Irreversible Electroporation versus Radiofrequency Ablation: A Comparison of Local and Systemic Effects in a Small-Animal Model.

Purpose To compare both periablational and systemic effects of two mechanistically different types of ablation: thermal radiofrequency (RF) ablation and electroporative ablation with irreversible electroporation (IRE) in appropriately selected animal models. Materials and Methods Animal experiments were performed according to a protocol approved by the Animal Care Committee of Hebrew University. Female C57BL/6 mice (n = 165) were randomized to undergo either RF or IRE ablation of noncancerous normal liver. The inflammatory response, cell proliferation, interleukin 6 (IL-6) levels, and intactness of vessels in the liver were assessed at 6, 12, and 24 hours and at 3, 7, and 14 days after ablation (n = 122 for mechanistic experiments). Systemic effects were then assessed by comparing tumor formation in an Mdr2-knockout (KO) mouse model (n = 15) and tumor growth in a remote BNL 1ME hepatoma xenograft tumor (n = 28). Results were averaged and evaluated by using two-tailed t tests. Results Although RF ablation was associated with a well-defined periablational inflammatory rim, for IRE, the infiltrate penetrated the ablation zone, largely along persistently patent vessels. Peak IL-6 levels (6 hours after ablation) were 10 and three times higher than at baseline for IRE and RF, respectively (P < .03). Mdr2-KO mice that were treated with IRE ablation had more tumors that were 3 mm or larger than mice treated with RF ablation or sham operation (mean, 3.6 ± 1.3 [standard deviation] vs 2.4 ± 1.1 and 2.2 ± 0.8, respectively; P < .05 for IRE vs both RF ablation and sham operation). For BNL 1ME tumors, both RF and IRE liver ablation reduced tumor growth, with a greater effect noted for IRE (1329 mm(3) ± 586 and 819 mm(3) ± 327 vs 2241 mm(3) ± 548 for sham operation; P < .05) that was accompanied by more infiltrating lymphocytes compared with sham operation (7.6 cells per frame ± 1.9 vs 11.2 ± 2.1 vs 0.3 ± 0.1; P < .05). Conclusion Persistent patency of vasculature within the coagulated zone from IRE increases the area and accumulation of infiltrative cells that is associated with a higher serum IL-6 level than RF ablation. These local changes of IRE induce more robust systemic effects, including both tumorigenic and immunogenic effects. (©) RSNA, 2016 Online supplemental material is available for this article.

Oncogenesis: An "Off-Target" Effect of Radiofrequency Ablation.

PURPOSE: To compare hepatocellular carcinoma (HCC) development after radiofrequency (RF) ablation, partial surgical hepatectomy, and a sham operation and to inhibit HCC recurrence after RF ablation in a mouse model of spontaneously forming HCC in the setting of chronic inflammation (ie, the MDR2 knockout model). MATERIALS AND METHODS: Animal experiments were performed according to an approved animal care committee protocol. The authors compared the survival of MDR2 knockout mice (an inflammation-induced HCC model) that underwent RF ablation, 35% partial hepatectomy (ie, left lobectomy), or a sham operation (controls) by using Kaplan-Meier survival curve analysis. Tumor load and tumor frequency in mice that underwent sham operation were further compared with those of mice treated with RF ablation at 1 month after therapy by using a two-tailed Student t test. Liver slices from mice treated with RF ablation were stained for α-smooth muscle actin and Ki-67 to establish the role of liver regeneration in the tumorigenic effect of RF ablation. Finally, tumor load and tumor incidence were evaluated in mice treated with a c-met inhibitor after RF ablation by using the Mann-Whitney U test. RESULTS: Ablation of 3.5% ± 0.02 of the MDR2 knockout mice liver induced increased tumor load (P = .007) and reduced survival (P = .03) in comparison to that of controls, with no significant difference to the 10-fold volume removal of partial hepatectomy. Seven days after RF treatment, the border zone of the coagulation zone was surrounded by α-smooth muscle actin-positive activated myofibroblasts. A significant elevation of hepatocyte proliferation was also seen 7 days after RF ablation in the distant liver (ablated lobe: P = .003; untreated lobe: P = .02). A c-met inhibitor significantly attenuated HCC development in MDR2 knockout mice treated with RF ablation (P = .001). CONCLUSION: Liver regeneration induced by RF ablation facilitates c-met/hepatocyte growth factor axis-dependent HCC tumor formation after treatment in the MDR2 knockout model. Blockage of the c-met/hepatocyte growth factor axis attenuates HCC recurrence, raising the potential for therapeutic intervention to reverse this potentially deleterious tumorigenic effect.

Radiofrequency Ablation: Inflammatory Changes in the Periablative Zone Can Induce Global Organ Effects, including Liver Regeneration.

PURPOSE: To determine the kinetics of innate immune and hepatic response to the coagulation necrosis area that remains in situ after radiofrequency (RF) ablation, the cytokine profile of this response, and its local and global effect on the whole organ in a small-animal model. MATERIALS AND METHODS: A standardized RF ablation dose (70°C for 5 minutes) was used to ablate more than 7% of the liver in 91 C57BL6 mice (wild type) according to a protocol approved by the animal care committee. The dynamic cellular response in the border zone surrounding ablation-induced coagulation and in the ablated lobe and an untreated lobe were characterized with immunohistochemistry 24 hours, 72 hours, 7 days, and 14 days after ablation (the time points at which cells migrate to necrotic tissues). After characterization of the cellular populations that reacted to the RF treatment, cytokines secreted by these cells were blocked, either by using interleukin-6 knockout mice (n = 24) or c-met inhibitor PHA 665752 (n = 15), to elucidate the key factors facilitating the wound healing response to RF ablation. Statistical significance was assessed with nonparametric analysis of variance. RESULTS: RF ablation induces a strong time-dependent immunologic response at the perimeter of the necrotic zone. This includes massive accumulation of neutrophils, activated myofibroblasts, and macrophages peaking at 24 hours, 7 days, and 14 days after ablation, respectively. In correlation with myofibroblast accumulation, RF ablation induced hepatocyte proliferation in both the ablated lobe and an untreated lobe (mean, 165.15 and 230.4 cyclin-dependent kinase 47-positive cells per ×20 field, respectively, at day 7; P < .02). Blockade of either IL-6 or c-met significantly reduced global hepatocyte proliferation (P < .05 for both), with the former reducing the accumulation of both macrophages and myofibroblasts surrounding the coagulation necrosis area (42.9 and 113.6 vs 7.3 and 46.6 macrophages and activated myofibroblasts per ×20 field, respectively; P < .036 for both). CONCLUSION: Hepatic RF ablation induces not only a local periablational inflammatory zone but also more global proliferative effects on the liver. These IL-6- and/or c-met-mediated changes could potentially account for some of the local and distant tumor recurrence observed after treatment.

Iron, ferritin and proteins of the methionine-centered redox cycle in young and old rat hearts.

Progressive oxidation of cellular components constitutes a major mechanism of the aging process. An emerging paradigm of redox signaling suggests that low level oxidants activate protective pathways resulting in prolonged cell survival. This report centers on the study of cardiac muscle in young and old rats, including (i) the expression of ferritin (Ft) the major iron storage protein, and (ii) the expression of the major proteins of the methionine-centered redox cycle (MCRC), which controls the cellular methionine redox status. Total amounts of Ft (protein) and its mRNA encoding for Ft L-subunit (Ft-L) were higher in the aged hearts, indicating that the iron-binding capacity of myocardial Ft increased with age. Among the proteins of the MCRC, methionine sulfoxide reductases A and B (MsrA, MsrB) and MsrA mRNA were significantly higher in hearts of old rats with a significant decrease in MsrA activity. The observed up-regulation of the expression of Msr and Ft-L could represent a protective response to the increased oxidative stress in the aging myocardium.

Heart protection by ischemic preconditioning: a novel pathway initiated by iron and mediated by ferritin.

Ischemic preconditioning is a well-known procedure transiently protecting the heart against injury associated with prolonged ischemia, through mechanism/s only partly understood. The aim of this study was to test whether preconditioning-induced protection of the heart involves an iron-based mechanism, including the generation of an iron signal followed by accumulation of ferritin. In isolated rat hearts perfused in the Langendorff configuration, we measured heart contractility, ferritin levels, ferritin-iron content, and mRNA levels of ferritin subunits. Ischemic preconditioning caused rapid accumulation of ferritin, reaching 359% of the baseline value (set at 100%). This was accompanied by a parallel decline in ferritin-bound iron: from 2191+/-548 down to 760+/-34 Fe atoms/ferritin molecule, p<0.05. Ferritin levels remained high during the subsequent period of prolonged ischemia, and returned to nearly the baseline value during the reperfusion phase. Selective iron chelators (acetyl hydroxamate or Zn-desferrioxamine) abrogated the functional protection and suppressed ferritin accumulation, thus demonstrating the essentiality of an iron signal in the preconditioning-induced protective mechanism. Moreover, introduction of an iron-containing ternary complex, known to import iron into cells, caused a three-fold accumulation of ferritin and simulated the preconditioning-induced functional protection against prolonged myocardial ischemia. The ischemic preconditioning-and-ischemia-induced increase in ferritin levels correlated well with the accumulation of ferritin L-subunit mRNA: 5.44+/-0.47 vs 1.23+/-0.15 (units) in the baseline, p<0.05, suggesting that transcriptional control of ferritin L-subunit synthesis had been activated. Ischemic preconditioning initiates de novo synthesis of ferritin in the heart; the extra ferritin is proposed to serve a 'sink' for redox-active iron, thus protecting the heart from iron-mediated oxidative damage associated with ischemia-reperfusion injury. The present results substantiate a novel iron-based mechanism of ischemic preconditioning and could pave the way for the development of new modalities of heart protection.

CXCR4 Promotes Neuroblastoma Growth and Therapeutic Resistance through miR-15a/16-1-Mediated ERK and BCL2/Cyclin D1 Pathways.

CXCR4 expression in neuroblastoma tumors correlates with disease severity. In this study, we describe mechanisms by which CXCR4 signaling controls neuroblastoma tumor growth and response to therapy. We found that overexpression of CXCR4 or stimulation with CXCL12 supports neuroblastoma tumorigenesis. Moreover, CXCR4 inhibition with the high-affinity CXCR4 antagonist BL-8040 prevented tumor growth and reduced survival of tumor cells. These effects were mediated by the upregulation of miR-15a/16-1, which resulted in downregulation of their target genes BCL-2 and cyclin D1, as well as inhibition of ERK. Overexpression of miR-15a/16-1 in cells increased cell death, whereas antagomirs to miR-15a/16-1 abolished the proapoptotic effects of BL-8040. CXCR4 overexpression also increased miR-15a/16-1, shifting their oncogenic dependency from the BCL-2 to the ERK signaling pathway. Overall, our results demonstrate the therapeutic potential of CXCR4 inhibition in neuroblastoma treatment and provide a rationale to test combination therapies employing CXCR4 and BCL-2 inhibitors to increase the efficacy of these agents.Significance: These results provide a mechanistic rationale for combination therapy of CXCR4 and BCL-2 inhibitors to treat a common and commonly aggressive pediatric cancer.Cancer Res; 78(6); 1471-83. ©2017 AACR.

Single Dose of the CXCR4 Antagonist BL-8040 Induces Rapid Mobilization for the Collection of Human CD34+ Cells in Healthy Volunteers.

Purpose: The potential of the high-affinity CXCR4 antagonist BL-8040 as a monotherapy-mobilizing agent and its derived graft composition and quality were evaluated in a phase I clinical study in healthy volunteers (NCT02073019).Experimental Design: The first part of the study was a randomized, double-blind, placebo-controlled dose escalation phase. The second part of the study was an open-label phase, in which 8 subjects received a single injection of BL-8040 (1 mg/kg) and approximately 4 hours later underwent a standard leukapheresis procedure. The engraftment potential of the purified mobilized CD34+ cells was further evaluated by transplanting the cells into NSG immunodeficient mice.Results: BL-8040 was found safe and well tolerated at all doses tested (0.5-1 mg/kg). The main treatment-related adverse events were mild to moderate. Transient injection site and systemic reactions were mitigated by methylprednisolone, paracetamol, and promethazine pretreatment. In the first part of the study, BL-8040 triggered rapid and substantial mobilization of WBCs and CD34+ cells in all tested doses. Four hours postdose, the count rose to a mean of 8, 37, 31, and 35 cells/µL (placebo, 0.5, 0.75, and 1 mg/kg, respectively). FACS analysis revealed substantial mobilization of immature dendritic, T, B, and NK cells. In the second part, the mean CD34+ cells/kg collected were 11.6 × 106 cells/kg. The graft composition was rich in immune cells.Conclusions: The current data demonstrate that BL-8040 is a safe and effective monotherapy strategy for the collection of large amounts of CD34+ cells and immune cells in a one-day procedure for allogeneic HSPC transplantation. Clin Cancer Res; 23(22); 6790-801. ©2017 AACR.

The knockdown of H19lncRNA reveals its regulatory role in pluripotency and tumorigenesis of human embryonic carcinoma cells.

The function of imprinted H19 long non-coding RNA is still controversial. It is highly expressed in early embryogenesis and decreases after birth and re-expressed in cancer. To study the role of H19 in oncogenesis and pluripotency, we down-regulated H19 expression in vitro and in vivo in pluripotent human embryonic carcinoma (hEC) and embryonic stem (hES) cells. H19 knockdown resulted in a decrease in the expression of the pluripotency markers Oct4, Nanog, TRA-1-60 and TRA-1-81, and in the up-regulation of SSEA1; it further attenuated cell proliferation, decreased cell-matrix attachment, and up-regulated E-Cadherin expression. SCID-Beige mice transplanted with H19 down-regulated hEC cells exhibited slower kinetics of tumor formation, resulting in an increased animal survival. Tumors derived from H19 down-regulated cells showed a decrease in the expression of pluripotency markers and up-regulation of SSEA-1 and E-cadherin. Our results suggest that H19 oncogenicity in hEC cells is mediated through the regulation of the pluripotency state.

The bitter fate of the sweet heart: impairment of iron homeostasis in diabetic heart leads to failure in myocardial protection by preconditioning.

Cardiovascular dysfunction is a major complication of diabetes. Examining mechanistic aspects underlying the incapacity of the diabetic heart to respond to ischemic preconditioning (IPC), we could show that the alterations in iron homeostasis can explain this phenomenon. Correlating the hemodynamic parameters with levels of ferritin, the main iron storage and detoxifying protein, without and with inhibitors of protein degradation, substantiated this explanation. Diabetic hearts were less sensitive to ischemia-reperfusion stress, as indicated by functional parameters and histology. Mechanistically, since ferritin has been shown to provide cellular protection against insults, including ischemia-reperfusion stress and as the basal ferritin level in diabetic heart was 2-fold higher than in controls, these are in accord with the greater resistance of the diabetic heart to ischemia-reperfusion. Additionally, during ischemia-reperfusion, preceded by IPC, a rapid and extensive loss in ferritin levels, during the prolonged ischemia, in diabetic heart but not in non-diabetic controls, provide additional substantiation to the explanation for loss of respond to IPC. Current research is shedding light on the mechanism behind ferritin degradation as well, suggesting a novel explanation for diabetes-induced loss of cardioprotection.

Cardiac protection by preconditioning is generated via an iron-signal created by proteasomal degradation of iron proteins.

Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC) was shown to be mediated by a transient 'iron-signal' that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this 'iron signal' and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after ex vivo ischemia-and-reperfusion (I/R) were found in hearts from in vivo hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min ('delay') prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the 'delay'. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the 'iron-signal' by IPC, ensuing de-novo apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the 'delay'. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.

Aging is an organ-specific process: changes in homeostasis of iron and redox proteins in the rat.

Organ-specific changes of iron- and redox-related proteins occur with age in the rat. Ferritin, the major iron storage and detoxifying protein, as well as the proteins of the methionine-centered redox cycle (MCRC) were examined in old and young animals, and showed organ-dependent changes. In spleens and livers of aged rats, ferritin (protein) levels were greater than in young ones, and their iron saturation increased, rendering higher ferritin-bound iron (FtBI). Iron saturation of the ferritin molecule in the tongues and sternohyoids of old rats was lower but ferritin level was higher than in young rats, resulting in increased FtBI with age. Ferritin level in the esophagus of older rats was lower than in young rats but its molecular iron content higher thus the total FtBI remained the same. In the larynx, both ferritin and its iron content were the same in young and old animals. MCRC proteins were measured in livers and spleens only. With aging, methionine sulfoxide reductase A and B (MsrA and MsrB) levels in livers and spleens decreased. Thioredoxin1 (Trx) and Trx-reductase1 were elevated in old spleens, but reduced in livers. Aged spleens showed reduced Msr isozyme activity; but in the liver, its activity increased. mRNA changes with age were monitored and found to be organ specific. These organ-specific changes could reflect the different challenges and the selective pathways of each organ and its resultant capacity to cope with aging.

Zinc-desferrioxamine attenuates retinal degeneration in the rd10 mouse model of retinitis pigmentosa.

Iron-associated oxidative injury plays a role in retinal degeneration such as age-related macular degeneration and retinitis pigmentosa. The metallo-complex zinc-desferrioxamine (Zn/DFO) may ameliorate such injury by chelation of labile iron in combination with release of zinc. We explored whether Zn/DFO can affect the course of retinal degeneration in the rd10 mouse model of retinitis pigmentosa. Zn/DFO-treated animals showed significantly higher electroretinographic responses at 3 and 4.5 weeks of age compared with saline-injected controls. Corresponding retinal (photoreceptor) structural rescue was observed by quantitative histological and immunohistochemical techniques. When administered alone, the components of the complex, Zn and DFO, showed a lesser, partial effect. TBARS, a marker of lipid peroxidation, and levels of oxidative DNA damage as quantified by 8-OHdG immunostaining were significantly lower in Zn/DFO-treated retinas compared with saline-injected controls. Reduced levels of retinal ferritin as well as reduced iron content within ferritin molecules were measured in Zn/DFO-treated retinas. The data, taken together, suggest that the protective effects of the Zn/DFO complex are mediated through modulation of iron bioavailability, leading to attenuation of oxidative injury. Reducing iron-associated oxidative stress using complexes such as Zn/DFO may serve as a "common pathway" therapeutic approach to attenuate injury in retinal degeneration.