD-L1 protein in the three ablation tumor tissues were similar.In the first three weeks after ablation,there were downregulation trend of different degrees,the node of immunological change appeared in the 3-4 weeks,and the expression was upregulated in the fourth week.ConclusionIn the HCC mouse model,RFA,MWA and CRYO can downregulate the expression of TIGIT/CD155 signaling pathway proteins.Among them,cryoablation downregulate the expression of protein most significantly,but there is no significant difference between radiofrequency ablation and microwave ablation.Meanwhile,the expression of TIGIT and PD-L1 proteins in tumors after ablation is consistent,and the time node of immune change occurs at 3-4 weeks,which provides a strong theoretical support for us to explore the timing of ablation combined with immune checkpoint inhibitors.