RFA, a standard ablative and first-line therapy for small-sized HCC, is more cost-effective than hepatic resection (10). RFA can achieve tumor necrosis at 375–480 kHz and > 60°C (11). However, traditional monopolar RFA is limited in tumors less than 2–3 cm or near vessels due to heat sink effect, which is also related to recurrence (12). Novel techniques are developed to improve ablation efficacy. No-touch multibipolar RFA can be used to tumors up to 5 cm with similar disease-free survival (DFS) and overall survival (OS) rates compared with resection (13). However, insufficient RFA (iRFA) is one of the major reasons for recurrence after RFA. iRFA could lead to HCC with a more aggressive phenotype, drug resistance and worse prognosis ( Table 1 ). The ablative margin assessed by computed tomography (CT) after RFA can be an important predictor of local tumor progression (LTP) and overall recurrence. A study indicated that insufficient ablative margin (<5 mm) was associated with higher rates of LTP and overall recurrence in HCC, but the sensitivity values were only 36.4% and 46.2%, respectively (26). iRFA could promote the proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and angiogenesis of residual tumors through the transcriptional and epigenetic regulation. Some signaling pathways associated with tumor growth and progression, such as the Akt signaling pathway involved in cellular proliferation, survival and angiogenesis are activated after iRFA (20, 22, 24, 25).

Several strategies have been used to counter iRFA. One of which is to improve the accuracy of imaging guidance for the specific identification of tumor boundaries, especially with the application of nanotechnology. Jiang and colleagues developed a nanobubble conjugated with colony-stimulating factor 1 receptor (CSF-1R), called NBCSF-1R, for HCC margin detection (27). NBCSF-1R provided a non-invasive effective ultrasound imaging capabilities for evaluating therapy response of RFA through the high specificity targeting of CSF-1R-overexpressing macrophages and HCC tumor margin. Another strategy is the combination therapy for salvage. For instance, sorafenib and IFN-α combined with herbal compound inhibited the EMT of HCC cells after iRFA (28, 29); bevacizumab inhibited the tumor growth and angiogenesis induced by iRFA (30); and CTLA-4 blockade suppressed the growth of residual tumors and improved survival in a subcutaneous murine HCC model (31). Other agents include metformin (32) and hydroxychloroquine (HCQ) (33). However, one study demonstrated that ATPase inhibitory factor 1 (IF1) increased HCC cells’ resistance to sorafenib after iRFA (16). These results indicated that the application of systemic therapy or immunotherapy could cope with the adverse impacts of iRFA but the choice of agents could be limited by iRFA-induced resistance.

MWA could provide higher temperature with expanded ablation zone and shorter ablation time because of its higher frequency (900–2,450 MHz) (11). A recent study showed that MWA provided more excellent tumor control than RFA for patients with perivascular HCC (34, 35). In addition, a meta-analysis of randomized-controlled trials demonstrated that MWA seemed to benefit disease-free survivals at 5 years compared with RFA (36). New microwave thermosphere ablation (MTA) may provide a safer and more effective ablation with shorter time than RFA with the developments of novel MWA systems (37).

Cryoablation is also a thermal technique that could be more effective and safer for tumors not suitable for RFA or MWA, such as perivascular HCC. The goal of cryoablation is to destroy tumor tissue by alternating freezing and thawing on the basis of the Joule-Thomson effect, which benefits low risk vascular complications (38). Moreover, a multicenter randomized controlled trial demonstrated that cryoablation achieved lower local tumor progression than RFA with similar OS and DFS rates (39). IRE is a non-thermal ablative technique that mediates cell damage by changing cell permeability and cellular homeostasis, which lead to cell death (40). Although IRE is a relatively new technique and few clinical studies have been conducted, its safety and efficacy have been proven (41, 42). Similar to cryoablation, one of advantages of IRE is that this technique can be used for tumors not suitable for RFA or MWA, such as perivascular HCC (43).

In a word, ablation take an indispensable place in the clinical treatment of HCC. A series of new techniques have been developed to improve the ablation efficacy and zone to benefit more patients. However, these technologies are image-guided, and their efficacy is closely related to the skills of operators to some extent. This factor is a major barrier to application and an interfering factor that is difficult to eliminate in comparative studies.

Phototherapy (e.g., PDT and PTT) is an emerging and prospective cancer therapeutic strategy. Phototherapy kills cancer cells through photochemical or photophysical effects to achieve therapeutic effects. Various photosensitizers (PSs), such as porphyrin-based PDT (44), 5-aminolaevulinic acid-PDT (45) and Radachlorin-PDT (46), could be applied for HCC. However, several factors need to be improved before these methods could be clinically used. First, light (laser) is one of the most indispensable elements in PDT and PTT, on which the therapeutic effect mostly depends. PSs and photothermal agents can be activated only when the light wavelength is in a specific range, known as therapeutic window. Moreover, light wavelength also determines the depth of tissue penetration, which limits percutaneous application of phototherapy to tumors in abdominal parenchymal organs, especially in deep parts. The rapid development of endoscopic techniques and biomedical materials gave rise to strategies to overcome the depth dependence. For example, Li et al. reported laparoscopic-assisted photothermal ablation method based on superparamagnetic iron oxide (SPIO) and new indocyanine green (ICG), called IR820@PEG-SPIO (47). More surprisingly, IR820@PEG-SPIO completely ablated orthotopic liver cancer in nude mice model, as well as detect early-stage HCC (diameter < 2 mm) via fluorescence, photoacoustic and magnetic resonance (MR) imaging. Compared with visible light, near infrared (NIR) light and X-Ray can provide deeper penetration (48–51). Besides, MHT, an alternative strategy, has been proposed to further overcome the limitation of penetration depth. Qian and colleagues developed a ferrimagnetic silk fibroin hydrogel (FSH) and demonstrated that FSH-mediated MHT, without depth limitation, could be more suitable for treating liver tumors compared with traditional PTT (52).

Nanoplatforms have stood out because they have improved therapeutic effects and reduced adverse effects, provide precise operation with optimized imaging guidance, and combine therapeutic strategies for synergistic anti-tumor effects. Zhu et al. designed a nanoparticle (ZnPc/SFB@BSA) that combined PDT, PTT and sorafenib with increased efficacy and decreased side effects of sorafenib (53). Jin and colleagues reported another nanoparticle loaded with sorafenib/indocyanine for PDT/PTT/chemotherapy, which could provide synergistic effects against HCC (54). Liu’s group has been devoted to designing different nanoplatforms for combined phototherapy/chemotherapy by aptamer (TLS11a) modification to enhance HCC-specific targeting (55–57). Nanoplatforms may provide more detailed and comprehensive information about tumor size, anatomical structure, and location and realize precise theranostic guidance by applying dual- or multimodal images that integrate optical and traditional medical images (e.g., CT and MR images). For instance, Qi et al. synthesized a NIR-II photoacoustic (PA) CT imaging-guided nanoagent for HCC theranostic strategy, called Pt@PDA-c (58). Pt@PDA-c had deep tissue penetration and high resolution, which provided accurate location of deep (~4 mm intraabdominal depth) and small (diameter < 5 mm) HCC lesions. Moreover, Pt@PDA-c-mediated PTT could eliminated HCC without recurrence under the guidance of real-time PACT.

Ablation has long been considered a local treatment. However, growing evidence shows that ablation does more than physically eliminating tumors; it can also play a considerable role in distant lesions through immune effects, also known as abscopal effect. Changes in circulating immune cells/factors and tumor immune microenvironments have been explored by analyzing peripheral blood and tumor models. In 2005, Michael Geissler and colleagues found that local tumor ablation (percutaneous ethanol injection [PEI]/RFA) increased HCC immunogenicity in patients thus to promote endogenous adjuvants release and dendritic cell (DC) activation (59). Besides, RFA induced systemic immune variation in innate immune cells (including natural killer (NK) cells and plasmacytoid DCs) and adaptive immune cells (including tumor-specific T cells, antigen-presenting cells [APCs] and CD8 central memory T cells) (60–62). De novo or enhanced tumor-specific immune responses could be observed in patients with HCC after MWA (63). Wu and colleagues observed that neutrophil, monocyte and NK cell were increased to induce innate immune response and immunosuppressive lymphocyte was decreased in patients with HCC post-IRE (64). Moreover, their results indicated an ideal treatment window for immunotherapy (3–14 days post-IRE) to further control tumor recurrence and metastasis. Moreover, the expression of immune checkpoints (programmed cell death protein-1 [PD-1] and PD-1 cognate ligand [PD-L1]), which are associated with HCC tumor size, blood vessel invasion, and BCLC staging, can be downregulated by cryoablation but upregulated at recurrence (65).

The results observed in patients have also been further validated in various animal models. RFA increased CD8+ T cells, memory CD8+ T cells, and DCs and decreased regulatory T (Treg) cells in a unique murine model developed through a combination of intrasplenic inoculated oncogenic hepatocytes and carbon tetrachloride (66). Dai et al. reported that IRE could increase anti-tumor CD8+ T cells to prevent local tumor regrowth and distant metastasis and decrease immunosuppressive Treg and PD-1+ T cells in C57BL/6J mouse model bearing subcutaneous H22 hepatoma (67). Fong’s group demonstrated that IRE induced tumor antigens and facilitated granulocyte macrophage colony-stimulating factor plasmid transfer to achieve local and systemic anti-tumor responses in Yorkshire pig models (68, 69). Similarly, in other solid tumors, RFA can not only reduce the proportions of immunosuppressive cells (including Treg cells, tumor-associated macrophages and neutrophils), but increase the T cell infiltration as well as expression of the immune checkpoints (PD-1/PD-L1 and lymphocyte-activation gene 3 [LAG3]) in RFA-treated tumors and distant non-RFA tumors (70, 71). Moreover, serum and intra-tumoral cytokines, such as IFN-γ, IL-1α/β, IL-2/6/8/10, and TNFα/β, were also increased or activated (64, 72–75).

Increasing evidence have proved that ablation therapy could activate systemic anti-tumor immunological effects and inhibit immunosuppressive effects ( Figure 2 ). However, RFA could also increase PD-1/PD-L1 expression, which was repressed by sunitinib with activation of immune response (66). This effect may facilitate checkpoint inhibitor therapy by constructing an immune-supportive microenvironment. Thus, combining ablation with immunotherapy is rational to achieve augmented and longer anti-tumor effects and prevent HCC progression with improved outcomes.

Table 2 reviews the finished clinical trials of the combinations of ablation and immunotherapy. Nivolumab and pembrolizumab, which are PD-1 blockades, received quick approvals as second line therapy for patients with HCC after sorafenib failure on the basis of CheckMate-040 (93) and KEYNOTE-224 (97). A recent proof-of-concept clinical trial suggested that the application of RFA or MWA enhanced the anti-tumor effects and response rates (from 10% to 24%) of nivolumab and pembrolizumab (135). The explanation for synergistic effects may be found in Section 2.3 in this review. In brief, the critical roles of RFA in T cell infiltration/response and PD-1 expression may be one of rationales for combining RFA with PD-1 blockade. Besides, the combination of RFA with tremelimumab (CTLA-4 blockade) have been also explored (134, 135).

Various studies have demonstrated the safe and effective to applicate adjuvant adoptive cellular immunotherapies to patients with HCC post-ablation with improved RFS and OS (102, 139–141). For patients with metastatic HCC, the combination of cryoablation and DC-CIK cell immunotherapy also achieved a significantly higher OS (median: 32 months) than cryoablation (median: 17.5 months) and the untreated group (median: 3 months) (144). Moreover, the multiple treatment modality for cryo-immunotherapy could provide better prognosis than the single one.

Notably, Tetsuya Nakatsura’s team found that RFA stood out among other locoregional therapies (including surgical resection and TACE) by referring to GPC3-specific T-cell-mediated immune response for HCC (145). Compared with resection, RFA significantly induced GPC3-specific CTLs, especially in patients with GPC3-overexpressing HCC. Consequently, the phase II study of GPC3 peptide vaccine for adjuvant immunotherapy was carried out, laying a foundation of antitumor efficacy of GPC3 peptide vaccine and induced GPC3-specific CTL (105). Although the combination of resection or RFA with GPC3 peptide vaccine decreased the 1-year recurrence rate (142), whether different local strategies had an impact on the prognosis of the combination treatment remained unclear. However, another randomized phase II study showed that adjuvant immunotherapy with tumor associated antigen (TAA)-pulsed DC vaccine prolonged the RFS of patients with complete remission in non-RFA (including surgical resection, TACE, and PEI) groups compared with those in the RFA group (102). These results suggest that combination strategy benefits patients, but the choice of optimal combinations is thought provoking.

While the clinical trials are in full force, the combination of ablation and immunotherapy is also attracting the attention of scientists in basic medicine and biomedical materials. The development and application of multi-functional nanoplatforms have enabled synergistic ablative-immunotherapy strategy to flourish, instead of the sequential combination. On the one hand, a nanoplatform can deliver multiple drugs with optimized drug performance and therapeutic efficiency, as well as reduced drug toxicity. On the other hand, nanoplatforms can apply imaging technology to identify and locate tumors, guide ablation procedures, as well as monitor drug responses and therapy efficacy. Moreover, such a combination strategy may maximize the synergistic anti-tumor effects and thus achieve a greater therapeutic efficacy than the mere sum of the parts.

First, nanoplatforms can improve the targeting ability of agents through the innate enhanced permeability and retention effect and modifications with specific targets to enhance the anti-tumor effects (8). A mesoporous silica based nanosystem co-loading ICG and sorafenib, named (ICG+S)@mSiO2, was developed for synergetic PTT/immuno-enhanced therapy (146). (ICG+S)@mSiO2 improved endocytosis of HCC cells and photothermal efficiency. Active targeting deliveries were achieved in SP94-PB-SF-Cy5.5 nanoparticles (NPs) (147) and PCN-ACF-CpG@HA NPs (148) by conjugated with HCC specific targeting peptide (such as SP94) and HA (targeting CD44 receptor-overexpressed HCC cells), respectively. Moreover, SP94-PB-SF-Cy5.5 and PCN-ACF-CpG@HA, in combination with PD-L1 blockade and an immunologic adjuvant (CpG), enhanced the PTT- and PDT-induced weak immunogenic cell death of cancer cells. Similarly, these strategies for enhanced immune responses also applied sonodynamic immunotherapy as recently reported by Tan et al. (149) and Lin et al. (150). Moreover, anti-TGF-β antibody modification is an active targeting strategy that enhances cell endocytosis for improved PTT and an immunotherapeutic strategy for immune activation (151). Besides, ICG/ICG-SF-Gel-based photothermal-immunotherapy inhibited primary and distal tumor growth, with improved survival time with the help of Ganoderma lucidum polysaccharides (GLP) for enhancing the antitumor immunity (152). These combined anti-tumor effects led to the application of TAAs in in situ vaccination to eliminate residual and distant lesions, as well as inhibit tumor recurrence and metastasis.

Biomimetic nanotechnology, which integrates advantages of nanoplatform delivery and cellular immunotherapy, provides novel strategies for synergistic ablative immunotherapy. On the one hand, biomimetic nanoplatforms are ideal for targeted drug delivery because of their superior biological characteristics. For instance, Wang et al. developed a photothermal immunotherapy nanoplatform based on synthetic high-density lipoprotein (sHDL) (153). The higher expression of sHDL in HCC cells than in other normal cells of liver facilitates the preferential delivery of agents into the cytosol of HCC cells. Ma and colleagues designed a CAR-T cell membrane-coated mesoporous silica NP, which specifically recognized GPC3+ HCC cells (154). On the other hand, a programmable therapeutic strategy based on engineered immune cells provide a possibility for the synergy of ablation and cellular immunotherapy. Zhang et al. constructed an artificial engineered NK cell decorated with TLS11a (a HCC-specific targeting aptamer) for photothermal immunotherapy (56).