Insufficient Radiofrequency Ablation Research Articles

Bioactive Antiangiogenic Lipid Nanoparticles for Inhibiting Residual Tumor Growth after Insufficient Radiofrequency Ablation

Bioactive Antiangiogenic Lipid Nanoparticles for Inhibiting Residual Tumor Growth after Insufficient Radiofrequency Ablation

Melatonin enhances the efficacy of anti-PD-L1 by improving hypoxia in residual tumors after insufficient radiofrequency ablation

The hypoxic microenvironment and inflammatory state of residual tumors caused by insufficient radiofrequency ablation (iRFA) are major reasons for rapid tumor progression and pose challenges for immunotherapy. We retrospectively analyzed the clinical data of patients with hepatocellular carcinoma (HCC) treated with RFA and observed that iRFA was associated with poor survival outcomes and progression-free survival. Using an orthotopic HCC mouse model and a colorectal liver metastasis model, we observed that treatment with melatonin after iRFA reduced tumor growth and metastasis and achieved the best outcomes when combined with anti-programmed death-ligand 1 (anti-PD-L1) therapy. In mechanism, melatonin inhibited the expression of epithelial–mesenchymal transitions, hypoxia-inducible factor-1α and PD-L1 in tumor cells after iRFA. Flow cytometry revealed that melatonin reduced the proportion of myeloid-derived suppressor cells and increased the proportion of CD8+ T cells. Transcriptomic analysis revealed an upregulation of immune-activated function-related genes in residual tumors. These findings demonstrated that melatonin can reverse hypoxia and iRFA-induced inflammation, thereby overcoming the immunosuppressive tumor microenvironment, and enhancing the efficacy of immunotherapy.

Aberrantly expressed HIF-1α enhances HCC stem cell-like traits via Wnt/β-catenin signaling activation after insufficient radiofrequency ablation.

Radiofrequency ablation has become a favorable treatment modality for small hepatocellular carcinoma (HCC) recently; however, insufficient radiofrequency ablation (RFA) was shown to lead to enhanced invasiveness and metastasis of HCC in our previous study, while the underlying molecular mechanism has not been understood. In order to explore the influence of the hypoxic microenvironment on residual cancer and cancer stem cell (CSC)-like characteristics of HCC cells in this process, an in vitro hypoxic model and an insufficient RFA mouse model were established with HCC cancer cell lines. Immunochemistry staining and western blot were used to examine the expression of hypoxia-inducible factor (HIF)-1α and liver CSC markers. The 3D colon formation assay, tumor cell invasion assay, and gene transfection assays were applied to test the change in liver CSC stemness and HCC cell invasion. After insufficient RFA treatment, the upregulated HIF-1α expression was associated with an increase in the CSC-like population in residual cancer. In vitro, hypoxic tumor cells showed aggressive CSC-like properties and phenotypes. Wnt/β-catenin signaling activation was shown to be necessary for the acquisition of liver CSC-like characteristics under hypoxic conditions. Overall, the aberrantly enhanced HIF-1α expression enhanced the liver CSC-like traits via abnormal Wnt/β-catenin signaling activation after insufficient RFA, and the overexpressed HIF-1α would be a vital factor and useful biomarker during the HCC recurrence and metastasis.

HSP70 via HIF-1 α SUMOylation inhibits ferroptosis inducing lung cancer recurrence after insufficient radiofrequency ablation.

Radiofrequency ablation (RFA) is an effective and feasible therapy for lung cancer, but accelerated progression of residual non-small cell lung cancer (NSCLC) after incomplete radiofrequency ablation (RFA) has frequently been reported. A previous study reported that HSP70 and HIF-1α were highly expressed in areas with incomplete RFA. Therefore, we sought to elucidate the regulatory effect of the HIF-1α/HSP70 pathway on lung cancer recurrence after incomplete radiofrequency ablation. In this study, we found that knockdown of HSP70 can reduce sumo 1, sumo 2/3 (marker of SUMOylation) of HIF-1α and inhibit A549 cell proliferation and migration under heat stress conditions (used to simulate incomplete RFA in vitro). We observed that knockdown of HSP70 altered the expression of ferroptosis-related proteins and genes (SLC7A11 and ACSL3), and the RNA-seq results showed that knockdown of HSP70 activated the ferroptosis pathway, further confirming that HSP70 regulates ferroptosis. In summary, HSP70, via HIF-1α SUMOylation, inhibited ferroptosis, inducing lung cancer recurrence after radiofrequency ablation. The study reveals a new direction for further research on therapeutic targets to suppress lung cancer recurrence and provides a theoretical foundation for further clinical studies.

GSDME has prognostic and immunotherapeutic significance in residual hepatocellular carcinoma after insufficient radiofrequency ablation

BackgroundHeat stress can induce programmed cell death (PCD). Pyroptosis is a gasdermin-mediated PCD. This study hypothesized that insufficient radiofrequency ablation (IRFA) induced pyroptosis in hepatocellular carcinoma (HCC) and investigated its underlying mechanism and clinical significance. MethodsThermostatic water bath was used to stimulate IRFA in vitro. Cell viability was assessed by MTT assay. IL-1β and HMGB1 were measured by ELISA assay. LDH level was measured by LDH cytotoxicity detection kit. Permeability of cell membrane was assessed by Hoechst33342/PI fluorescence staining. RNA expression was evaluated by qRT-PCR, and protein was assessed by Western Blotting or immunofluorescence or immunohistochemistry. Gene expression with clinicopathological characteristics from HCC patients treated by RFA were analyzed for associations between GSDME expression and prognosis. ResultsOur study revealed that IRFA induced pyroptosis in HCCLM3 and HepG2 cells. GSDME, rather than GSDMD, was cleaved in heat stress-induced pyroptosis in HCCLM3 and HepG2 cells due to caspase-3 activation. However, GSDME overexpression promoted HCC growth in vivo and predicted poor PFS and OS in HCC patients treated by RFA. Heat stress modulated gene expression related to PD-L1 signaling and caspase inhibitors inhibited heat-induced PD-L1 expression in residual HCC after IRFA. Gsdme overexpression caused resistance to PD-L1 inhibitor in residual HCC after IRFA by increasing infiltrating of CD3+PD-1+ or CD3+CTLA-4+ exhausted T cells. ConclusionsThis study indicated that GSDME could serve as a potential prognostic biomarker and help to prescribe personalized sequential immunotherapy for HCC patients receiving RFA.

Size-Tunable Nanoregulator-Based Radiofrequency Ablation Suppresses MDSCs and Their Compensatory Immune Evasion in Hepatocellular Carcinoma.

Radiofrequency ablation (RFA) is a widely used therapy for hepatocellular carcinoma (HCC). However, in cases of insufficient RFA (iRFA), nonlethal temperatures in the transition zone increase the risk of postoperative relapse. The pathological analysis of HCC tissues shows that iRFA-induced upregulation of myeloid-derived suppressor cells (MDSCs) in residual tumors is critical for postoperative recurrence. Furthermore, this study demonstrates, for the first time, that combining MDSCs suppression strategy during iRFA can unexpectedly lead to a compensatory increase in PD-L1 expression on the residual MDSCs, attributed to relapse due to immune evasion. To address this issue, a novel size-tunable hybrid nano-microliposome is designed to co-deliver MDSCs inhibitors (IPI549) and αPDL1 antibodies (LPIP) for multipathway activation of immune responses. The LPIP is triggered to release immune regulators by the mild heat in the transition zone of iRFA, selectively inhibiting MDSCs and blocking the compensatory upregulation of PD-L1 on surviving MDSCs. The combined strategy of LPIP + iRFA effectively ablates the primary tumor by activating immune responses in the transition zone while suppressing the compensatory immune evasion of surviving MDSCs. This approach avoids the relapse of the residual tumor in a post-iRFA incomplete ablation model and appears to be a promising strategy in RFA for the eradication of HCC.

Incomplete radiofrequency ablation following transarterial chemoembolization accelerates the progression of large hepatocellular carcinoma.

To examine post-operative progression and risk impact of insufficient radiofrequency ablation (RFA) following transarterial chemoembolization (TACE) for the prognosis of large hepatocellular carcinoma (HCC). From January 2014 to January 2021 were analyzed. A total of 343 patients with large HCC (diameter >5 cm) who received TACE combined with RFA were enrolled and were divided into two groups: complete ablation (CA, n = 172) and insufficient ablation (IA, n = 171). Overall survival (OS) and progression-free survival (PFS) were determined by the Kaplan-Meier curve and compared with the log-rank test. To find parameters influencing OS and PFS, clinicopathological variables underwent univariate and multivariate analysis. The cumulative 1-, 3-, and 5-year OS and PFS rates of the CA group were significantly higher than that of the IA group (P < 0.001). 25 (41%) patients in local tumor progression (LTP), 36 (59%) in intrahepatic distant recurrence (IDR), and 0 (0%) in extrahepatic distant recurrence (EDR) in the CA group. 51 (32.1%) patients in LTP, 96 (60.4%) patients in IDR, and 12 (7.5%) cases in EDR in the IA group. The recurrence patterns of the two groups were statistically significant difference (P = 0.039). In multivariate analysis, inadequate ablation and conjunction with TKIs were both significant risk factors for OS and PFS. Apart from these, older age and >7 cm of tumor size were indicators of poor OS and multiple tumors were indicators of poor PFS. Insufficient ablation causes a poor survival outcome of TACE combined with RFA for large HCC, particularly, which can promote IDR.

Perfluorocarbon nanodrug induced oxygen self-enriching sonodynamic therapy improves cancer immunotherapy after insufficient radiofrequency ablation.

Residual lesions and undetectable metastasis after insufficient radiofrequency ablation (iRFA) are associated with earlier new metastases and poor survival in cancer patients, for induced aggressive tumor phenotype and immunosuppression. Programmed cell death protein 1(PD-1) blockade has been reported to enhance the radiofrequency ablation-elicited antitumor immunity, but its ability to eliminate incompletely ablated residual lesions has been questioned. Here, we report a combined treatment modality post iRFA based on integrating an oxygen self-enriching nanodrug PFH-Ce6 liposome@O2 nanodroplets (PCL@O2)-augmented noninvasive sonodynamic therapy (SDT) with PD-1 blockade. PCL@O2 containing Ce6 as the sonosensitizer and PFH as O2 reservoir, was synthesized as an augmented SDT nanoplatform and showed increased ROS generation to raise effective apoptosis of tumor cells, which also exposed more calreticulin to induce stronger immunogenic cell death (ICD). Combining with PD-1 blockade post iRFA, this optimized SDT induced a better anti-tumor response in MC38 tumor bearing mouse model, which not only arrested residual primary tumor progression, but also inhibited the growth of distant tumor, therefore prolonging the survival. Profiling of immune populations within the tumor draining lymph nodes and tumors further revealed that combination therapy effectively induced ICD, and promoted the maturation of dendritic cells, tumor infiltration of T cells, as well as activation of cytotoxic T lymphocytes. While iRFA alone could result in an increase of regulatory T cells (Tregs) in the residual tumors, SDT plus PD-1 blockade post iRFA reduced the number of Tregs in both primary and distant tumors. Moreover, the combined treatment could significantly initiate long-term immune memory, manifesting as elevated levels of CD8+ and CD4+ central memory cells. Therefore, this study establishes the preclinical proof of concept to apply oxygen self-enriching SDT to augment cancer immunotherapy after iRFA.

Mitochondrial ROS driven by NOX4 upregulation promotes hepatocellular carcinoma cell survival after incomplete radiofrequency ablation by inducing of mitophagy via Nrf2/PINK1

BackgroundThe recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) remains a major clinical problem. Cells that survive the sublethal heat stress that is induced by incomplete RFA are the main source of HCC relapse. Heat stress has long been reported to increase intracellular reactive oxygen species (ROS) generation. Although ROS can induce apoptosis, a pro-survival effect of ROS has also been demonstrated. However, the role of ROS in HCC cells exposed to sublethal heat stress remains unclear.MethodsHepG2 and HuH7 cells were used for this experiment. Insufficient RFA was performed in cells and in a xenograft model. ROS and antioxidant levels were measured. Apoptosis was analyed by Annexin-V/PI staining and flow cytometry. Protein expression was measured using western blotting. Colocalization of lysosomes and mitochondria was analyzed to assess mitophagy. Corresponding activators or inhibitors were applied to verify the function of specific objectives.ResultsHere,we showed that sublethal heat stress induced a ROS burst, which caused acute oxidative stress. This ROS burst was generated by mitochondria, and it was initiated by upregulated NOX4 expression in the mitochondria. n-acetylcysteine (NAC) decreased HCC cell survival under sublethal heat stress conditions in vivo and in vitro. NOX4 triggers the production of mitochondrial ROS (mtROS), and NOX4 inhibitors or siNOX4 also decreased HCC cell survival under sublethal heat stress conditions in vitro. Increased mtROS trigger PINK1-dependent mitophagy to eliminate the mitochondria that are damaged by sublethal heat stress and to protect cells from apoptosis. Nrf2 expression was elevated in response to this ROS burst and mediated the ROS burst-induced increase in PINK1 expression after sublethal heat stress.ConclusionThese data confirmed that the ROS burst that occurs after iRFA exerted a pro-survival effect. NOX4 increased the generation of ROS by mitochondria. This short-term ROS burst induced PINK1-dependent mitophagy to eliminate damaged mitochondria by increasing Nrf2 expression.

HMGB1 released from dead tumor cells after insufficient radiofrequency ablation promotes progression of HCC residual tumor via ERK1/2 pathway

Background Radiofrequency ablation (RFA) is a first-line treatment for early-stage hepatocellular carcinoma (HCC). However, the recurrence after RFA remains an urgent challenge. Current studies have shown that residual tumor after RFA is an important cause of recurrence. Objective We hypothesized that the products of dead tumor cells after RFA have direct effects on the development of residual tumors. Further, we investigated the underlying mechanisms. Methods The proliferation and invasion ability of HepG2 and Huh7 cells were assessed using CCK-8, colony formation, EdU, transwell invasion and migration assay. Immunofluorescence and western blotting were used to show HMGB1 released from dead tumor cells. The levels of MMP2, MMP9, CyclinE1 and pERK1/2 were determined using western blotting. Finally, in vivo validation was performed in BALB/c nude mice xenograft tumor models. Results The products of dead tumor cells after thermal treatment can promote the proliferation and invasion of residual HCC cells. Dead tumor cells could release high-mobility group box 1 (HMGB1) after thermal treatment. Similar to the products of dead tumor cells, the recombinant protein of HMGB1 can promote the proliferation and invasion of residual HCC cells. Moreover, HMGB1 could bind to receptor of advanced glycation end-products. Then, it activated the ERK1/2 pathway and significantly upregulated the expressions of MMP2, MMP9, and CyclinE1. Conclusion Our study reveals that HMGB1 released by dead tumor cells after thermal treatment can promote the proliferation and invasion of residual HCC cells. Hence, the HMGB1/RAGE/ERK1/2 pathway is a potential target for improving the prognosis of HCC after radiofrequency ablation.

A well-matched marriage of immunotherapy and radiofrequency ablation to reduce the relapse and progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains a health challenge with increasing incidence worldwide. Radiofrequency ablation (RFA) is a potentially curative option for patients with early-stage HCC. However, the high rate of tumor recurrence limits long-term survival when the tumors are larger than 2 cm and undergoing insufficient RFA (iRFA). Notably, in situ tumor necrosis due to thermal ablation is assumed to be a source of antigens that induce antitumor immunity. Therefore, mounting studies and trials have attempted to provide a rational and effective therapeutic strategy combining RFA and immunotherapy to treat HCC. Nowadays, many controversies and challenges with this combined therapeutic strategy remain to be resolved, such as the indications for adjuvant immunotherapy along with RFA in early HCC, the sequence of the two treatments in advanced HCC, and the optimal timing of immunotherapy before or after RFA. In addition, individualized treatment strategies need to be perfected for patients with HCC.

HIF-2α regulates proliferation, invasion, and metastasis of hepatocellular carcinoma cells via VEGF/Notch1 signaling axis after insufficient radiofrequency ablation.

Background and AimsAlthough insufficient radiofrequency ablation (RFA) promotes the recurrence and metastasis of liver cancer, the underlying mechanism remains unclear. This study aimed to investigate the role and mechanism of HIF-2α in hepatocellular carcinoma cells (HCCs) after Insufficient RFA.MethodsWe established a model of insufficient RFA in MHCC97H hepatoma cells and screened for stable sublines. We inhibited HIF-2α expression in the Insufficient RFA group using PT2385 and assessed the resulting changes in proliferation and biological function of HCCs. Cell viability and proliferation were detected by the MTT method, and scratch and Transwell chamber invasion tests detected migration and invasion abilities of HCCs. The mRNA and protein expression levels of VEGF, HIF-2α, and Notch1 were detected using qPCR, immunofluorescence, and western blotting.ResultsCompared with normal HCCs without RFA treatment, insufficient RFA enhanced the proliferation and invasion abilities of hepatocellular carcinoma subline MHCC97H (P < 0.001), as well as their migration ability (P = 0.046). The HIF-2α-specific inhibitor PT2385 downregulated the migration (P = 0.009) and invasion (P < 0.001) of MHCC97H cells but did not affect cell proliferation (P > 0.05). Insufficient ablation increased the mRNA and protein expression of VEGF, HIF-2α, and Notch1 in HCCs, whereas inhibition of HIF-2α reversed these changes.ConclusionsInsufficient RFA increases the proliferation, migration, and invasion of HCCs via the HIF-2α/VEGF/Notch1 signaling axis; HIF-2α is a potential target for novel treatments of HCC after insufficient RFA.

Targeting N7-methylguanosine tRNA modification blocks hepatocellular carcinoma metastasis after insufficient radiofrequency ablation

Radiofrequency heat ablation is an ideal radical treatment for hepatocellular carcinoma (HCC). However, insufficient radiofrequency ablation (IRFA) could lead to high recurrence of HCC. N7-methylguanosine (m7G) on tRNAs, an evolutionally conservative modification in mammals and yeast, modulates heat stress responses and tumor progression, while its function in HCC recurrence after IRFA remains unknown. Here, we found that IRFA significantly upregulates the level of m7G tRNA modification and its methyltransferase complex components METTL1/WDR4 in multiple systems including HCC patient-derived xenograft (PDX) mouse, patients' HCC tissues, sublethal-heat-treated models of HCC cell lines, and organoids. Functionally, gain-/loss-of-function assays showed that METTL1-mediated m7G tRNA modification promotes HCC metastasis under sublethal heat exposure both invitro and invivo. Mechanistically, we found that METTL1 and m7G tRNA modification enhance the translation of SLUG/SNAIL in a codon frequency-dependent manner under sublethal heat stress. Overexpression of SLUG/SNAIL rescued the malignant potency of METTL1 knockdown HCC cells after sublethal heat exposure. Our study uncovers the key functions of m7G tRNA modification in heat stress responses and HCC recurrence after IRFA, providing molecular basis for targeting METTL1-m7G-SLUG/SNAIL axis to prevent HCC metastasis after radiofrequency heat ablation treatment.

Circular BANP knockdown inhibits the malignant progression of residual hepatocellular carcinoma after insufficient radiofrequency ablation.

Circular RNAs (circRNAs) are endogenous non-coding RNAs, some of which have pathological roles. The current study aimed to explore the role of circRNA BTG3-associated nuclear protein (circ-BANP) binding with let-7f-5p and its regulation of the toll-like receptor 4 (TLR4)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in residual hepatocellular carcinoma (HCC) after insufficient radiofrequency ablation (RFA). Circ-BANP, let-7f-5p, and TLR4 expressions in HCC samples were assessed using reverse transcription- quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Bioinformatics prediction, RNA pull-down assay, and dual luciferase reporter gene assay were used to analyze the relationships among circ-BANP, let-7f-5p, and TLR4. Huh7 cells were used to generate an in vitro model of residual HCC, defined as Huh7-H cells, which were transfected with either a plasmid or the sequence of circ-BANP, let-7f-5p, or TLR4. Expression of circ-BANP, let-7f-5p, and TLR4 mRNA was determined by RT-qPCR. TLR4, STAT3, p-STAT3, vascular endothelial growth factor A, vascular endothelial growth factor receptor-2, and epithelial-mesenchymal transformation (EMT)-related factors proteins were determined by Western blotting. Cell proliferation was determined by cell counting kit-8 and 5-Ethynyl-2'-deoxyuridine (EdU) assay and cell migration and invasion by Transwell assay. Animal studies were performed by inducing xenograft tumors in nude mice. Circ-BANP and TLR4 mRNAs were upregulated in HCC tissues (the fold change for circ-BANP was 1.958 and that for TLR4 was 1.736 relative to para-tumors) and expression further increased following insufficient RFA (fold change for circ- BANP was 2.407 and that of TLR4 was 2.224 relative to para-tumors). Expression of let-7f-5p showed an opposite tendency (fold change for let-7f-5p in HCC tissues was 0.491 and that in tumors after insufficient RFA was 0.300 relative to para-tumors). Competitive binding of circ-BANP to let-7f-5p was demonstrated and TLR4 was identified as a target of let-7f-5p (P < 0.01). Knockdown of circ-BANP or elevation of let-7f-5p expression inhibited the TLR4/STAT3 signaling pathway, proliferation, invasion, migration, angiogenesis, and EMT in Huh7 and Huh7-H cells (P < 0.01). The effects induced by circ-BANP knockdown were reversed by let-7f-5p inhibition. Overexpression of TLR4 reversed the impact of let-7f-5p upregulation on the cells (P < 0.01). Silencing of circ-BANP inhibited the in vivo growth of residual HCC cells after insufficient RFA (P < 0.01). Knockdown of circ-BANP upregulated let-7f-5p to inhibit proliferation, migration, and EMT formation in residual HCC remaining after insufficient RFA. Effects occur via regulation of the TLR4/STAT3 signaling pathway.

Arsenic trioxide inhibits angiogenesis of hepatocellular carcinoma after insufficient radiofrequency ablation via blocking paracrine angiopoietin-1 and angiopoietin-2

Objectives Angiogenesis occurs during tumor progression of hepatocellular carcinoma (HCC) after insufficient radiofrequency ablation (RFA). Arsenic trioxide (ATO) shows promising therapeutic potential in advanced HCC. Whether ATO regulates angiogenesis and can be used to prevent tumor progression in HCC after insufficient RFA is still unknown. Methods Insufficient RFA was simulated using a water bath. MTT assay and tube formation assay were used to evaluate the effects of ATO on viability and proangiogenic abilities of SMMC7721 and HepG2 cells after insufficient RFA in vitro. The molecular changes with the treatment of ATO were evaluated through Western blot. An ectopic nude mice model was used to evaluate the effect of ATO on the tumor of SMMC7721 cells in vivo after insufficient RFA. Results In this study, HepG2 and SMMC7721 cells after insufficient RFA (named HepG2-H and SMMC7721-H, respectively) showed higher proliferation than the untreated cells and promoted tube formation of endothelial cells in a paracrine manner. ATO eliminated the difference in proliferation between untreated and RFA-treated cells and suppressed angiogenesis induced by HCC cells after insufficient RFA through the Ang-1 (angiopoietin-1)/Ang-2 (angiopoietin-2)/Tie2 pathway. Hif-1α overexpression abolished the inhibitory effect of ATO on angiogenesis in HCC after insufficient RFA. ATO inhibited tumor growth and angiogenesis in HCC after insufficient RFA. Conclusions Our results demonstrate that ATO blocks the paracrine signaling of Ang-1 and Ang-2 by inhibiting p-Akt/Hif-1α and further suppresses the angiogenesis of HCC after insufficient RFA.

Eliminating METTL1-mediated accumulation of PMN-MDSCs prevents hepatocellular carcinoma recurrence after radiofrequency ablation.

Radiofrequency ablation (RFA) is an important curative therapy in hepatocellular carcinoma (HCC), but recurrence rate remains as high as all the other HCC therapeutic modalities. Methyltransferase 1 (METTL1), an enzyme for m 7 G tRNA modification, was reported to promote HCC development. Here, we assessed the role of METTL1 in shaping the immunosuppressive tumor microenvironment after insufficient RFA (iRFA). By immunohistochemistry and multiplex immunofluorescence (mIF) staining, we showed that METTL1 expression was enhanced in post-RFA recurrent HCC, accompanied by increased CD11b + CD15 + polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and decreased CD8 + T cells. Mechanistically, heat-mediated METTL1 upregulation enhanced TGF-β2 translation to form the immunosuppressive environment by induction of myeloid-derived suppressor cell. Liver-specific overexpression or knockdown of Mettl1 significantly affected the accumulation of PMN-MDSCs and subsequently affected CD8 + T cell infiltration. Complete RFA successfully eliminated the tumor, whereas iRFA-treated mice exhibited enhanced tumor growth and metastasis with increased PMN-MDSC accumulation and decreased CD8 + T cells compared to sham surgery. Interrupting METTL1-TGF-β2-PMN-MDSC axis by anti-Ly6G antibody, or knockdown of hepatoma-intrinsic Mettl1 or Tgfb2 , or TGF-β signaling blockade significantly mitigated tumor progression induced by iRFA and restored CD8 + T cell population. Our study sheds light on the pivotal role of METTL1 in modulating an immunosuppressive microenvironment and demonstrated that interrupting METTL1-TGF-β2-PMN-MDSC axis could be a therapeutic strategy to restore antitumor immunity and prevent HCC recurrence after RFA treatment, meriting further clinical studies.

An Overview of Hepatocellular Carcinoma After Insufficient Radiofrequency Ablation.

Radiofrequency ablation (RFA) is a commonly used treatment for hepatocellular carcinoma (HCC), however, various complex conditions in clinical practice may lead to insufficient radiofrequency ablation (IRFA), allowing residual HCC to survive. In clinical practice and laboratory models, IRFA plays an important role in rapid tumor progression. Therefore, targeting the residual HCC and avoiding IRFA were worthwhile methods. A deeper understanding of IRFA is required; IRFA contributes to the improvement of proliferative activity, migration rates, and invasive capacity, and this may be due to the involvement of multiple complex processes or proteins, including epithelial mesenchymal transitions (EMTs), cancer stem cells (CSCs), autophagy, heat shock proteins (HSPs), changes of non-tumor cells and extracellular matrix, altered immune microenvironment, hypoxia-inducible factors (HIFs), growth factors, epigenetic alterations, and metabolic reprogramming. We focus on the processes of the above mechanisms and possible therapeutic approach, with a review of the literature. Additionally, we recapitulated the construction methods of various experimental models of IRFA (in vivo and in vitro).

Targeting PI3Kγ/AKT Pathway Remodels LC3-Associated Phagocytosis Induced Immunosuppression After Radiofrequency Ablation.

Residual tumors after insufficient radiofrequency ablation (IRFA) shows accelerated progression and anti‐PD‐1 resistance. It is also reported that macrophages infiltrating into residual tumors leads to anti‐PD‐1 resistance. Elements of autophagy have been detected to conjugate LC3 to be increasingly expressed in residual tumors. The underlying mechanisms between LC3 and macrophages are aimed to be investigated, and explore further ways to enhance immunotherapy in treating residual tumors. In mice models and patients, macrophages demonstrate increased infiltration into residual tumors, especially surrounding the ablated zone. Single‐cell transcriptome demonstrates enhancement of immunosuppression function in macrophages after IRFA. It is shown that macrophages engulf heat‐treated cells through LC3‐associated phagocytosis (LAP), enhance IL‐4 mediated macrophage programming through the PI3Kγ/AKT pathway, and suppress T cell proliferation. Blockade of the PI3Kγ/AKT pathway enhances the antitumor activity of PD‐1 blockades, inhibits malignant growth, and enhances survival in post‐IRFA models. In conclusion, in mice models and patients, macrophages demonstrate increased infiltration around ablated zones in residual tumors. Blockade of the PI3Kγ/AKT pathway suppresses the growth of residual tumors in subcutaneous and orthotopic models. The results illustrate the translational potential of PI3Kγ inhibitors to enhance anti‐PD‐1 therapy for the treatment of residual tumors after IRFA.

Circular BANP knockdown inhibits the malignant progression of residual hepatocellular carcinoma after insufficient radiofrequency ablation.

Background:Circular RNAs (circRNAs) are endogenous non-coding RNAs, some of which have pathological roles. The current study aimed to explore the role of circRNA BTG3-associated nuclear protein (circ-BANP) binding with let-7f-5p and its regulation of the toll-like receptor 4 (TLR4)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in residual hepatocellular carcinoma (HCC) after insufficient radiofrequency ablation (RFA).Methods:Circ-BANP, let-7f-5p, and TLR4 expressions in HCC samples were assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Bioinformatics prediction, RNA pull-down assay, and dual luciferase reporter gene assay were used to analyze the relationships among circ-BANP, let-7f-5p, and TLR4. Huh7 cells were used to generate an in vitro model of residual HCC, defined as Huh7-H cells, which were transfected with either a plasmid or the sequence of circ-BANP, let-7f-5p, or TLR4. Expression of circ-BANP, let-7f-5p, and TLR4 mRNA was determined by RT-qPCR. TLR4, STAT3, p-STAT3, vascular endothelial growth factor A, vascular endothelial growth factor receptor-2, and epithelial-mesenchymal transformation (EMT)-related factors proteins were determined by Western blotting. Cell proliferation was determined by cell counting kit-8 and 5-Ethynyl-2’-deoxyuridine (EdU) assay and cell migration and invasion by Transwell assay. Animal studies were performed by inducing xenograft tumors in nude mice.Results:Circ-BANP and TLR4 mRNAs were upregulated in HCC tissues (the fold change for circ-BANP was 1.958 and that for TLR4 was 1.736 relative to para-tumors) and expression further increased following insufficient RFA (fold change for circ- BANP was 2.407 and that of TLR4 was 2.224 relative to para-tumors). Expression of let-7f-5p showed an opposite tendency (fold change for let-7f-5p in HCC tissues was 0.491 and that in tumors after insufficient RFA was 0.300 relative to para-tumors). Competitive binding of circ-BANP to let-7f-5p was demonstrated and TLR4 was identified as a target of let-7f-5p (P < 0.01). Knockdown of circ-BANP or elevation of let-7f-5p expression inhibited the TLR4/STAT3 signaling pathway, proliferation, invasion, migration, angiogenesis, and EMT in Huh7 and Huh7-H cells (P < 0.01). The effects induced by circ-BANP knockdown were reversed by let-7f-5p inhibition. Overexpression of TLR4 reversed the impact of let-7f-5p upregulation on the cells (P < 0.01). Silencing of circ-BANP inhibited the in vivo growth of residual HCC cells after insufficient RFA (P < 0.01).Conclusions:Knockdown of circ-BANP upregulated let-7f-5p to inhibit proliferation, migration, and EMT formation in residual HCC remaining after insufficient RFA. Effects occur via regulation of the TLR4/STAT3 signaling pathway.

Identification of circRNAs involved in the development of hepatocellular carcinoma after insufficient radiofrequency ablation.

Previous studies have reported that circular RNAs (circRNAs) play a key role in the pathogenesis and progression of various diseases. In the present study, we aimed to identify potential circRNAs associated with the progression of hepatocellular carcinoma (HCC) after insufficient radiofrequency ablation (IRFA). A xenograft mouse IRFA model was initially established, and immunohistochemical staining (IHC) and polymerase chain reaction (PCR) were performed to confirm the expression of programmed cell death-ligand 1 (PD-L1) and vascular endothelial growth factor receptor-1 (VEGFR-1). CircRNA expression alterations were screened by next-generation sequencing (RNA-seq). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to predict the function of genes coding differentially expressed circRNAs. The selected circRNAs were validated utilizing PCR and Sanger sequencing. The relationships between circRNAs, microRNAs, PD-L1, and VEGFR-1 were predicted by bioinformatics. Overall, a total of 612 circRNAs were differentially expressed in IRFA-treated subcutaneous tumorigenesis tissue. Among them, 435 circRNAs were significantly upregulated and 177 circRNAs were downregulated. GO and KEGG analyses were employed to predict the functions of these circRNAs. Thereafter, quantitative reverse transcription PCR (qRT-PCR) assays determined that these seven circRNAs were overexpressed in the IRFA group, which was consistent with the RNA-seq results. Based on the bioinformatic analysis, seven circRNAs confirmed by Sanger sequencing were predicted to likely regulate PD-L1 and VEGFR-1 expression levels by functioning as sponges for microRNAs (miRNAs) and forming a circRNA-miRNA-PD-L1/VEGFR-1 regulatory network. Finally, IHC and qRT-PCR of PD-L1 and VEGFR-1 confirmed the activation of this pathway. Taken together, we report that differentially expressed circRNAs might simultaneously regulate PD-L1 and VEGFR-1 in the IRFA tissues, which provides a novel view of circRNAs in HCC progression after the IRFA procedure.