First identified in radiotherapy, the abscopal effect describes the regression or reduction of tumors outside the irradiated field (1). Owing to its unpredictable nature, this phenomenon has mostly been observed in case reports and early-phase single-arm clinical trials (2). The emergence of immunotherapy has enabled the combination of local and systemic treatments, thus increasing the incidence of the abscopal effect (3) and generating enthusiasm for using combination therapies in both localized and metastatic cancers (4, 5). However, the exact mechanisms underlying the abscopal effect remain unclear because of the variety of local treatment methods.

Different therapies may trigger distinct processes, such as the production of tumor cell fragments, release of tumor antigens, activation of the immune system, or alteration of the tumor microenvironment. For example, radiotherapy can damage tumor cell DNA, leading to the release of tumor antigens and cytokines, which subsequently activate dendritic cells and T cells, resulting in a systemic anti-tumor response (6–8). As a precise and non-invasive local treatment modality, focused ultrasound (FUS) can effectively destroy tumor cells while also activating an immune response (9, 10); thus, it has been widely applied in treating of solid tumors. However, FUS alone is often insufficient to elicit a robust anti-tumor immune response capable of inducing a significant abscopal effect (11, 12). Preclinical models by Han et al. have demonstrated that FUS treatment has minimal impact on the immune microenvironment of distant tumors (13), with no significant inhibition of tumor growth observed post-treatment. This has led to an interest in combination therapies.

FUS could induce the release of tumor-associated antigens, activating anti-tumor immunity. Immunotherapy could enhances the recognition and processing of these antigens, prompting immune cells to target and destroy tumor cells (14–16). Together, these treatments work synergistically to reduce the likelihood of tumor recurrence and metastasis. However, studies investigating the abscopal effect of combined FUS and immunotherapy are relatively sparse, and the precise mechanisms and consistency of this effect remain largely unconfirmed, which hinders the clinical application of combined treatments. Therefore, this study aims to conduct a systematic review and meta-analysis of existing preclinical animal models of solid tumors to evaluate the efficacy and consistency of FUS combined with immunotherapy in achieving the abscopal effect. Our findings provide a more robust theoretical foundation and data support for future clinical research.

This systematic review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and the Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) tool for animal studies. The protocol has been registered with PROSPERO (registration no. CRD42023460710).

The abscopal effect, a promising phenomenon closely linked to immune activation, occurs as a synergistic therapeutic effect when localized treatment is combined with systemic immunotherapy. We conducted a systematic review and meta-analysis to evaluate the abscopal effect and its immune microenvironment influence in solid tumor animal models treated with Uncombined with immunotherapy. All included studies reported positive therapeutic effects of this combination therapy on mice. Following combined treatment, improvements were observed in distant tumor volume, survival time, and treated tumor volume. Additionally, the studies elucidated the effect of the immune microenvironment by which FUS combined with immunotherapy induces the abscopal effect ( Figure 4 ). To our knowledge, this is the first meta-analysis to assess the abscopal effect of FUS combined with immunotherapy in solid tumor animal models.

FUS is a non-invasive, image-guided local tumor treatment technique. Under the monitoring of ultrasound or Magnetic resonance imaging, a focused transducer external to the body concentrates ultrasound energy to a focal point within the tissue, akin to an “invisible ultrasound scalpel,” enabling precise cutting treatment. FUS is an important supplement or alternative to traditional cancer treatments such as surgery, chemotherapy, and radiotherapy. It can modulate the tumor immune microenvironment, transforming immunologically “cold” tumors into reactive “hot” ones, and enhancing anti-tumor responses when combined with immunotherapy (29, 30). Among the 12 included studies, eight reported that FUS increased the number of activated T cells within the treated tumor microenvironment, with upregulation of CD8⁺ T cells and increased expression of IFN-γ and granzyme B. IFN-γ is closely related to transcription factor regulation, macrophage activation, and expression of antigen processing and presentation molecules, promoting ferroptosis in tumor cells and enhancing the cytotoxic activity of CD8⁺ T cells (31). Moreover, this immune-enhancing effect is not only limited to the treated site but also extends to distant tumors.

Moreover, exploration of the immune microenvironment in distant tumors revealed a significant increase in the number of T cells following combination therapy, accompanied by a decrease in that of myeloid-derived suppressor cells (MDSCs) and an increase in M1-type macrophage polarization. This suggests the systemic mobilization and activation of anti-tumor immunity. Originating from the bone marrow, MDSCs promote tumor escape by generating immunosuppressive cytokines, inducing regulatory T cells, and degrading key amino acids essential for T cell responses (32, 33). Additionally, MDSCs interfere with natural killer cell functions, altering the tumor microenvironment to support tumor growth and metastasis (34). Combination therapy effectively triggers the release of damage-associated molecular patterns (DAMPs) from injured and dying cells. Following treatment, calreticulin exposure on the cell membrane peaked at 6 h, and the levels of ATP and HMGB1 increased significantly (27, 35), indicating that combination therapy induces immunogenic cell death, thereby inhibiting tumor recurrence and metastasis.

Subgroup analyses provided several intriguing insights. First, the differing mechanisms of action of FUS have been a focal point of interest. The distinct mechanisms of thermal and mechanical effects are believed to elicit varying immunological responses. The thermal effect elevates the temperature of the targeted tissue (up to 60°C), leading to coagulative necrosis. Meanwhile, the mechanical effect disrupts cells, causing rupture and necrosis (36), and generates a cellular homogenate that releases more tissue debris, which is thought to induce a more robust immune response, thereby enhancing antitumor efficacy (24, 37). Our results reinforce that combination therapy in the mechanical-effect group offers a clear survival advantage over immunotherapy alone, a benefit not observed with the thermal effect. This distinction is attributed to the unique mechanism of action in mechanical ablation. Unlike thermal effects, which rely on heat, mechanical effects employ acoustic cavitation to physically disrupt tumor cells and their internal structures, leading to cell membrane rupture and death. This mechanical disruption generates a broader spectrum of tumor-associated antigens and damage-associated molecular patterns DAMPs, which effectively recruit and activate antigen-presenting cells such as dendritic cells and macrophages. Subsequently, this stimulates a more robust anti-tumor immune response, particularly through the infiltration of cytotoxic T cells into the tumor microenvironment (38, 39). Nevertheless, the limited number of studies included in the survival outcome analysis necessitates a cautious interpretation of these findings.

The sequence of FUS and immunotherapy administration was also explored. Silvestrini et al. found that the order of these treatments affects the immune efficacy of the combination therapy (25). FUS can break down tumor tissue into a rich mixture of DAMPs, which are recognized by Toll-like and Nod-like receptors, thus activating anti-tumor immunity (12, 40). Pre-administration of immunotherapeutic agents can create a pre-activated immune microenvironment, which promptly recognizes DAMPs post-ultrasound treatment, thereby initiating immune activation. However, our results presented a contrasting conclusion regarding the treatment sequence. The survival benefit of immunotherapy followed by FUS showed borderline statistical significance, indicating no substantial difference attributable to the treatment sequence. We attribute this to the fact in three of the included studies (23, 25, 28), the survival curves of the combination and immunotherapy groups did not show statistical differences, although there was significant difference in tumor volume. Similar to the previous subgroup analysis of ultrasound effects, we advise caution in interpreting this conclusion. In other subgroups, such as different numbers of immunotherapeutic agents, tumor types, and tumor transplantation sites, the combination therapy group consistently demonstrated improved efficacy. This consistency suggests that FUS combined with immunotherapy can exert abscopal effects across various tumors, treatments, and models.

In this meta-analysis, we observed substantial heterogeneity in the measurement of treated tumor growth volume, even when using a random-effects model. Subgroup analyses suggest that this heterogeneity may stem from the different types of immunotherapeutic agents and tumor models utilized. The use of single immunotherapeutic agents and various tumor models contributed to the observed heterogeneity. Therefore, further analysis of the specific types of drugs or tumors might help elucidate the sources of this heterogeneity. However, owing to the limited sample size of the included studies, additional experiments are required to explore these variations. Nevertheless, sensitivity analysis confirmed the robustness of our findings. Despite performing an extensive literature search, we cannot exclude the possibility that some studies, such as conference abstracts and supplements, were unavailable or that some negative results remain unpublished. Given that results from animal experiments cannot fully replicate the complex pathophysiology of clinical settings, large-scale clinical trials are required to validate these findings before clinical extrapolation.

This study has some limitations. First, despite our efforts to minimize potential biases, the limited number of experiments and heterogeneity may preclude strong evidence from subgroup and immune environment analyses. Therefore, the results of these analyses should be interpreted with utmost caution. Second, our study focused primarily on the thermal and mechanical effects of FUS combined with immunotherapeutic agents, and excluded emerging techniques such as sonodynamic therapy, which may limit the generalizability of our conclusions. Finally, the limitations of the animal models and immunotherapeutic agents included necessitate further diverse studies to confirm these results.

In summary, this systematic review and meta-analysis of preclinical animal experiments evaluated the abscopal effect of combined FUS and immunotherapy in mouse models of solid tumors. FUS can modulate the tumor immune microenvironment and enhance anti-tumor immunity, demonstrating an abscopal effect and improving overall survival when combined with immunotherapeutic agents. These findings provide deeper insights into the synergistic effects of FUS and immunotherapy, promoting their combined use in clinical applications and offering a new therapeutic paradigm for treating advanced tumors.