Preclinical Study of Locoregional Therapy of Hepatocellular Carcinoma by Bioelectric Ablation with Microsecond Pulsed Electric Fields (ŒºsPEFs)
Authors: Xinhua Chen
Jihong Sun
Xinmei Chen
Chenguo Yao
Shusen Zheng
Jianwen Jiang
Zhigang Ren
Dianbo Zhou
Fei Guo
Chengxiang Li
Journal: Scientific Reports (2015)
Discipline: Multidisciplinary
Keywords: Unresectable hepatocellular carcinoma; Microsecond Pulsed Electric Fields; Xenograft tumor model; Tumor volume; Mitochondria function
Affiliations: 1] The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310003, China [2] Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, Zhejiang, 310003, China., The State Key Laboratory of Power Transmission Equipment &System Security and New Technology, Chongqing University, Chongqing, 400030, China., The Department of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, 250014, China., The Department of Radiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310003, China.

Abstract:  Unresectable hepatocellular carcinoma (HCC) needs locoregional ablation as a curative or downstage therapy. Microsecond Pulsed Electric Fields (ŒºsPEFs) is an option. A xenograft tumor model was set up on 48 nude mice by injecting human hepatocellular carcinoma Hep3B cells subcutaneously. The tumor-bearing mice were randomly divided into 3 groups: ŒºsPEFs treated, sham and control group. ŒºsPEFs group was treated by ŒºsPEFs twice in 5 days. Tumor volume, survival, pathology, mitochondria function and cytokines were followed up. ŒºsPEFs was also conducted on 3 swine to determine impact on organ functions. The tumors treated by ŒºsPEFs were completely eradicated while tumors in control and sham groups grew up to 2 cm(3) in 3 weeks. The ŒºsPEFs-treated group indicated mitochondrial damage and tumor necrosis as shown in JC-1 test, flow cytometry, H&amp;E staining and TEM. ŒºsPEFs activates CD56+ and CD68+ cells and inhibits tumor proliferating cell nuclear antigen. ŒºsPEFs inhibits HCC growth in the nude mice by causing mitochondria damage, tumor necrosis and non-specific inflammation. ŒºsPEFs treats porcine livers without damaging vital organs. ŒºsPEFs is a feasible minimally invasive locoregional ablation option. 

Retracted: No

PubPeer comments:
 #1 There was some trouble in the last posting. Not sure why. Some more context in this reply. At least according to these UK-based guidelines, the tumours should not exceed 1.5 cm diameter and necrotic mice should be terminated within two days https: www.ncbi.nlm.nih.gov pmc articles PMC2883160 It beggars belief that the referees and the handling editor could have accepted a paper that claims a total cancer cure by pulsed electromagnetic fields? Surely it is now very well known that electromagnetic fields are supposed to be causing cancer instead of curing it? https: pubpeer.com publications 86E709835E96908738330ED4B849CC The previous nude mouse presentation in this series introduced human iPS cell xenografts that had induced a stereo pair of ginormous teratomas https: pubpeer.com publications 486E79344CA66E3E96F53994703A27 , 
 #2 If we ignore, for the moment, the implications of the nude mouse image duplications, then the reported cancer-curing mechanism is that *u201cmicrosecondPEFs inhibits HCC growth in the nude mice by causing mitochondria damage, tumor necrosis and non-specific inflammation.u201d* I would be interested in a second opinion on the suggested mechanism. Can we rule out that the tumours were not simply cooked by the electric current between the pair of electrodes, rather than any low temperature effects of the u201celectric fieldsu201d? Does anyone know the specific heat capacity of xenografts in nude mice? And the electrical resistance, thereof, if that is also needed? Electric current was delivered as 90 pulses, once per second, each pulse lasting 100 microseconds. The pulsed electric current was 50 Amps, delivered at 3000 volts. My calculation of max electrical power is that this is a whole bunch of Watts to deliver in 90 seconds between two electrodes inserted lt;10 mm apart into a blob of tumour. How much of that electrical power would convert to heat energy? And what would be the rise in temperature for a tumour volume of say 1 cm3? I ask because, in my opinion, a cooked tumour attached to a nude mouse would tick several boxes: It would have damaged mitochondria; it would ulcerate; and there might also be some non-specific inflammation. Iu2019m actually quite surprised that the mice could even survive the very strong electric shocks they were being subjected too - but this is way outside my expertise and I am not minded to undertake validation experiments. Apart from anything else, I canu2019t see our ethical review committee being convincedu2026 , 
 The busy Fig. 2D presented to us by the UKu2019s flagship Nature Publishing Group has a lot to see in it. There are tumours that are too large; there are tumours that are claimed to have rapidly and fully necrosed (which canu2019t have been a nice experience for the mice); and there are reused images of several nude mice, some of which manage to perform as both control and treated  , 
 #1 uff081uff09Response: The maximum tumour size in our protocol was set as 2 cm to keep the same criteria as our previous studies in US. The permitted tumor size varies according to the criteria of different research institutes. In 2010, a British research team issued a criterion that no more than 1.2 centimeters should be used in mice, whereas the criteria of American research institutes generally do not exceed 2 centimeters. When making our animal experiment protocol, we followed 3Rs u2014 which refers to replacing and reducing animals in experiments, and refining procedures to make them less harmful. We respect the 3Rs in the way of understanding the special characters of the tumor ablation machined used in this study- pulsed electric field with high voltage. Without a safe space between two electrode needles, high voltage pulsed electric field can breakdown, causing harm both to human and animal. In this study, the tumor must be 1 cm at least to begin the treatment. As shown in figure 1b, the 1.0 cm- tip needle needs enough space. Previous studies confirmed that it required an adequate treatment margin of 2.0 cm. The reference was quoted as the following. u201cWe commonly will place the most lateral needle (probe 1u2014Figure 5) within the pancreatic at the most lateral extent. Then using spacers at 2.0 cm intervals we build off that initial needle to ensure adequate treatment margin(s). Once this margin(s) is obtained, one or two needles are then placed anterior in order to obtain complete bracketing of the tumor while allowing the normal non-tumor bearing tissueu2014that being the posterior aspect of the stomach anteriorly, the duodenum laterally and the transverse mesocolon inferiorlyu2014to be left in place.u201d u201cCare should also be undertaken that the maximum needle exposure to perform safe IRE of the pancreas should be 1.0 to 1.5 cm because of the significant fibrotic nature of these tumors and a larger needle exposure will not be tolerated by the gland or the underlying soft tissue to be treated. We have previously published that a greater probe exposure leads to high current conditions and the potential for thermal damage if these high current conditions are allowed to persist. Thus the maximum probe exposure should be 1.5 cm or less (16).tu201c Bower M, Sherwood L, Li Y, et al. Irreversible electroporation of the pancreas: definitive local therapy without systemic effects. J Surg Oncol 2011;104:22-8. Robert C. G. Irreversible electroporation of stage 3 locally advanced pancreatic cancer: optimal technique and outcomes. J Vis Surg. 2015; 1: 4. In our protocol, when tumor is bigger than 2 cm, it is the criteria to euthanize the animal for its welfare. In the control group, when we found there were three mice with tumor size exceeding the 2 cm criteria, we made the record and euthanized them immediately. Due to the 2 cm criteria, the mice in the control group were euthanized. As shown in the surviving curve, the control group ended much earlier than treatment group. In the treatment group, after treatment, the tumor shrinks dramatically and no mice in the treatment group exceed 2 cm, which is why the follow-up can be continued. The 2 cm criterion was strictly kept during post-treatment follow-up. With the 2 cm criterion, we kept safe space between two electrode needles to ensure adequate energy. Because the adequate energy delivered, the tumors in the treatment group were eradicated. If not doing so, the tumor cannot be ablated. Here are the results from other team. They kept the tumor very small (less than 1 cm). To avoid electric arc breakdown, the electric field strength was decreased. As a consequence, even the tumor was smaller; they cannot be completely ablated. Even worse---they got residue tumor and thermal damage. The animals suffer more with the metastasis and secondary treatment. uff082uff09Response: From the initial submission in 2012 to the publication in 2015, we have not found that the pictures have been reused. We were surprised and shocked until you told us that there was a duplication problem in the picture. We immediately checked the original experimental records and data pictures, found the original correct version, and re-updated the representative pictures. New representative pictures are shown in Figures 1 and 2 below. You can see every mouse at every time point had its own original record. Our lab still kept the original record and data between 2010 and 2012. The lab committeemen had checked record. The tumor measurement, record calculation and statistics were straightforward and accurate. When select picture for show, among almost 200 mice pictures at all-time points, the graduate student messed up two similar pictures from the same mouse. The Figure 2d is to combine some representative pictures, but it did not change the statistic result and conclusion. It is a showcase for the some selected mice (it is not showing all experiment animals). With or without this showing picture, the measurement and statistic are the same. As the rule of our lab, we kept all experiment animal pictures, measurement records with scale, follow up records and calculation. According to our original experiment record and data, we found the original figures in this study from 2010 to 2012. Therefore, we corrected the showcase of the bearing-HCC mice, as shown in the following figure 1. Meanwhile, we also presented the original picture when we performed animal experiment and took photos, as shown in the following figure 2. uff083uff09In closing this mail, I would like to highlight the real meaning of doing the animal experiment. In 2011, we designed this animal experiment to test the treatment effect of irreversible electroporation (IRE) therapy on the mice. In 2015, we performed preclinical study in porcine in vivo to test the feasibility and safety of IRE. In 2018, the machine was used in clinic practice to save human life. The machine saved 40 patients with tumor. In these patients, 38 of 40 patients have tumors bigger than 1 cm. Our recent publications on irreversible electroporation (IRE) therapy are listed as follows: [1] Chen X, Ren Z, Li C, Guo F, Zhou D, Jiang J, Chen X, Sun J, Yao C, Zheng S. Preclinical Study of Locoregional Therapy of Hepatocellular Carcinoma by Bioelectric Ablation with Microsecond Pulsed Electric Fields (musPEFs). Sci Rep 2015;5:9851. [2] Chen X, Ren Z, Zhu T, Zhang X, Peng Z, Xie H, Zhou Lin, Yin S, Sun J, Zheng S. Electric Ablation with Irreversible Electroporation (IRE) in Vital Hepatic Structures and Follow-up Investigation. Sci Rep 2015 Nov 9;5:16233. [3] Dong S, Wang H, Zhao Y, Sun Y, Yao C. First Human Trial of High-Frequency Irreversible Electroporation Therapy for Prostate Cancer. Technol Cancer Res Treat. 2018 Jan 1;17 Table 1 The Patients Treatment Information. NotNumber of ElectrodestMaximum Size of Tumor, cmtNumber of PatientstPatients AgetTreatment Time, min 1t2t 1t3t64, 76, 79t 8 2t3t1.0-1.5t13t81, 71, 59, 71, 82, 74, 69, 59, 79, 67, 51, 58, 63t 20 3t4t1.5-2.0t10t76, 68, 73, 57, 65, 81, 84, 85, 73, 78t 30 4t5t2.0-2.5t8t68, 51, 65, 74, 68, 76, 82, 75t 40 5t6t2.5-3.0t6t75, 66, 75, 64, 72, 77t 45 Thanks for your kind email. We appreciate the comments on our research paper. We are open for any criticism and totally understand that higher standard is the key for better research practice. With best regards! , 
 #4 Thank you for replying. This reminds me to pass on another problem I have with Fig. 2D which is that several different rulers have been used to measure the xenografts. For the impressively bulky xenografts of the 32 days **Control Sham**, a solid ruler was used, whereas, for the 32 days necrotic xenografts of the **msPEFs** mice, a transparent ruler was used. And the rulers were differently placed on the mice. The logical inference is that these experimental animals were not measured by the same staff, on the same day, in the same facility, which would imply that the controls were not conducted in parallel with the treatments. And, were that to be the case, they would then not be true controls. , 
 Thank you very much for your attention and reminder. In fact, two different rulers have been used to measure the xenografts on the same mice. We have requested to make a corrigendum and correct the improper figures. Figure 2d was misassembled during the preparation of the manuscript: four tumor images that were selected as the representative pictures among almost 200 mice pictures at all-time points were messed up and presented incorrectly. The corrected Figure 2d appears below as Figure 1. The results and conclusions of the article are unaffected by this correction. The Authors apologize for the error and any inconvenience caused. The corrected Fig. 2D can be found as follows: Figure 1. Robust efficacy of msPEFs against Hep3B xenografts in nude mice. The dynamic process of msPEFs ablation tumor compared with the control and sham groups. The volume presented as mean tumor volume (mm3) at each time point. Two different rulers have been used to measure the xenografts on the same mice, and the original images can be found as follows: Figure 2. Two different rulers have been used to measure the xenografts on the same mice. Thank you very much for your attention and support. Best Regards.  ,