Immune checkpoint inhibitors, including antibodies to programmed death cell death protein 1 (PD-1) and its ligand PD-L1 have revolutionized the treatment of patients with non-small cell lung cancer over the past decade. However, response rates to these therapies remain suboptimal even when the tumor demonstrates significant PD-L1 expression. Thus, combination immunotherapy approaches which use immune checkpoint inhibitors in combination with other therapeutic interventions have been proposed with the goal of improving overall clinical responses.

The cancer-immunity cycle has been proposed as a framework of seven steps required for the killing of cancer cells by the immune system (1). Specifically, the steps include: 1) release of tumor associated antigens and immunomodulatory danger signals, (2) cancer antigen presentation, (3) priming and activation of immune cells ,(4) trafficking and (5) infiltration of T cells into tumors, (6) recognition of cancer cells by T cells, and (7) immune-mediated cell death leading to further tumor antigen and danger signal release.. Each of these steps has been targeted by investigators for its potential to combine with current immunotherapies to improve response rates.

Release of cancer cell antigens is pivotal to generation of an anti-tumor immune response and may be augmented in several ways. For example, local treatments (tumor ablation, intratumoral injection, radiotherapy) and systemic therapies have been proposed as strategies to increase tumor antigen release and presentation to the immune system. Additionally, an increasing body of evidence suggests that immunogenic cell death may sensitize patients to PD-1/PD-L1 blockade (2). One particular local therapy, cryoablation, has been studied for its ability to generate an anti-tumor immune response and may have advantages over several of the other proposed methods.

In this review, we will discuss the proposed immunologic mechanisms of cryoablation and highlight pre-clinical and clinical investigations using cryoablation as part of a combination immunotherapy strategy.

Cryoablation is a thermal ablation technique in which cold temperature is used to induce tissue injury. Although the therapeutic properties of cold have been utilized for centuries, the modern era of cryosurgery began in the 1960s when the neurosurgeon Dr. Irving Cooper developed a modern cryosurgical system using a probe for treatment of brain tumors and movement disorders (3). Since then, cryoablation has been utilized for local treatment of several malignancies including liver, kidney, breast, prostate, lung and bone (4).

To achieve low temperatures at the tip of the probe, a compressed gas (i.e., cryogen) is passed through a valve where it rapidly expands, resulting in cooling via the Joule-Thomson effect. A variety of cryogens are utilized clinically including liquid nitrogen, argon, and carbon dioxide, which produce a range of cold temperatures at the tip of the probe within seconds of application (5). By direct application of this temperature to target tissues, tissue injury and cell death are locally induced. It is postulated that this tissue injury in turn leads to antigen presentation and the development of an antitumor immune response both locally and at distant sites (7). This will be further discussed in Section 6, Immune Response to Cryoablation.

Cryoablation results in cell death via an immediate direct cell injury, as well as delayed effects due to vascular thrombosis and potentially immune-mediated pathways (5). First, the direct application of cold damages target tissues and causes crystallization of extracellular water, leading to a hyperosmotic extracellular space resulting in osmotic injury to cells. With application of colder temperatures, direct intracellular crystallization impairs function of membranes and organelles, leading to cellular dysfunction and death. Following this direct effect, microvascular stasis occurs resulting in thrombosis, local ischemia and additional cell injury. While cells not subjected to lethal injury may be able to recover, temperatures of -20°C to -50°C may induce complete necrosis, which notably depends on the nature of the frozen tissue (6). For example, neoplastic cells may be more resistant to lethality from cold temperature than benign tissues. The effects of cold injury depend on many factors, including the tissue frozen, rapidity and duration of freeze and thaw cycles, number of freeze-thaw cycles (4, 5).

The optimal “ablative dose” is not known and many have investigated different protocols to optimize cryosurgical techniques (8). Thus, several authors have proposed a target temperature of -40 C for at least one minute in order to achieve a lethal effect in neoplastic tissues (4). In general, a rapid cooling phase, followed by a slow thaw phase, and repeated freeze-thaw cycles increase the amount of cell death with cryoablation (4). In a model of pulmonary cryoablation, three freeze-thaw cycles were suggested to be superior to two cycles for overall efficiency and visualization of the cryoablation zone. In particular, the first freeze-thaw cycle may improve thermal conductivity of lung tissue and tumor to amplify the freeze effect during subsequent cycles (9). Further investigation is needed to determine optimal ablation protocols.

In comparison with other modalities of image-guided thermal ablation such as radiofrequency ablation (RFA) and microwave ablation (MWA), cryoablation offers several possible advantages as part of an immune stimulating strategy. Certain tissues have an intrinsic cryo-resistance due to low water content (including cartilage, a component of the bronchial wall) which may minimize damage to normal tissues surrounding a tumor (10). Thus, cryoablation is the preferred image-guided ablation technique for tumors near the large airways, diaphragm or pleura (11). A meta-analysis comparing thermal ablative therapies for lung cancer reported no statistically significant differences in adverse reactions between cryoablation, radiofrequency ablation (RFA), and microwave ablation (MWA) (12). Additionally, cryoablation may be less painful that other thermal modalities as cold temperatures have an analgesic effect on tissues (13). Finally, cryoablation differs from RFA and MWA in that it does not produce extreme heat which is known to denature tumor antigens, and therefore may better preserve tumor antigens for processing by the immune system.

Studies dating back to the 1960s have suggested a potential immune response to cryoablation. Specifically, it was initially recognized that cryoablation caused formation of auto-antibodies against the frozen tissues (43). Characterization of the immune response was limited by the laboratory techniques at the time. However, there were several case reports in the 1970s of regression of distant lesions (“abscopal” effect) following cryoablation of prostate tissue (44, 45). Since then, technologies for assessment of the immune response have improved and allowed for further investigation into potential mechanisms of immune response to cryoablation, although the understanding remains incomplete.

Much of the data on immune response to cryoablation are based on pre-clinical models. Briefly, after cryoablation there is an influx of acute inflammatory cells to the cryoablation zone followed by antigen presenting cells. Dying cells release a variety of pro-inflammatory cytokines (e.g., IL-12, IFN-gamma and TNF-alpha) from necrotic tissues at the center of the ablation zone, which serve as “danger signals” to alert the immune system of cellular injury. Antigen presenting cells take up cellular debris including key tumor antigens and then migrate to regional lymph nodes where they interact with T and B-lymphocytes to initiate a cellular and/or humoral immune response ( Figure 1 ). It is important to note that cells at the periphery of the ablation zone, where temperatures are not sufficient to induce necrosis, may undergo apoptosis, potentially resulting in release of immune suppressive cytokines (e.g., IL-10, TGF-beta) (7).

Subsequent to cryoablation, anti-tumor antibodies may be formed against ablated tissues, as demonstrated in several pre-clinical cancer models (46–48). It is thought that these antibodies are a marker of an anti-tumor immune response, due to their specificity to tumor tissues. Additionally, murine models demonstrated resistance to re-challenge by the same tumor (but not different tumor lines) after cryoablation which may be due to both humoral- and cell-mediated immunity. Furthermore, the resistance to tumor challenge was demonstrated to be tumor specific (49). Further details of pre-clinical studies of the immunological effects of tumor cryoablation has been covered in detail in previously published manuscripts (7). It is not known how some of these mechanisms may interact with immunotherapies, including immune checkpoint inhibitors.

Notably, not all studies have demonstrated an anti-tumor immune response to cryoablation. In fact, some investigations have suggested an immunosuppressive effect of cryoablation (50–52). Sabel and colleagues proffered that the immune response to cryoablation is dependent upon the cytokine profile triggered by cryoablation, the availability of antigens that can be processed by antigen presenting cells, the mechanism of cell death (specifically necrosis versus apoptosis), and the subsets of phagocytes that process ablated cells (i.e., dendritic cells versus macrophages) (7). Further investigation is needed to understand mechanisms of immune response following cryoablation.

Multiple pre-clinical and clinical studies in non-small cell lung cancer have suggested positive outcomes with cryoablation. Several of these studies are have been reported in a previously published review by our group (53). A list of published studies involving cryoablation for the induction of immune response (i.e., cryo-immunotherapy) are included in Table 1 .

Additionally, more recently Xu et al. studied the clinical outcomes and immune response of patients with lung cancer who received systemic therapies with or without bronchoscopic cryoablation for endobronchial disease (60). Bronchoscopic cryotherapy was applied via cryoprobe via a single freeze-thaw cycle lasting 3 to 4 minutes. Improved response rate was noted in the cryoablation arm and there was suggestion of decreased serum tumor markers (e.g., carcinoembryonic antigen) and changes in CD4 and CD8 T-cell subsets in the peripheral blood following cryotherapy. It is important to note that the methodology in this study was limited by absence of standardization of systemic therapy, and that patients in this study received systemic chemotherapy or molecular targeted therapy rather than immunotherapy. However, the concept of combining cryoablation, in this study delivered bronchoscopically, with systemic therapies – particularly immune checkpoint inhibitors – is an approach that warrants further investigation.

There are theoretical benefits of combining cryoablation with immune checkpoint inhibitors, as they work on different elements of anti-tumor immunity. Based on a proposed mechanism, releasing tumor antigens while the immune system is under the influence of immune checkpoint blockade may prevent T-cell exhaustion or anergy and reinstate anti-tumor immunity ( Figure 2 ). At least one case report has demonstrated a robust clinical response due to the combination of bronchoscopic cryoablation and camrelizumab, a humanized monoclonal antibody against programmed cell death protein 1 (PD-1), in a patient with advanced sarcomatoid carcinoma of the lung (32).

Leppelman et al. performed a single-center retrospective cohort study evaluating the safety of locoregional therapies in patients receiving systemic immune checkpoint inhibitors for several malignancies including non-small cell lung cancer (12 of 65 patients) (58). Notably, locoregional therapies included thermal ablation, embolization, or selective internal radiation therapy, with cryoablation accounting for 28% of procedures. Immune-related adverse events occurred in 10.7% of patients, all less than or equal to grade 3, with none occurring in those with non-small cell lung cancer. It is important to recognize the potential for adverse effects from combining systemic and locoregional therapies. At least in this experience, there were no safety signals for combination therapies noted with cryoablation.

Feng et al. investigated the safety and efficacy of percutaneous cryoablation with or without nivolumab in patients with stage IIIB or IV non-small cell lung cancer (59). The combination of cryoablation with nivolumab versus nivolumab alone, was associated with an increase in the number of immune effector cells such as total CD4+ and CD8+ T cells and natural killer (NK) cells and the serum levels of inflammatory cytokines (i.e., IL-2, TNF-beta, IFN-gamma) in the cryotherapy plus nivolumab group. Adverse events were all less than or equal to grade 3 and without significant differences among the two groups. The combination of nivolumab with cryoablation resulted in significantly reduced circulating tumor cells and decreases in the tumor markers CYFRA21-1 and neuron-specific enolase (NSE). This study was limited by its retrospective nature and unconventional treatment strategies.

Further data regarding the safety and efficacy of the combination of cryoablation with immune-checkpoint inhibitors is needed. Several studies are currently investigating cryoablation in combination with systemic immunotherapies in non-small cell lung cancer (see Table 2 ). Additionally, studies which characterize both the tumor microenvironment and peripheral immune cells before and after cryoablation will be crucial to further understanding potential mechanisms of synergy.

Historically, cryoablation has been a means of local tumor control in patients who are unfit for surgery. However, there is growing interest in cryoablation as a means to induce an immunologic effect which is specific to the ablated tumor. With recent development of immunotherapies for lung cancer, cryoablation has entered a new era, with the potential to synergize with systemic checkpoint inhibitors. Several important questions arise regarding the role of cryoablation in non-small cell lung cancer based on this review of recent literature.

First, what is the optimal ablative dose and number of lesions to target? Despite increasing knowledge and experience with various cryoablation protocols, knowledge of the best strategy remains limited. As multiple studies have suggested that immune response is variable after cryoablation (61, 62), determining the most effective dose for immune stimulation (which may be different from achieving maximal cell death) is of utmost importance.

Second, is there any role for cryoablation of tumor-draining lymph nodes? Although at present in limited capacity, some have postulated nodal cryoablation as a means to combat immune checkpoint inhibitor resistance. Thoracic lymph nodes in non-small cell lung cancer may be readily accessible for EBUS-mediated bronchoscopic cryoablation, and may be therefore present an opportunity for future investigation into this topic.

Additionally, is there any potential neoadjuvant role of cryoablation and immunotherapy prior to resection of early stage non-small cell lung cancer? Impressive pathological responses have been observed with neoadjuvant checkpoint inhibitors prior to resection (63), and the addition of cryoablation to such a regimen at the time of bronchoscopy could theoretically further enhance this effect, particularly in those lung cancers with “cold” baseline immune microenvironments (low PD-L1 expression and absence of infiltrating T cells).

Accumulating evidence suggests that cryoablation may generate an anti-tumor immune response at the cellular level by potentiating the cancer-immunity cycle, especially when used in combination with other immunotherapies. At the clinical level, limited data suggest a potential improvement in disease regression and progression-free survival when combination of therapy and cryoablation is used. More clinical studies are needed to further determine how cryoimmunotherapy translates to clinical outcomes.

Due to technical limitations, cryoablation has largely been performed via percutaneous approach, although recent progress in the field of interventional pulmonology may allow for routine bronchoscopic cryoablation in the near future ( Figure 3 ). Additionally, further research into discovering optimal dose and mechanisms of cryoablation will be essential to maximize this potential therapy.

AV, AD, and DS designed this study. AV performed the initial literature search and wrote the first draft of the manuscript. AD and DS revised the manuscript and provided additional sources. All authors contributed to the article and approved the submitted version.