Cryoablation remodels the immune microenvironment in hepatocellular carcinoma: From mechanistic insights to clinical translation in combination immunotherapy

Abstract

Hepatocellular carcinoma remains a persistent therapeutic challenge, in part due to an immunosuppressive tumor microenvironment that renders it a “cold” tumor and limits the efficacy of immune checkpoint inhibitors. Local cryoablation transcends its direct cytotoxic role by eliciting systemic immunomodulatory effects. This review discussed the dual role of cryoablation in reprogramming the hepatocellular carcinoma tumor microenvironment. Mechanistically: Cryoablation induces immunogenic cell death, which serves as an in situ vaccine to release tumor antigens and danger signals to activate dendritic cells and prime tumor-specific T-cell responses. Nonetheless, this immunogenic response is normally offset by an attendant, transient immunosuppressive backlash. We review the preclinical and clinical data on the combination of cryoablation and immune checkpoint inhibitors, and a strategy that is aimed at blocking this inhibitory feedback and synergistically transforming the so-called “cold” tumors into immunologically “hot” tumors. The completeness of ablation, technical parameters and the host immune status are key determinants of therapeutic outcome. Current translational issues, especially with regard to stratification of patients, are discussed. Future progress of this promising combinatorial strategy will depend on the development of predictive biomarkers and on fine-tuning of combination regimens.

Key Words: Hepatocellular carcinoma; Cryoablation; Tumor microenvironment; Immunotherapy; Immunogenic cell death; Combination therapy

Core Tip: Cryoablation exerts a dual and exploitable immunomodulatory effects in hepatocellular carcinoma. Acting as an in situ vaccine, cryoablation induces immunogenic cell death and simultaneously triggers a transient immunosuppressive effect. This duality is strategically used by combining cryoablation with immune checkpoint inhibitors to remodel the immunosuppressive “cold” tumor microenvironment. Ablation completeness, technical parameters, and host immune status are critical determinants of therapeutic success. In order to fully achieve its clinical potential, future initiatives ought to be directed towards overcoming the difficulties of patient stratification and treatment sequencing by developing predictive biomarkers and optimized combination regimens.

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the leading causes of the global cancer burden, with an estimated annual incidence of over 684659 new cases and approximately 597434 deaths, according to the latest GLOBOCAN 2022 data[1]. HCC usually occurs in the background of chronic hepatitis (caused by hepatitis B or C virus) or alcoholic liver disease, both of which can lead to cirrhosis. This chronic inflammatory and fibrotic microenvironment prompts the development of a very complex and immunosuppressive tumor microenvironment (TME)[2,3]. In this TME, there is functional exhaustion or depletion of cytotoxic CD8⁺ T cells. The cells of the immune system that suppress immune responses are numerous: (1) Regulatory T cells (Tregs); (2) Tumor-associated macrophages (TAMs) (specifically the M2 phenotype); and (3) Myeloid-derived suppressor cells (MDSCs). These cells secrete inhibitory cytokines like transforming growth factor-beta (TGF-β) and interleukin (IL)-10, collectively establishing an “immune-desert” landscape conducive to tumor growth and immune evasion[2,3].

Immune checkpoint inhibitors (ICIs) such as programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors have become the standard systemic therapy for advanced HCC. They work by inhibiting T-cell suppression, thus partially recovering anti-tumor immunity[4]. Nevertheless, the objective response rate (ORR) of ICI monotherapy is still low at around 15%-20% with most patients developing primary or acquired resistance[3,4]. Although tyrosine kinase inhibitors (e.g., sorafenib, lenvatinib) and combination targeted-immunotherapy regimens have been developed, the performance of advanced HCC patients is suboptimal. Meta-analyses and key trials suggest that ORR continues to be modest with median overall survival (mOS) oftentimes reported to be approximately 20 months, highlighting a persistent therapeutic plateau[5-8]. This underscores the inadequacy of simply providing the T-cell “brake” to conquer the immunosuppressive ecosystem that is so deeply rooted in HCC.

Elaborate studies have found the highly immunosuppressive TME as the fundamental impediment to successful ICI therapy in HCC. As a specialized immunological organ, the liver is physiologically predisposed to immune tolerance to prevent excessive immune responses against gut-derived antigens. Hepatocarcinogenesis exploits and amplifies this intrinsic tolerance, establishing a complex network of diverse immunosuppressive cells (e.g., Tregs, MDSCs, and M2-TAMs) and inhibitory molecules (e.g., PD-L1, TGF-β, and IL-10). This network constitutes a formidable ”immune-excluded” barrier[9-11]. This immunological exclusion from immune surveillance and effector attack is reflected in the low response rate to ICI monotherapy in HCC – less than 20%[12]. Therefore, the most critical advance in improving the efficacy of HCC immunotherapy lies in developing strategies to overcome this immunosuppressive barrier and to remodel the TME, thus converting the so-called “cold” tumors into “hot” ones[13,14].

It is against this background that local ablative therapy, such as radiofrequency ablation (RFA), microwave ablation (MWA), or cryoablation is established as an option in curative efforts in the early stages of HCC. These methods have several advantages compared to surgery and include accurate visual guidance, low invasiveness, safety, and good treatment efficacy. They also have significant applications in combination therapy using intermediate stage HCC or in palliative care of advanced cases[15-18]. The paradigm has evolved during the last twenty years, which was traditionally considered as the purely local physical destructive technique. There is emerging evidence that ablation-induced tumor cell death can release a large number of tumor-specific antigens and danger signals, which have the potential to activate the anti-tumor immune system of the host. This can result in a systemic abscopal effect, which is basically an “in situ vaccine”[13,14,19,20]. Cryoablation, among other methods of ablation, has received particular interest in immuno-oncology due to its unique freeze-thaw cycle, based on the Joule-Thomson effect (argon gas to freeze, helium gas to thaw)[21-23]. In contrast to traditional thermal ablation methods (RFA and MWA), which generate high temperature, cryoablation induces cell necrosis and apoptosis caused by a combination of different mechanisms, including the formation of ice crystals in the cell, interruption of lipid-protein complexes of the cell membrane, osmotic shock and microvascular thrombosis[22]. More to the point, there is a growing body of evidence that cryoablation may also have some specific benefits in terms of regulating immune response and affecting the cellular composition of the TME and cytokine profiles. The potential benefits of this include the ability to induce immunogenic cell death (ICD), a clearly visualized “ice ball” boundary for safety, and a relatively lower risk of thermal-related injury of the other major vascular structures[23-25]. Thus, cryoablation is no longer considered simply as a local therapy device but as a powerful immunomodulatory device with significant potential for deep synergy with systemic immunotherapy to overcome the fortress of immunotherapy resistance in HCC[14,26-29].

We propose a mechanistic framework of this dual immunomodulatory role and its therapeutic implication as illustrated in Figure 1. In brief, ICD released by cryoablation-induced damage to the tumor initiates a systemic anti-tumor immune response (left panel). At the same time, the process is accompanied by counter-regulatory immunosuppression, which is characterized by the attraction of suppressive cells and the upregulation of immune checkpoints (right panel). The inhibitory axis can be blocked by the concomitant administration of ICIs, thus shifting the balance to successful immunity and providing the rationale for combination therapy (central intervention).

Figure 1 The dual immunomodulatory effects of cryoablation and the rationale for combination with immunotherapy in hepatocellular carcinoma. Schematic illustration of the dual immunomodulatory effects of cryoablation and the synergistic rationale for its combination with immune checkpoint inhibitors in hepatocellular carcinoma. Central trigger: Cryoablation induces immunogenic cell death in hepatocellular carcinoma cells, leading to the release of damage-associated molecular patterns and tumor-associated antigens. Left panel (immune activation): The released damage-associated molecular patterns/tumor-associated antigens promote dendritic cell maturation and antigen presentation. Mature dendritic cells migrate to draining lymph nodes to prime and activate naïve CD4⁺ and CD8⁺ T cells. These activated T cells differentiate into effector and memory subsets, which traffic to and attack both the primary and distant tumor sites. Right panel (concomitant immunosuppression): In parallel, cryoablation can trigger counter-regulatory mechanisms that foster an immunosuppressive tumor microenvironment. These include the recruitment of immunosuppressive cells (e.g., regulatory T cells, myeloid-derived suppressor cells, M2-type tumor-associated macrophages), the secretion of inhibitory cytokines (e.g., transforming growth factor-beta, interleukin-10), and the upregulation of immune checkpoint molecules [e.g., programmed death-ligand 1 (PD-L1)] on tumor and immune cells. Therapeutic intervention and rebalancing (central motif): The centrally placed balance scale symbolizes the dynamic equilibrium between the concurrently induced immune activation and suppression. The administration of immune checkpoint inhibitors (e.g., anti-programmed death-1/PD-L1 antibodies) to block the programmed death-1/PD-L1 pathway acts as a decisive intervention that tips the balance in favor of productive anti-tumor immunity, thereby overcoming resistance and enabling systemic tumor control. ATP: Adenosine triphosphate; CALR: Calreticulin; DAMP: Damage-associated molecular pattern; DC: Dendritic cell; HMGB1: High mobility group box 1; ICD: Immunogenic cell death; IL: Interleukin; MDSC: Myeloid-derived suppressor cell; PD-1: Programmed death-1; PD-L1: Programmed death-ligand 1; TAA: Tumor-associated antigen; TAM: Tumor-associated macrophage; Treg: Regulatory T cell; TGF-β: Transforming growth factor-beta.

To this end, this review comprehensively synthesizes the immunomodulatory role of cryoablation in HCC management. We will initially deconstruct the dual mechanisms by which cryoablation positively and negatively influences the HCC immune microenvironment at the cellular and molecular levels. We will then systematically synthesize evidence on animal models and clinical studies to assess the effectiveness and safety of cryoablation, alone or in combination with immunotherapy. Based on this, we shall examine some of the major determinants of the effectiveness of its immunomodulatory effects and combination strategies. Lastly, we will determine the existing gaps in knowledge and challenges in the clinical translation, and come up with future innovative research directions to further improve this area.

MOLECULAR AND CELLULAR MECHANISMS OF CRYOABLATION IN REGULATING THE HCC IMMUNE MICROENVIRONMENT: A DUAL-EFFECT PERSPECTIVE

Cryoablation destroys tumors through a dynamic pathophysiological process. Though it induces physical cell inactivation, which is accomplished by extreme cold, its effects on the immune system are far from a unidirectional activation; rather, it initiates a complex network that concurrently encompasses both immune stimulation and potential immunosuppression[30]. Understanding this duality is crucial for optimizing therapeutic strategies. To give a systematic review of this dual immunomodulatory profile, the key processes, comparison with thermal ablation, and implications of this clinical safety are summarized in Table 1[13,14,19,20,27,30-56].

Table 1 Dual immunomodulatory effects of cryoablation on the hepatocellular carcinoma microenvironment.

Immunomodulatory direction	Core mechanisms and events	Key effects/findings
Positive immune activation	Induction of immunogenic cell death: Releases intact tumor antigens and DAMPs via unique physical injury (ice crystal formation, cell rupture)[30-33,35]	Functions as an in situ vaccine, initiating systemic anti-tumor immunity. Antigen accumulation within DCs may be higher than with thermal ablation
	DAMP release and antigen-presenting cell activation: Releases key DAMPs (high mobility group box 1, adenosine triphosphate, calreticulin, heat shock proteins), promoting DC maturation and antigen presentation via receptors like Toll-like receptor 4 and P2X7[19,32,34,36]	Provides “danger signals”, bridging innate and adaptive immunity
	Initiation of adaptive immunity: DCs capture antigens, migrate to lymph nodes, and activate tumor-specific CD4+ and CD8+ T cells[13,14,20,35]	Preclinical models confirm increased numbers and activity of T cells and NK cells locally and systemically, with tumor-specific antibody production
	Effector T cell infiltration and abscopal effect: Activated CTLs home to tumors. Enrichment of CD8+ T cells and NK cells is also observed in untreated distant lesions[27,37-39]	Combination with immune adjuvants or anti-PD-L1 significantly enhances T cell infiltration and function, yielding synergistic activity
	Formation of immunological memory: Induces expansion of effector memory T cell and central memory T cell, particularly potent neoantigen-specific CD4+ T-cell responses[40-46]	Clinical studies show increased memory T cell subsets in peripheral blood post-procedure. Combination with immune checkpoint inhibitors or adjuvants significantly enhances the strength and durability of memory responses
Potential immunosuppression	Release of immunosuppressive cytokines: Necrotic areas release TGF-β, interleukin-10, vascular endothelial growth factor, etc. May induce more intense pro-inflammatory cytokine release compared to MWA/radiofrequency ablation[30,47-50]	TGF-β strongly inhibits CTL/NK function, promotes Treg differentiation, and M2 macrophage polarization
	Recruitment of immunosuppressive cells: Recruits myeloid-derived suppressor cells, M2-type tumor-associated macrophages, and regulatory T cells to the tumor site. May polarize tumor-associated neutrophils to an immunosuppressive N2 phenotype, forming physical barriers via NETs[20,37,50-53]	These cells inhibit effector T cells via mechanisms like depletion of essential amino acids and secretion of inhibitory factors. NET formation is a novel mechanism limiting efficacy
	Upregulation of immune checkpoints: As an adaptive resistance mechanism, induces upregulation of checkpoint molecules (e.g., programmed death-1/PD-L1) in distant tumor tissues[54]	May lead to rapid T cell exhaustion and limit the abscopal effect. Studies show it may induce higher PD-L1 upregulation than MWA
	Antigen overload and immune tolerance[55,56]	Massive short-term antigen release in a suppressive milieu may lead to T cell deletion/anergy, inducing immune tolerance

CTL: Cytotoxic T lymphocyte; DAMP: Damage-associated molecular pattern; DC: Dendritic cell; MWA: Microwave ablation; NET: Neutrophil extracellular trap; NK: Natural killer; PD-L1: Programmed death-ligand 1; TGF-β: Transforming growth factor-beta; Treg: Regulatory T cell.

Positive immunological activation: Initiating a systemic anti-tumor immune response

Cryoablation, as a physical therapy, can use its innate immunological potency by inducing the formation of ICD, thereby acting as a natural in situ vaccine. This offers an immunological rationale to the long-term tumor control and recurrence prevention[31].

ICD is a specific form of cell death characterized by the release or exposure of damage-associated molecular patterns (DAMPs) with immunoadjuvant functions, thereby activating adaptive immune responses[19,32,33].

In the TME, ICD restarts anti-tumor immunity via the subsequent fundamental processes. These released DAMPs are recognized by antigen-presenting cells (APCs) such as dendritic cells (DCs) to trigger their maturation and increase their capacity to effectively phagocytose and process tumor antigens from dead cells. This is followed by migration of the activated DCs towards lymph nodes where antigens are presented to naïve T cells, ultimately inducing antigen-specific cytotoxic T-cell responses[34]. Phosphorylation of eukaryotic translation initiation factor 2 alpha and activation of autophagy are some of the key molecular events accompanying this process. All these events ensure the effective transmission of immunogenicity from dying cells, thereby laying the foundation for systemic anti-tumor immunity[31,32].

Release of DAMPs (the “danger signal” for immune response): The release of ICD and DAMPs is caused by the distinct physical injury mechanism of cryoablation that exhibits spatial heterogeneity in the ablation zone. The cells immediately surrounding the probe experience direct mechanical rupture as a result of fast freezing leading to the formation of ice crystals in the cells. In the periphery of the ablation zone, cells experience slow freezing, where the formation of extracellular ice crystals results in intracellular dehydration and increase in the concentration of solutes, causing damage. Then, during the thawing phase, cells begin to swell and burst suddenly as the extracellular ice melts and forms a local hypotonic environment[30,35]. This necrotic form is the most effective in preserving the native conformation of tumor antigens[25].

Following cell rupture, a large number of endogenous “danger signals” are released[30]. The key DAMPs released and their immunostimulatory pathways include: (1) High mobility group box 1: As a critical DAMP, high mobility group box 1 binds to Toll-like receptor (TLR) 4 on APCs like DCs, promoting DC maturation and the secretion of pro-inflammatory cytokines (e.g., IL-1β, IL-6, tumor necrosis factor-alpha)[19], while directly enhancing their antigen processing and presentation capabilities[32,34]; (2) Extracellular adenosine triphosphate: This important pro-inflammatory factor activates the NLRP3 inflammasome via the P2X7 receptor, driving the processing and release of cytokines like IL-1β and amplifying the local inflammatory response[19]. Adenosine triphosphate can also directly recruit and activate APCs[32]; (3) Surface-exposed calreticulin: Acting as a classic “eat me” signal, calreticulin directly promotes the phagocytosis of dead cells by APCs and their subsequent antigen presentation function[32]; (4) Heat shock proteins (e.g., heat shock protein 70): Acting as molecular chaperones, they can form complexes with tumor antigens. These complexes are efficiently internalized and processed by DCs via receptors like CD91, significantly enhancing cross-presentation of antigens[19,36]; and (5) Other regulatory molecules: Such as annexin A1 exerting immunoadjuvant effects, and type I interferons participating in and amplifying anti-tumor immune responses[32]. Notably, studies suggest that antigen accumulation within DCs post-cryoablation may be higher than after thermal ablation[30], further supporting its potential advantage in inducing specific immune responses.

Tumor antigen exposure, presentation, and initiation of adaptive immunity: Tumor-specific or tumor-associated antigens are released when massive lysis of tumor cells is performed using cryoablation. These are sniffed by locally infiltrating DCs. Having been stimulated by the danger signal co-stimulation (DAMPs), DCs reach maturity and migrate to regional draining lymph nodes. There, they present processed antigen peptides to CD8⁺ and CD4⁺ T cells via major histocompatibility complex class I and II molecules, thus triggering antigen-specific adaptive immune responses[13,14].

In woodchuck HCC models, post-cryoablation, the CD3+ T lymphocytes and natural killer (NK) cells significantly increased in number at the periphery of the ablation zone, which is an indication of local recruitment and activation of immune cells[20]. Histological examination also demonstrated the neutrophil inflammation in tumor tissue after cryoablation, with subsequent recruitment of massive macrophage inflammation. Moreover, an enhanced activity of NK cells, tumor-specific T-cell responses in regional lymph nodes, and high levels of circulating T cells systemically have been reported after cryoablation[35]. Cryoablation has also been demonstrated to cause a systemic response in the production of tumor-specific antibodies[35].

Effector-T-cell activation, tumor infiltration, and abscopal effect: Activated tumor specific CD8⁺ T cells expand and differentiate into cytotoxic T lymphocytes (CTLs), which in turn home back to the primary site and possible metastatic sites via the blood stream. This is directly evidenced in preclinical studies. The populations of CD8⁺ T cells, CXCR3-expressing CD8⁺ T cells (CXCR3 is a chemokine receptor mediating migration to sites of inflammation), and interferon-gamma (IFN-γ)-producing CD8⁺ T cells in the residual tumor were significantly increased[37]. This systemic change, whereby NK cell enrichment is also found in remote, untreated tumors[20,38]. Furthermore, this effect can be further enhanced by cryoablation in combination with immune adjuvants (e.g., CpG) which can substantially boost the degree of intratumoral cytotoxic T-cell infiltration[27]. Cryoablation in combination with immune checkpoint blockade (e.g., anti-PD-L1) can lead to synergistic anti-tumor response. The PD-1⁺ CD8⁺ T cells produced following combination therapy have more robust effector functions[39].

Formation and consolidation of immunological memory: By inducing ICD, cryoablation not only directly kills tumor cells but also releases large numbers of tumor antigens and DAMPs. DCs capture and present these substances, which is capable not only of triggering a potent effector immune response but also inducing the development of long-term immunological memory. This process allows cryoablation to prevent local recurrence, produce an abscopal effect, and achieve long-term immune surveillance, ultimately establishing long-term protective anti-tumor immunity[34]. ICD also serves as the bridge between conventional tumor therapy and modern immunotherapy[57].

Clinical prospective studies have shown that compared to surgical resection, cryoablation significantly increases the number of CD69⁺ CD4⁺/CD8⁺ T cells and memory CD8⁺ T cells in peripheral blood at 3 months post-procedure, indicating its capacity to induce durable immune responses[40]. The immune memory that is evoked by the cryoablation is characterized by the proliferation of the T-cell subsets. Research has established that cryoablation can greatly boost the percentage of effector memory T cells and central memory T cells[40,41]. Notably, modified cryo-thermal therapy has been found to induce strong neoantigen-specific CD4⁺ T-cell responses, which form the basis of long-term, robust immune protection[42].

Combining cryoablation with other immunomodulatory strategies can significantly amplify and consolidate immune memory, demonstrating a “1 + 1 > 2” synergistic effect. It has been shown that cryoablation with anti-CTLA-4 therapy can raise the percentage of effector and effector memory CD4⁺/CD8⁺ T cells in both distant tumors and blood, and in effect, increase the development of systemic immunological memory[41]. The cryoablation remodeled TME can be used to synergize with the ICIs to achieve more durable anti-tumor memory responses[43]. CpG and saponin adjuvants combined with cryoablation can substantially increase the ratio of polyfunctional T cells and enhance the capability of tumor-specific CD8⁺ T cells to produce IFN-γ and, thus, induce more robust long-term immune memory[44]. With the development of functional nanovaccines to entrap tumor debris formed by cryoablation, effective induction of the DC activation and T-cell differentiation can be obtained. This combined approach in animal models has high rates of primary tumor suppression and long-term survival, which significantly delays recurrence[45].

The animal model studies with the different angles confirmed that the cryoablation and its modified approaches can induce solid protective immune memory. In particular, the combination of cryoablation and immunotherapy can slow tumor growth and metastasis, increase survival, and, more importantly, induce anti-tumor memory which protects mice against rechallenge[46]. Cryoablation can generate long-term anti-tumor memory responses within the TME, an effect that can be further enhanced by combination with ICIs[43]. Also, technically optimized cryo-thermal therapy, in comparison to traditional RFA, not only induces antigen-specific CD8⁺ T-cell responses but, more importantly, generates powerful neoantigen-specific CD4⁺ T-cell responses. This immune response, primarily mediated by CD4+ T cells, confers resistance to tumor rechallenge[42].

In short, as an in situ vaccine, cryoablation efficiently induces specific immunity and expands the memory T cell repertoire. It also can be combined with ICIs, adjuvants, or vaccine strategies to further boost immune memory strength and duration synergistically. Collectively, these findings provide a strong scientific rationale for considering cryoablation as an effective inducer of immunological memory and a promising strategy for cancer therapy.

Potential immunosuppressive responses: Post-treatment negative feedback regulation

Although it stimulates anti-tumor immunity, cryoablation can also induce endogenous negative feedback regulatory mechanisms, resulting in the development or maintenance of an immunosuppressive microenvironment. This paradoxical phenomenon is an important reason for recurrence or progression in some patients after treatment[58].

This comorbid immunosuppression, as pictured on the right of Figure 1, mainly entails the following aspects.

Release of immunosuppressive cytokines: Necrotic tissues in the ablation zone and infiltrating immune cells can produce high levels of TGF-β, IL-10, and vascular endothelial growth factor. Notably, compared to MWA and RFA, cryoablation may induce higher release of pro-inflammatory cytokines (e.g., IL-1, IL-6, tumor necrosis factor-alpha), accompanied by more significant elevation of inflammatory and hepatocellular injury markers[30]. In advanced stages of cancer progression, the TGF-β signaling pathway promotes the formation of an immunosuppressive microenvironment through multiple mechanisms[47]. TGF-β is one of the strongest known immunosuppressive factors. It directly inhibits the function of CTLs and NK cells[48], promotes Treg differentiation and inhibits the activation and function of effector T cells[49], and also induces macrophage polarization towards the M2 phenotype[27,37,50].

Recruitment and activation of immunosuppressive cells: The inflammatory response following cryoablation recruits various immunosuppressive cells, including MDSCs, TAMs, and Tregs, to the tumor site or around residual lesions[51]. These cells collectively maintain an immunosuppressive microenvironment by secreting inhibitory cytokines and expressing immune checkpoint molecules[52]. In particular, TGF-β can promote the recruitment and activation of MDSCs, which suppress T cell function through mechanisms involving arginase, nitric oxide synthase, etc[50].

In mouse models, although the combination of incomplete cryoablation with a matrix metalloproteinase inhibitor increased CD8⁺ T cell infiltration, an increase in CD206⁺ M2-type TAMs and matrix metalloproteinase 9⁺ cells was also observed[37]. Another woodchuck study found that the number of Tregs was higher in distant, non-ablated liver tumors than in the primary site or control tumors[20]. These cells directly inhibit effector T cell function through various mechanisms, such as depleting essential amino acids (tryptophan, arginine), secreting inhibitory cytokines, and expressing inhibitory ligands (e.g., PD-L1). Notably, the remodeling of the immunosuppressive microenvironment post-cryoablation may be strongly influenced by neutrophils. Studies indicate that cryoablation can cause a phenotypic shift in tumor-associated neutrophils, from the anti-tumor active N1 type to the immunosuppressive N2 type. The mechanism is related to increased intracellular Ca²⁺ concentration in neutrophils induced by cryoablation, which subsequently activates PAD4-dependent neutrophil extracellular trap (NET) formation. NETs can form a physical barrier preventing cytotoxic T cells from contacting the tumor, and the proteolytic enzymes and inflammatory factors they release further suppress T cell function, exacerbating immune escape[53]. This finding provides a new perspective for understanding treatment failure in some patients.

Dynamic reprogramming of macrophage states (beyond the M1/M2 paradigm): The post-ablation recruitment and activation of macrophages is a central immunosuppressive event, but their biology is more complex than the classic M1/M2 dichotomy. Cryoablation induces a significant influx of monocyte-derived macrophages while also affecting tissue-resident Kupffer cells. These macrophages can adopt diverse, context-dependent functional states that promote immunosuppression. Key states relevant to post-cryoablation rebound include: (1) Immunosuppressive/T cell-suppressive state: Characterized by high expression of PD-L1, CD206, and arginase-1, and secretion of IL-10 and TGF-β, directly inhibiting T cell function[27,37,50]; (2) Wound-healing/fibrotic state: Associated with tissue repair pathways that can promote tumor stroma formation and create a physical barrier[59-61]; and (3) Angiogenic state: Involved in secreting vascular endothelial growth factor and other factors to support revascularization of residual tumor[62-64]. This state-based framework explains the limitations of targeting generic ”M2-TAMs” and points toward more precise therapeutic interventions. Rational combination strategies could aim to reprogram these macrophages towards an anti-tumor, antigen-presenting state by targeting specific receptors (e.g., CSF1R for depletion, CD47-SIRPα axis for phagocytosis blockade), signaling pathways (e.g., PI3K-γ inhibition), or metabolic programs (e.g., adenosine signaling).

Upregulation of immune checkpoint molecules: Cryoablation also drives an immunosuppressive mechanism: The upregulation of key immune checkpoints (see the right panel of Figure 1). Studies show that cryoablation significantly upregulates the expression of the PD-1/PD-L1 signaling pathway in distant tumor tissues during the later stages of treatment. For example, in a bilateral breast tumor mouse model, PD-1 and PD-L1 expression increased in contralateral tumor tissue after cryoablation. This was closely associated with a local immunosuppressive state and ultimately limited the abscopal effect of cryoablation[58]. Therefore, although cryoablation can induce specific T cell responses, the concomitant immunosuppressive microenvironment, particularly the activation of the PD-1/PD-L1 pathway, may weaken or even counteract the initial immunological gain. This can lead to an insufficient anti-tumor immune response, failing to effectively prevent recurrence and metastasis[58]. This reveals the duality and complexity of cryoablation’s immunomodulatory effects.

As an adaptive resistance mechanism, the inflammation and IFN-γ release induced by cryoablation can induce the upregulation of immune checkpoint molecules like PD-L1 on tumor and immune cells[54]. While this provides a target for combination with ICI therapy, it also implies that newly activated T cells may rapidly enter an exhausted state if immune checkpoint blockade is not applied synchronously. Research indicates that cryoablation induces higher levels of tumor PD-L1 expression than MWA[54]. When the immune system is already influenced by immune checkpoint blockade, the tumor antigens released by cryoablation can help prevent T cell exhaustion or dysfunction, restoring anti-tumor immune responses[54].

Antigen overload and immune tolerance: The massive release of tumor antigens shortly after cryoablation, if lacking appropriate co-stimulatory signals or occurring within a strongly immunosuppressive environment, may lead to T cell “flooding” and induce their deletion or anergy, thereby promoting immune tolerance. Furthermore, if APCs are functionally insufficient or the T-cell response is too weak, the release of large amounts of antigen might theoretically trigger “high-zone tolerance” prompting the body to develop immune tolerance rather than an immune response, creating conditions for tumor immune escape[55,56].

In summary, the anti-tumor immunity triggered by cryoablation is always accompanied by a dynamic immunosuppressive negative feedback process, which is key to limiting its efficacy. This process involves the release of inhibitory molecules (e.g., TGF-β, PD-L1), recruitment of inhibitory cells (e.g., Tregs, MDSCs), T cell anergy due to antigen overload, and the formation of physical barriers like NETs. The combined effects of these mechanisms are to reverse the initial immune activation and to favor immune escape. Consequently, a combination of a cryoablation technique with a set of strategies aimed at disrupting such immunosuppression is the key to fundamentally improving its therapeutic effect.

Considering this dualistic profile, the net immunomodulatory effect of cryoablation, which is the balance between immune stimulation and suppression is highly context-dependent, varying across tumor models, patient populations, and technical protocols. Such variability highlights the need to use personalized treatment plans and predictive biomarkers. This natural duality offers the basic justification for combining cryoablation with ICIs.

Comparison of immunomodulatory properties with thermal ablation

As summarized in Table 1, the immunomodulatory profile of the cryoablation process is dualistic, and it is different in several key aspects compared to conventional thermal ablation[13,14,19,20,27,30-56]. ICD and some immune responses can also be triggered by RFA and MWA, which are the most widely used thermal ablation techniques[13,14,25]. Nevertheless, increasing amounts of comparative research indicate that the ablation modalities with various energy sources can determine different immune landscapes.

Immune induction properties of thermal ablation: Joule heating produced by high-frequency current can be effectively used to induce ICD by RFA, although its immunostimulatory activity depends on a variety of factors[65]. Research discovered that RFA treatment of liver metastases can induce large quantities of tumor antigens, triggering transient immune responses to multiple tumor antigens. This RFA-induced immune effect is not always adequate to prevent tumor recurrence[65]. Conversely, MWA has superior heat conduction efficiency, shorter ablation times, and superior constant intratumoral temperature properties, which could influence the strength and direction of the immune response it causes[66].

It is interesting to note that the immune response that is caused by thermal ablation is dual. On the one hand, the tumor debris produced by ablation is used as an unbiased source of tumor antigens, which is used as a basis of in situ cancer vaccine to the immune system[67]. On the other hand, immunosuppressive components of the TME may mix with the antigen reservoir, weakening the immune effect[67]. Thus, the immune environment created by various ablation technologies will rely not only on the physical properties of these technologies but also on various factors including tumor type, TME characteristics, and treatment parameters[68].

Immunomodulatory differences between cryoablation and thermal ablation: The immunomodulatory differences between cryoablation and thermal ablation are supported by direct comparative studies. A study that directly compares RFA with MWA and cryoablation in a mouse model of HCC has found that all three methods induce immune changes at 3-4 weeks. Cryoablation induced the greatest increase in CD8⁺ T cell percentage and the most significant decrease in Tregs and PD-L1 protein levels within the treated tumor bed at this specific time point[25]. This suggests potential early local immunostimulation. Conversely, other studies focusing on later time points or distant sites report contrasting findings. Another comparative study in a liver cancer mouse model further revealed that, compared to MWA, cryoablation induces higher tumor PD-L1 expression particularly in residual or distant lesions, triggers more T cell infiltration, and is accompanied by less infiltration of PD-L1-high CD11b⁺ myeloid cells. When combined with anti-PD-L1 antibody therapy, the cryoablation regimen demonstrated superior therapeutic efficacy[54]. Another mechanistic study showed that cryoablation combined with anti-PD-L1 antibody, compared to MWA combination therapy, could more effectively stimulate responses from CTLs and NK cells. This is partly because cryoablation leads to different changes in myeloid cell subsets[54].

Clinical observations also support this difference. One study indicated that both cryo-thermal therapy and RFA could induce antigen-specific CD8⁺ T-cell responses, but only cryo-thermal therapy strongly induced neoantigen-specific CD4⁺ T-cell responses[42]. Another study confirmed a significant increase in tumor-infiltrating CD8⁺ T cells and a significant decrease in the CD4⁺/CD8⁺ ratio after cryoablation, suggesting that cryoablation can unleash T-cell responses against cancer[69].

The immunomodulatory differences may be explained by the following mechanism. The high temperature of thermal ablation easily denatures proteins, potentially damaging antigenic epitopes. In contrast, cryoablation relatively gently preserves the native conformation of antigens, which is more conducive to recognition and presentation by DCs[70]. Additionally, the coagulative necrosis boundary formed by cryoablation is clear, aiding in the complete encapsulation and release of antigens. Recent research shows that the combination of ablation techniques with ICIs is becoming an important strategy in cancer immunotherapy. RFA can significantly enhance the therapeutic effect of PD-1 blockers, a synergistic effect confirmed in both preclinical and clinical studies[71,72]. Meanwhile, the application of local immunomodulators like granulocyte-macrophage colony-stimulating factor (GM-CSF)-BCG hydrogel can further enhance the immune-activating effect of RFA, achieving complete cure of local and distant tumors[71]. These findings suggest that optimizing the combination strategy of ablation technology and immunotherapy may provide new breakthroughs for improving cancer immunotherapy outcomes.

Technical and clinical considerations (acknowledging the limitations of cryoablation): Despite the promising immunomodulatory profile outlined above, a balanced perspective necessitates acknowledging certain technical and clinical limitations of cryoablation compared to thermal ablation techniques. First, achieving complete and uniform ablation for larger tumors (> 3-4 cm) can be technically challenging with cryoablation. The growth of the “ice ball” may be constrained by tissue perfusion and thermal mass, potentially leading to less predictable ablation margins in large volumes compared to MWA, which is less susceptible to heat-sink effects[73,74]. Second, there has been some inconsistency in the rate of local tumor progression of cryoablation when compared to radiofrequency or MWA when applied against HCC of some sizes and locations, although the data are not all consistent and may be subject to learning curves and technical regimes[75,76]. This highlights the significance of operator skills and strict technique in achieving the best oncologic results, which forms the basis of any subsequent immune gain. Lastly, most cryoablation systems are more complicated and expensive than the typical RFA or MWA devices and require special gas systems (argon/helium)[21,22]. This complexity will translate into a steeper learning curve and can restrict its general availability in resource-limited environments. As such, the decision regarding ablation modality should be a combination of potential immunological response and practicality with regard to tumor size, location, local expertise, cost-effectiveness and most importantly, the ultimate goal, which is to attain durable local control.

In conclusion, the unique physical processes of RFA, MWA and cryoablation result in distinctive antitumor immune profiles. These immunological properties, as well as important clinical-technical parameters, are systematically compared in Table 2[21-25,28,30,33,37,38,54,58,65,70,76-83]. Therefore, selecting an ablation modality for combination immunotherapy necessitates a holistic evaluation that carefully balances its immunogenic potential against the proven reliability of achieving durable local control and the practical technical feasibility for the specific tumor and patient.

Table 2 Comparison of immunomodulatory and clinical-technical properties among radiofrequency ablation, microwave ablation, and cryoablation.

Comparison parameter		RFA	MWA	Cryoablation	Putative mechanisms and clinical implications
Immunological parameter	Primary damage-associated molecular pattern release	HMGB1, heat shock proteins	HMGB1, ATP	HMGB1, ATP, CALR[23,33] (possibly a broader spectrum)	The freezing process better preserves antigens and facilitates the in situ exposure of “eat-me” signals like CALR[30]
	Antigen preservation integrity	Moderate/high (high temperature may denature some epitopes)	Moderate (rapid high temperature may alter antigen conformation)	High (low temperature better maintains native antigen conformation[25,70]	More intact antigens may help elicit high-affinity T-cell responses
	T-cell infiltration trend	Can increase, but may coincide with substantial Treg recruitment	Can increase, with heterogeneous effects	Significant increase in CD8+ T cells, some studies show Treg reduction[25,37]	May be related to a more favorable cytokine/chemokine profile
	Immune checkpoint induction	Induces PD-L1 upregulation	Induces PD-L1 upregulation	Potently induces PD-L1 upregulation[54,58]	Provides a clear target for combination with immune checkpoint inhibitors, but also suggests limited efficacy as monotherapy
	Risk to adjacent vasculature/bile ducts	High (thermal injury)	High (thermal injury)	Low (less affected by “heat sink” effect, collagen structure preserved)[24,76]	Makes cryoablation more suitable for tumors in high-risk locations, allowing more aggressive ablation for antigen release[80,81]
	Reported clinical abscopal effect	Case reports[65]	Rare	Relatively more preclinical evidence[28,38] and case reports[82,83]	Suggests its potential for inducing systemic immunity might be more pronounced
Technical and clinical parameters	Local efficacy for large tumors (> 3-4 cm)	Limited by “heat sink” effect	Generally more effective, less susceptible to “heat sink” effect[76,77]	Technically challenging; ice ball growth constrained by perfusion, leading to less predictable margins[76,77]	Durable local control is the prerequisite for any immune benefit. MWA may offer advantages in ablating large volumes
	Comparative LTP rate	Baseline standard, variable	Often comparable or superior to RFA in studies	Some studies suggest a potentially higher LTP rate, though data are inconsistent and influenced by learning curve[78,79]	Highlights the critical importance of operator expertise and optimal technique in cryoablation for foundational oncologic outcomes
	Technical complexity and cost	Relatively simple, lower cost	Moderately complex, intermediate cost	More complex and costly; requires specialized gas systems and often multi-probe setups[21,22]	Influences the learning curve and may limit widespread availability in resource-constrained settings

It summarizes key comparative findings from preclinical studies; clinical validation of these observations is ongoing. Representative references are indicated by superscript numbers. Risk assessments (low/high) denote the relative probability of major complications. The “putative mechanisms” column integrates hypotheses from the cited literature to explain the differences observed. When used in this table and the text, the terms “M1/M2” or “M2-like” serve as simplified descriptive shorthand for pro-inflammatory/anti-tumor and immunosuppressive/pro-tumor functional states, respectively. It is acknowledged that macrophage phenotypes in vivo are heterogeneous, plastic, and context-dependent. ATP: Adenosine triphosphate; CALR: Calreticulin; HMGB1: High mobility group box 1; LTP: Local tumor progression; MWA: Microwave ablation; PD-L1: Programmed death-ligand 1; RFA: Radiofrequency ablation; Treg: Regulatory T cell.

Unique impact on vasculature and bile ducts: Clinical safety and immunomodulatory implications

Cryoablation causes less damage to connective tissues like collagen fibers, and the “heat sink” effect is relatively more controllable during the freezing process compared to thermal ablation. This theoretically lowers the risk of serious complications such as vascular thrombosis, bile duct stricture, or hepatic infarction when treating tumors adjacent to major blood vessels or bile ducts[24,73]. This safety profile not only makes cryoablation suitable for HCC in “high-risk locations” but also creates conditions for activating anti-tumor immunity by performing more extensive ablation to release more antigens[77-79,84].

Evidence for vascular and biliary safety: Clinical studies demonstrate the significant safety of cryoablation in treating tumors adjacent to vasculature. A study by Wang et al[79] on 57 patients with subcapsular HCC undergoing computed tomography-guided percutaneous cryoablation showed that only 2 patients (3.5%) experienced major complications, and all patients successfully completed treatment. Research by Ma et al[85] further confirmed the safety of cryoablation for HCC in special locations. They treated 69 tumors adjacent to surrounding structures (including liver capsule, gallbladder, blood vessels, diaphragm, intestine, and adrenal gland) with a technical success rate of 100% and no severe complications[85].

The protective effect of cryoablation on biliary structures is particularly notable. A report by Kalra et al[86] showed no procedure-related complications in the cryoablation of 9 early-stage liver tumors. This finding aligns with the cryoprotective mechanism of cryoablation. The temperature range of -20 °C to -40 °C protects vascular endothelial cells but causes necrosis of tumor cells[87]. Conversely, Anand and Acharya observed in a review that the conventional thermal ablation methods such as RFA have difficulties in treating tumors close to biliary structures since bile ducts are particularly vulnerable to thermal damage[80]. This selective destruction characteristic of cryoablation gives it a clear advantage in treating HCC located in “high-risk” locations.

Clinical and immunological significance of the safety advantage: The vascular safety advantage of cryoablation is also reflected in its real-time monitoring capability. Compared to thermal ablation, cryoablation allows identification of the ablation zone as a hypoattenuating ice ball extending beyond the ablated tissue under computed tomography guidance. This enables the operator to confidently identify anatomical structures at risk of thermal injury[81]. This visualization capability is particularly important for tumors near critical blood vessels, as an editorial comment by Jennings[81] points out that in cryoablation, nerve damage begins when temperatures drop below 10 °C, with complete motor and sensory loss occurring between 0 °C and 5 °C.

This unique safety characteristic has important immunological implications. Evidence indicates that cryoablation can stimulate systemic anti-tumor immunity, a mechanism primarily attributed to the release of tumor antigens[88]. A review by Cohen et al[74] further supports the unique advantages of cryoablation, noting its ability to necrotize larger tissue volumes, increasing the chance of ablating satellite lesions. This is particularly important for larger tumors adjacent to major blood vessels[74]. This characteristic makes it an important complement to thermal ablation techniques and provides the possibility of maximizing the in situ vaccine effect through more aggressive ablation strategies. This may potentially lead to stronger synergistic effects when combined with immunotherapy.

SYNERGISTIC EFFECTS OF CRYOABLATION COMBINED WITH IMMUNOTHERAPY: FROM BENCH TO BEDSIDE

Based on the complementary mechanisms of cryoablation releasing tumor antigens and triggering in situ immunity, and ICIs relieving TME immunosuppression[58], the conceptual synergy between cryoablation as an “igniter” and ICIs as an “amplifier” is visually captured in the central intervention of Figure 1. The core logic is that cryoablation acts as the ”igniter”, releasing tumor antigens and danger signals. At the same time, immunotherapy functions as the “amplifier”, blocking inhibitory pathways, and maintaining the anti-tumor immune response. Table 3 (clinical studies) and Table 4 (preclinical studies) summarize selected studies, now structured with standardized key fields (e.g., model/patient details, ablation completeness, treatment sequencing, endpoints) to facilitate comparative analysis[27-29,37,38,82,83,89-94].

Table 3 Summary of selected clinical studies on cryoablation combined with immunotherapy for liver cancers.

Design	Patient population (line of therapy)	Intervention details	Key efficacy and survival outcomes (assessment criteria)	Key baseline confounders (tumor burden, PVTT, liver function)	Key immunological findings	Ref.
Case report	Advanced HCC, post-multiline therapy (post-resection, post-lenvatinib failure)	Bevacizumab + immune checkpoint inhibitors + intratumoral cryoablation (of a single metastasis)	mRECIST, sustained CR lasting > 24 months	Tumor burden: High (multifocal intrahepatic metastases + lymph node involvement); PVTT: Yes (noted in primary tumor); liver function: NR (HBV-positive, well-controlled on therapy)	Tumor mutational burden increased from 3 Muts/Mb to 18.67 Muts/Mb post-cryoablation (biopsy of a separate, non-ablated lesion)	Li et al[91], 2021
Case report	Advanced, multifocal HCC, post-sorafenib and nivolumab (human immunodeficiency virus/HBV co-infection)	Cryoablation (partial, of 2 sites) + nivolumab - liver transplant	The mRECIST, durable CR; bridged to successful liver transplantation > 4 years post-treatment	Tumor burden: High (multifocal, bilobar, infiltrative progression); PVTT: NR; liver function: Cirrhosis (Child-Pugh score NR, HBV-DNA undetectable)	Potent “abscopal effect”: Cryoablation of partial lesions triggered a systemic immune response, leading to regression of untreated, multifocal intrahepatic tumors and reversal of immunotherapy resistance	Lucas et al[92], 2024
Phase II study (preliminary results) NCT04724226	Advanced HCC with PVTT; first-line therapy	Cryoablation (to inactivate lesions as much as possible) –within 48 hours, start combination therapy with camrelizumab (anti-PD-1) + apatinib	ORR (mRECIST): 71.4%; ORR (RECIST v1.1): 14.3%; mPFS (mRECIST): 4.63 months; mOS: 19.0 months	Tumor burden: Limited (“up-to-7” criteria); PVTT: Yes (100% of patients, 57.1% with Vp3/4); liver function: Child-Pugh A (all patients, scores of 5 or 6)	Increase in CD8+ T cells; decrease in regulatory T cells and myeloid-derived suppressor cells	Gao et al[82], 2025
CASTLE-01 single-arm, phase II NCT05010668	Locally advanced or metastatic ICC; post first-line gemcitabine + cisplatin (second-line)	Partial cryoablation of one intrahepatic lesion, followed by combination therapy with sintilimab (anti-PD-1) + lenvatinib	ORR: 75.0% (21/28, including 2 CR); disease control rate: 100%; mPFS: 16.8 months; mOS: 25.4 months (RECIST v1.1)	Tumor burden: High (79% TNM stage IV, advanced/metastatic); PVTT: Not specifically reported; liver function: Predominantly well-preserved (all enrolled had Child-Pugh class A; 11% had cirrhosis)	Cryoablation triggered recruitment and clonal expansion of CD8+ PD-1hi effector T cells into the TME and enhanced tumor immunogenicity (increased antigen presentation and IFN signaling). Lenvatinib promoted tumor vasculature normalization (increased postcapillary venule ECs, decreased angiogenic tip/stalk ECs), facilitating the influx of novel T cell clones. Combination therapy reshaped a “cold” TME into an inflamed “hot” one, increased CD4+ CXCL13+ T follicular helper cells, and was associated with tertiary lymphoid structures formation	Gu et al[83], 2026
Single-arm study NCT03183219	Advanced HCC/ICC	Locoregional therapy (incl. cryoablation) + allogeneic γδ T-cell adoptive transfer	HCC: The mPFS 8.0 months vs 4.0 months (combo vs mono), mOS 13.0 months vs 8.0 months; ICC: MPFS 8.0 months vs 4.0 months	Mixed population (HCC and ICC); detailed burden/function: NR	Expansion/persistence of donor γδ T cells; elevated serum IFN-γ, tumor necrosis factor-alpha	Zhang et al[93], 2022
Retrospective study	Metastatic HCC; advanced patients unsuitable for or refusing surgery/chemotherapy (not first-line, mostly after prior treatments)	Four groups compared: (1) Comprehensive cryoablation + immunotherapy (cryo-immunotherapy): Complete cryoablation of intrahepatic primary and accessible extrahepatic metastatic lesions, followed by adoptive dendritic cell and cytokine-induced killer cell immunotherapy cell immunotherapy (4 infusions); (2) Cryotherapy only group; (3) Immunotherapy only group; and (4) Untreated/supportive care group	The mOS: Cryo-immunotherapy group: 32.0 months; cryotherapy only group: 17.5 months; immunotherapy only group: 4.0 months; untreated group: 3.0 months; statistical significance: Overall survival in the cryo-immunotherapy group was significantly longer than in the cryotherapy only group (P = 0.024) and the untreated group (P < 0.01; assessment criteria: Revised RECIST 1.1)	Tumor burden: High. All patients had metastatic disease (bone, lung, or multiple organs). High intrahepatic primary tumor burden (24 cases with single lesion, avg. diameter 6.5 cm; 21 cases with multiple lesions, total 71 lesions). PVTT: Not explicitly mentioned. Liver function: All patients were Child-Pugh class A (25 cases) or B (18 cases); 43/45 (95.6%) had cirrhosis	In the combination therapy group (cryo-immunotherapy), patients showed an increased proportion of CD3+ CD4+ T cells in peripheral blood and elevated serum levels of interleukin-2 and IFN-γ	Niu et al[94], 2013

Efficacy was assessed according to modified Response Evaluation Criteria in Solid Tumors for hepatocellular carcinoma unless stated otherwise. Sample size (n) indicates the number of patients or evaluable lesions. Survival outcomes (median progression-free survival, median overall survival) are expressed in months. CR: Complete response; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; ICC: Intrahepatic cholangiocarcinoma; IFN: Interferon; mOS: Median overall survival; mPFS: Median progression-free survival; mRECIST: Modified Response Evaluation Criteria in Solid Tumors; NR: Not reported; ORR: Objective response rate; PD-1: Programmed death-1; PVTT: Portal vein tumor thrombus; RECIST: Response Evaluation Criteria in Solid Tumors; TME: Tumor microenvironment.

Table 4 Summary of key preclinical studies on cryoablation combined with immunomodulatory strategies.

Ref.	Disease model	Ablation completeness	Combination regimen and sequencing	Sample size	Key immunological and therapeutic outcomes
Mandt et al[27], 2023	Murine HCC	Incomplete	CpG (Toll-like receptor 9 agonist) + αPD-1, administered after cryoablation	n = 63 total	Synergistically enhanced systemic antitumor immunity, characterized by increased intratumoral CD8+ T cell infiltration and a robust Th1-type cytokine response, leading to inhibited distal tumor growth and prolonged survival
Gu et al[28], 2024	Murine HCC	Complete	Anti-PD-1 + anti-CTLA-4 (dual immune checkpoint inhibitor), concurrent with/after cryoablation	n = 40 total, n = 10/group	Most effectively remodeled the TME by promoting CD8+ and CD4+ T cell infiltration while reducing regulatory T cells and myeloid-derived suppressor cells, resulting in potent inhibition of distant tumors and a synergistic effect
Qian et al[29], 2024	Bilateral subcutaneous HCC	Complete	αPD-1 + αCTLA-4, after cryo-thermal ablation	Not specified	Converted immunologically “cold” tumors to “hot” by inducing immunogenic cell death and enhancing DC activation, with the triple combination eliciting the strongest abscopal effect
Shewarega et al[37], 2024	Murine HCC	Incomplete	Matrix metalloproteinase inhibitor, administered after cryoablation	n = 40 total, n = 6/group	Specifically increased intratumoral CD8+ T cell infiltration, providing a model to study the immune effects of subtotal ablation
Ghani et al[38], 2023	Murine HCC	Incomplete	CPMV priming, followed by cryoablation, and then a CPMV boost	n = 44-56 total, n = 11-14/group	Functioned as an in situ vaccine, where CPMV acted as a potent innate immune stimulant, synergizing with cryoablation-released antigens to enhance antigen-presenting cell recruitment and cross-priming
Yang et al[89], 2025	Murine cervical cancer	Complete	αPD-1, administered after cryoablation	Bilateral model; typical n = 3-4/group for assays	Induced an effective abscopal effect by remodeling the TME, increasing cytotoxic lymphocyte activity, and upregulating programmed death-ligand 1 expression, thereby sensitizing tumors to checkpoint blockade
Wang and Guo[90], 2025	Murine colorectal cancer liver metastasis	Complete (described as tumor fully ice-covered)	Cryoablation + granulocyte-macrophage colony-stimulating factor (administered immediately post-ablation)	n = 80 total, n = 20/group	Enhanced antitumor immune response: Significantly and persistently increased intratumoral infiltration of CD11c+ DCs; promoted a Th1-biased immune response with sustained high serum interferon-gamma levels and suppressed interleukin-4 levels; associated with prolonged survival in the combination group

CPMV: Cowpea mosaic virus; CTL: Cytotoxic T lymphocyte; DC: Dendritic cell; HCC: Hepatocellular carcinoma; PD-1: Programmed death-1; TME: Tumor microenvironment.

Strong preclinical evidence

Animal models present a perfect platform for validating the synergistic effects and mechanisms of combination therapy. Comparative analysis of significant studies (Table 3) indicates certain trends[82,83,91-94]. Multiple studies confirm that combining cryoablation with ICIs (such as anti-PD-1 and/or anti-CTLA-4 antibodies) can produce powerful systemic anti-tumor immunity. In bilateral tumor-bearing mouse models, combination therapy not only controls the ablated primary tumor but also significantly inhibits the growth of contralateral untreated tumors (i.e., producing an abscopal effect) and prolongs overall survival (OS)[28,29,89]. Mechanistically, combination therapy remodels the immune microenvironment, manifested by increased intratumoral infiltration and enhanced function (high expression of IFN-γ, granzyme B) of CD8⁺ and CD4⁺ T cells, alongside a reduction in immunosuppressive components like Tregs and MDSCs[28,29,89], demonstrating in vivo the rebalancing of the immune microenvironment toward activation, as proposed in Figure 1.

To further enhance immune priming, studies have explored combining cryoablation with various immunomodulators. For example, combination with the TLR 9 agonist CpG more effectively increases intratumoral cytotoxic T cell infiltration and serum IFN-γ levels[27]. Combination with cowpea mosaic virus nanoparticles can additively activate innate immunity, inducing a stronger abscopal effect[38]. Combination with matrix metalloproteinase inhibitors has been shown to increase intratumoral CD8+ T cell infiltration[37]. Moreover, cryoablation combined with GM-CSF exerts a pronounced stimulatory effect on CD8⁺ T-cell functionality and improves survival in tumor-bearing animals[90]. These studies demonstrate from multiple angles the great potential of combination strategies in initiating and enhancing anti-tumor immunity.

Accumulating clinical evidence and translational implications

Though phase III trials are being conducted on a large-scale basis, emerging early clinical evidence offers strong support to combination therapy and presents a clear translational path.

Early clinical exploration and case evidence: Case reports give initial hints that combination therapy can reverse immunotherapy resistance. An example is that a report indicated considerable tumor regression in a patient with advanced HCC who initially did not respond to immunotherapy but was treated with intratumoral cryoablation[91]. Another case of multifocal HCC achieved sustained complete response and successfully bridged to liver transplantation with long-term disease-free survival after combination therapy with cryoablation and ICIs[92].

Preliminary validation from prospective clinical studies: Preliminary prospective study data further confirm the feasibility of combination strategies. A phase II study (NCT04724226) in patients with advanced HCC and portal vein tumor thrombus showed that cryoablation followed by combination therapy with camrelizumab (a PD-1 inhibitor) and apatinib achieved an ORR of 71.4%, a median progression-free survival of 4.63 months, and a mOS of 19.0 months, with manageable safety[82]. Based on this promising prospect, several phase II trials (e.g., ChiCTR2500097463, NCT06530784) aimed at evaluating this combination strategy, particularly its potential to reverse immunotherapy resistance, are underway, aiming to provide higher-level evidence-based medical evidence for clinical practice.

A key paradigm shift: Breakthrough evidence from intrahepatic cholangiocarcinoma research

Importantly, the CASTLE-01 study in intrahepatic cholangiocarcinoma (ICC) provided breakthrough proof-of-concept and a mechanistic paradigm for the “local ablation + immunotherapy + targeted therapy” triplet regimen[83]. This study applied “cryoablation + sintilimab (a PD-1 inhibitor) + lenvatinib” in patients with advanced ICC who failed first-line chemotherapy, achieving outstanding efficacy with an ORR of 75%, median progression-free survival of 16.8 months, and mOS of 25.4 months. Their detailed multi-omics analysis clearly showed that there was an operational model of synergy between cryoablation (release of antigens), lenvatinib (remodelling of the vasculature and stroma) and PD-1 inhibitors (preservation of T cell functionality) for optimising combination strategies in HCC[82]. While these results are transformative for the field, they await confirmation in large-scale, randomized phase III trials.

Exploration of combinations with other therapeutic modalities: Exploration of combination therapy has expanded to other modalities. For example, local ablation (including cryoablation) combined with allogeneic γδ T cell adoptive transfer showed a trend towards prolonged progression-free survival in patients with HCC and cholangiocarcinoma[93]. This suggests that the inflammatory environment created by cryoablation is conducive to the homing and function of adoptive cells. Combination of cryoablation with adoptive cell therapies (e.g., DC-CIK, CAR-T) has shown synergistic effects in some studies[94]. Furthermore, transarterial chemoembolization (TACE) combined with cryoablation has been widely used to treat larger (> 3 cm) or unresectable HCC, showing survival benefits superior to TACE alone[95-98]. Meta-analyses confirm that TACE-CRA can significantly improve ORR, disease control rate, and 1-year and 3-year survival rates[95-97]. TACE itself can induce tumor ischemic necrosis and antigen release; its combination with cryoablation can be considered an “enhanced” form of local immunogenic injury. This lays a practical foundation for further combining TACE-CRA with systemic immunotherapy (constituting a “triplet mode”), with related explorations ongoing[99].

Application value in HCC at special sites and oligometastatic disease: Due to its real-time monitorable “ice ball” boundary and relatively lower risk of damage to adjacent critical structures, cryoablation offers unique advantages in treating HCC located in high-risk anatomical sites (e.g., adjacent to the pericardium, diaphragm, gastrointestinal tract, major bile ducts, or large blood vessels)[73,77,79,84,100]. Local radical treatment and possibly the breaking of the local immunosuppressive barrier can be achieved by successful ablation of these lesions, which traditionally are considered no-go zones during thermal ablation. In addition, image-guided cryoablation may be an effective local consolidative treatment for oligometastatic lesions of the HCC (e.g., isolated adrenal, lung, or bone metastases). It is hoped that this local control, when combined with systemic immunotherapy, will further extend the duration of disease stabilization, and provide new treatment options for patients with advanced disease[101,102].

In short, evidence of theoretical mechanisms, animal experiments, and multi-level clinical studies all presents a cumulative and largely complementary body of evidence in support for the synergistic effect of cryoablation combined with immunotherapy. Preclinical studies have not only validated its potent synergistic anti-tumor effect and abscopal effect but also revealed multiple strategies to enhance immune priming. The combination strategy of local ablation + immunotherapy ± targeted therapy has systematically demonstrated the feasibility, excellent efficacy, and potential to reverse resistance of the combination therapy, which evolved over time as a result of initial case recommendations and preliminary validation of the combination therapy in prospective studies to breakthrough paradigm studies in ICC. Significantly, the effectiveness of cryoablation in special-site HCC and oligometastatic disease further underlines the exceptional value of this combination strategy in complicated clinical situations. Taken all together, this evidence suggests that cryoablation with immunotherapy can not only be used as a new synergistic model in the treatment of liver cancer but may also redefine the treatment landscape of some of the advanced patients, by overcoming local and systemic immunosuppression, which is one of the most promising directions in the current treatment of HCC.

Although the accruing clinical data are optimistic, it is important to take them with caution due to the predominance of early-phase trials, potential publication bias, and heterogeneity in treatment protocols. These constraints directly highlight the main issues that need to be resolved to bring this combination strategy to the normal clinical practice.

KEY VARIABLES INFLUENCING EFFICACY AND CHALLENGES IN CLINICAL TRANSLATION

The effectiveness of cryoablation as a combination therapy with immunotherapy depends on an enormous number of variables due to the dualistic and context-dependent nature of immunomodulation of cryoablation. It ultimately depends on the interaction of intricate factors in several dimensions, such as technical operation, tumor biological characteristics, and host immune status. At the same time, the innovative approach faces several challenges in its development towards the standard clinical practice, such as the level of evidence, safety management, and efficacy assessment. The goal of this chapter is to analytically examine these major variables and fundamental challenges, which will aid in optimizing treatment strategies and promoting clinical translation. The multifactorial determinants of efficacy and the main translational hurdles are summed up in Table 5 as a framework for this analysis[27,37,78,94,103-120].

Table 5 Summary of the multifactorial determinants influencing outcomes and the current translational hurdles for cryoablation combined with immunotherapy in hepatocellular carcinoma.

Category	Core variable/challenge	Specific elements and impact	Clinical implication/optimization direction
Key efficacy variables	Technology-related variables[37,78,94,103-111]	Ablation mode and completeness: Contradiction between preclinical “incomplete ablation” for immune stimulation and clinical need for local control. Current consensus favors radiologically complete ablation when combined with effective systemic immunotherapy. Technical parameters: Probe layout, freezing rate, minimum temperature, and number of freeze-thaw cycles influence cell death mode, damage-associated molecular pattern release, and the intensity/quality of the immune response	Paradigm shift from “radical ablation” to “strategic immuno-ablation”. Standardize and optimize parameters to maximize the in situ vaccine effect and immune benefits
	Baseline host and tumor variables[103,112-117]	Host status: Systemic immune capacity, immunodeficiency, severity of background liver disease (Child-Pugh grade, albumin-bilirubin score), cirrhosis, and hepatitis B virus/hepatitis C virus infection status. Tumor features: Intrinsic immunogenicity (tumor mutational burden, neoantigen load), baseline immune cell infiltration level (immune score), programmed death-ligand 1 expression. Conventional metrics like tumor burden and alpha-fetoprotein levels also correlate with efficacy	Foundation for personalized therapy. Pre-identification of “advantaged populations” via molecular imaging or liquid biopsy is key. The combination may potentiate the conversion of “immune-cold” tumors
	Treatment strategy and dynamic monitoring variables[27,94,118]	Timing and sequence of combination: The sequence (neoadjuvant, concurrent, adjuvant) and interval are core points of controversy for strategy optimization. Management of immunosuppressive complications: Complications (e.g., massive pleural effusion, “cryoshock”) may induce systemic immunosuppression. Dynamic peripheral blood immune monitoring: Dynamic changes in lymphocyte subsets, neutrophil-to-lymphocyte ratio, T-cell receptor clonal expansion, and cytokine profiles serve as a real-time, non-invasive “liquid biopsy” window	Dynamic strategy adjustment based on baseline conditions. Active complication management to preserve efficacy. Utilize blood-based monitoring for early response assessment and efficacy prediction
Current major challenges	Lack of high-level prospective evidence[78,118,119]	Current evidence is primarily from retrospective analyses or small single-arm studies, limited by selection bias, confounding factors, and protocol heterogeneity. Conclusions are hypothesis-generating	Future phase III randomized controlled trials must employ prospective randomized design, stratified randomization, standardized protocols, and appropriate control arms (e.g., atezolizumab + bevacizumab) to confirm survival benefit
	Immunosuppressive risk and combination safety[118]	Ablation itself may stimulate immunosuppression. Overlapping toxicities (e.g., immune-related hepatitis vs post-ablation injury) require careful management. Significant heterogeneity exists in patient selection	Need for strategies to counteract ablation-induced immunosuppression and to differentiate/manage overlapping toxicities. Establishing universal patient selection criteria is difficult
	Evolution of efficacy evaluation standards[120]	Traditional size-based criteria (e.g., Response Evaluation Criteria in Solid Tumors) may fail to capture delayed responses or pseudoprogression induced by immunotherapy	Comprehensive evaluation incorporating functional imaging (e.g., contrast-enhanced magnetic resonance imaging, positron emission tomography) and immune-related response criteria is necessary

Key efficacy variables: Refined and individualized regulation from “ablation” to “immuno-ablation”

Technical, host, and tumor factors determine the efficacy of cryoablation combined with immunotherapy, which necessitates a paradigm shift from the traditional objective of achieving a radical ablation to a strategic one of achieving an immuno-ablation. Interestingly, however, recent prospective clinical trials (e.g., the CASTLE-01 study[83]) and advanced preclinical comparative experiments[25,28,54] have not only confirmed the synergistic therapeutic effects but have also, through multi-omics analyses, provided an in-depth insight into the dynamic process by which the in situ vaccine cooperates with systemic therapy to remodel the TME. This forms the mechanistic basis for the combination strategy.

Technology-related variables (refinement of immuno-ablation): Ablation mode and completeness: “Incomplete ablation” or “subtotal ablation” is often used in preclinical models to maximize antigen release while preserving some tumor vascular structure to facilitate immune cell infiltration[37,121]. However, in the clinic, it is clearly associated with higher local tumor progression rates[75]. Therefore, balancing the achievement of sufficient ablation margins (an ablation volume/tumor volume ratio ≥ 10 is considered an important indicator[103]) for local control with preserving adequate immunostimulatory intensity is a clinical decision-making challenge. The current consensus tends to favor striving for radiologically complete ablation when combined with effective systemic immunotherapy.

Therefore, the clinical optimization strategy is not to compromise local control, but to refine ablation parameters within the goal of complete ablation to maximize the in situ vaccine effect – i.e., pursuing “immuno-ablation” rather than mere tumor destruction. Appropriate multiple freeze-thaw cycles may be more effective than a single large-volume ablation in enhancing immune function[103-105].

Cryoablation technical parameters: Factors such as probe layout, freezing rate, minimum temperature, and number of freeze-thaw cycles, all influence the mode of cell death, DAMP release, and ultimately the intensity and quality of the immune response[22,104]. Studies suggest that rapid freezing may induce stronger CD4⁺/CD8⁺ T-cell responses[104]. Passive thawing causes a greater degree of cell death than active thawing; thus, multiple freeze-thaw cycles can further increase cell death[106,107] and significantly enhance pro-inflammatory cytokine secretion and abscopal anti-tumor effects[108]. Ensuring that at least a 1 cm peritumoral zone reaches the “lethal temperature” of -40 °C to -50 °C is the basis for achieving complete ablation[109,110]. Standardizing and optimizing these parameters to maximize immune benefits is the direction for future technical development.

Baseline host and tumor variables (the basis for individualized therapy): Host systemic and hepatic immune status: The patient’s systemic immune capacity, presence of immunodeficiency, and severity of background liver disease (Child-Pugh grade, albumin-bilirubin score) are all crucial[111-113]. Concomitant cirrhosis and hepatitis B/C infection status directly affect liver reserve function, systemic immune status, and treatment safety, and are core considerations in decision-making[114]. Effective antiviral therapy is also fundamental for maintaining immune homeostasis in patients with HBV-related HCC[115].

Tumor intrinsic immunogenicity and burden: The tumor’s own gene mutation burden, neoantigen quantity, baseline immune cell infiltration level (immune score), and PD-L1 expression status all influence its responsiveness to antigen release and immunotherapy[121]. “Immune typing” based on gene expression or immunohistochemistry (e.g., immune-enriched/hot vs immune-desert/cold) is considered an important predictor of ICI efficacy. Cryoablation combined with immunotherapy may have stronger potential to convert “cold” tumors into “hot” ones[116]. Furthermore, conventional clinical indicators such as baseline tumor burden and alpha-fetoprotein levels are also related to efficacy. Patients with excessive tumor burden may require multiple needles, multiple sessions, or combination with other local therapies for debulking before effective systemic immunity can be induced. For example, the generally high tumor burden of patients enrolled in the CASTLE-01 study precisely highlights the value of local therapy in controlling burden and converting the immune microenvironment[83]. How to use molecular imaging or liquid biopsy technologies to pre-identify “advantaged populations” more likely to benefit from combination therapy is key to achieving precision medicine.

Treatment strategy and dynamic monitoring variables (optimization and navigation): Under given baseline conditions, the choice and dynamic adjustment of treatment strategy are crucial.

Timing and sequence of combination therapy: The sequence of cryoablation and immunotherapy administration (neoadjuvant, concurrent, adjuvant) and the interval time may significantly affect the intensity and quality of the immune response and are currently a core point of controversy in optimizing strategy. The optimal approach – whether to create a “hot spot” with ablation first and then apply ICI, use ICI pretreatment before ablation, or perform them concurrently – remains to be explored[27].

Management of immunosuppressive complications: Complications that may arise after cryoablation, such as massive pleural effusion, intrahepatic hematoma, or infection, as well as treatment-related systemic inflammatory responses (e.g., “cryoshock”, which is rare but severe[117]) may all affect efficacy by inducing systemic immunosuppression or depleting immune resources. Active prevention and management are necessary to ensure therapeutic efficacy.

Dynamic monitoring of peripheral blood immunity: Peripheral blood immune status, as a form of “liquid biopsy”, also contains rich information for efficacy prediction. Dynamic changes in peripheral blood lymphocyte subsets, neutrophil-to-lymphocyte ratio, T-cell receptor (TCR) clonal expansion, and cytokine profiles provide a real-time, non-invasive “liquid biopsy” window, aiding in early assessment of immunotherapy response and prediction of efficacy, as demonstrated by TCR clonal tracking in the CASTLE-01 study[83].

Current main challenges

Lack of high-level prospective evidence: Currently, the majority of evidence supporting combination therapy originates from retrospective analyses, small-sample single-arm studies, or case reports[76,118,119]. Although these studies suggest promise, they are generally limited by: (1) Selection bias: Retrospective studies often select patients with better performance status and relatively lower tumor burden, potentially overestimating real-world efficacy; (2) Confounding factors: It is difficult to fully balance the influence of baseline characteristics (e.g., etiology, liver reserve function, prior lines of therapy) on outcomes; and (3) Heterogeneity: The lack of standardization in ablation technical parameters, types of ICIs, and timing of combination leads to results that are challenging to compare directly. Consequently, their conclusions primarily serve to generate hypotheses rather than provide confirmatory evidence.

The future phase III randomized controlled trials that should seek to validate survival benefits must strive to address these biases. The key design considerations should include: (1) A prospective, randomized design should be used, which is the main design that is used to eliminate selection bias; (2) The combination treatment protocol should be predefined and implemented using stratified randomization based on key prognostic factors, such as Barcelona Clinic Liver Cancer stage, vascular invasion, and baseline alpha-fetoprotein level; (3) Clearly defining and standardizing the combination treatment protocol is needed, including criteria for ablation completeness, specific ICI agents and dosages, and treatment sequence (neoadjuvant/concurrent/adjuvant); and (4) A proper control arm should be used; for advanced HCC, the control arm should be the current standard systemic therapy (e.g., atezolizumab plus bevacizumab). It is only with such rigorous design that the central query whether combination therapy truly provides a survival benefit for patients can be definitively answered. To fill this gap, it is necessary to conduct rigorously designed phase III trials as described in the future perspectives [phase II: Protocol optimization and proof-of-concept (randomized phase II studies)].

Immunosuppressive risk and combination safety: As mentioned, ablation itself may stimulate immunosuppression. If the combined immunotherapy regimen cannot effectively counteract this aspect, it may lead to counterproductive results. Furthermore, the risk of overlapping toxicities from the two treatment modalities, particularly the differentiation and management of immune-related hepatitis vs post-ablation liver injury, requires high attention.

Heterogeneity in patient selection: HCC is highly heterogeneous. Responses to combination therapy may vary greatly among patients with different etiologies, stages, and liver reserve functions. Establishing universally applicable patient selection criteria is very difficult.

Evolution of efficacy evaluation standards: Conventional size-based solid tumor efficacy evaluation criteria (e.g., Response Evaluation Criteria in Solid Tumors) might not be sufficient to reflect delayed responses or pseudoprogression of immunotherapy. Following combination therapy, there is a requirement of extensive examination including functional imaging (e.g., contrast-enhanced magnetic resonance imaging, positron emission tomography) and immune-related response criteria[120].

Altogether, the effectiveness of cryoablation with immunotherapy is a complicated phenomenon which is controlled by numerous factors. To upgrade the current paradigm of ablating the tumor to a new paradigm of immuno-ablation of the tumor, refined technical control, individualized patient selection and dynamic treatment strategy adjustment need to be introduced into clinical practice. Despite the practical considerations surrounding combination therapy which include lack of high-level evidence and complex safety management, systematic identification and control of these key variables have the potential to maximize synergy, which ultimately brings definitive survival benefits to the HCC patients.

FUTURE PERSPECTIVES AND RESEARCH DIRECTIONS

The strong research roadmap to overcome the outlined challenges and transform the promise of cryoablation-immunotherapy combinations into precision clinical practice is clear and stepwise. The future endeavors will be organized into three consecutive stages, each stage having certain research priorities as discussed below.

Phase I: Mechanistic exploration and biomarker-driven research (exploratory phase I/II studies)

The first step will help to further mechanistically elucidate the combination therapy and determine predictive biomarkers.

Developing and validating predictive biomarkers: Use technologies such as single-cell sequencing, spatial transcriptomics, TCR sequencing, and liquid biopsy (e.g., circulating tumor DNA). The aim is to find the credible biomarkers that can be used to predict response or resistance and dynamically monitor the efficacy of treatment, answering the key question of which patients benefit the most[83,122,123]. Particularly, circulating tumor DNA has a wide potential in treatment response monitoring and facilitation of early detection of mechanisms of resistance[124]. Also, a non-invasive method to dynamically measure the changes in the immune landscape within the TME is the use of molecular imaging, such as positron emission tomography/magnetic resonance imaging probes targeting CD8⁺ T cells or a specific immune checkpoint[121]. The analysis should focus on defining immune signatures associated with response to treatment, including clonal growth of specific T-cells, or changes in myeloid cell subsets, or distinct chemokine phenotypes. The work will be the basis of accurate stratification of the patient.

To standardize the assessment of macrophage reprogramming – a key therapeutic axis highlighted in “Dynamic reprogramming of macrophage states: Beyond the M1/M2 paradigm” – we propose a minimal set of markers and functional endpoints for preclinical and translational studies (Table 6)[20,27,32,34,37,50,54,59,60,63,64,83,121].

Table 6 Proposed minimal marker set and endpoints for assessing macrophage reprogramming in preclinical and translational studies.

Category	Key markers / endpoints	Rationale and interpretation	Suggested supporting reference
Phenotypic and characteristic markers	Pro-inflammatory/anti-tumor (M1-like) phenotype: Surface: CD86, MHC-II (human leukocyte antigen-DR isotype); Cytokine: IL-12high, TNF-αhigh	Indicates antigen-presenting capacity and Th1-type immune activation. Increased ratio of these markers to M2 markers suggests successful reprogramming	CD86/MHC-II: Noy et al[63] and De Palma et al[64]; IL-12/TNF-α: Mills et al[59] and Wynn et al[60]
	Immunosuppressive/pro-tumor (M2-like) phenotype: Surface: CD206 (MRC1), CD163; enzyme: Arg1; immune checkpoint: PD-L1	Associated with T-cell suppression, tissue repair, and angiogenesis. Reduction post-treatment indicates attenuation of immunosuppressive tumor microenvironment	CD206/Arg1: Mills et al[59] and Wynn et al[60]; PD-L1: Tan et al[54]
	Phagocytic and “eat-me” signals: Phagocytosis receptor: FcγR; pro-phagocytic signal: CALR exposure on tumor cells	Essential for antibody-dependent cellular phagocytosis and immunogenic cell death. Upregulation enhances tumor cell clearance	CALR/phagocytosis: Fucikova et al[32] and Zhou et al[34]
Functional and prognostic endpoints	In vitro co-culture suppression assay: Inhibition of T-cell proliferation or interferon-gamma production	Direct functional readout of macrophage-mediated immunosuppression. Decreased suppression indicates functional reprogramming	Mandt et al[27], Shewarega et al[37], and Wang et al[50]
	Spatial context (multiplex immunohistochemistry/immunofluorescence): Co-localization with CD8+ T cells (permissive vs excluded)	Defines the physical interaction between macrophages and effector cells, critical for predicting immunotherapy response	Shewarega et al[37] and Santana et al[121]
	Correlation with treatment outcome: Inverse correlation with CD8+ T-cell infiltration: Direct correlation with tumor growth or survival in vivo	Validates the clinical relevance of the macrophage phenotype. Successful reprogramming should correlate with improved T-cell infiltration and survival	Mauda-Havakuk et al[20], Tan et al[54], and Gu et al[83]

It proposes a framework for standardizing the assessment of macrophage polarization states beyond the simplistic M1/M2 dichotomy, focusing on functional relevance in the context of cryoablation and immunotherapy combinations. These markers and endpoints are recommended for use in preclinical studies (murine models) and translational research (analysis of human biopsy samples pre- and post-treatment) to evaluate the efficacy of combination strategies in reprogramming the immunosuppressive macrophage compartment. A shift in the balance or ratio of pro-inflammatory to immunosuppressive markers is often more informative than absolute changes in a single marker. Spatial analysis (e.g., via multiplex imaging) is crucial for understanding the functional impact within the tissue architecture. Arg1: Arginase-1; CALR: Calreticulin; IL: Interleukin; MHC: Major histocompatibility complex; PD-L1: Programmed death-ligand 1; TNF-α: Tumor necrosis factor-alpha.

Exploring novel mechanisms and expanded applications: Investigate the impact of cryoablation on the tumor metabolic microenvironment (e.g., extracellular acidification) and its immunomodulatory significance[125]. Concurrently, explore the therapeutic value of the combination in neoadjuvant (downstaging for surgery or liver transplantation) and adjuvant (eradicating micrometastases to prevent recurrence) settings, as well as in extremely high-risk scenarios like portal vein/inferior vena cava tumor thrombi[126,127]. Furthermore, developing personalized, low-dose, and safe regimens for special populations, such as the elderly or frail patients, is an important aspect of expanding its application. The development of translational models that more closely mimic the human HCC immune microenvironment (e.g., PDX models) is crucial to accelerate preclinical research and investigate primary and acquired resistance mechanisms as proposed in Table 6[20,27,32,34,37,50,54,59,60,63,64,83,121].

Local immuno-engineering post-cryoablation: Nanoparticles, virus-like particles, and scaffold-based strategies

The post-ablation niche, rich in tumor debris and inflammatory signals, presents a unique opportunity for local immuno-engineering. Advanced biomaterials can be deployed to capture, present, and amplify the immunogenic signal, transforming the ablation cavity into a sustained immunostimulatory ”micro-factory”[128-130]. Key strategies include: (1) Functional nanovaccines: Nanoparticles designed to capture tumor-associated antigens released from cryoablated tissue and co-deliver immune adjuvants (e.g., TLR agonists, STING agonists) to APCs. This ensures spatiotemporal co-localization of antigen and danger signals, dramatically enhancing DC cross-priming and subsequent T-cell activation[45]. Recent work demonstrates that such nanovaccines can efficiently promote DC maturation and CTL differentiation, leading to potent abscopal effects and long-term immune memory[131]; (2) Macrophage-reprogramming nanomedicines: Nanoparticles loaded with agents targeting immunosuppressive myeloid pathways (e.g., CSF1R inhibitors, SHP2 inhibitors) can be locally released to alter the polarization of TAMs from a pro-tumor to an anti-tumor phenotype[132,133], thereby breaking a major axis of post-ablation immunosuppression; and (3) Virus-like particles (VLPs) as programmable scaffolds: VLPs, such as those based on hepatitis B core antigen, are non-infectious, self-assembling protein structures that mimic native viruses[134,135]. Their inherent immunogenicity and capacity for high-density antigen display make them ideal scaffolds for creating personalized or multi-antigen vaccines. Post-cryoablation, VLPs engineered to carry tumor neoantigens can be administered locally or systemically to powerfully boost the breadth and durability of the antigen-specific response, acting as a potent ”booster shot” to the in situ vaccine[45,133,134]. Their programmable nature allows for targeting specific immune cells, enhancing safety and efficacy.

Phase II: Protocol optimization and proof-of-concept (randomized phase II studies)

Guided by preliminary biomarkers, this phase aims to shift to optimizing the combination treatment protocol.

In-depth exploration of optimal combination strategies: Multi-dimensional, systematic research is required. To begin with, it is important to optimize the one-to-one combination of available ICIs and cryoablation. Systematically compare the optimal mode, timing, sequence, and dosage of synergy between cryoablation and various ICI agents (e.g., anti-PD-1, anti-CTLA-4, anti-LAG-3) using single or combination agents. Second, explore the opportunities of combination therapy by expanding the armamentarium of immunomodulatory approaches (e.g., NK cells, DC vaccines, GM-CSF) and novel immunotherapies (e.g., bispecific antibodies, CAR-T therapy). The success of the triplet regimens such as the CASTLE-01 study[83] can be taken as an example of this phase. The ultimate objective would be to go beyond the existing paradigm of cryoablation plus ICI and establish a new standard of next-generation combination immunotherapy by integrating cryoablation with agents targeting immunosuppressive pathways (e.g., TGF-β, adenosine) or sequential use with oncolytic viruses or personalized neoantigen vaccines.

Optimizing synergy between local ablation and immune adjuvants: Consider intraoperative or postoperative local inoculation of sustained-release immune adjuvants (e.g., TLR agonists, STING agonists, cytokine nanoparticles), oncolytic viruses, or engineered bacteria at the location of cryoablation. This would concentrate on forming a sustained, controllable immunostimulatory “micro-factory” in situ, maximizing the stimulation of local and subsequent systemic immune responses[128].

Phase III: Confirmatory efficacy validation (pivotal phase III randomized controlled trials)

Based on the earlier steps and, the main aim is to confirm the OS benefit of the optimized combination regimen vs standard therapy in a well-defined target population (on the basis of validated biomarkers).

Conducting rigorously designed pivotal trials: This will require the promotion of multicenter, randomized, controlled phase III trials. Such trials should compare the effectiveness of cryoablation together with ICIs head-to-head with the current standard-of-care (e.g., TACE, ICI alone, or ablation alone) in HCC patients at various stages. The study design should include biomarker-based patient stratification, use OS as the primary endpoint, and have an appropriate control arm (e.g., atezolizumab plus bevacizumab in advanced HCC). The final aim is to offer level I evidence to guide clinical practice.

Promoting technological intelligence and standardization: To facilitate the generalizability and success of these large-scale trials, parallel programs are needed to develop image-guided, artificial intelligence-assisted ablation planning systems so as to achieve accurate tumor coverage and optimum immune-stimulating margins. To reduce inter-operator variability, it is necessary to establish technical specifications and quality control standards of cryoablation procedures.

CONCLUSION

In oncology, cryoablation is fundamentally repositioned – not a purely local cytodestructive method but a powerful in situ immunomodulatory platform. This paradigm shift, from “ablating tumors” to strategic “immuno-ablation”, represents the central thesis of this review and the most promising pathway for enhancing HCC therapy. Through strong induction of ICD and the complex remodeling of the TME, cryoablation offers a unique mechanism to overcome the profound immunosuppression that characterizes HCC and limits the efficacy of systemic immunotherapy alone. The synergistic effect of cryoablation with ICIs and other systemic agents is based on this principle: The ablation serves as an “igniter” to release the tumor antigens and trigger a response, while immunotherapy serves as an “amplifier” to sustain it. The uniformity of indications of efficacy through preclinical and early-phase clinical studies is a solid outline of the transformative potential of this combination. In the future, the way ahead in achieving this potential is through a structured, biomarker-driven translational roadmap. The next steps that are indispensable are to further mechanistic knowledge, optimize combination regimens, and use predictive biomarkers to stratify patients precisely. In addition, it is important to actively develop the combinatorial armamentarium of immunomodulators, targeted agents, and adoptive cell therapies. With the combination immunotherapy led by pioneering studies such as CASTLE-01 and through the multidisciplinary collaboration, which is precision-focused, personalized and technically standardized, cryoablation-based combination immunotherapy is poised to transform the therapeutic landscape for HCC. Such a strategy has the potential of transforming short-term tumor control to long-term survival, and ultimately, have a curative potential on a wider range of patients.