Systemic immune response induced by radiofrequency ablation versus microwave ablation at varying powers in murine models of hepatocellular carcinoma

Introduction

Primary liver cancer is the sixth most common malignancy and ranks third in cancer mortality globally, with notably higher incidence in developing countries and regions undergoing socioeconomic transition (1,2). The current treatment strategies for liver cancer place equal emphasis on multidisciplinary collaboration and diversified treatment models, with many international guidelines having endorsed various treatments for liver cancer, including resection, liver transplantation, and local ablation for early-stage tumors, trans-arterial chemoembolization (TACE) for mid-stage tumors, and immunotherapy for advanced liver cancer (3-5), among which ablation is considered a curative treatment option for small liver cancers, akin to surgical resection (3,6).

Radiofrequency ablation (RFA) and microwave ablation (MWA) are two prevalent thermal ablation methods for liver cancer, offering similar efficacy and safety (7-9). Additionally, they can achieve effectiveness comparable to surgery for early-stage cases. RFA induces tissue coagulative necrosis by generating heat (60–100 ℃) via high-frequency current-induced ionic oscillation; in contrast; MWA employs high-frequency electromagnetic fields to induce violent vibration in polar molecules (e.g., water molecules and proteins), causing collisions and friction that convert part of the kinetic energy into heat, leading to rapid, uniform tissue necrosis and solidification at temperatures above 100 ℃ (4,10).

Unlike surgeries, thermal ablation can release tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) after in situ inactivation of tumor cells, which will be ingested by antigen-presenting cells (APCs) and presented to tumor-draining lymph nodes. T cells specifically recognize TAAs presented by the major histocompatibility complex (MHC) via T cell receptors (TCR) and become activated and clonally expanded with costimulatory signals; finally, T cells migrate to the tumor microenvironment via circulation and kill residual tumor cells (11,12). Notably, both RFA and MWA can activate and amplify tumor-specific T cell responses, potentially inducing systemic anti-tumor immunity (13,14). This would principally favor the concept of complementing local ablation (RFA or MWA) with adjuvant immune checkpoint inhibitor therapy (15). However, the combination of atezolizumab (anti-PD-L1) and bevacizumab (anti-VEGF) did not enhance long-term recurrence-free survival in patients who underwent surgical or ablative therapy for hepatocellular carcinoma (HCC) (15,16). Clinical trials with other immunotherapies as an adjuvant therapy for local ablation are ongoing, emphasizing the need to advance our understanding of immune responses elicited by ablative tumor therapies.

Tumor cells that have undergone immunogenic cell death (ICD) release different DAMPs, which not only promote the recruitment and activation of APCs but also enhance the adjuvant effects of dead tumor cells to trigger adaptive immunity (17,18). As the modulation of the tumor immune microenvironment emerges as a pivotal therapeutic target in precision HCC management, ablation technologies are undergoing a paradigm shift from local tumor eradication to in situ immune vaccination strategies. Although an animal study has revealed that MWA and RFA with different energy parameters (powers or durations) elicit varying levels of local inflammation (19), the mechanisms by which these parameters affect systemic immunity remain unclear. The murine subcutaneous HCC model provides an ideal platform for investigating the ablation-immunity interplay, owing to its standardized tumor burden and highly reproducible experimental workflow. This model enables precise parametric control of ablation settings, facilitating systematic exploration of immunomodulatory effects induced by thermal energy delivery. This study was designed to explore the influence of RFA and MWA with varying powers and durations on systemic T cell subset dynamics and cytokine profiles in mice, with an attempt to offer immunological evidence for optimized ablation strategies. Our working hypothesis posits that high-power ablation protocols enhance systemic immune activation through the rapid release of TAAs and pro-inflammatory cytokines. We present this article in accordance with the ARRIVE reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-915/rc).

Methods

Cell lines and culture

Human HCC SMMC7721 cells (Pricella, Wuhan, China) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% mix of penicillin-streptomycin. The cultures were kept at 37 ℃ in an incubator with 5% CO₂. Cells in logarithmic phase were harvested, counted, adjusted to 5×10⁷ cells/mL, and mixed 1:1 with Matrigel before they were injected into the right axillae of mice.

Animal models and treatments

A total of 50 4-week-old C57BL/6 female mice weighing 19.9±1.1 g were purchased from SiPeiFu (Suzhou, China). The animals were acclimated for 7 days before inoculation with 200 µL (5×10⁶ cells) of tumor cells each. The inoculated mice were housed in specific-pathogen-free (SPF) conditions (22±1 ℃, 50%±10% humidity, and 12 h light-dark cycle) (Hanjiang Biotch, Jiangsu, China). All animal experiments were performed in the laboratory facilities of Jiangsu Hanjiang Biotechnology Co., Ltd. Tumor volume (V = length × width² ×0.5) was measured thrice weekly using a caliper, and thermal ablation was performed once the tumor volume reached about 150 mm³. The individual mouse was considered the experimental unit within the study.

Mice were anesthetized with Telazol and xylazine hydrochloride (administered intraperitoneally) before ablation. RFA was performed using an RFA-II system (Bolaide, Beijing, China). The tumor area was adequately scraped and attached with a grounding pad. The tumor site was sterilized with 75% alcohol. A Mindray Resona R9S system (Mindray, Shenzhen, China) was employed, with the tumor being located using an L14-3WU transducer. The ablation needle was percutaneously inserted (Figure 1A). The MWA needle (ECO-100B3, YiGao Medical, Hangzhou, China), which was operated at 2,450 MHz, was also inserted into the target tumor under ultrasound.

Figure 1 Ultrasound evaluation of the thermal ablation process in murine models. (A) Ablation needle (arrow) is inserted into a tumor under ultrasound guidance. (B) Ultrasound of a subcutaneous tumor in a mouse model. Tumor maximum diameter measured by 2D ultrasound: left-right = “1”, upper-lower = “2” (left); tumor blood supply assessed by color Doppler ultrasound (right). (C) After the ablation, 2D ultrasound reveals uniform hypoechoic zones with needle tract visible (arrow) (left). CEUS confirms no contrast perfusion at the ablation site (right). 2D, two-dimensional; CEUS, contrast-enhanced ultrasound.

A total of 50 mice were randomly divided into five groups (n=10 per group), including: a control group (without any treatment), a high-power MWA group (10 W MWA for 30 s), a low-power MWA group (5 W MWA for 60 s), a high-power RFA group (10 W RFA for 30 s), and a low-power RFA group (5 W RFA 60 s). The sample size in this study (n=10 per group) was determined based on effect size estimates derived from prior similar ablation immunology research (20), with rigorous randomization protocols implemented to minimize selection bias and confounding variables. Random numbers were generated using the standard RAND (function in Microsoft Excel). The testing order was randomized each time. Since a pilot experiment had indicated that 5 W MWA for 60 seconds could completely ablate tumors sized 6–8 mm in diameter in mouse models, we adjusted the ablation durations for tumor volume in our current experiments.

Experiments were performed under a project license (No. HJSW-24072501) granted by the Institutional Animal Care and Use Committee of Jiangsu Hanjiang Biotechnology Co., Ltd., in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Contrast-enhanced ultrasound (CEUS)

The outcomes of ablation were assessed using CEUS immediately after the procedures. Sulphur hexafluoride microbubbles (SonoVue^®, Bracco Suisse SA, Shanghai, China) were used as a contrast agent. After the contrast agent (0.1 mL) was mixed well with 5 mL of saline, it was injected via the mouse’s tail vein after the tail had been swabbed with alcohol. The ablation was deemed complete if the tumor area was cavitated without the presence of contrast agent.

Flow cytometry

The anticoagulated blood was diluted with red blood cell (RBC) lysate at a ratio of 10:1, incubated at room temperature in the dark for 5–10 minutes, and centrifuged at 500 rpm for 10 minutes. The precipitate was harvested after the supernatant was discarded. If the RBC precipitate persisted, the lysis and centrifugation steps were repeated. The precipitate was resuspended in phosphate-buffered saline (PBS) and added with 2 µL of CD3 [allophycocyanin (APC) hamster anti-mouse CD3e], CD4 [fluorescein isothiocyanate (FITC) rat anti-mouse CD4], and CD8 [phycoerythrin (PE) rat anti-mouse CD8a] antibodies before staining for 30 minutes. Simultaneously, single-stain and blank control tubes were used, in which 500 µL of 1× PBS was added. After adequate mixture, filtration was performed to remove aggregates or large particles before testing on the system. Flow cytometry was performed using a BD Calibur flow cytometer. Data were acquired with BD FACS software (Becton, Dickinson, and Co., Franklin Lakes, NJ, USA) and analyzed in FlowJo software. Peripheral CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were quantified by flow cytometry.

Enzyme-linked immunosorbent assay

Blood samples were collected from the orbital vein on days 14 and 28 post-surgery and centrifuged at 4,000 rpm for serum separation. The sera were stored at −80 ℃ for analysis. The concentrations of cytokines were measured following kit instructions [interleukin (IL)-12-ml037868, IL-4-ml064310, IL-10-ml037873, and interferon-gamma (IFN-γ)-ml002277M; Mlbio, Shanghai, China]. Dual wells and standard curves were used. The absorbance was read at 450 nm with a microplate absorbance reader.

Histopathological examination

The thawed tumor tissue was processed into paraffin sections and deparaffinized through xylene I/II (10 min each), absolute ethanol I/II (5 min each), and a gradient of alcohols (95%, 90%, 80%, and 70% for 3 or 2 min each), followed by a 2-minute distilled water soak. Slices were hematoxylin-stained for 5–7 minutes, rinsed with 1% HCl-alcohol for 2–5 seconds, then rinsed to blue with tap water. Slices were eosin-stained for 2 minutes, then rinsed in tap water for 30 seconds. Sections were then dehydrated in absolute ethanol, cleared in xylene, mounted with neutral gum after air drying, and observed microscopically. It was found that the nuclei appeared blue, with cytoplasm, muscle fibers, collagen fibers, and erythrocytes showing various shades of red.

Statistical analysis

SPSS 27.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analysis and GraphPad Prism 10.1.2 (GraphPad Software, San Diego, CA, USA) for graphing. The continuous data were tested for normality using the Shapiro-Wilk test and for variance homogeneity using the Levene test. One-way analysis of variance (ANOVA) was performed to assess inter-group differences under the assumptions of normality and homogeneity of variances. Post-hoc analyses were conducted using Tukey’s honestly significant difference test for equal variances or Bonferroni correction when variances were unequal. All statistical tests were two-tailed, and P values <0.05 were deemed statistically significant.

Results

Results of tumor size measurement and two-dimensional (2D) ultrasound (Figure 1B)

All mice were shaved. Tumor morphology and size were evaluated by 2D ultrasound, and vascularization was analyzed using color Doppler flow imaging (CDFI).

The efficacy of MWA and RFA

On days 14 and 28, tumor volumes (mm³) in all thermal ablation groups were significantly smaller than those in the control group (P<0.001), demonstrating the efficacy of ablation in reducing tumor burden (Table 1).

Outcomes of MWA and RFA

CEUS following MWA or RFA showed no enhancement in the arterial phase of the ablated tumor area, indicating complete tumor necrosis (Figure 1C) (3).

Regulatory effects of RFA and MWA on T cell subsets

We assessed the impacts of MWA and RFA on systemic immunity by measuring CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell proportions in mouse blood samples on post-ablation days 14 and 28. On day 14, flow cytometry revealed a significant increase in CD3⁺CD4⁺ T percentage in peripheral blood across all ablation groups versus controls (P<0.001 or P=0.01) (Figure 2A). The CD3⁺CD4⁺ T cell percentage was significantly higher in the high-power MWA group than in the low-power MWA group (P=0.008) and the high-power RFA group also significantly outpaced the low-power RFA group (P<0.001); however, no significant difference was seen between high-power MWA and RFA groups (Figure 2B). The CD3⁺CD8⁺ T cell percentage was significantly elevated in the ablation groups versus the control group (P=0.008 or P<0.001) except for low-power RFA, with the high-power RFA group also significantly exceeding the low-power RFA group (P<0.001) (Figure 2C). By day 28 post-ablation, CD3⁺CD4⁺ T cell percentage was significantly higher in all groups except the low-power RFA group versus controls. CD3⁺CD8⁺ T cells remained significantly elevated only in high-power MWA and RFA groups, with a significant decrease from day 14 (P<0.001) (Figure 3).

Figure 2 Representative flow cytometry showing the percentage of T cells at day 14 post-ablation. (A) CD3⁺, CD4⁺, and CD8⁺ T cells; (B) CD3⁺CD4⁺ T cells; (C) CD3⁺CD8⁺ T cells. APC, allophycocyanin; FITC, fluorescein isothiocyanate; MWA, microwave ablation; PE, phycoerythrin; RFA, radiofrequency ablation; SSC, side scatter.

Figure 3 Changes of peripheral blood immune T cell levels at days 14 and 28 post-ablation. (A,B) CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell percentages 28 days post-ablation; (C,D) CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell percentages at days 14 and 28 post-ablation. Data were plotted individually with P values calculated by paired t-test (P<0.05). MWA, microwave ablation; ns, not significant; RFA, radiofrequency ablation.

Temporal change of T helper (Th)1/Th2 cytokines after ablation

Levels of Th-associated cytokines in peripheral blood were assessed on days 14 and 28, with heat maps revealing different Th cell-related cytokine profiles between ablation groups and the control group (Figure 4A). On day 14 post-thermal ablation, IL-12 levels significantly rose in all ablation groups except for the low-power RFA group (all P<0.001), along with significant reductions in IL-4 and IL-10 levels (all P<0.001). Additionally, the IFN-γ level showed no significant change. No significant differences were observed between the high-power MWA and RFA groups (Figure 4B-4E). On day 28 post-ablation, IL-12 and IFN-γ returned to the baseline levels, whereas IL-4 remained significantly lower in the high-power MWA group than in the control group (P<0.001) and IL-10 was persistently suppressed across all ablation groups (P=0.003 or P<0.001) (Figure 4F-4I).

Figure 4 Immune-related cytokine levels across various groups. (A) Heatmaps of four Th-related cytokines in serum at days 14 and 28 post-ablation across groups. (B-I) Histograms on day 14 (B-E) and day 28 (F-I) post-ablation. IL, interleukin; IFN-γ, interferon-gamma; MWA, microwave ablation; RFA, radiofrequency ablation; Th, T helper.

Histopathology of the ablation areas

Tumor cells in the control group were arranged in nests, with round/oval nuclei and visible mitosis. Focal necrosis and erythrocyte exudation were observed. In the ablation groups, massive coagulative necrosis (featured by condensed nuclei and absence of structures) was observed in the central area, with scattered focal necrosis in the marginal areas. Fibrous tissue hyperplasia with lymphocyte and macrophage infiltration was seen around cancer nests (Figure 5).

Figure 5 A typical lesion in the control group (left) and after ablation (right). HE staining; scale bar: 100 µm. HE, hematoxylin and eosin.

Discussion

Thermal ablation has been widely applied for the treatment of solid tumors, including liver, lung, breast, and thyroid cancers (7,21,22), offering curative potential for early-stage disease and symptom relief and quality of life improvement for advanced tumors by reducing tumor load (23,24). den Brok et al. [2004] demonstrated that RFA-treated orthotopic tumors could serve as an antigen source to elicit anti-tumor immune responses (25), underpinning the immunomodulatory potential of thermal ablation.

In our present study, we created C57BL/6 mouse models with unilateral subcutaneous tumors and ablated the tumors with two different ablation methods at varying powers and durations; subsequently, the changes in systemic T cells and cytokines were compared across different power settings and ablation methods. It was found that high-power RFA and MWA elicited a stronger adaptive immune response, with similar immune changes post-ablation for both ablation methods. Notably, it is still not clear which ablation modality provokes a stronger immune response: although some studies have suggested MWA induces a weaker adaptive response than cryoablation and RFA (18,20,23), others have argued that both MWA and RFA elicit notable local and systemic immune stimulation (12,13,26).

Similar to our present study, Qian et al. (27) found that high-power MWA boosted T cell response and Th1 polarization in VX2 rabbit models of breast cancer, and Yu et al. (28) showed MWA at 3 W for 3 minutes enhanced systemic and intratumoral anti-tumor immunity and stimulated an abscopal effect in colorectal cancer mice. Nevertheless, the “optimal” ablation parameters may vary by tumor types and need to be validated in conjunction with specific tumor traits (e.g., benign/malignant nature and thermal sensitivity).

The immune response induced by thermal ablation correlates tightly with the cell death pattern, with the necrosis-to-apoptosis ratio of tumor cells significantly impacting the anti-tumor response (17,18,29,30). The molecular pathways that trigger ICD include necrosis, apoptosis, necroptosis, and pyroptosis. Necrosis typically only engages innate immunity (17) and rarely induces adaptive immunity (31). Pyroptosis is an inflammatory cell death triggered by intracellular sensors (30). Necroptosis is highly immunogenic due to the massive release of DAMPs [including DNA, RNA, high mobility group protein B1 (HMGB1), histones, and heat shock proteins (HSPs) released from the nuclei or mitochondria] and inflammatory factors and thus plays a key role in the activation of adaptive immunity (32). The ablated lesions can be divided into three zones according to the thermal gradient: (I) a central zone, which develops coagulative necrosis due to the high temperature of needle tip; (II) an intermediate or transition zone, in which the sublethal heat is conducted from the central zone—this zone experiences the ongoing apoptosis and can recover from reversible injuries; and (III) a peripheral zone, in which the normal tissues will not be affected by the ablation (29,33). The “dying” cells in the transition zone dominate the activation of systemic immune by releasing DAMPs, a process not fully accounted for by the traditional necrosis–apoptosis dichotomy (34). Notably, incomplete ablation may lead to immune escape and tumor recurrence (35). In our present study, the ablation range was strictly controlled under CEUS, which helped to avoid incomplete ablation. It was found that the immune response induced by high-power MWA and RFA was stronger than that in the low-power groups, possibly due to the broader transition zone created by the high-power ablation.

We also found CD4⁺ T cell levels significantly increased in all ablation groups versus controls but with no significant change over time (from days 14 to 28). CD8⁺ T cells decreased at day 28, potentially due to effector T cell depletion or transition to memory cells. Zhang et al. reported that CD8⁺ T cells peaked at week 3 after cryoablation or RFA, with gradual increases post-MWA peaking at week 4 before declining (18). In contrast, Takaki et al. noted a transient rise in peripheral blood cytotoxic T lymphocytes (CTLs) at day 14 after ablation (36). Kan et al. discovered a decrease in peripheral CD8⁺ T cells with CD4⁺ T cell dominance in liver cancer patients following MWA, indicating that thermal ablation might modulate the immune microenvironment by shifting T cell subset balance (37). The cytotoxic function of CD8⁺ T cells is pivotal in anti-tumor immunity; upon completion of the primary killing task, activated CD8⁺ T cells are progressively reduced through apoptosis. In contrast, CD4⁺ T cells are essential in initiating and executing the immune response (38,39). Given that CD8⁺ T cells are the primary effector cells of anti-tumor immunity, their declining numbers or depletion may limit long-term efficacy.

To further investigate the impacts of MWA and RFA at different powers on the immunity, we analyzed the levels of T cell-related cytokines on days 14 and 28 post-ablation. The Th1 cells, based on the secreted cytokines and surface markers during their activation, can be primarily categorized into subsets Th1 and Th2. Early elevation of IL-12, a key Th1 differentiation inducer, may drive Th1 polarization via the STAT1/STAT4 pathway (40). Suppressing IL-4 and IL-10 may reduce the recruitment of immunosuppressive cells such as M2 macrophages and myeloid-derived suppressor cells (MDSCs), thereby mitigating the immunosuppressive microenvironment (41,42). Our present study revealed that MWA and RFA reduced IL-4 and IL-10 expression at days 14 and 28, with high-power MWA and RFA enhancing IL-12 expression at day 14, whereas the IFN-γ levels remained unchanged. Despite immune system activation, the lack of change in IFN-γ level suggested that Th1 effector function might be limited by local immunosuppression [e.g., regulatory T cell (Treg) infiltration or upregulation of checkpoint molecules (43)], justifying the use of combined immune checkpoint inhibitors. Both MWA and RFA resulted in a Th1-biased immune signature, augmenting the anti-tumor immunity of the body, which was particularly obvious in the high-power groups.

Although high-power ablation shows stronger potential for immune activation, its clinical application still faces challenges. It has been shown that although thermal ablation can temporarily activate the immune system, it fails to suppress tumor progression continuously (44). The possible mechanisms underlying such a failure may include the following: (I) the residual tumors recruit MDSCs into the tumor microenvironment to inhibit immune responses (35); (II) the depletion of effector T cells causes the loss of immune memory, leading to rapid tumor recurrence (44); and (III) the tumor inflammatory microenvironment experiences reprogramming after the ablation (45). Future studies should further optimize the ablation parameters (e.g., power-duration matching and multi-needle strategies) and employ combination therapies (e.g., immunoadjuvants and adoptive cell therapy) to prolong immune responses.

There were several limitations in our study. First, the subcutaneous xenograft model cannot fully replicate the human immune microenvironment. We recognize that the observed peripheral immune cell levels may underestimate the complex immune regulatory mechanisms in clinical settings, particularly by neglecting tissue-specific immune interactions within the tumor microenvironment. Future studies should adopt endogenous and orthotopic HCC models [e.g., diethylnitrosamine (DEN)], especially in fibrotic or cirrhotic livers, to better mirror the immune landscape of clinical HCC patients. Second, this study has a modest sample size (10 mice per group, total n=50), which may compromise statistical power and elevate the likelihood of type II errors (false negatives). Future investigations should prioritize larger cohorts and pre-experimental power analyses to ensure robust statistical validity and mitigate the risk of erroneous interpretations in clinical translation strategies. Third, we did not specifically exclude natural killer T (NKT) cell effects. The study focused on T cell analysis via CD4 and CD8 staining, without considering the potential regulatory role of NKT cells in T cell subset dynamics. Fourth, assessments of T cell numbers and cytokine levels were conducted only on days 14 and 28 post-ablation, lacking baseline comparisons. Consequently, transient increases induced by procedural effects on day 14 might have been overlooked. Additionally, the relatively short follow-up period (28 days) limits conclusions regarding long-term immune memory or sustained therapeutic efficacy. Finally, reporting T cell subsets as percentages without absolute counts could obscure true biological variations, particularly when inter-group differences in total lymphocyte counts exist.

Conclusions

By comparing the impacts of the two ablation modes at different powers and durations on mouse models of HCC, we found that high-power MWA and RFA were more effective in activating systemic anti-tumor immunity and Th1 response. Thermal ablation not only lies in local tumor control but may also improve prognosis by reshaping systemic immune status. Further investigations are warranted to elucidate the molecular drivers of these dynamic immune changes and translate them into precision strategies for clinical immunotherapy synergy.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-915/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-915/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-915/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-915/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. HJSW-24072501) granted by the Institutional Animal Care and Use Committee of Jiangsu Hanjiang Biotechnology Co., Ltd., in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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