Radiofrequency Ablation and Immunotherapy: Orchestrating the Immune Microenvironment for Improved Hepatocellular Carcinoma Control

Radiofrequency Ablation and Immunotherapy: Orchestrating the Immune Microenvironment for Improved Hepatocellular Carcinoma Control | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Immunology This is a preprint and has not been peer reviewed. Data may be preliminary. 9 January 2025 V1 Latest version Share on Radiofrequency Ablation and Immunotherapy: Orchestrating the Immune Microenvironment for Improved Hepatocellular Carcinoma Control Authors : Liu Yang 0009-0008-0636-2180 , Shuhang Wei , Zongxin Liu , Qiqi Liu , Zhen Yu , Yuemin Feng , and Qiang Zhu [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173641571.10217985/v1 Published Immunology Version of record Peer review timeline 433 views 267 downloads Contents Abstract RFA + Immune Checkpoint Inhibitors (ICIs) Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Radiofrequency ablation (RFA) is a radical treatment modality for early stage hepatocellular carcinoma (HCC). In addition to direct elimination of tumor cells, RFA induces changes in infiltrating cells within the liver tumor immune microenvironment (TIME), thereby eliciting anti-tumor immune effects. Moreover, incomplete RFA (IRFA) leads to tumor recurrence and metastasis by inducing an immunosuppressive microenvironment. Immunotherapy is a systemic treatment that enhances anti-tumour immune responses to treat HCC. So, influencing the TIME makes it possible to combine RFA and immunotherapy, which may significantly enhance the anti-tumor immune function to attack residual tumor cells. This may become one of the important means to reduce the recurrence rate after RFA. This review discusses the impact of RFA on TIME of HCC, and the immune-related mechanisms leading to tumor cell survival and invasion after IRFA. Finally, we summarize the alterations in the TIME and treatment outcomes of combining RFA with immunotherapy in HCC, aiming to provide new insights and references for improving the effectiveness of RFA. Radiofrequency Ablation and Immunotherapy: Orchestrating the Immune Microenvironment for Improved Hepatocellular Carcinoma Control Liu Yang 1 , Shuhang Wei 1 , Zongxin Liu 1 , Qiqi Liu 1 , Zhen Yu 2 , Yuemin Feng 2 *, Qiang Zhu 1,3 * 1Department of Gastroenterology, Shandong Provincial Hospital, Shandong University, 324, Jing 5 Rd, Jinan, Shandong, 250021, China. 2Department of Gastroenterology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, 250021, China. 3Department of Infectious Disease, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, 250021, China. *These authors contributed as co-corresponding authors. Email: [email protected] , [email protected] . Abstract Radiofrequency ablation (RFA) is a radical treatment modality for early stage hepatocellular carcinoma (HCC). In addition to direct elimination of tumor cells, RFA induces changes in infiltrating cells within the liver tumor immune microenvironment (TIME), thereby eliciting anti-tumor immune effects. Moreover, incomplete RFA (IRFA) leads to tumor recurrence and metastasis by inducing an immunosuppressive microenvironment. Immunotherapy is a systemic treatment that enhances anti-tumour immune responses to treat HCC. So, influencing the TIME makes it possible to combine RFA and immunotherapy, which may significantly enhance the anti-tumor immune function to attack residual tumor cells. This may become one of the important means to reduce the recurrence rate after RFA. This review discusses the impact of RFA on TIME of HCC, and the immune-related mechanisms leading to tumor cell survival and invasion after IRFA. Finally, we summarize the alterations in the TIME and treatment outcomes of combining RFA with immunotherapy in HCC, aiming to provide new insights and references for improving the effectiveness of RFA. Key Words Hepatocellular Carcinoma; Radiofrequency ablation; Immunotherapy; Combined therapy; Tumer immune microenvironment Introduction According to the latest statistics, HCC ranks sixth in incidence and third in mortality among malignant tumours [1]. In the 2022 update of BCLC management strategy, local ablation plays a leading role in radical treatment of early-stage HCC [2]. RFA is the most widely used local treatment which is performed by inserting an electrode into tumor tissue under the guidance of the imaging technology. The electrode generates a temperature between 60°C and 100°C with high-frequency alternating current at 375-480 kHz, thereby causing tumor necrosis [3]. However, the intrahepatic recurrence rate at 1, 3 and 5 years after RFA is significantly higher than SR [4]. So the main obstacle to RFA in clinical practice is high local and distant recurrence, which is mainly due to IRFA. It’s worth noting that the thermal damage from RFA contributes directly to tumor cell death through protein denaturation and may alter the TIME, mediating the weak and short-lived antitumor immune effect. However, if HCC cells are incompletely ablated, sub-lethal heat stimulation will form an immunosuppressive microenvironment by releasing pro-tumor factors, thereby leading to the resistance to immunotherapy and the progression of residual tumors. Therefore, taking advantage of this characteristic that RFA can alter the TIME, combination with immunotherapy becomes a promising option. This article reviews the current status and progress of RFA, and discusses the potential mechanism of “1+1>2”: whether the combination of complete RFA and immunotherapy can further augment the anti-tumor immune responses; whether the combination of IRFA and immunotherapy can overcome the immunosuppressive microenvironment, thereby improving the prognosis of HCC patients. Finally, the article prospects the development trend and prospects of the combination of the two, and points out the direction of future research. The immune microenvironment in HCC The liver is regarded as an immune organ because of its unique structure, which is rich in immunoreactive cells that modulate immune responses and maintain immune tolerance to self and foreign antigens [5]. HCC usually develops due to chronic inflammation, viral and alcohol damage, which causes abnormal liver “soil” and vascular regulation, then normal cells become cancerous. Consequently, the “soil” in which cancer cells live is the TIME. Apart from tumour cells, other cellular components include immune cells, hepatic stellate cells (HSC) and endothelial cells (EC). Its non-cellular components include extracellular matrix, cytokines, vascular and lymphatic networks. HCC is an immunogenic tumour that occurs in immunosuppressive microenvironment. This is due to the inherent immune tolerance when the liver is exposed to various antigens. Many immune cells accumulate in TIME, and the suppressive immune microenvironment of HCC is mainly driven by activated regulatory T-cells (Tregs) and myeloid-derived suppressor cells (MDSCs) [6]. MDSCs, being immature myeloid cells, exert their inhibitory effects by suppressing NK cells and specific T cells’ functions, secreting pro-inflammatory cytokines and inducing Tregs [7]. Meanwhile, NK cells, killer CD8+ T cells and CD4+ T cells of pro-inflammatory Th1 phenotypes exert anti-tumour effects [8]. Additionally, cancer-associated fibroblasts and EC promote cancer cell growth, immune evasion and angiogenesis through growth factors [9]. In summary, HCC has a complex immune microenvironment. The imbalance of various cells and matrices in TIME may all result in the progression and drug resistance of HCC. The immune microenvironment changes induced by RFA 1. Increased release and presentation of tumour-associated antigens (TAAs) The series of immune responses induced by RFA can not occur without the release of TAAs from cellular damage. RFA destroys the structure of tumor cells through thermal damage. Proteins and sugar molecules on cell membrane are released into microenvironment and recognized as ”exogenous” antigens, thus activating immune system. Immune cells are attracted to the antigens, phagocytose them and present them, then initiate anti-tumor immune responses. Then the apoptosis of cells will further release and present TAAs. Meanwhile, damage-associated molecular patterns (DAMPs) are also released in central ablated area, including DNA, RNA, HMGB1, HSP and ATP. These substances are recognized as dangerous signals and captured by dendritic cells (DCs), activating surface molecules of DCs and other immune cells. Moreover, they can activate different immune cells, such as NK cells and monocytes, enhancing the anti-tumor immune effect. DAMPs also act on receptors, such as toll-like receptors, to stimulate inflammatory responses and antigen presentation [10]. It can be concluded that TAA is the most critical factor of anti-tumour immune effect induced by RFA. Its release and presentation leads to subsequent activation of immune. 2. Functional enhancement of DCs As the most functional antigen-presenting cell (APC), the function of DC is strengthened after RFA. Ali et al. first demonstrated that local ablation resulted in elevated TNF-α and IL-1β, which activated myeloid dendritic cells (MDCs) to activate [11]. Furthermore, another study showed that tumour destruction after RFA induced DCs infiltration and suggested that combining with systemic immunomodulatory drugs could create an effective ”in situ DC-vaccine” [12]. Dromi et al. found that TAAs released after RFA enhanced antigen presentation by APC. They found complete tumour regression in mice, a phenomenon associated with intratumoural DCs infiltration [13]. 3. Tumor-specific T lymphocytes and NK cells functional activation After antigen presentation by APCs, effector immune cells are successfully activated and exert cytotoxic effects to help eliminate tumor cells [14]. Wissniowski et al. reported the first study to identify RFA-mediated tumour-specific T lymphocytes activation [15]. Moreover, Zerbini et al. first demonstrated such immunostimulatory effects in tumors in vivo [16]. Follow-up studies have also observed an increase in peripheral T cells after RFA, especially CD8+ T cells, which improves the prognosis of HCC [17, 18]. It has been shown that antigens produced by tumor have been shown to alter the number, phenotype and function of different immune cell subpopulations associated with patient survival. For instance, Mizukoshi et al. found that RFA could enhance various TAA-specific T-cell responses. What’s more, a positive correlation has been found between the increase in CD8 T cells and the reduction in MDSCs after RFA and recurrence-free survival (RFS) [19]. Besides, RFA also increases Th1 cytokines (such as IFN-γ and IL-2) in HCC patients, while significantly reducing the levels of Th2 cytokines (such as IL - 4, IL - 6 and IL - 10), which may contribute to enhanced T-cell responses [20]. These results indicate that RFA increases T lymphocytes infiltration and activation in the liver and generate anti-tumor T-cell responses, but it has almost no effect on B cells [21]. Also, RFA enhances cytotoxicity of NK cells. After RFA, a high level of activated NK receptors can be found in HCC patients’ peripheral blood, and the number of NK cells with enhanced cytotoxicity also significantly increases [22]. In addition, Mo et al. concluded that RFA could enhance NK-mediated antitumour activity. And they found that RFA raised the expression of NKG 2D on the surface of NK cells. NKG 2D regulates lymphocyte activation by recognising ligands on tumour cells, then promoting antitumour immune responses [23]. In summary, RFA enhances specific immune cell responses, especially T cells, associated with the release of TAAs. Moreover, the strong TAA-specific CD8+ T cell response prolongs recurrence-free interval after treatment [18]. Nevertheless, HCC is inherently immunosuppressive. Tumor quickly overcomes the immune responses by inhibiting the function of CD8+ and CD4+ T cells, thereby driving a shift to higher regulatory T-cell to Teff ratio, and upregulating PD-L1/PD-1 expression. The number of TAA-specific T-cells after RFA was inversely correlated with the frequency of MDSCs [16, 19, 24, 25]. Based on these findings, these results suggest that the anti-tumour immune effect induced by RFA alone cannot adequately resist the tumor immunosuppressive state existing in HCC and is insufficient to fully prevent HCC recurrence. Figure 1 Created in BioRender Mechanism of antitumor TIME formation after RFA. RFA can activate the immune responses and promote the infiltration of various immune cells, including APCs (e.g. DCs), tumor-killing cells (e.g. CD8+ T and NK cells). Most of these immune cells are suppressed by MDSCs. The immune microenvironment changes induced by IRFA IRFA stimulates complex pathophysiological processes, including inflammatory responses, immune responses, oxidative stress responses, hypoxia, and more. In addition to altering tumour cell epigenetics or activating signal transduction pathways to promote tumour progression, IRFA produces cytokines, growth factors and inflammatory mediators which possess potentially pro-tumourigenic properties. Consequently, various factors and immune cells in microenvironment interact with each other to create an overall suppressive immune microenvironment after IRFA, which accelerates the progression of residual HCC cells. 1. Release of pro-tumor factors Research has demonstrated that IRFA can generate pro-tumor factors through cellular inflammatory injury, including HIF-1, HSP, HGF, IL-6, TGF-β and VEGF [26-29]. In addition, Kong’s team found that tumour-associated endothelial cells (TAECs) promoted the growth of residual cells after RFA [30]. It was also found that the expression of ICAM-1 in TACEs is elevated, which activates platelets and increases endothelial permeability, leading to the progression of HCC [31]. And Liu’s team found that the Sumo2 expression and activation of SUMOylation increased after IRFA, then obstructing IFN-1 signaling in HCC cells. This obstruction, in turn, hindered the anti-tumor immunity mediated by DCs and CTLs [32]. Based on these elements, the balance in the TIME is profoundly tilted toward protumor inflammation, and then suppressing the function of immune cells [33]. Apart from forming the suppressive TIME, these variou factors are also extensively involved in angiogenesis, cell survival, proliferation, and cell migration after IRFA. 2. The recruitment of inflammatory cells and infiltration of MDSCs In acute cell injury, pro-inflammatory cytokines and chemokines are released from ablated tumour, causing neutrophils, macrophages and activated myofibroblasts in microenvironment to recruit towards the ablated area, resulting in an inflammatory response around the necrotic lesion [3, 34]. Shi et al. found tumour-associated inflammation induced by IRFA could improve the production of CCL2 by tumour cells, thereby causing the accumulation of monocytes and tumour-associated macrophages (TAM). However, the inflammatory response within residual tumour after IRFA is characterised by a large infiltration of MDSCs. MDSCs can interact with Kupffer cells in TIME and inhibit function of TILs by up-regulating PD-L1 expression, hindering the efficacy of inhibitors. Moreover, MDSCs also secrete IL-10 and VEGF, which further promotes immunosuppression and tumour progression [35-37]. Researchers found that IRFA could activate the upstream caspase-3 protein, leading to a high expression of GSDME. This change could convert the death of HCC cells from apoptosis to pyroptosis, and also up-regulate the expression of PD-L1 in HCC residual cells [38]. In addition, Zeng et al. elucidated the crucial role of methyltransferase 1 (METTL1) in regulating immunosuppressive microenvironment. The expression of METTL1 was up-regulated in recurrent tumor tissues after IRFA. It promoted the translation of TGF-β2 and induced the expansion of PMN-MDSC, then subsequently reduced CD8+ T cell infiltration [39]. 3. The M2 differentiation of macrophages The macrophages in TIME differentiate into pro-inflammatory M1 (immune-activating) or anti-inflammatory M2 (immune-suppressing) macrophages depending on the mediator [40-42]. In HCC after suffering acute subthermal heat injury, the M2 differentiation of macrophages is promoted. Specifically, Liu et al. revealed that macrophages phagocytose heat-treated cells through LC3-associated phagocytosis. Then, they activate the PI3Kγ/AKT pathway. Subsequently, this promotes IL-4 mediated macrophage M2 programming, which inhibits T-cell proliferation by expressing anti-inflammatory cytokines [43]. HCC is accompanied with chronic liver injury. In such a hypoxic environment, liver cells undergo apoptosis and necrosis, releasing specific DAMPs, which then activate HSC. Under the influence of TGF-β, HSC are transformed into cancer-associated fibroblasts (CAF) [44, 45]. Then CAF promote HCC progression by recruiting macrophages and converting them to the M2 phenotype, while simultaneously producing immunosuppression by secreting VEGF [46]. 4. Immune microenvironment changes in recurrent HCC after RFA. Recently, researchers found that the microenvironment changes of recurrent HCC after RFA are like those of IRFA, possibly involving epigenetic and immunological alterations. Shi et al. studied the transcriptomic and proteogenomic aspects of HCC patients with early recurrence after RFA. They discovered that PRTN3’s differentially-expressed genes and proteins in these patients were constantly up-regulated and linked to OS. The tumorigenic effect of PRTN3 was also shown in animals, promoting tumor growth through multiple oncogenic factors and signaling pathways like PI3K/AKT and P38/ERK [47]. Zhao et al. found that serum components from RFA might have a dual impact on the immune system in recurrent HCC patients. They can enhance monocytes’ antigen-presenting ability and help activate the immune system, but also suppress anti-cancer immune responses by reducing CD8 effector and memory T cells, inhibiting T-cell activation, and down-regulating CD161 and CD5 expressions in T cell subpopulations. These components also contribute to an immunosuppressive microenvironment by promoting VEGF secretion in monocytes, Tregs, B cells, and CD4 naive T cells [48]. FIGURE 2 Created in BioRender Mechanisms of immunosuppressive microenvironment formation post-IRFA: (A) Some factors (e.g. CCL2, ICAM-1, IL-4, IL-6, METTL1, IL-10, VEGF, TGF-β) release after IRFA which contribute to immunosuppressive microenvironment. (B) The exhaustion markers (e.g. PD-1/PD-L1) on TILs/MDSCs are upregulated, enabling immune evasion. (C) MDSCs suppress the antitumor responses of TILs and NK cells. (D) M2-like macrophage polarization contributes to the suppressive tumor immune microenvironment. (E) Reduced DC cell maturation weakens antigen-presenting functions. (F) The overexpression of Sumo2 and activating of SUMOylation play a critical role in shaping suppressive TIME following IRFA RFA in combination with immunotherapy Previous studies have suggested that RFA impacts the immune system. Moreover, immunotherapy can regulate tumor microenvironment by increasing the infiltration of effector T cells and inhibiting immunosuppressive cells, further enhancing the anti-tumor immune responses. Therefore, the combination of RFA and immunotherapy may play a significant role in modulating the immune microenvironment, and it is highly likely that such modulation will contribute to strengthening and consolidating the anti-tumor immune effect. At present, many clinical trials have been launched to probe into the safety and efficacy of the combined treatment of RFA and immunotherapy for HCC. Nevertheless, it still awaits determination whether the combination of RFA and immunotherapy can enhance anti-tumor immune effect by modifying the TIME. The details are presented in Table 1. RFA + Immune Checkpoint Inhibitors (ICIs) Immune checkpoint molecules are key regulators of anti-tumor T-cells responses, which can be expressed by T cells, APCs and tumor cells. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death receptor 1 (PD-1) are the major inhibitory immune checkpoint receptors. Mechanistically, the interaction between PD-1 and its ligand, PD-L1, leads to pan-dephosphorylation of T-cell-activated kinases, resulting in the inactivation of T-cells. Besides, CTLA-4 can interact with CD80/86 molecules expressed on APC membrane to prevent antigen presentation and the activation of effector T-cells. Moreover, CTLA-4 acts as an effector molecule of Tregs to impair anti-tumor immune responses and accelerate immune escape of tumor cells [49, 50]. ICIs are monoclonal antibodies that block interaction between checkpoint receptors and their ligands. This disruption allows the immune cells to escape from the inhibitory signals and regain their anti-tumor functions, thus reactivating immune cells to exert anti-tumor effects [51, 52]. Several ICIs have achieved remarkable results in clinical applications, and CTLA-4 and PD-1 inhibitors have been successfully used in clinical treatment of melanoma, HCC, and non-small cell lung cancer [49]. When the efficacy of ICI monotherapy in advanced HCC was initially demonstrated [53-57], many researchers have recently shifted focus to ICI-centered combination therapy. Nowadays, local ablation combined with ICIs is gradually becoming a hotspot. More and more studies have shown that applying ICIs to HCC patients suitable for RFA treatment may reduce the recurrence rate and improve prognosis, and can also reduce adverse reactions related to ICIs. It is expected to become a more effective first-line treatment method. A study has demonstrated that the anti-tumor immune responses elicited by RFA can be augmented by α-CTLA-4, contributing to durable tumor protection [58]. Positive clinical outcomes were observed in patients with advanced HCC treated with the α-CTLA-4 (Tremelimumab) in combination with RFA. Moreover, this combination increased CD8+ T cell infiltration in tumors and was accompanied by a significant reduction in HCV viral load [59]. The results of another study revealed that, in mouse models, the combination therapy of RFA and α-PD-L1 could enhance the efficacy of CD8+ T cell while decreasing the infiltration of Tregs, thereby prolonging the survival time of mice [24]. Although there are limited relevant studies, some clinical studies still initially demonstrate the safety and effectiveness of this combined therapy for HCC. The synergistic mechanism of RFA in conjunction with ICI is as follows: RFA can elicit TAA-specific anti-tumor immune effects, and when combined with ICI, it can relieve the immunosuppression induced by immune checkpoints, thereby reactivating immune cells to exert strong anti-tumor effects. RFA + Adoptive Cell Therapy (ACT) ACT is a active immunotherapy based on host cells, aiming to confer durable anti-tumor immunity to lymphocytes. The process generally involves first extracting lymphocytes from the body, then sensitizing and/or expanded them in vitro, and finally injecting these cells into the host to exert cytotoxic effects on autologous tumor cells [60]. RetroNectin activated killer cell (RAK) immunotherapy is a biological treatment using the body’s immune cells strengthened in vitro and then transfused back. Ma et al. combined RFA with autologous RAK to treat HCC. After the combined treatment, the percentage of CD3+ CD8+ T cells and the concentration of IFN-γ in peripheral blood of patients increased significantly. It was concluded that CD8+ T cells played a role in long-term inhibition of HCC recurrence and metastasis [61]. CIK cells, a heterogeneous immune cell population, are generated by co-culturing lymphocytes with cytokines like IFN-γ and IL-2. They exert cytotoxic effects on target tumor cells through high-affinity binding between leukocyte function-associated antigen-1 and its ligands as well as other pathways [62]. Weng et al. recruited HCC patients who had undergone TACE and RFA. Autologous CIK cells were transfused into patients through hepatic artery. The numbers of CD3+, CD4+, CD56+, CD3+ CD56+ cells and the CD4+/CD8+ ratio increased significantly after infusion, leading to a reduction in recurrence [63]. Similarly, a study recruited HCC patients who received SR, RFA, or percutaneous ethanol injection. After the test group was administered with CD3/CD56 CIK cells, a significant improvement in the median RFS was observed [64]. In another trial, the RFA/CIT group had significantly longer PFS and OS than RFA alone. Moreover, the percentages of CIK and NK cells were higher, and the HCV viral load was decreased [65]. Another study concluded that RFA treatment followed by injection of autologous CIK cells prolonged RFS in HCC patients [66]. In HCC, studies on DCs and T-cells adoptive transfer have been carried out. Peng et al. explored the combination of neoantigen-based DC vaccination and adoptive T-cell transfer for HCC patients post-RFA or SR. This therapy successfully induced neoantigen-specific immunity and prolonged DFS of responding patients. So, it indicates that neoantigen-based combined immunotherapy is feasible, safe, and has the potential to reduce the recurrence of HCC after radical treatment [67]. Therefore, ACT immunotherapy can be regarded as sending the immune cells for “enhanced training” outside the patients. After enhancing their functions, they “repay” to the body to further eliminate tumors. And it also enhances anti-tumor immune effect caused by RFA, thereby reducing the postoperative recurrence rate. RFA + other other immune modulators The immune environment of HCC is complex. Some novel immunotherapy methods have come to the fore, and several studies have investigated the efficacy of RFA combined with these therapies. CpG B oligonucleotide, a toll-like receptor 9 agonist, has undergone testing in the rabbit VX2 HCC model. The results showed that CpG treatment enhanced the anti-tumor efficacy of T cells induced by RFA and also increased IL-8 and IL-10 levels. In addition, the combination significantly reduced tumor load and even prevented subsequent tumor metastasis, thereby increasing survival rates [68]. Resiquimod (R848), a toll-like receptor 7/8 agonist, was combined with RFA in a study to treat HCC mice. Tumor-infiltrating immune cells, total T cells, the ratio of CD8 T and NK cells to CD45 cells and functional NK cells were significantly elevated after the combination compared to RFA or R848 monotherapy. The data suggested that the combined treatment could stimulate stronger antitumor immune responses and effectively inhibite HCC progression in a NK cell-dependent manner [69]. Some chemokines can attract immune cells to boost anti-tumor immunity and be applied in tumor treatment. For example, injecting ECI301 (the active variant of CC chemokine ligand 3) after RFA inhibited the growth of contralateral non-RFA-treated tumors. ECI301 enhanced RFA-induced antitumor immune responses by increasing the number of CCR1+ immune cells in peripheral blood and ablated tumors, but it couldn’t induce antitumor effects alone [70]. In the tumor microenvironment, the DNA released upon tumor cell death can activate the cGAS-STING signaling pathway, and then promotes the production of inflammatory factors to increase the infiltration of immune cells, efficiently eliminating tumor cells [71]. An advanced in situ nanovaccine formed by layered double hydroxides carrying cGAMP (STING agonist) (LDHs-cGAMP) and adsorbed TAAs was designed to potentiate the RFA-induced antitumor immune responses. The infiltration of cytotoxic lymphocytes (CTLs) and activated DCs in tumors and lymph nodes was significantly enhanced after nanovaccine treatment, which markedly inhibited the progression and metastasis of HCC. This strategy also dramatically altered tumor immune microenvironment, facilitated the response efficiency of anti-PD-L1 immunotherapy, and prevented the progression of poorly immunogenic Hepa1-6 HCC [72]. The combination of RFA and radioimmunotherapy, which involves killing cancer cells through radiation damage by combining high-energy radionuclides with monoclonal antibodies, has also shown promise for HCC. A study demonstrated a beneficial treatment effect of [ 131 I] metuximab (a CD147-targeted therapeutic strategy) after RFA on prevention of tumor recurrence in patients with HCC [73]. Another similar combination therapy trial also achieved positive results [74]. From these test results, it can be seen that these emerging immune-targeted drugs will ameliorate tumor immunosuppressive microenvironment and further enhance anti-tumor immune effect caused by RFA, achieving the inhibition of HCC progression and metastasis. This is also the underlying principle for future research team to develop new drugs and is expected to be applied in clinical practice as soon as possible. Table 1. The effects of RFA+Immunotherapy Ref Species Immunotherapy Combined immune effect Outcome Adverse effect [59] Human α-CTLA-4 CD8+ T cells ↑ Enhanced systemic antitumor immunity Reduced HCV viral load Pruritus [24] Human Mouse α-PD-1 Tumor-specific T cells ↑ Teff to Treg ratio ↑ Enhanced systemic antitumor immunity Inhibited growth of distant tumor — [61] Human RAK CD3+ CD8+ T cells ↑ IFN-γ ↑ No recurrences or deaths — [63] Human CIK CD3+, CD4+, CD56+, and CD3+ CD56+ cells ↑ CD4+/CD8+ ratio ↑ Improved RFS Reduced recurrence rate Pyrexia [64] Human CIK Antitumor toxicity of immune cells ↑ Improved RFS Chills, pyrexia, cough [65] Human NK cells γδT cells CIK Antitumor toxicity of immune cells ↑ Improved PFS and OS Reduced HCV viral load Pyrexia [66] Human CIK Antitumor toxicity of immune cells ↑ Improved RFS Fatigue, pyrexia [67] Human DC vaccine CIK Neoantigen-specific T cell responses ↑ Improved DFS Dizziness, cough, gum bleeding [73] Human [ 131 I] metuximab Tumor cells death ↑ Antitumour immune function ↑ Reduced recurrence rate Improved OS Pain, fever Leukocytosis Thrombocytopenia [74] Human (131) I-chTNT Tumor cells death ↑ Antitumour immune function ↑ Improved PFS and OS Leukocytosis [68] Rabbit CpG B Antitumour T cell responses and cytotoxicity ↑ Reduced tumour spread Improved survival — [69] Mouse R848 Tumor-infiltrating immune cells ↑ Total T cells, CD8/CD45, NK/CD45, and NK cells ↑ Inhibited tumor angiogenesis Promoted apoptosis of tumor cells — [70] Mouse ECI301 CD4+, CD8+, and CD11c+ cells ↑ CCL3, CCL4 ↑ Inhibited growth of non-ablated tumors — [72] Mouse LDHs-cGAMP TAAs α-PD-L1 Tumor-specific immune responses ↑ Improved immune microenvironment Inhibited tumor progression — IRFA in combination with immunotherapy Previous studies have shown that IRFA can hinder the immune responses by inducing an immunosuppressive microenvironment. Moreover, after IRFA, immune cells, especially tumor-infiltrating T cells, lose their effector functions and become exhausted in a short time, thereby resulting in rapid tumor relapse. Therefore, a combination of immunotherapy and IRFA has emerged as a promising approach for treating local recurrences. The details are presented in Table 2. 1. IRFA+ICI Notably, following IRFA, there was a significant upregulation of the immune checkpoint molecule PD-L1 on the surfaces of tumor cells [38]. This highlighted the insufficiency of relying solely on α-PD-1/PD-L1 therapy to reverse the immunosuppressive state within the HCC microenvironment after IRFA, thereby limiting its potential to inhibit HCC progression and metastasis. Consequently, it was necessary to solve the clinical difficulty of poor sensitivity to α-PD-1/PD-L1 therapy. In a particular study, IRFA initiated an elevation of MDSCs within residual tumors and restrained the function of T-cells, ultimately impeding the action of ICI [35]. Another subsequent study demonstrated that combining MDSCs inhibitors during IRFA can counterintuitively lead to a compensatory increase in PD-L1 expression on residual MDSCs. To address this issue, Tang et al. designed a size-adjustable hybrid nano-microliposome for co-delivery of MDSCs inhibitor (IPI549) and α-PD-L1. This nano-microliposome can selectively suppress MDSCs and block the PD-L1 up-regulation of surviving MDSCs, effectively inhibiting tumor recurrence [75]. In addition, Sun et al. proposed a new treatment strategy, that is, combining OK-432 with α-PD-1 for the treatment of residual tumors after IRFA. They found OK-432 significantly enhanced DCs maturation. When combined with α-PD-1, it could strengthen the infiltration and function of CD8+ T cells and significantly reduce the number of FoxP3 regulatory T cells in the residual tumors. Moreover, the triple-therapy group (IRFA+OK-432+α-PD-1) exhibited the smallest tumor volume and the longest survival period [76]. A recently published study observed that IRFA combined with α-PD-L1 followed by melatonin reduced tumour growth and metastasis. In mechanism, melatonin inhibited the EMT, as well as the expression of HIF-1α and PD-L1 in tumor cells after IRFA. It was also revealed that melatonin reduced proportion of MDSCs and increased the proportion of CD8 T cells. These findings proposed the strategies that can overcome the TIME and enhance the efficacy of immunotherapy [77]. IRFA + other immune modulators To address the mild protective autophagy triggered by sublethal thermal stimulation in residual tumor cells, researchers often combine immunomodulators after IRFA to further promote immunogenic cell death (ICD) of cancer cells. LTX-315 is a tumorolytic peptide capable of inducing ICD. Zhou et al. investigated the feasibility of interventional oncolytic immunotherapy with LTX-315 for residual tumors after IRFA of VX2 liver tumors. Compared with the control group, a significant increase in CD8+ T cells and HSP70 and a significant decrease in Tregs were observed in residual tumors of the combination group. Specifically, the weakened activity of Tregs led to their gradual apoptosis, which further contributed to the alteration of the tumor microenvironment in favor of anti-tumor immune responses [78]. Zhang et al. constructed active targeting zeolitic imidazolate framework-8 (ZIF-8) nanoparticles (NPs) loaded with STF62247 or both STF62247 and BMS202. It was found that the NPs inhibited proliferation and stimulated apoptosis of residual tumor cells exposed to sublethal thermal injury in an autophagy-dependent manner and induced ICD. This significantly promoted maturation of DCs, which then activated the anti-tumor immune microenvironment, and effectively inhibited the growth of residual tumors [79]. Secondly, the TIME of IRFA features the polarization of M2 macrophages, which in turn inhibits antigen presentation by DCs and reduces CTL infiltration. Interestingly, MSA-2, a STING agonist, can reorganize M2 pro-tumorigenic macrophages into M1 anti-tumorigenic ones, improving the immunosuppressive state. To optimize the therapeutic effect of MSA-2, Ao et al. designed an ALG@MSA-2 injectable hydrogel. And the results confirmed that ALG@MSA-2 mediates STING pathway activation and promotes a favorable TIME, which also resulted in a complete regression of contralateral tumors and widespread liver metastases in vivo [80]. Zhu et al. have engineered a delivery platform for GW4869 and amlodipine (AM), aiming at inhibiting exosome secretion, and further degrading the PD-L1, and remodeling the post-IRFA TIME. This design strengthened the activation and proliferation of diverse functional T-cell subsets after IRFA, especially CD8 T cells, IFN-γ CD8 cytotoxic T cells and NK cells. It also decreased the infiltration of immunosuppressive cells such as Tregs and MDSCs. Such beneficial remodeling of TIME greatly inhibited the progression and metastasis of HCC [81]. As Liu et al. first revealed, the heat stress from IRFA activates SUMOylation in residual tumors, creating a suppressive TIME. To address this, they developed a TAK-981-loaded nanocomposite hydrogel (BT-NPs@PLEL) designed to target SUMOylation. This notably stimulates the DCs and CTLs-mediated antitumor immune response in residual tumors while maintaining biosafety. Nonetheless, TAK-981 can increase PD-L1 expression. Their data further demonstrated that combining with α-PD-L1 treatment synergistically eradicated residual tumors and suppresses distant tumors [32]. Yu et al. Have found a ruthenium complex Ru, which can effectively inhibit the HIF-1α-related autophagy pathway. In addition, it can activate the immune system and reverse the tumor immune suppression microenvironment after IRFA [82]. From the above results, it can be seen that such combinatorial therapies can target the immunosuppressive molecules and pro-tumor factors generated by IRFA. These novel drugs can also reverse immunosuppressive state and inhibiting tumor progression. Table 2. The effects of IRFA+Immunotherapy Ref Species Therapy Combined immune effect Outcome [75] Mouse IPI549 α-PD-L1 MDSCs ↓ PD-L1 on MDSCs ↓ Activated immune responses Improve the sensitivity to ICI [76] Mouse OK-432 α-PD-1 DCs, cDC1s ↑ CD8+, CD8/TNF-α, and CD8/IFN-γ T cells ↑ IFN-γ, TNF-α ↑ IL-10, TGF-β, and Tregs ↓ Inhibited tumor growth Enhanced antitumor immunity Improve the sensitivity to ICI [77] Mouse Melatonin α-PD-L1 MDSCs, Tregs ↓ T cells, B cells, and CD8+ T cells ↑ IFN-γ, TNF-α, IL-12, and IL-2 ↑ IL-4, IL-10, and HIF-1α ↓ MMP-2, MMP-9 ↓ Improved OS Inhibited tumor growth and metastasis Reshaped immune microenvironment Improve the sensitivity to ICI [78] Rabbit LTX-315 Tregs ↓ CD8+ T cells, HSP70 ↑ Inhibited tumor growth Reshaped immune microenvironment [79] Mouse SZP NPs/ SBZP NPs Autophagosomes ↑ CD4+, CD8+ T cells ↑ CD11C+ DCs ↑ TGF-β, IL-6, and TAMs ↓ IFN-γ, TNF-α, and DAMPs ↑ Induced ICD Inhibited tumor growth Reshaped immune microenvironment Improve the sensitivity to ICI [80] Mouse ALG@MSA-2 M1-like macrophages, DCs, and NK cells ↑ CD8+, Memory T cells ↑ IFN-γ, GrzmB, and TNF-α ↑ M2-like macrophages, Tregs ↓ Inhibited tumor growth and metastasis Reshaped immune microenvironment [81] Mouse AM/GW@PDA PD-L1, TGF-β1↓ CD8, IFN-γCD8 cytotoxic T cells ↑ NK cells, ILCs ↑ MDSCs, Tregs ↓ IFN-γ, IL-6, IL-12, and TNF-α ↓ Inhibited tumor growth and metastasis Reshaped immune microenvironment Enhanced antitumor immunity [32] Mouse BT-NPs@PLEL α-PD-L1 SUMOylation ↓ IFN-1 signaling ↑ CDs, CTLs ↑ Inhibited tumor growth and metastasis Enhanced antitumor immunity Discussion Combining RFA with immunotherapy showed promise in improving HCC treatment responses through affecting the tumor immune microenvironment. Various components of TIME undergo complex changes after RFA. While RFA removes tumor burden, it also releases TAAs to stimulate the antigen-presentation process of relevant APC, inducing T-cells or cytotoxic cells to exert anti-tumor immune effects. However, numerous studies have shown that the immune responses induced by RFA falls short of triggering a powerful and sustainable immune effect. Moreover, IRFA can also lead to a more aggressive phenotype of tumor cells and cause an inflammatory-related immunosuppressive environment. In addition, HCC itself has a strong immunosuppressive property, which enables cancer cells to evade the surveillance of immune system. These phenomena can reduce the efficacy of the ablation procedure, contributing to HCC recurrence and metastasis. With the development of immunotherapy, it has been found that monotherapy using immunotherapy alone is less effective than combination therapy. Therefore, comprehensive treatment and individualized treatment are the future trends of HCC treatment. Immunotherapy aims to enhance the antigen-presenting ability regarding TAAs and activate anti-tumor immune effect. Currently, multiple research teams have also developed novel immunomodulatory drugs to regulate the immune microenvironment of HCC to further eliminate residual and distant metastatic lesions. Judging from the current research results, the combination of RFA and immunotherapy exhibits superior efficacy than either therapy alone. This treatment strategy can consolidate and prolong the anti-tumor responses induced by RFA. However, IRFA can trigger compensatory expression of immune checkpoints, causing tumor to develop resistance to ICI. So adding other immunomodulators can resolve this issue and allow ICI to better exert its therapeutic efficacy. These immunomodulators can also regulate the tumor immunosuppressive microenvironment, relieve the immunosuppressive state and restore the immune system’s function in killing tumor cells. From the clinical perspective, RFA can reduce the tumor burden for subsequent immunotherapy. Immunotherapy, in turn, can further enhance the patient’s anti - tumor immune function, thereby reducing the risk of HCC recurrence. Future research directions should focus on optimizing treatment strategies, exploring new therapeutic combinations, revealing molecular mechanisms, and developing precision medicine and personalized treatments. With the release of the data from the latest IMbrave050 trial, the comprehensive treatment of HCC patients with a high risk of recurrence remains in need of innovation, and future research should also be validated with respect to long-term benefits. And the pro-tumor immune mechanisms caused by local treatment still need to be further explored. Meanwhile, more high-quality clinical studies are required to evaluate the sequence of immunotherapy and RFA, the efficacy of combined therapy on HCC at different BCLC stages, biomarkers that can predict treatment outcomes, and the management of adverse events. It’s still necessary to develop more new types of immunological drugs to better enhance the efficacy of RFA, such as molecular-targeted therapy, nanoparticle-mediated therapy, etc. In conclusion, the combination of RFA and immunotherapy for liver cancer can enhance tumor control, survival rate, and quality of life, which also brings hope for patients with other solid cancers. Acknowledgements This work was supported with the National Natural Science Foundation of China (NO. 82203658; 82160124; 82100641), Science and Technology Department of Xinjiang Uygur Autonomous Region (NO. 2022E02044), Science and Technology Bureau of Xinjiang Production and Construction Corps (NO. 2022AB024), the Natural Science Foundation of Shandong Province (NO. ZR2021QH276). Credit Author Statement Liu Yang, Yuemin Feng, and Qiang Zhu contributed to the conception, writing, and discussion of this manuscript. Shuhang Wei, Zongxin Liu, Qiqi Liu, and Zhen Yu equally contributed and reviewed the revised version of the manuscript. All authors have approved the final version of the manuscript. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References 1. 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Recent Pat Anticancer Drug Discov, 2024. Information & Authors Information Version history V1 Version 1 09 January 2025 Peer review timeline Published Immunology Version of Record 26 Jun 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Immunology Keywords immune homeostasis immunotherapy liver tumour immunology Authors Affiliations Liu Yang 0009-0008-0636-2180 Shandong University View all articles by this author Shuhang Wei Shandong University View all articles by this author Zongxin Liu Shandong University View all articles by this author Qiqi Liu Shandong University View all articles by this author Zhen Yu Shandong Provincial Hospital View all articles by this author Yuemin Feng Shandong Provincial Hospital View all articles by this author Qiang Zhu [email protected] Shandong Provincial Hospital View all articles by this author Metrics & Citations Metrics Article Usage 433 views 267 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Liu Yang, Shuhang Wei, Zongxin Liu, et al. Radiofrequency Ablation and Immunotherapy: Orchestrating the Immune Microenvironment for Improved Hepatocellular Carcinoma Control. Authorea . 09 January 2025. 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