Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein(RNP)electroporation

Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising cost.Furthermore,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in cattle.In this point of view,electroporation in embryos has been risen as an alternative.Results First,editing efficiency of our electroporation methods were tested for embryos.Presence of mutation on embryo was confirmed by T7E1 assay.With first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,respectively.In case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for BLG.Afterwards,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing.Thirteen recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double KO.Ten surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both genes.Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied.Finally,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.

Irreversible electroporation for the management of pancreatic cancer: Current data and future directions

Pancreatic cancer is currently the seventh leading cause of cancer death(4.5%of all cancer deaths)while 80%-90%of the patients suffer from unresectable disease at the time of diagnosis.Prognosis remains poor,with a mean survival up to 15 mo following systemic chemotherapy.Loco-regional thermal ablative techniques are rarely implemented due to the increased risk of thermal injury to the adjacent structures,which can lead to severe adverse events.Irreversible electroporation,a promising novel non-thermal ablative modality,has been recently introduced in clinical practice for the management of inoperable pancreatic cancer as a safer and more effective loco-regional treatment option.Experimental and initial clinical data are optimistic.This review will focus on the basic principles of IRE technology,currently available data,and future directions.

Irreversible electroporation of the pancreas-A decade on

Irreversible electroporation(IRE) employs the use of an electric field to cause irreversible permeability of the cell membrane, inducing apoptosis. The use of IRE for locally advanced pancreatic cancer(LAPC) was first described in 2012. The crucial advantage of IRE compared with other devices employing thermal ablation is the safety around vital structures such as vessels and ducts. This makes it an attractive option for use in the pancreas due to the close proximity of multiple major vascular structures, biliary ducts, and adjacent gastrointestinal organs. Over the past decade, IRE has established itself as a useful treatment adjunct and may soon become the standard of care, particularly for LAPC. This article will explore the current evidence and provide a concise summary of pertinent issues, including patient selection, preoperative management, clinical outcomes, radiological response and future prospects of IRE in pancreatic cancer.

Transformation of Metallothionein Gene into Mushroom Protoplasts by Application of Electroporation

Metallothionein gene (MT) has been transferred into mushroom protoplasts by electroporation. It is a low molecular weight, cysteine-rich and metal-binding protein. MT can bind metals. Its synthesis is induced by Zn ion. Thus the expression of MT gene in mushroom can improve the accumulation of Zn in this fungus. This transgenic mushroom, consumed as a kind of vegetable, can supply the necessary Zn to people who are short of the element. When protoplasts were prepared, the concentration (C) of protoplasts is 6.745 x 10(6) /mL. After protoplast electroporation, the transformation rate of protoplasts is 0.01 %. Polymerase chain reaction (PCR) analysis showed that the gene had been integrated into the mushroom chromosome, SDS-PAGE, Western blot analysis indicated that the MT gene had been expressed in the transgenic mushroom. The expressing level, detected by ELISA, is 0.6 % - 0.8 %. Tested for metal resistance, the wild-type mushroom growth was inhibited on die medium containing 1.0 - 1.2 mmoL/L ZnSO4. While the transgenic mushroom was inhibited on the medium containing 1. 5 - 2.0 mmol/L ZnSO4. The mycelium can develop into hymenophore in the medium of rice bran: sawdust = 1: 3, and not in the medium of rice bran: sawdust = 1: 4.

Gene Transfer into Young Embryos via Electroporation and Regeneration of Plantlets in Rice

Green fluorescent protein ( GFP ) gene was expressed transiently in 2-3 d old rice embryos by electroporation with the aid of a specially designed loading net. Under suitable conditions (500 μF capacitance, 300 V/cm Voltage, 100 μg/mL plasmid DNA), the percentage of embryos expressing GFP was up to 35%. The highest electroporation efficiency (40%) was obtained at pH 5.8 of the electroporation buffer. The GFP gene driven by the Ubi promoter produced the highest efficiency. Thus, on the basis of optimizing electroporation conditions, a transformation system has been developed for young embryos in rice. The electroporated 4-6 d old embryos regenerated plantlets under the controlled cultural conditions. Fluorescence microscopic observations indicated that GFP gene expressed in their calli and R0 plantlets.

Is irreversible electroporation safe and effective in the treatment of hepatobiliary and pancreatic cancers?

Background: Irreversible electroporation(IRE) is a novel ablative technique for hepatobiliary and pancreatic cancers. This review summarizes the data regarding the safety and efficacy of IRE in the treatment of hepatobiliary and pancreatic cancers. Data sources: Studies were identified by searching Pub Med and Embase for articles published in English from database inception through July 31, 2017. For inclusion, each clinical study had to report morbidity and survival data on hepatobiliary and pancreatic cancers treated with IRE and contain at least 10 patients. Studies that met these criteria were included for analysis. Two authors assessed each clinical study for data extraction. The controversial parts were resolved through discussion with seniors. Results: A total of 24 clinical studies were included. Fourteen focused on hepatic ablation with IRE comprising 437 patients with 666 lesions of different tumor types. Two patients(0.5%) died after the IRE procedure. Morbidity of hepatic ablation with IRE ranged from 7% to 35%. Most complications were mild. Complete response for hepatic tumors was reported as 57%–97%. Ten studies with 455 patients focused on pancreatic IRE. The overall mortality of IRE in pancreatic cancer was 2%. Overall severe morbidity of IRE in pancreatic cancer ranged from 0 to 20%. The median overall survival after IRE ranged from 7 to 23 months. Patients treated with IRE combined with surgical resection showed a longer overall survival. Conclusions: IRE significantly improves the prognosis of advanced hepatobiliary and pancreatic malignances, and companied with less complications. Hence, IRE is a relatively safe and effective non-thermal ablation strategy and potentially recommended as an option for therapy of patients with hepatobiliary and pancreatic malignances.

Postoperative inflammation as a possible cause of portal vein thrombosis after irreversible electroporation for locally advanced pancreatic cancer

Portal vein thrombosis(PVT) is a rare but serious postoperative complication associated with irreversible electroporation(IRE). We report a case of postoperative PVT in a 54-year-old woman who underwent IRE for locally advanced pancreatic cancer. Drain removal and discharge of the patient from the hospital were scheduled on postoperative day(POD) 7; however, a magnetic resonance imaging scan revealed the presence of PVT. We suspected postoperative inflammation in the pancreas as the main cause of PVT. However, the patient did not undergo any medical treatment because she did not have any clinical symptoms, and she was discharged on POD 8.

Optimization of Electroporation Parameters for Immature Embryos of indica Rice (Oryza sativa)

To obtain a suitable condition for electroporation transformation in indica rice, the 10-day-old immature embryos were selected for optimization experiments. The results showed that one pulse at 850 V/cm, 950μF capacitance, 200 μL electroporation buffer with 70 mmol/L sodium glutamate, 100 μg/mL plasmid, 50μg/mL carrier DNA, 20 embryos per cuvette, 0℃ treatment and CC medium were the best parameters, which not only improved the transformation efficiency to 30.89%, but also ameliorated the embryo survival ratio to 95.92%. To further verify the practicability of this condition, the embryos from another indica rice variety and a rice type Ⅱ metallothionein-like gene （OsMT2bL） promotec：mgfp5：：gusA construct were tested, and specific GUS expression on the embryos was visualized by histochemical staining. The results showed that the GUS expression on the embryos activated by the OsMT2bL promoter was mainly concentrated on the apical point of the plumule whereas the expression driven by CaMV35S promoter was distributed on nearly all areas of the electroporated tissues. These results indicated that the optimized embryo electroporation conditions could be used not only in genetic transformation of indica rice but also in assay of gene regulation on embryos.

Phase Ⅱb trial of in vivo electroporation mediated dualplasmid hepatitis B virus DNA vaccine in chronic hepatitis B patients under lamivudine therapy

AIM To assess the efficacy and safety of in vivo electroporation(EP)-mediated dual-plasmid hepatitis B virus(HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine(LAM) in patients with chronic hepatitis B. METHODS Two hundred and twenty-five patients were randomized to receive either LAM + vaccine(vaccine group, n = 109) or LAM + placebo(control group, n = 116). LAM treatment lasted 72 wk. Patients received the DNA vaccine or placebo by intramuscular injection mediated by EP at weeks 12(start of treatment with vaccine or placebo, SOT), 16, 24, and 36(end of treatment with vaccine or placebo, EOT). RESULTS In the modified intent-to-treat population, morepatients had a decrease in HBV DNA > 2 log10 IU/m L in the vaccine group at week 12 after EOT compared with the control group. A trend toward a difference in the number of patients with undetectable HBV DNA at week 28 after EOT was obtained. Adverse events were similar. In the dynamic per-protocol set, which excluded adefovir(ADV) add-on cases at each time point instantly after ADV administration due to LAM antiviral failure, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 and 28 after EOT compared with the control group. More patients with undetectable HBV DNA at week 28 after EOT in the vaccine group were also observed. Among patients with a viral load < 1000 copies/mL at week 12, more patients achieved HBeA g seroconversion in the vaccine group than among controls at week 36 after EOT, as well as less virological breakthrough and YMDD mutations. CONCLUSION The primary endpoint was not achieved using the HBV DNA vaccine. The HBV DNA vaccine could only be beneficial in subjects that have achieved initial virological response under LAM chemotherapy.

Delivery of CatSper2 siRNA into Rat Sperms by Electroporation Repressed Ca^(2+) Influx During Sperm Hyperactivation

CatSper is a unique Ca2＋ channel-like protein family exclusively expressed in the testis and sperm, and plays important roles in sperm motility, capacitation, acrosome reaction and sperm-egg interactions. Here we studied the mechanism of regulation of CatSper2-dependent Ca〉 influx, extracellular and intracellular Ca2＋on sperm hyperactivated motility. The siRNA duplexes were transfected into the sperm cells by electroporation （EP） to silence the expression of CatSper2. The results for targeted disruption of CatSper2 showed the highest silence efficiency 77.7% （P〈0.05）, the hyperactivated sperm rate calculated by computer-assisted semen analysis （CASA） analysis of interferenced sperm was significantly lower 11.1% than the control 99.2%. Flow cytometry （FCM） detection of the intracellular Ca2＋ concentration of interferenced sperm was 50 nmol L-t higher than the normal. It was remarkably lower than hyperactivated sperm with 200-1 000 nmol L-1 （P〈0.05）. It was not sufficient to evoke hyperactivation. To trigger hyperactivation, the sperm were incubated in 50 pmol L-t thimerosal or 5 mmol L-1 procaine, it sharply increased the intracellular Ca2＋ via two different channels. CASA and FCM detection indicated that intracellular Ca〉 is required for initiating hyperactivation while extracellular Ca2＋ is essential to maintain the process. We concluded that to mediate sperm hyperactivation, it is essential to inhibit Ca〉peak present with targeted disruption of CatSper2 expression. This provides important prospective for further exploration of signal channel of sperm hyperactivated motility, potential factors for male infertility and provide further reference to the exploration of male contraceptive drug targets of male reproduction.

Electroporation: A New Approach Enhancing Antitumor Effects of Cytoxan

Electrochemotherapy (ECT) is a novel cancer treatment in which electric pulses (EPs) inducing cell membrane pored (electroporation) are used as a means of delivering antitumor drugs to the cytoplasm of cancer cells. In vitro, with scan electromicroscope (SEM) and Trypan blue staining examination, the best parameter of EPs of electroporation is studied by the S-180 cells exposed to EP with various voltages, pulses , capacitance. The best parameter of EP of electroporation is 600V/cm, 6 pulses, 10 μF. In the in vivo study, ECT is studied with the Cytoxan (CTX) injected directly into the tumor followed immediately by a local EP at the tumor site. Four parameters, which include the tumor inhibitory ratio, the curing ratio and the vas capillare of tumor, the tumor’s histopathological characteristics are determined and compared among the ECT group, the control group, the EP-only group and the drug-only group. The results indicate that the antitumor effect of CTX is significantly enhanced by electroporation.

ETM study of electroporation influence on cell morphology in human malignant melanoma and human primary gingival fibroblast cells

Objective:To estimate electroporation(EP) influence on malignant and normal cells.Methods: Two cell lines including human malignant melanoma(Me-43) and normal human gingival fibroblast(HCFs) were used.EP parameters were the following:230,1000,1 730,2 300 V/cm;30 μ s by 3 impulses for every case.The viability of cells after EP was estimated by MTT assay. The ullrastructural analysis was observed by transmission electron microscope(Zeiss EM 900). Results:In the current study we observed the intracellular effect following EP on Me-43 and HGF cells.At the conditions applied,we did not observe any significant damage of mitochondrial activity in both cell lines treated by EP.Conversely,we showed that EP in some conditions can stimulate cells to proliferation.Some changes induced by EP were only visible in electron microscopy.In fibroblast cells we observed significant changes in lower parameters of EP(230 and 1 000 V/cm).After applying higher electric field intensities(2 300 V/cm) we detected many vacuoles,myelin-like bodies and swallowed endoplasmic reticulum.In melanoma cells such strong pathological modifications after EP were not observed,in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters of EP.Conclusions:We can claim that EP conditions are cell line dependent.In terms of the intracellular morphology,human fibroblasts are more sensitive to electric field as compared with melanoma cells.Optimal conditions should be determined for each cell line.Summarizing our study,we can conclude that EP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.

Gemcitabine plus concurrent irreversible electroporation vs gemcitabine alone for locally advanced pancreatic cancer

BACKGROUND Locally advanced pancreatic cancer(LAPC)is a common malignant digestive system tumor that ranks as the fourth leading cause of cancer-related death in the world.The prognosis of LAPC is poor even after standard treatment.Irreversible electroporation(IRE)is a novel ablative strategy for LAPC.Several studies have confirmed the safety of IRE.To date,no prospective studies have been performed to investigate the therapeutic efficacy of conventional gemcitabine(GEM)plus concurrent IRE.AIM To compare the therapeutic efficacy between conventional GEM plus concurrent IRE and GEM alone for LAPC.METHODS From February 2016 to September 2017,a total of 68 LAPC patients were treated with GEM plus concurrent IRE(n=33)or GEM alone(n=35).Overall survival(OS),progression free survival(PFS),and procedure-related complications were compared between the two groups.Multivariate analyses were performed to identify any prognostic factors.RESULTS There were no treatment-related deaths.The technical success rate of IRE ablation was 100%.The GEM+IRE group had a significantly longer OS from the time of diagnosis of LAPC(19.8 mo vs 9.3 mo,P<0.0001)than the GEM alone group.The GEM+IRE group had a significantly longer PFS(8.3 mo vs 4.7 mo,P<0.0001)than the GEM alone group.Tumor volume less than 37 cm3 and GEM plus concurrent IRE were identified as significant favorable factors for both the OS and PFS.CONCLUSION Gemcitabine plus concurrent IRE is an effective treatment for patients with LAPC.

Arterial complication of irreversible electroporation procedure for locally advanced pancreatic cancer

Irreversible electroporation(IRE) is a non-thermal ablation technique used especially in locally advanced pancreatic carcinomas that are considered surgically unresectable.We present the first case of acute superior mesenteric artery(SMA) occlusion secondary to pancreatic IRE procedure that has not been reported before in the literature.A 66-year-old man underwent neoadjuvant chemoradiotherapy for locally advanced pancreatic ductal adenocarcinoma.IRE procedure was applied to the patient during laparotomy under general anesthesia.After finishing the procedure,an acute intestinal ischemia was detected.A conventional vascular angiography was performed and a metallic stent was successfully placed to the SMA and blood flow was maintained.It is important to be careful in such cases of tumor involvement of SMA when evaluating for IRE procedure of pancreatic tumor.

Safety and feasibility of irreversible electroporation for the pancreatic head in a porcine model

BACKGROUND Irreversible electroporation(IRE)is a local non-thermal ablative technique which has been suggested as a potential cancer therapy.However,the specific anatomic characteristics of the pancreatic head make it challenging to perform any local ablation in this region.Therefore,the safety and feasibility of IRE in the pancreatic head region should be further explored.AIM To evaluate the safety of IRE in pancreatic head region including its effects on pancreatic ducts,vessels,and adjacent gastrointestinal organs.METHODS Eight landrace miniature pigs underwent IRE of pancreatic head tissue successfully,with a total of 16 lesions created.Laboratory testing including white blood cell(WBC)count and serum amylase before IRE with follow-up laboratory analysis and pathological examination at 1,7,14,and 28 d postablation were performed.RESULTS All pigs tolerated the ablation procedure without serious perioperative complications.Transiently elevated WBC count and amylase were observed at 24 h post-IRE,suggesting an acute pancreatic tissue damage which was confirmed by pathological observations.Vascular endothelial cells and pancreatic duct epithelial cells in ablation zone were also positive in terminal deoxynucleotidyl transferase dUTP nick end labeling staining.There was extensive duodenum mucosa damage with local hemorrhage 24 h after ablation,while regeneration of new villous structures were observed at 7 and 28 d post-IRE.Masson’s trichromatic staining showed that the extracellular matrix was still intact in vessels and pancreatic ducts,and even in the duodenum.CONCLUSION IRE ablation to the pancreatic head may be safe and feasible without long-term damage to the surrounding vital structures.However,risks of stress injuries in acute phase should be taken into consideration to prevent severe perioperative complications.

Transdermal Drug Delivery by Electroporation： The Effects of Surfactants on Pathway Lifetime and Drug Transport

Electroporation creates aqueous pathways by short high-voltage pulses resulting in a transient perme- abilization of stratum corneum and an increase in the transdermal delivery rate.However the aqueous pathways will reseal after pulsing,which leads to the rapid drop of transdermal flux.In the present study,the surfactants were added to the donor solution to hinder the shrinkage and resealing of the electropore,and to prolong the lifetime of the aqueous pathways with the consideration that the surfactants could reduce the surface energy of the electropore. These effects of surfactants were demonstrated by the dynamic electrical resistance of the skin and the fluorescent imaging of the local transport regions.Piroxicam(PIX)was transported percutaneously in the presence of surfac- tants in vitro.Owing to the longer lifetime of aqueous pathways,together with the promotion of PIX availability at the barrier exterior and the improvement in the partition of PIX into the aqueous pathways,the presence of surfac- tants led to a remarkable increase in the transdermal delivery rate during electroporation and a significant growth of the accumulative transdermal amount of PIX.

Genetic transformation of marine cyanobacterium Synechococcus sp.CC9311(Cyanophyceae) by electroporation

Synechococcus sp.CC9311 is a marine cyanobacterium characterized by type IV chromatic acclimation(CA).A genetic transformation system was developed as a first step to elucidate the molecular mechanism of CA.The results show that Synechococcus sp.CC9311 cells were sensitive to four commonly used antibiotics:ampicillin,kanamycin,spectinomycin,and chloramphenicol.An integrative plasmid to disrupt the putative phycoerythrin lyase gene mpeV,using a kanamycin resistance gene as selectable marker,was constructed by recombinant polymerase chain reaction.The plasmid was then transformed into Synechococcus sp.CC9311 via electroporation.High transformation efficiency was achieved at a field strength of 2 kV/cm.DNA analysis showed that mpeV was fully disrupted following challenge of the transformants with a high concentration of kanamycin.In addition,the transformants that displayed poor growth on agar SN medium could be successfully plated on agarose SN medium.

Irreversible electroporation for liver metastasis from pancreatic cancer: A case report

BACKGROUND Pancreatic cancer has a poor prognosis;40%–50% of patients have liver metastases at the time of initial diagnosis and only 15%–20% undergo surgical resection. Irreversible electroporation(IRE) is a new, non-thermal local ablation method for solid tumors, which can induce cell membrane permeabilization,resulting in unrecoverable nanoscale perforation and apoptotic cell death without damaging the structural components of tissues.CASE SUMMARY We report the case of a 66-year-old female patient with liver metastasis from pancreatic cancer with a pathological diagnosis of poorly differentiated adenocarcinoma. Carbohydrate antigen 19-9 was elevated to 420.3 U/m L.Computed tomography showed a pancreas mass of 2.7 cm × 2.5 cm and single liver metastasis of 1.4 cm × 1.1 cm in the S6 area. The patient underwent IRE and arterial infusion chemotherapy and received tegafur. The therapeutic effect of the combination treatment has been evaluated as complete response. To date, the patient has survived for > 12 mo and is receiving tegafur as maintenance therapy(at the time this case report was written).CONCLUSION IRE plus arterial infusion chemotherapy and tegafur may be synergistic,providing a reference for treating liver metastasis from pancreatic cancer.

Irreversible electroporation versus radiofrequency ablation for malignant hepatic tumor:A prospective single-center double-arm trial

Objective:Irreversible electroporation(IRE)is a nonthermal ablation technique for the treatment of malignant liver tumors.IRE has demonstrated efficacy and safety in the treatment of malignant liver tumors and its unique advantages in the treatment of nearby vascular lesions.This study aimed to compare the efficacy,safety,and intermediate-term outcomes of IRE and radiofrequency(RF)therapy in malignant liver tumors.Methods:Twenty-four patients with primary or secondary liver malignancies were included in this prospective,double-arm clinical trial.Patients were randomly divided into the IRE and RF groups.The primary outcome was the efficacy(local ablation control evaluation at 90 days).The secondary outcomes were safety(procedure-related complications at≤90 days)and intermediate-term survival(at 24 months).Results:The ablation assessment at 90 days after surgery with m RECIST for IRE versus RF were 70%,20%,0%,and 10%versus 92.9%,7.1%,0%,and 0%(CR,PR,SD,and PD,respectively).The complication rates of IRE versus RF with Clavien-Dindo classification were 16.7%,25%,0%,8.3%,and 8.3%versus 8.3%,50%,0%,0%,and 0%(Grade I,II,Ⅲ,IV,and V,respectively).The average overall survival(OS)was 17.55 months in the IRE group(95%CI 15.13-22.37)and 18.75 months in the RF group(95%CI 12.48-22.61).There was no statistical difference between the IRE and RF groups in terms of efficacy(p=0.48),safety(p=0.887),or 24-month OS(p=0.959).Conclusions:IRE ablation revealed similar efficacy and safety in a short-term follow-up,and similar OS in midterm survival as RF ablation in treating malignant hepatic tumors.

Effectiveness and safety of irreversible electroporation for recurrent hepatocellular carcinoma ineligible for thermal ablation after surgery

Objectives:To preliminarily evaluate the clinical effectiveness and safety of computed tomography(CT)imageguided irreversible electroporation(IRE)for the treatment of recurrent hepatocellular carcinoma(HCC)after surgical resection.Methods:From January 2016 to February 2018,18 patients diagnosed with recurrent HCC after surgical resection received IRE under CT image guidance for 22 tumors.Patients were enrolled for IRE when ineligible for thermal ablation due to tumor location.Clinical records and imaging data were reviewed to assess complete ablation rate,local tumor progression free rate(LTPFR),local tumor progression free survival(LTPFS)and complications after a median follow-up time of 14 months.Results:Successful complete ablations were achieved in 20/22(90.1%)tumors.Mean LTPFS was 10.5?9.4 months.Overall 3-,6-and 12-months LTPFR in 22 tumors following IRE were 68.2%(95%confidence interval[CI]:45%–83%),59.1%(95%CI:33%–76%)and 36.4%(95%CI:17%–56%),respectively.Complications included pneumothorax(2/18,11.1%),localized pain(3/18,16.7%),bile duct dilation(1/18,5.6%)and transient hypertension(1/18,5.6%).No major complications or treatment-related deaths were observed.The alphafetoprotein levels of two patients decreased to the normal range at 3 and 4 months,respectively.Conclusions:This study showed that percutaneous CT image-guided IRE can serve as a safe and effective treatment for recurrent HCC not suitable for thermal ablation.