Immunological effect of local ablation combined with immunotherapy on solid malignancies

Recent comprehensive investigations clarified that immune microenvironment surrounding tumor cells are deeply involved in tumor progression, metastasis, and response to treatment. Furthermore, several immunotherapeutic trials have achieved successful results, and the immunotherapeutic agents are available in clinical practice. To enhance their demonstrated efficacy, combination of immunotherapy and ablation has begun to emerge. Local ablations have considerable advantages as an alternative therapeutic option, especially its minimal invasiveness. In addition, local ablations have shown immune-regulatory effect in preclinical and clinical studies. Although the corresponding mechanisms are still unclear, the local ablations combined with immunotherapy have been suggested in the treatment of several solid malignancies. This article aims to review the published data on the immune-regulatory effects of local ablations including stereotactic body radiotherapy, cryoablation, radiofrequency ablation, and high-intensity-focused ultrasound. We also discuss the value of local ablations combined with immunotherapy. Local ablations have the potential to improve future patient outcomes; however, the effectiveness and safety of local ablations combined with immunotherapy should be further investigated.

Although local ablations including stereotactic body radiotherapy (SBRT) and cryoablation are relatively new, they have become alternative, minimally invasive local therapeutic options for several solid malignancies [23][24][25][26][27][28]. Advances in these novel ablations should be reevaluated in terms of possible immunological advantages [22,29,30]. In addition, recent studies have demonstrated the possible synergistic effect of these local ablations when combined with immunotherapy [31][32][33]. This raises the following questions: (1) whether combining local ablations with immunotherapy improves the survival outcomes of patients with solid tumors, (2) whether it is possible to differentiate the immunologic effect from abscopal effect of local ablations, and (3) which local ablation is the most likely to be effective when combined with immunotherapy. In answering these questions it would be ideal to focus on specific type(s) of cancer; however, there is a severe lack of data if our purview is limited to one specific type of solid tumor. To this end, we aim to review all published data available at PubMed on the immunological anti-tumor effects of local ablations and their possible additive effects when combined with immunotherapy.

Stereotactic body radiotherapy
Ablative radiation technique, termed SBRT, has been developed in the last few decades on the basis of knowledge and experience of stereotactic radio-surgery for brain tumors [34]. With image-guided target localization and multiple precision beams, SBRT minimizes damage to normal tissue around the tumor, which allows increased tumoricidal dose and reduced fractionation. Generally, local therapeutic effect of radiotherapy is well recognized as tumor cell death through DNA damage which renders proliferating tumor cells more sensitive to radiation than normal tissues [35]. Previous studies have mostly focused on DNA damage and the repair capacity of cells. In recent years, however, other molecular pathways contributing to cell stress, including those associated with immunologic anti-tumor effect, have been elucidated [36].

Immunological effect of irradiation
Several studies have investigated the changes in expression of major histocompatibility complex (MHC) class I and antigen presentation after irradiation both in vitro and in vivo [37][38][39]. Radiation beam exposure can enhance immune anti-tumor response through up-regulation of MHC class I in a dose-dependent manner [39]. The MHC class I up-regulation could be mediated by mammalian target of rapamycin (mTOR) activation and antigen presentation [39]. More recently, interferon beta was shown to stimulate MHC class I expression when exposed to radiation in combination with chemotherapeutic agents in vitro [40]. Gameiro et al. [41] suggested that radiation-induced release of high mobility group box 1 (HMGB1), one of the key regulators of systemic inflammatory response [42], activate antigen-presenting cells and subsequent antigen-specific T-cell response. Barrio et al. [43] demonstrated that the phagocytosis of dying, radiated tumor cells by both dendritic cells and macrophages could equally induced specific CD8 + T-cell cross presentation. In addition, the "abscopal effect" defined as tumor regression outside of irradiation field, was reported in patients treated with irradiation including SBRT [44]. Furthermore, a relatively higher dose of radiation per fraction may induce more significant antitumor immune effects in experimental models [45,46]. Based on this data, the immunological anti-tumor effect of irradiation is potentially translatable to clinical practice and is the target of ongoing clinical trials [47,48].

Effect of irradiation in combination with immunotherapy
Since several immune-regulatory agents including checkpoint inhibitors were shown to have significant efficacy on several solid malignancies [8][9][10][11][12][13], a combination strategy has now emerged. Previous literature has suggested the potential enhanced immunological anti-tumor effect of these agents in combination with irradiation [49,50]. Local radiation-induced immune-regulatory effects may synergistically activate specific immunologic antitumor response and immune checkpoint inhibitors if they overcome immunosuppressive nature of local tumor microenvironment. In preclinical model, CD8 + T cells were considered to play a key role in regulating immune response by producing interferon gamma [51][52][53]. In another study, the immune-regulatory effect of radiotherapy in combination with an immune checkpoint inhibitor may be largely regulated through the mTOR pathway via MHC class I expression of tumor cells, dendritic cell activation, and CD8 + T cell function [54]. This suggested mechanism may activate an antigen-specific, systemic immune-regulatory response. Assuming the underlying mechanisms of the tumor microenvironment response to irradiation are similar, other local ablations may have the potential to replace irradiation when combined with immune-regulatory agents. Additionally, other classes of novel immune-regulatory agents might have synergistic effect with local radiotherapy. For instance, Tolllike receptor, which is expressed by antigen-presenting cells as well as effector B and T cells, is an alternative in combination with irradiation [54]. These classes of novel immune-regulatory agents in combination with SBRT may have the potential to drive even more efficient antitumor immunity.

Cryoablation
Cryoablation therapy, which destroys tumor tissue through several cycles of extremely cold temperatures and thawing, has recently emerged as an option of minimally invasive treatment of various solid tumors [24]. It relies on controlled and local freezing, resulting in tissue damage by removing thermal energy from tumor cells. The subsequent necrosis and apoptosis are the basic mechanisms of cryoablation therapy [24]. The cryoablation "dose" is difficult to define differently from radiation dose, even though numerous preclinical and clinical reports attempted to elucidate "dose" needed for complete tumor destruction. On the other hand, thermal distribution was sharply distinguishable [55]. Despite this, the magnitude of tissue damage did not correlate to absolute temperatures [56]. There are multiple possible reasons for this discrepancy as many factors contribute to artificial freeze damage, such as the number of cycles, duration of freezing, nadir temperature, and the susceptibility of tumor cells.
The cell death is primarily induced by mechanical tissue injury through ice crystal formation. The cryo-probe, which is placed inside of tumor tissues, delivers extremely cold temperatures. Thus, the tissue damage depends on the distance from the cryo-probe to great vessels, which can largely affect degree of tissue temperature. Subsequent microcirculation failure leads to intracellular dehydration. Ice crystal formation also damages integrity of cell membrane [57]. Secondary intracellular ice crystal formation causes vascular stenosis and tissue ischemia that largely decrease cell viability. These two mechanisms synergistically contribute to tumor cell death. Cryoablation-induced cell deaths also relies on the apoptosis cascade which includes mitochondrial dysfunction mediated by decreased Bcl-2 expression and increased expression of Bax, a pro-apoptotic protein [58,59].
Since the early 1970s, cryoablation has been thought to have abscopal immune-regulatory effects on remote tumors [60,61]. However, another study reported that cryoablation alone did not always induce an abscopal effect and it may require specific circumstances to be present [62]. Recent literature showed that approximately three-fourths of tumor cell death by cryoablation may be caused by increased proinflammatory cytokine levels, which in turn contribute to decreased tumor growth rate and favorable survival outcomes, in mice [63]. Several clinical studies have further demonstrated similar findings that T-cell response was activated by increased serum cytokines [64,65]. In addition, several researchers documented the increased immunological anti-tumor effect in combination with dendritic cells [66] as well as several immune-regulatory agents including lipopolysaccharide [67] and anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibody [68]. Taken together, these data suggest an immunological advantage and promising translatability of this ablative procedure. However, to date no clinical trials have investigated the efficacy of cryoablation in combination with immune-regulatory agents.

Radiofrequency ablation
Radiofrequency ablation (RFA) has been applied to treat several solid malignancies such as kidney [69], breast [70], and liver cancers [71,72]. It has also been applied to treat metastatic tumors of the liver [73][74][75][76]. This modality destroys target tumor tissue by applying thermal energy which leads to a temperature above 50 °C [77,78]. The radiofrequency probe generates resistive heat by electric current, causing destruction of tissue [23]. Therefore, local therapeutic effects are largely affected by tumor size and RFA probe location. Clinically, RFA is mostly restricted to small-sized tumor especially those <3 cm in diameter [24].
While apoptotic cell death is not usually harmful for the host immune system, necrotic cell death releases signal molecules which can sometimes lead to dangerous reactions. Among the signal molecules, increased cell surface expression of heat shock proteins (HSPs) has been shown to activate dendritic cells [79], and several serum proinflammatory cytokines have been demonstrated to contribute to the clinical immunomodulation following RFA [80,81].
Several preclinical studies have reported the potential effect of combining RFA with immunotherapy. CTLA-4 blocking antibody showed additive enhancement of antigen-specific CD8 + T-cell induction after RFA in vivo [82]. PD-1-blocking antibody was also reported to boost anti-tumor immunity elicited by RFA [83]. Antibodyconjugated interleukin-2 [84] as well as dendritic cell injection [85,86] also enhanced the therapeutic effect of RFA in experimental models.

High-intensity-focused ultrasound
High-intensity-focused ultrasound (HIFU) is a local ablation which applies acoustic cavitation without invasive procedures; it has been applied for solid tumors including breast cancer [87].
In previous literature, HIFU demonstrated antigenspecific immune-regulatory effect mediated by increased HSP-70 [87] and proinflammatory cytokines [88]. HIFU has garnered attention as a promising local ablation which may enhance anti-tumor immunity [33]. It may also have possible additive effect in combination with immunotherapy [33].

Other ablative therapies
Microwave ablation provides high thermal energy by excitation of water molecules, causing tissue injury and tumor cell necrosis [89,90], which has been clinically used to treat liver, lung, breast, and bone cancers [91]. Dendritic cell injection with microwave ablation improved anti-tumor immune cell subsets in patients with hepatocellular carcinoma [92].
Laser-induced thermal ablation causes coagulation necrosis by a refraction of laser light on the tumor tissue. This high-energy therapeutic modality led to tissue temperature over 60 °C [93]. In clinical observations, laser ablation increased the levels of serum proinflammatory cytokines including interleukin-6 in patients with liver tumors [94].

Conclusions and perspectives
Recent advances in ablative engineering and its computed tomography-guided procedure have provided basis to conduct the minimally invasive local treatment of solid tumors. These ablations have been clinically used as alternative local approaches for treatment of various solid malignancies especially in unresectable or medically complexed cases. These modalities have secondary advantages including minimal invasiveness, patient comfort, and cosmetic outcome. This is in addition to the aforementioned immune-regulatory effects. It should be noted that the data reviewed in this article are mostly preclinical and there has been no definitive data to address whether local ablations in combination with immunotherapy can improve survival outcome of patients with solid tumors. The preclinical data reviewed in the current study may provide rationale for future clinical trial. SBRT in particular appears to demonstrate positive immunological advantages, and some clinical trials are evaluating its utility. However, further analysis is required to address which ablation is the most promising as no studies have compared the immunological advantages of different ablative modalities yet. Furthermore, the immune effect of local ablations may vary according to many factors related with tumor characteristics and treatment. Although the effectiveness and safety of combination of local ablations and immunotherapy should be investigated, they have the potential to improve further patient outcomes.
Authors' contributions YT, NM, TN, HD, HU, and MK contributed to the conceptualization and writing of the manuscript. All authors read and approved the final manuscript.