The study of direct and indirect effects of radiofrequency ablation on tumor microenvironment in liver tumor animal model.

BACKGROUND: Direct and indirect effects of radiofrequency ablation (RFA) on tumor microenvironment of the liver tumor have been noted, which was reported to be related to a variety of tyrosine protein kinase or cytokinetic pathway, but have not been thoroughly investigated and conclusive. PURPOSE: To elucidate direct and indirect effects of RFA on tumor microenvironment in the liver tumor model, and to explore the role of the specific inhibitor in tumor growth by targeting the key pathway of RFA. MATERIALS AND METHODS: One hundred and ten mice with H22 liver tumor were used in animal experiments. Eighty-four mice were randomized into three groups: control, direct RFA and indirect RFA (a block slide was inside the middle of the tumor). The growth rate of the residual tumor after RFA was calculated (n = 8 each group) and the pathologic changes at different time points (6 h, 24 h, 72 h and 7d after RFA) were evaluated (n = 5 in each subgroup). After semi-quantitative analysis of the pathological staining, the most significant marker after RFA was selected. Then, the specific inhibitor (PHA) was applied with RFA and the tumor growth and pathological changes were evaluated and compared with RFA alone. The Kruskal-Wallis test was used for evaluating the significance of different treatments in the pathological positive rate of specific markers in tumor. The two-way analysis of variance was used to determine the significance of treatment in tumor growth or body weight. RESULTS: The growth rate of the residual tumor in the direct RFA group was faster than the indirect RFA group (P = 0.026). The pathological analysis showed the expression of HSP70 (73 ± 13% vs 27 ± 9% at 24 h, P < 0.001), SMA (70 ± 18% vs 18 ± 7% at 6 h, P < 0.001) and Ki-67 (51 ± 11% vs 33 ± 14% at 7d, P < 0.001) in the direct RFA group was higher than those in the indirect RFA group after RFA. On the other hand, the expression of c-Met (38 ± 11% vs 28 ± 9% at 24 h, P = 0.01), IL-6 (41 ± 10% vs 25 ± 9% at 24 h, P < 0.001) and HIF-α (48 ± 10% vs 28 ± 8% at 24 h, P < 0.001) in the indirect RFA group was higher than those in the direct RFA group. And the expression of c-Met increased mostly in both direct and indirect RFA group compared to the baseline (53 and 65% at 72 h). Then the specific inhibitor of c-Met-PHA was applied with RFA. The growth rate of the tumor was significantly slower in the RFA + PHA group than the RFA alone group (1112.9 ± 465.6 mm3 vs 2162.7 ± 911.1 mm3 at day 16, P = 0.02). CONCLUSION: Direct and indirect effects of RFA on tumor microenvironment changed at different time points and resulted in increased residual tumor growth in the animal model. It can be potentially neutralized with specific inhibitor of related pathways, such as tyrosine-protein kinase c-Met.

Percutaneous Radiofrequency Ablation Is an Effective Method for Local Control of Liver Metastases From Lung Cancer.

Objective: To investigate the clinical value of percutaneous radiofrequency ablation (RFA) for liver metastasis from lung cancer (LCLM). Materials and Methods: We retrospectively enrolled 58 patients who underwent RFA for LCLM between January 2014 and December 2019. Primary lung cancer histology included 38 adenocarcinomas, 15 squamous carcinomas, and 5 small cell carcinomas. For 83 metastatic lesions (mean tumor diameter 3.3 ± 1.1 cm, range 0.9-5.0 cm), 65 RFA sessions were performed. Before RFA, 17 and 41 patients presented no and stable extrahepatic metastasis, respectively, whereas 18 and 40 patients had synchronous and metachronous liver metastasis, respectively. Survival was analyzed using the Kaplan-Meier method. Cox proportional hazards model was used for multivariable analysis. Results: The technical success rate was 96.3% (80/83 lesions). Local tumor progression was observed in 8 (9.8%, 8/82) lesions of 57 (14.0%, 8/57) patients at 4-12 months after RFA. New liver metastases occurred in 27 (46.6%) patients. The overall survival (OS) rates at 1, 2, 3, and 5 years after RFA were 55.2%, 26.0%, 22.0%, and 14.4%, respectively. The median OS after RFA and after liver metastasis were 14.0 ± 1.6 and 20.0 ± 1.5 months, respectively. Based on the univariable analysis, tumor size (p=0.017), histological type (p=0.015), and timing of liver metastasis (p=0.046) were related to OS. In further multivariable analyses, squamous carcinoma (hazard ratio= 2.269, 95% confidence interval: 1.186-4.339, p=0.013) was an independent unfavorable prognostic factor for OS. Based on the univariable analysis, histological type (p=0.010) was identified as parameters significantly related to local tumor progression (LTP)-free survival. Further multivariable analyses revealed that squamous carcinoma (hazard ratio=2.394, 95% confidence interval: 1.260-4.550, p=0.008) was an independent unfavorable prognostic factor for LTP-free survival. Conclusion: RFA is a safe therapeutic option for LCLM with acceptable local tumor control, especially in patients with a tumor size ≤3 cm, adenocarcinoma/small cell carcinoma, and metachronous liver metastases.

Can two-step ablation combined with chemotherapeutic liposomes achieve better outcome than traditional RF ablation? A solid tumor animal study.

Objectives: To determine whether two-step ablation using sequential low and high temperature heating can achieve improved outcomes in animal tumor models when combined with chemotherapeutic liposomes (LP). Materials and methods: Balb/c mice bearing 4T1 tumor received paclitaxel-loaded liposomes followed 24 h later by either traditional RFA (70 °C, 5 min) or a low temperature RFA (45 °C, 5 min), or two-step RFA (45 °C 2 min + 70 °C 3 min). Intratumoral drug accumulation and bio-distribution in major organs were evaluated. Periablational drug penetration was evaluated by pathologic staining and the intratumoral interstitial fluid pressure (IFP) was measured directly. For long-term outcomes, mice bearing 4T1 or H22 tumors were randomized into five groups (n = 8 per group): control (no treatment), RFA alone, LP + RFA (45 °C), LP + RFA (70 °C) and LP + RFA (45 + 70 °C). End-point survivals were compared among the different groups. Results: The greater intratumoral drug accumulation (3.35 ± 0.32 vs. 3.79 ± 0.29 × 108 phot/cm2/s at 24 h, p = 0.09), deeper periablational drug penetration (45.7 ± 5.0 vs. 1.6 ± 0.5, p < 0.001), and reduced off-target drug deposition in major organs (liver 96.1 ± 31.6 vs. 47.4 ± 1.5 × 106 phot/cm2/s, p < 0.001) were found when combined with RFA (45 °C) compared to drug alone. For long-term outcomes, 4T1 tumor growth rates for LP + two-step RFA (45 + 70 °C) were significantly slower than those of LP + RFA (70 °C), LP + RFA (45 °C), and RFA alone (P < 0.01 for all comparisons). End point survival for LP + RFA (45 + 70 °C) was also longer than that for LP + RFA (70 °C) (median 16 vs. 10 days, p = 0.003) or LP + RFA 45 °C (11 days, p = 0.009) and RFA alone (8.3 days, p < 0.001) in 4T1 tumor models. The intratumoral IFP after RFA (45 °C) was significantly lower than baseline RFA (3.3 ± 0.8 vs. 19.2 ± 3.1 mmHg, p < 0.001), but was not measurable after RFA (70 °C). Conclusions: A two-step ablation combined with chemotherapeutic liposomes can achieve better survival benefit compared to traditional RFA in animal models.

Specific Inhibitor of Matrix Metalloproteinase Decreases Tumor Invasiveness After Radiofrequency Ablation in Liver Tumor Animal Model.

OBJECTIVE: To determine whether the specific inhibitor of matrix metalloproteinase (MMP)-batimastat (BB-94)-could decrease the progression of liver tumor after radiofrequency ablation (RFA) and achieve better therapeutic efficacy in an animal model. METHODS: In vitro experiments, the proliferation of H22 liver tumor cells was detected by CCK 8 assay and cell migration was detected by Transwell method. In vivo experiments, H22 murine liver tumors were used. First, 32 mice with one tumor were randomized into four groups (n = 8 each group): control (PBS only), RFA alone (65°C, 5 min), BB-94 (30 mg/kg), RFA+BB-94. The growth rate of the residual tumor and the end point survival were calculated and the pathologic changes were evaluated. Secondly, a total of 48 tumors in 24 animals (paired tumors) were randomized into three groups (n = 8 each group): control, RFA alone, RFA+BB-94. Each mouse was implanted with two tumors subcutaneously, one tumor was treated by RFA and the other was evaluated for distant metastasis after applying BB-94. RESULTS: In vitro, the proliferation assay demonstrated higher proliferation ability after heat treatment (0.82 ± 0.07 vs 1.27 ± 0.08, P = 0.008), and it could be inhibited by BB-94 (1.27 ± 0.08 vs 0.67 ± 0.06, P = 0.001). In the cell migration assay, the H22 cells demonstrated enhanced tumor invasiveness in the heat group than the control group (33.7 ± 2.1 vs 19.7 ± 4.9, P = 0.011). And it could be significantly suppressed after BB-94 incubation (33.7 ± 2.1 vs 23.0 ± 4.6, P = 0.009). With one tumor animal, the growth rate of the residual tumor in the BB-94+RFA group was slower than that in the RFA alone group (P = 0.003). And combination of BB-94 could significantly prolong the survival of the mice (40.3 ± 1.4d vs 47.1 ± 1.3d, P = 0.002). The expression of CD31 and VEGF at the coagulation margin were decreased after combined with BB-94. With two tumors animal, the growth of metastasis tumor in the BB-94+RFA group was slower than that in the RFA group (P < 0.001). CONCLUSION: BB-94 combined with RFA reduced the invasiveness of the liver tumor and improved the end-point survival. Our data suggested that targeting the MMP process with the specific inhibition could help to increase overall ablation efficacy.

Palliative Radiofrequency Ablation Accelerates the Residual Tumor Progression Through Increasing Tumor-Infiltrating MDSCs and Reducing T-Cell-Mediated Anti-Tumor Immune Responses in Animal Model.

Previous studies showed that radiofrequency ablation (RFA) has a favorable treatment efficacy for hepatocellular carcinoma (HCC) or colorectal liver metastases (CRLMs). Palliative RFA (pRFA) resulting from larger HCC or multiple CRLMs further accelerated the progression of potential residual tumor, yet its mechanism was still unknown. This study investigated the influence of myeloid-derived suppressor cells (MDSCs) on T-cell immune responses and tumor recurrence after pRFA. CT26 tumor models were used. The percentage of MDSCs in peripheral blood was analyzed by flow cytometry after pRFA. The level of Th1 and Th2 cytokines were measured by ELISA through different treatments (n = 4/group). The tumor-infiltrating MDSCs, dendritic cells, and intracellular cytokines level were analyzed by IHC staining after different treatments. The functional CD8+ T cells were confirmed by the co-localization immunofluorescence staining. The long-term outcomes were also evaluated through CT26 and 4T1 tumor models. The results showed that tumor models treated with pRFA displayed significant increases in the percentage of MDSCs of peripheral blood and tumor infiltration. The expression level of TGF-ß and IL-6 after pRFA was higher than that before pRFA by ELISA and IHC staining. After depleting MDSCs by combining with Abs, the pRFA + Abs group achieved a higher level of Th1 cytokines and greatly enhanced the percentage of tumor-infiltrating functional CD8+ T cells when compared with pRFA alone. The depletion of MDSCs through combination with Abs also resulted in tumor regression. In conclusion, pRFA accelerates the residual tumor progression through increasing tumor-infiltrating MDSCs and reducing T-cell-mediated anti-tumor immune responses, which could provide a potential approach for delaying tumor recurrence caused by pRFA.

Combination of intratumoural micellar paclitaxel with radiofrequency ablation: efficacy and toxicity in rodents.

OBJECTIVES: To determine whether radiofrequency ablation (RFA) is more effective when combined with intratumoural injection (IT) than with intravenous injection (IV) of micelles. MATERIALS AND METHODS: Balb/c mice bearing 4T1 breast cancer were used. The tumour drug accumulation and biodistribution in major organs were evaluated at different time points after IT, IV, IT+RFA and IV+RFA. Periablational drug penetration was evaluated by quantitative analysis and pathologic staining after different treatments. For long-term outcomes, mice bearing tumours were randomised into six groups (n = 7/group): the control, IV, IT, RFA alone, IV+RFA and IT+RFA groups. The end-point survival was estimated for the different treatment groups. RESULTS: In vivo, intratumoural drug accumulation was always much higher for IT than for IV within 48 h (p < 0.001). The IT+RFA group (3084.7 ± 985.5 µm) exhibited greater and deeper drug penetration than the IV+RFA group (686.3 ± 83.7 µm, p < 0.001). Quantitatively, the intratumoural drug accumulation in the IT+RFA group increased approximately 4.0-fold compared with that in the IV+RFA group (p < 0.001). In addition, compared with the IT treatment, the IT+RFA treatment further reduced the drug deposition in the main organs. Survival was longer in the IT+RFA group than in the IV+RFA (p = 0.033) and RF alone (p = 0.003) groups. CONCLUSION: The use of IT+RFA achieved much deeper and greater intratumoural drug penetration and accumulation, resulting in better efficacy, and decreased the systemic toxicity of nanoparticle-delivered chemotherapy. KEY POINTS: • Association of IT+RFA achieved much deeper and greater intratumoural drug penetration than of IV+RFA, leading to better therapeutic efficacy. • Compared with IV or IT chemotherapy alone, the combination with RFA decreased toxicity, especially in the IT+RFA group.

The Role of a Curved Electrode with Controllable Direction in the Radiofrequency Ablation of Liver Tumors Behind Large Vessels.

PURPOSE: To investigate the role of a novel curved radiofrequency ablation (RFA) electrode with controllable direction in the ablation of tumors behind large hepatic vessels in ex vivo bovine and in vivo canine liver experiments. MATERIALS AND METHODS: Approval from the institutional animal care and use committee was obtained. In ex vivo experiments, conventional multi-tines expandable electrodes, conventional monopolar straight electrodes and novel curved electrodes were used in the ablation of the bovine liver (n = 90). The ablated area, parallel axis, vertical axis and shape of different electrodes were compared. Then, 24 beagle dogs (10 months old, female) were used for in vivo experiments. Visual tumor targets deeply located in the portal vein were established, and ultrasound-guided liver ablation was performed with different electrodes. The ablation range, target coverage rate, percentage of normal tissue injury and damage to adjacent vessels were evaluated. The Kruskal-Wallis test and the Chi-squared test were used for statistical analysis. RESULTS: For the ex vivo study with a 3-cm electrode, the ablation area of the multi-tines expandable electrode group (7.14 ± 0.16 cm2) was significantly larger than that of the novel curved electrode group (5.01 ± 0.30 cm2, P < 0.001) and the monopolar straight electrode group (5.43 ± 0.15 cm2, P < 0.001). The results obtained with the 4-cm electrode in the three groups were in accordance with those of the 3-cm electrode. In vivo, the normal tissue damage area of the novel curved electrode group was smaller than that of the multi-tines expandable electrode group (1.10 ± 0.18 cm2 vs. 4.00 ± 0.18 cm2, P < 0.001). The target coverage rate of the novel curved electrode group was better than that of the monopolar straight electrode group (100% vs. 80.86 ± 1.68%, P < 0.001). The hematoxylin and eosin (H&E) and TUNEL staining results showed that the ablation necrosis area was adjacent to large vessels, but the vascular wall was not significantly damaged in the novel curved electrode group. CONCLUSION: Our preliminary results showed that the novel curved RFA electrode with controllable direction could achieve accurate ablation for tumors behind large hepatic vessels, with a better target coverage rate and less damage to normal tissue, than conventional multi-tines expandable electrodes and monopolar straight electrodes.

Long-term outcomes and prognostic analysis of percutaneous radiofrequency ablation in liver metastasis from breast cancer.

OBJECTIVE: To evaluate the long-term efficacy and prognostic factors of ultrasound-guided percutaneous radiofrequency ablation (RFA) for breast cancer liver metastasis (BCLM). METHODS: Between 2000 and 2015, 69 patients who underwent ultrasound-guided percutaneous RFA for BCLM and had regular follow-up examinations were included. All patients had undergone resection of the primary breast cancer and had received chemotherapy, endocrine therapy or both after surgery. The sample included two males and 67 females with an average age of 50.3 ± 10.0 years (31-76 y). The mean maximum diameter of metastatic lesions in the liver was 2.9 ± 1.4 cm (1.0-6 cm). Thirty-five patients had a single metastasis, while 34 patients had multiple liver metastases (2-5 lesions). Survival results were generated using Kaplan-Meier estimates and a multivariate analysis was performed using the Cox regression model. RESULTS: In total, 92 RFA sessions were performed and 135 BCLM lesions were treated. Major complications occurred in one of the 92 sessions (1.1%). Technical efficacy was achieved in 92.6% of lesions (125/135 lesions). Local tumor progression occurred in 11.6% (8/69) of patients and new intrahepatic metastasis occurred in 55.1% (38/69) of patients. From the time of initial RFA, the median overall survival was 26 months, and the one-, two-, three- and five -year survival rates were 81.8, 50.1, 25.3 and 11.0%, respectively. Based on the multivariate analysis, the following three factors were identified as independent prognostic factors for overall survival: tumor size (p = .017), positive estrogen receptor status (p = .009) and extrahepatic metastatic disease (p = .001). The median progression-free survival was 24 months, and the one-, two-, three- and five -year survival rates after RFA were 77.4, 47.0, 23.7 and 8.5%, respectively. Additionally, the independent prognostic factors for progression-free survival included tumor size (p = .011), ER positivity (p = .001), margin size (p = .017) and extrahepatic metastatic disease (p < .001). CONCLUSION: The results of this study showed that RFA is a safe and locally effective method for the treatment of BCLM, especially in patients with lesions measuring less than 3 cm in diameter, a single liver metastasis, positive estrogen receptor status and no extrahepatic metastases. Also, patients with margin size >10 mm had no local tumor progression.

Protective and Heat Retention Effects of Thermo-sensitive Basement Membrane Extract (Matrigel) in Hepatic Radiofrequency Ablation in an Experimental Animal Study.

PURPOSE: To evaluate the protective effect of using thermo-sensitive basement membrane extract (Matrigel) for hydrodissection to minimize thermal injury to nearby structures and to evaluate its heat sink effect on the ablation zone in radiofrequency ablation (RFA) of the liver. MATERIALS AND METHODS: First, the viscosity profile and heat sink effect of Matrigel were assessed during RFA in vitro and ex vivo. Fresh pig liver tissue was used, and the temperature changes in Matrigel and in 5% dextrose in water (D5W) during RFA were recorded. Then, the size of the ablation zone in the peripheral liver after RFA was measured. Second, in an in vivo study, 45 Sprague-Dawley rats were divided into three groups of 15 rats each (Matrigel, D5W and control). In the experimental groups, artificial ascites with 10 ml of Matrigel or D5W were injected using ultrasound guidance prior to RFA. The frequency of thermal injury to the nearby organs was compared among the three groups, with assessments of several locations: near the diaphragm, the abdominal wall and the gastrointestinal (GI) tract. Finally, the biological degradation of Matrigel by ultrasound was evaluated over 60 days. RESULTS: First, Matrigel produced a greater heat retention (less heat sink) effect than D5W during ex vivo ablation (63 ± 9 vs. 26 ± 6 °C at 1 min on the surface of the liver, P < 0.001). Hepatic ablation zone volume did not differ between the two groups. Second, thermal injury to the nearby structures was found in 14 of 15 cases (93.3%) in the control group, 8 of 15 cases (53.3%) in the D5W group, and 1 of 15 cases (6.7%) in the Matrigel group. Significant differences in the thermal injury rates for nearby structures were detected among the three groups (P < 0.001). The most significant difference in the thermal injury rate was found in locations near the GI tract (P = 0.003). Finally, Matrigel that was injected in vivo was gradually degraded during the following 60 days. CONCLUSIONS: Using thermo-sensitive Matrigel as a hydrodissection material might help reduce the frequency of collateral thermal injury to nearby structures, especially in locations close to the GI tract, compared to conventional D5W. Additionally, Matrigel did not increase the heat sink effect on the ablation zone during ablation and was degraded over time in vivo.