Immunomodulatory effect of locoregional therapy in the tumor microenvironment

Abstract

Cancer immunotherapy appears to be a promising treatment option; however, only a subset of patients with cancer responds favorably to treatment. Locoregional therapy initiates a local antitumor immune response by disrupting immunosuppressive components, releasing immunostimulatory damage-associated molecular patterns, recruiting immune effectors, and remodeling the tumor microenvironment. Many studies have shown that locoregional therapy can produce specific antitumor immunity alone; nevertheless, the effect is relatively weak and transient. Furthermore, increasing research efforts have explored the potential synergy between locoregional therapy and immunotherapy to enhance the long-term systemic antitumor immune effect and improve survival. Therefore, further research is needed into the immunomodulatory effects of locoregional therapy and immunotherapy to augment antitumor effects. This review article summarizes the key components of the tumor microenvironment, discusses the immunomodulatory role of locoregional therapy in the tumor microenvironment, and emphasizes the therapeutic potential of locoregional therapy in combination with immune checkpoint inhibitors.

Graphical abstract

Locoregional therapy plays an important role in the immunomodulatory effect of the tumor microenvironment. The combination therapeutic strategy of locoregional therapy and immunotherapy has great potential to further augment antitumor effects and bring survival benefits for cancer patients.

Introduction

Immunotherapeutic agents, especially immune checkpoint inhibitors (ICIs), have shown sustained antitumor responses in various cancer types.¹ However, many patients with less immunogenic cancers do not benefit from immunotherapy. In addition, the tumor immunophenotype at baseline may affect the initial reaction, which determines the immunotherapy sensitivity.² The immunophenotype classification system consists of three aspects: (1) the abundance of effector and suppressor cells, (2) immune cell localization, and (3) immune cell activation state.³ Cold tumors are insensitive tumors that generally lack lymphocytes in the tumor microenvironment (TME). Researchers are working to rapidly convert cold tumors into inflamed, or so-called hot, tumors that are more susceptible to immune responses.

Previous studies have demonstrated that radiotherapy to a single lesion with metastatic disease occasionally results in the regression of non-targeted, distant lesions.⁴ The “abscopal effect” refers to the phenomenon of local therapy eliciting a distal antitumor response, which is mediated by the systemic anticancer immune response and can be amplified by ICIs.^5,6 The discovery of this effect has piqued the interest of researchers, and immunomodulatory interventions have been used to augment tumor immunogenicity. However, these phenomena have not been consistently demonstrated in clinical practice. A potential immunostimulatory limitation may be the insufficient release of immunogenic components.⁷ Furthermore, ICI application has altered T cell receptor (TCR) repertoires and expanded specific clones, such as de novo TCR repertoires in patients with melanoma that specifically recognize melanoma antigens after programmed death (PD)-1 therapy, suggesting the emergence of neo-epitope-targeting T cells. However, ICI monotherapy fails to unleash T cell antitumor immunity without eliciting and defining specific T cell responses.^8,9 Thus, there is evidence that pre-existing antitumor immunity is critical for subsequent ICIs, demonstrating the importance of locoregional therapy in improving immunotherapy efficacy.¹⁰ Therefore, further investigation into the enhancement of long-term systemic antitumor immunity through synergy between local therapy and immunotherapy is warranted.

Tumor progression is linked to specific TMEs, and locoregional therapy can remodel the TME and shape antitumor immunity.^7,11 Understanding how locoregional treatment changes and remodels the TME allows for developing new treatment strategies for refractory tumors and improving therapeutic outcomes. This article reviews the most critical TME components, discusses how locoregional therapy modulates the TME, emphasizes the therapeutic potential of locoregional therapy in combination with ICIs, and summarizes recently published literature on these topics.

Tumor microenvironment

The TME is highly complex and heterogeneous, dynamically modulating tumor activity and affecting the efficacy of therapeutic agents. TME is defined as the complex multicellular environment of tumor development, including various immune cell types, stromal cells (such as cancer-associated fibroblasts, stromal mesenchymal cells, and pericytes), stroma, blood and lymphatic vascular networks, extracellular matrix (ECM), and secreted molecules (including chemokines, cytokines and growth factors).^12,13 Tumor-infiltrating lymphocytes mainly include dendritic cells (DCs), natural killer (NK) cells, T cells, myeloid-derived suppressor cells (MDSCs), neutrophils, and tumor-associated macrophages (TAMs) and they can be divided into two categories: immunogenicity and immunotolerance.

DCs

DCs play a vital role in antigen presentation cells (APC). Intracellular components are released into surrounding tissues during the late stages of tumor cell apoptosis or necrosis. Dying tumor cells release a “find-me” signal, which attracts tissue-residual DCs and promotes phagocytosis by DCs through cell surface “eat-me” signals.¹⁴ Considering that, CD8⁺ T cells are the major effectors of antitumor immune response; therefore, the cross-presentation of DCs for the relevant tumor-associated antigens is paramount. DCs can be classified into three subtypes: plasmacytoid DCs (pDCs), monocyte-derived inflammatory DCs, and CD11c⁺ classical DCs (cDCs). cDCs are further classified into two types: cDC1 expressing CD11c, CD103, XCR1, and CLEC9A, and DC2 expressing CD11c, CD11b, CD1c, MHC-II, and CD172a (signal regulatory protein alpha, SIRPα) with different abilities to prime CD8⁺ and CD4⁺ T cells.^15,16,17

cDC1s are associated with high-quality cross-presented antigens on major histocompatibility complex (MHC) class I molecules to stimulate cytotoxic CD8⁺ T cells, and they can support T_H1 cell polarization. cDC1s secrete cytokines and chemokines that attract effector NK cells and T cells to tumors and keep them cytotoxic.¹⁸ In addition, cDC1 migration to tumor-draining lymph nodes (dLNs) contributes to tumor antigen delivery to naive CD8 T cells, eliciting tumor-specific immune responses. However, cDC1s are rarely detected in tumors; therefore, increased infiltration of cDC1s within tumors is associated with improved survival and immunotherapy response.¹⁹ cDC2 appears to be critical for stimulating CD4⁺ T cell responses via MHC class II molecules.²⁰

T cells

The current consensus is that T cells play a major role in tumor immunology because of their ability to kill tumors. T cells within tumors correlate with a better prognosis in various malignancies, such as colorectal, melanoma, and ovarian cancer.²¹ The main T cell types are CD4⁺ and CD8⁺ T cells. Cytotoxic CD8⁺ T cells serve as essential effector cells in tumor destruction by secretion of granzyme, interferon (IFN)-γ, and perforin. However, dysfunctional T cells exist in immunosuppressive environments and are distinguished by several upregulated immune receptors or inhibitory checkpoints, such as PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT, the ligands of which are expressed in tumor cells or APCs, thus inhibiting T cell functions.^22,23

Likewise, CD4⁺ T cells are crucial for tumor control. By recognizing human leukocyte antigen class II and releasing IFN-γ and tumor necrosis factor (TNF)-α, conventional CD4⁺ T cells modulate innate and adaptive immunity and inhibit tumor growth, thereby killing tumor cells through cytotoxic functions.^24,25 In addition, CD4⁺ T cells can attract APCs, which help recruit T cells and enhance the innate immune response.²⁶

Regulatory T cells (Tregs, CD3⁺ CD4⁺ FOXP3⁺ CD25⁺) are a typical adaptive immune response suppressor cell population that suppresses functional T cells in four ways as follows: (1) interleukin (IL)-2 depletion, immunosuppressive cytokine secretion, including IL-10, IL-35, and transforming growth factor (TGF)-β, and direct apoptosis induction of effector T cells or APCs via perforins and granzyme B^27,28,29,30; (2) Treg cells also express CTLA-4, which has a higher affinity for CD80/CD86 to APCs than CD28, inhibits co-stimulatory signaling, and affects APC maturation, thereby attenuating T cell stimulation³¹; (3) interaction between CTLA-4 with CD80/86 increases IDO secretion, resulting in T cell dysfunction³²; and (4) several immune checkpoint molecules, including LAG-3, expressed on Tregs inhibit cytotoxic T cells.³³

Murine models revealed that PD-1⁺ T cells comprise distinct subsets, and PD-ligand 1 (PD-L1) inhibition reactivated the intermediate expression of T-bet^hi PD-1^int T cells only. In contrast, Eomes^hi PD-1^hi T cells showed no response.^34,35 This result indicates that recruiting more T cells to tumors may increase T cell diversity, potentially enhancing ICI efficacy. More recently, single-cell RNA sequencing (scRNA-seq) and cytometry by time of flight (CyTOF) have provided additional information about T cell status. T cell reactivation with tumor-reactive TCRs is expected to have the greatest impact on antitumor immune response.³⁶

NK cells

NK cells exert cytotoxic functions without MHC specificity, which complements and synergizes with a cytotoxic T cell-mediated approach to MHC-restricted tumor killing. In addition to their cytotoxic effects, NK cells release cytokines that regulate adaptive immune responses.³⁷ NK cells can be separated into CD56^bright CD16⁻ NK cells and CD56^dim CD16⁺ NK cells.³⁸ The CD56^dim NK cells subset is considered a mature cytotoxic cell population that kills other cells when activated. CD56^bright is less mature and exerts an immunomodulatory effect when exposed to stimuli such as IL-1β, IL- 2, IL-12, IL-15, and IL-18, thereby releasing numerous cytokines.³⁹ However, studies revealed that NK cells in the TME exhibit dysfunction, diminished cytotoxic activity, and reduced secretion of pro-inflammatory cytokines.⁴⁰ This may partially be attributed to changes in vascularization and nutrient and oxygen limitations caused by hypoxia in the TME that constrain NK cell activities.⁴¹

TAMs

TAMs are often associated with a poor prognosis.⁴² Macrophage polarization and the M1 and M2 classifications define macrophages as pro-inflammatory (polarization by lipopolysaccharide, granulocyte macrophage colony stimulating factor [GM-CSF], IFN-γ, TNF-α, or pathogen-associated molecular patterns [PAMPs]) or anti-inflammatory (polarization by macrophage colony stimulating factor, TGF-β, IL-4, IL-10).^43,44,45 M1 macrophages upregulate gene expressions related to antigen processing, presentation, and co-stimulators that promote T cell activities, including MHC class II molecules, inducible nitric oxide synthase, and TNF. In contrast, M2 macrophages are regarded as pro-tumorigenic, with high expression of levels of CD206, CD163, Arg1, and IL-10, produce inhibitory TGF-β, and promote angiogenesis.^46,47

However, macrophages in vivo are transcriptomically dynamic and highly heterogeneous, as determined by the TME. The classification of M1 and M2 was oversimplified to describe the intrinsic properties of macrophages. TAMs, in general, contribute to an immunosuppressive microenvironment in hypoxia, fibrosis, and numerous cancer-associated fibroblasts (CAFs), resulting in the exclusion of cytotoxic T cells. Macrophages are programmed to drive inflammation by eliciting an adequate immune response and limiting the inhibitory environment.⁴⁸

MDSCs

MDSCs are a subgroup of immature myeloid cells. It remains controversial whether MDSCs are a distinct lineage or immature neutrophils; however, MDSCs are considered key contributors to the TME and are, thus, an obstacle to many cancer immunotherapies.^49,50 MDSCs induce strong immunosuppressive effects through (1) stimulating other immunosuppressive cells such as Tregs and M2 macrophages; (2) impeding TCR expression through the production of nitrogen species (RNS) and reactive oxygen species (ROS); (3) impairing lymphocyte homing; (4) depleting metabolites critical for T cell functions; (5) altering the expression of ectoenzymes regulating adenosine metabolism and reducing the expression of affected molecules such as TNF-α, perforin, and IFN-γ; and (6) modulating inhibitory immune checkpoint molecule expressions.⁵¹

Neutrophils

Tumor-associated neutrophils also exhibit the N1 (tumor-suppressive) and N2 (tumor-promoting) phenotypes. Their phenotype and function within the TME also depend on tumor status. In the early stages of tumor development, neutrophils are inflammatory and immunosuppressive. TANs regulate ECM by secreting neutrophil extracellular traps, including matrix metalloproteinases, cathepsin G, and neutrophil elastase, and promote angiogenesis, tumor progression, invasion, and metastasis.^52,53,54

Other components of the TME

Interactions between immune cells and other TME components are essential for immune response. For instance, the ECM stimulates and inhibits adaptive immune responses by promoting T cell infiltration in tumors or inhibiting T cell proliferation.⁵⁵ CAFs can be either pro-inflammatory or immunosuppressive. CAFs attract myeloid cells to tumors by secreting chemokines, including CCL2, CCL5, CXCL8, and CXCL12.⁵⁶ CAFs present antigens through MHC class I molecules, whereas the interaction between FAS and PD-1 on T cells with FASL and PD-L1/L2 on CAFs suppress CD8⁺ T cell functions.^57,58 CAFs polarize TAMs to an immunosuppressive phenotype by recruiting Tregs and MDSCs, reducing the DC presentation capacity, and secreting inhibitory cytokines, further promoting the inhibitory TME, leading to T cell dysfunction.⁵⁹ CAFs also inhibit T cells by producing TGF-β and CXCL12.⁶⁰ Aberrant vasculature and lymphoid tissue restrict T extravasation and lead to hypoxia through the hypoxia-inducible factor family (HIF-1α). HIF1α also increases PD-L1 expression. In addition, hypoxia causes the release of cytokines and growth factors that affect the proliferation, differentiation, and function of immune cells.⁶¹

Components in a tumor can be tumor-suppressive or tumor-supporting, based on the stage of cancer progression.¹² Conventional therapies, such as chemotherapy and radiotherapy, have increased the number of inhibitory TAMs, leading to cancer treatment evasion and recurrence.⁶² Furthermore, radiotherapy induces hypoxia and HIF-1α activation, reducing radiosensitivity.⁶³ CAFs can also be activated, leading to lysyl oxidase (LOX) secretion, favoring collagen deposition, and promoting tumor progression.⁶⁴ The complex interactions of the TME components influence tumor progression, invasion, and metastasis. Therefore, various therapeutic strategies against TME have been investigated to eliminate tumor-supporting cells or convert them into tumor-suppressive phenotypes.⁶⁵ However, the method for balancing the relationship between pro-tumor and antitumor effects induced by locoregional therapy and optimizing its efficacy needs to be studied further.

Immunotherapy

ICI therapy has revolutionized cancer treatment, and clinically approved agents are limited to the application of blockades targeting PD-1/PD-L1 and CTLA-4.⁶⁶ CTLA-4 was the first immune checkpoint molecule identified on T cells, which binds co-stimulatory B7 molecules B7.1 (CD80) and B7.2 (CD86) with higher affinity than CD28. This combination competitively inhibits T cell activation instead of producing a stimulatory signal, leading to T cell anergy.⁶⁷ Ipilimumab was approved by the Food and Drug Administration (FDA) in 2011 for patients with advanced-stage melanoma targeting the immune checkpoint molecule CTLA-4.⁶⁸ Numerous clinical trials investigating CTLA-4 monotherapy or combination therapy have shown promising results in non-small cell lung cancer (NSCLC) and metastatic melanoma.⁶⁹ However, CTLA-4 depletion causes severe immune dysfunction, which may be related to the high expression of CTLA-4 on Tregs.^70,71 PD-1 is another immune checkpoint molecule mainly expressed on the surface of activated T cells. PD-L1 or PD-L2, mainly expressed on tumor and myeloid cells, inhibit T cell activation and induce apoptosis by binding to PD-1. PD-1 blockade is effective in various types of cancer, including bladder cancer, NSCLC, renal cell carcinoma, and melanoma. Inhibitors targeting PD-L1 have also been approved by the FDA, including atezolizumab, durvalumab, and avelumab. Co-inhibitory targets (BTLA, TIGIT, TIM3, and LAG3) and co-stimulatory targets (ICOS 4-1BB, OX40, and GITR) have significant potential to complement the currently available ICIs and are currently being investigated for clinical applications.⁶⁶

Despite the fact that immunotherapy with ICIs has demonstrated significant clinical efficacy, only 12.46% of the patients can benefit from ICIs.⁷² Resistance to ICIs is related to the potential immunological characteristics within the TME. The presence of lymphocytes distinguishes “hot” tumors (especially many TILs). Increasing evidence suggests that characteristics correlated with inflamed tumors are potential prognostic biomarkers of ICI responses.⁷³ Therefore, researchers have made considerable efforts to transform cold tumors into hot ones. In addition to counteracting the intrinsic mechanisms of immune evasions, such as β-catenin signaling, in situ vaccination by locoregional therapy is certainly a promising approach.^66,74

Locoregional therapy

Locoregional therapy plays an increasing role in cancer treatment due to its low invasiveness, quick recovery, targeting, and repeatability.^75,76 In addition to killing cancer cells directly, locoregional therapy also plays a pivotal role in the immunomodulatory effects of TME, which is crucial in the current era of cancer immunotherapy.⁷⁷ Currently, there are several types of locoregional therapies with extensive clinical applications. Interventional therapies, such as energy-based ablation, including cryoablation, radiofrequency ablation, microwave ablation, cryo-thermal ablation, irreversible electronics, and high-intensity focused ultrasound, have been used in clinical practice as promising locoregional therapy.⁷⁸ Local ablation has been proposed as a potentially curative therapy for small liver malignancies and resection. Radiofrequency ablation is the standard ablative modality for early-stage hepatocellular carcinoma (HCC).⁷⁶ Intratumoral immunotherapy is another noteworthy locoregional treatment. Intratumoral administration increases in situ bioavailability and efficacy of immunotherapy. These immunotherapies include microbes, immunomodulatory monoclonal antibodies, cytokines, and synthetic compounds mimicking infectious agents.⁷⁹ A detailed introduction of each locoregional therapy is discussed in the following sections.

Immunomodulatory effects of locoregional therapy

Tumor cell immunogenicity is critical for cancer therapy efficacy. Locoregional therapy causes cancer cell death via immunogenic cell death (ICD), allowing the tumor to be available as an in situ tumor vaccine, and contributes to the activation and restoration of antitumor responses. Unlike classical apoptosis, ICD induces the release of damage-associated molecular patterns (DAMPs), including upregulated heat shock protein 70 (HSP70), passively released ATP (adenosine-5′-triphosphate), high-mobility group box protein 1 (HMGB1), and surface-exposed calreticulin (CRT).⁸⁰ Notably, DAMP secretion is due to cellular stress and does not always trigger antitumor responses.⁸¹ DAMPs phagocytized by DCs activate the nuclear factor (NF)-κB pathway and upregulate the co-stimulatory expression of CD80/86.^82,83

DCs (mainly cDC1s) migrate to dLNs, where they present antigen peptides via MHC molecules and display co-stimulation to T cells. Naive CD8⁺ T cells recognize the antigen peptides through TCR and co-stimulatory receptors CD28 and bind to the co-stimulatory molecules CD80/86 expressed on mature DCs. This stimulates the secretion of IL-2, an essential cytokine for T cell expansion, which stimulates tumor-specific immunity. Increased tumor-specific T cells ultimately migrate to tumors and distant lesions and directly kill tumor cells.⁸⁴ However, apoptosis did not result in the release of DAMPs. Without this, the NF-κB pathway cannot be activated, and CD80/86 expression is absent, resulting in T cell unresponsiveness or even clonal deletion, which induces immunosuppressive effects.⁸⁵ (Figures 1 and 2).

Figure 1.

Schematic overview of locoregional therapy

Schematic overview of locoregional therapy, including cryoablation, radiofrequency ablation, microwave ablation, cryo-thermal ablation, HIFU, irreversible electroporation, oncolytic virus, immune checkpoint inhibitors, and CAR-T cell therapy. Radiofrequency ablation, microwave ablation, and HIFU destroy tumors through thermal injury. M-HIFU also induces cell death in subcellular fragments. Cryo-thermal ablation takes advantage of heat and frozen ablation. Irreversible electroporation is a nonthermal method for generating tumor cell death by inducing membrane rupture. Oncolytic viruses prioritize infection and replication in cancer cells and lyse tumor cells. Intratumoral ICIs cause mAb accumulation in the TME and dLNs. Locoregional CAR-T cells delivery circumvents T cell homing barriers and reduces off-target tumor toxicity. These techniques relies on various physical or biological mechanisms to induce tumor necrosis and/or boost the immune response. CAR, chimeric antigen receptor; HIFU, high-intensity focused ultrasound; mAb, monoclonal antibodies; dLNs, draining lymph nodes.

Figure 2.

Overview of cancer-immunity cycle in post-locoregional therapy

Locoregional therapies generate cytotoxic CD8⁺ T cells that enhance the destruction of local and systemic cancers. Tumor destruction by locoregional therapy results in the release of several tumor antigens that can be processed by APCs and presented to naive T cells through MHC class I molecules. The simultaneous release of DAMPs induces an adaptive immune response. Tumor-specific cytotoxic T cells were activated and proliferated in the dLNs, then migrated into the circulatory system, destroyed the local tumor, and protected from distant metastases. APC, antigen-presenting cells; DAMPs, damage-associated molecular patterns; IFN-γ, interferon-gamma; Gzmb, granzyme B; TNF-α, tumor necrosis factor-alpha.

Cryoablation

Cryoablation uses repeated freeze/thaw cycles below −40°C to induce crystallization, microvascular damage, and ischemic and mechanical cell destruction.⁸⁶ Cellular necrosis occurs at the center of the probe, releasing inflammatory cellular contents and numerous antigens while promoting innate immunity by activation of Toll-like receptors. The surrounding tissue exposed to sublethal hypothermia undergoes apoptosis-mediated cell death due to cold-induced mitochondrial injury, which is associated with presentation of phagocytosed antigens.

Cryoablation is regarded as the optimal modality for inducing antitumor immune response because it resulted in intact antigen release with preserved cellular structure and increased plasma membrane permeability, rather than protein denaturation caused by hyperthermia.⁸⁷ Following cryoablation, an enhanced immune response was observed with infiltration of T cells, macrophages, and neutrophils within the tumors and secretion of IL-1, IL-6, IL-10, TNF-α, IFN-γ, and NF-κβ.⁷⁵ Kato et al. revealed that after cryoablation in patients with kidney cancer, certain T cells with tumors expanded. In addition, expression of immune genes, including CD8, CD4, granzyme A, and CD11c in the tumor increased after cryoablation.⁸⁸ However, few studies have reported no immune response or immunosuppression following cryoablation.^89,90 A possible explanation is that many apoptotic cells induced by cryoablation may cause T cell anergy and clonal deletion, suggesting the importance of the balance between apoptosis and necrosis following cryoablation.⁸⁵ Furthermore, Yang et al. observed that 1 week after oblation, patients with HCC had increased circulating PD-1/PD-L1 expression, indicating that the combination of checkpoint blockade enhances the antitumor response.⁹¹

Radiofrequency ablation

Radiofrequency ablation (RFA) is a technique that uses high-frequency electrical current to generate heat through vibration and friction of ions, resulting in coagulative necrosis of the tissue surrounding the electrodes. Heat conduction causes sublethal temperatures in the peripheral zone, leading to apoptosis. In addition, RFA causes the release of numerous intracellular components, including inflammatory cytokines, such as IL-1β, IL-6, IL-8, and TNF-α, which stimulate innate and adaptive immune responses.⁷⁵ Den Brok et al. found that RFA elicited a weak immune response against OVA in a murine B16-OVA melanoma model. However, adoptive transfer of splenocytes to untreated mice provided antitumor protection, suggesting that in situ tumor destruction by RFA offers antigen sources to boost antitumor immunity.⁹²

Furthermore, a previous study observed that RFA enhanced IFN-γ-producing tumor-specific T cell responses to tumor-associated antigens (TAAs), including de novo antigens, in 62.3% of patients with HCC. The expansion of TAA-specific T cells is associated with recurrence prevention; however, their numbers typically decrease 24 weeks after RFA, suggesting that RFA alone is insufficient to prevent tumor recurrence.⁹³ ELISPOT assays showed that lysates from untreated tumor cells or necrotic tumors also enhanced specific T cell responses following RFA.⁹⁴ RFA stimulates T cell immune responses; nevertheless, it is rapidly suppressed by T cell dysfunction with upregulated PD-1/PD-L1 expression, which may be related to the RFA-induced inflammatory TME.

Fei et al. performed scRNA-seq in a pancreatic cancer mouse model to investigate changes in tumor-infiltrating immune cells in distant lesions after RFA. The result showed that RFA decreased the proportion of suppressive immune cells in distal lesions, including Tregs, TAMs, and TANs, while increasing the number of functional DCs and T cells. Moreover, PD-1 and LAG-3 were upregulated in T cells, suggesting that RFA reconstituted the T cell immune microenvironment, and T cell exhaustion was common in distant lesions, underscoring the necessity of combined therapy.¹¹

Notably, incomplete RFA (iRFA) has been linked to tumor progression through autophagy.⁹⁵ Shi et al. demonstrated that iRFA also hindered the PD-1 therapy efficacy, increased MDSCs infiltration within residual tumors, and suppressed T cell function. CCL2/CCR2 prevents MDSC recruitment and restores the remaining tumor resistance to PD-1 treatment.⁹⁶

Microwave ablation

Microwave ablation (MWA) uses oscillating electromagnetic energy to induce agitation of polar molecules, generating lethal temperatures and destroying tumor tissues through coagulative necrosis.⁹⁷ The immunogenic profile of thermal ablation has mainly been described in RFA and cryoablation, while limited studies have focused on MWA. Following MWA, NK cells were stimulated in a breast cancer mouse model and exhibited enhanced cytotoxic functions to inhibit breast cancer metastasis through the macrophage/IL-15/NK cell axis. Depletion experiments confirmed that NK cells are a key population for prolonged survival. Furthermore, a weaker T cell response was observed with increased levels of IL-2 and IL-12. However, depleting CD4⁺ and CD8⁺ T cells did not affect survival, suggesting a limited T cell response in MWA that should be investigated further.⁹⁸

MWA increased T cell and NK cell infiltration in ablated lesions, adjacent normal tissue, and remote untreated tumors in patients with HCC, positively correlating with survival outcome.⁹⁹ Thirty percent of 23 patients with HCC treated with MWA exhibited de novo or enhanced tumor-specific immune responses (IFN-γ and IL-5) in their peripheral blood, which was strongly associated with a favorable prognosis.¹⁰⁰ An increase in IL-12 and CD4⁺ T cells was observed, with a decrease in IL-4 and IL-10, following MWA, indicating a Th2/Th1 deviation.¹⁰⁰ An increase in Th17 cells compared with the baseline level was positively correlated with tumor recurrence in HCC after MWA.¹⁰¹

Cryo-thermal ablation

Cryoablation-induced apoptosis has an immunosuppressive effect. In contrast, heat-induced coagulative necrosis, such as MWA and RFA, limits antigen release, reducing the magnitude of the immune responses.^85,87,102 Several animal models have recently used a technique termed thermal cryoablation, combining thermal ablation and cryoablation with alternating heating and freezing, avoiding the disadvantages of both treatment modalities.¹⁰³ Mechanistically, cryo-thermal ablation produces various DAMPs in situ and peripheral circulation, similar to other ablation modalities. Cryo-thermal ablation increased cytotoxic CD8⁺ T cells and induced CD8⁺ T cell differentiation into memory stem T cells. In addition, cryo-thermal ablation promotes CD4⁺ T cell development into dominant cytotoxic CD4⁺ T cells, Th1 cells, and follicular helper CD4⁺ T cells (Tfh). We found that cryo-thermal treatment relies heavily on CD4⁺ T cells to provide long-term antitumor immune response. Moreover, cryo-thermal ablation decreases the number of immunosuppressive MDSCs and Tregs.¹⁰³

Meanwhile, there was a significant increase in serum IFN-γ, boosting the antitumor immune effect.¹⁰³ Furthermore, cryo-thermal ablation causes macrophages to polarize toward the M1 phenotype, which is mediated by released HSP70, regulating T cell differentiation and proliferation and inducing sustained immunity.¹⁰⁴ Subsequently, the induction contributes to activating and maturing DCs and promotes CD4⁺ T cell differentiation into Th1 and cytotoxic T cells, and cytotoxic CD8⁺ T cells, reconstituting the immunosuppressive environment and inducing a long-term antitumor memory effect, and compared with RFA, cryo-thermal treatment elicited robust T cell-mediated antitumor immunity, especially neoantigen-specific CD4⁺ T cell responses.¹⁰⁵ Following cryo-thermal treatment, innate immunity is activated by generating DAMPs, IL-6-rich pro-inflammatory cytokines, and macrophage polarization.¹⁰⁶

Irreversible electroporation

Irreversible electroporation (IRE) creates nanopores in cellular membranes using high-voltage millisecond electric pulses, leading to programmed cell death. Intracellular components, extracellular collagenous matrix structures, and vessels are well preserved, enabling APCs to infiltrate lesions and transport apoptotic antigens to dLNs, where tumor-specific T cell responses begin.¹⁰⁷ It may be that IRE is more conducive to immune cell infiltration than thermal ablation. IRE may “reset” the TME from an immunosuppressive to an immunostimulatory state as suggested.^108,109

IRE promotes T cell priming by mediating DC activation and maturation and increasing CD40, CD86, and CCR7 expression levels.¹¹⁰ Periablation areas were infiltrated with more DCs and CD8⁺ T cells in the hepatoma mouse model after IRE. IRE-treated mice rejected the tumor rechallenge and exhibited increased splenic IFN-γ CD8⁺ T cells. Furthermore, IRE alleviated immunosuppression by reducing PD-1⁺ T cells and Tregs in the tumor and spleen.¹¹¹ A transient decrease in Tregs (including activated Tregs and resting Tregs) and an increase in peripheral PD-1⁺CD8⁺ and PD-1⁺CD4⁺ T cells after IRE was observed in advanced pancreatic cancer patients.¹¹² PD-1⁺ T cells have been identified as tumor-specific T cells, indicating enhanced antitumor T cell responses after IRE.¹¹³ Two of the three patients had improved specific T cell responses, and two patients had de novo T responses after IRE. Furthermore, IRE induces a transient increase in microvessel density (MVD), promotes permeability of tumor blood vessels, alleviates hypoxia, and causes immunosuppression.^110,114 Meanwhile, IRE softens the ECM temporarily by reducing some fibrotic stromal components, including fibroblast activation protein alpha-positive (FAP-α), LOX, and hyaluronic acid-binding protein 1 (HABP1), all of which impair the efficacy of anti-PD-L1 inhibitors by limiting T cell infiltration.^60,110,115

High-intensity focused ultrasound

High-intensity focused ultrasound (HIFU) is a noninvasive therapy that produces high-intensity ultrasound beams that are focused on a small region, increasing the energy density. After the tumor tissue absorbs energy, the temperature rises to 60–85°C, causing coagulative necrosis at the target lesion.¹¹⁶ Histotripsy is a novel nonthermal focused ultrasound therapy, using microsecond or millisecond ultrasound pulses to homogenize the targeted tissue by mechanically forming a cloud of cavitation bubbles. Heat generation is prevented by separating the pulses.¹¹⁷ Unlike focused thermal ultrasound, the so-called thermal HIFU (T-HIFU), nonthermal focused ultrasound is known as mechanical HIFU (M-HIFU).¹¹⁸ As heat-induced denaturation of tumor proteins may affect the systematic antitumor response after T-HIFU, M-HIFU induces cell death into subcellular fragments and disrupts the extracellular matrix to release DAMPs, including HMGB1, CRT, and HSP, and stimulates the innate immune system, thereby enhancing immunogenicity.^117,119,120 Furthermore, as the stroma is mechanically disrupted, more antigens migrate to the lymph nodes, allowing tumor-infiltrating T cell migration. Cytokine secretion upregulates the endothelial adhesion molecules expression and activates intratumoral T cells.^121,122

M-HIFU preserves subcellular component integrity by disrupting cell membranes and producing nonviable tumor homogenates containing peptide tumor antigens with retained immunogenicity rather than heat-based denaturation and necrosis.¹²³ In an immunologically “cold” neuroblastoma murine model, M-HIFU increased lymph node and splenic NK cells and circulating IL-2, GM-CSF, and IFN-γ, while reducing Tregs, IL-10, and vascular endothelial growth factor (VEGF)-A.¹²⁴ GM-CSF is associated with APC activation and maturation, whereas VEGF is associated with tumor angiogenesis, suggesting that M-HIFU may alleviate immunosuppressive TME.¹²⁵ Osada et al. demonstrated that M-HIFU induces a stronger antitumor response than T-HIFU, mediated by the activation of CD8⁺ T cells, TAM repolarization to pro-inflammatory, M1-biased phenotype, and DC maturation. Furthermore, the expression of inhibitory immune checkpoint molecules increases following M-HIFU, preventing antitumor immunity.¹²⁶ M-HIFU maintains major blood vessel integrity and induces sterile inflammation, potentially improving the responsiveness to ICIs.¹²⁷ M-HIFU releases more ATP and HSP 60 and stimulates a more potent antitumor immune response than T-HIFU.¹²⁸

Intratumoral immunotherapy

Systemic immunotherapy has revolutionized cancer treatment; nonetheless, obstacles in the immune system due to immunosuppressive elements in the TME have limited efficacy in many patients.¹²⁹ Therefore, intratumoral immunotherapy (IT-IT) was developed as an additional tool for locoregional therapy repertoire by percutaneous delivery of immunomodulatory agents into the tumor with increased intratumoral drug concentration and reduced unnecessary systemic exposure.^129,130 Moreover, IT-IT allows for rapid drug delivery into the dLNs, which is critical for initiating and maintaining immune response. It also offers direct access to the tertiary lymphoid structures (TLS) within the tumor.¹²⁹ Herein, we describe several intratumoral or tumor tissue-targeted immunotherapies.

Oncolytic virus

Oncolytic viruses (OV) infect and replicate preferentially in tumor cells before lysing them with enhanced localized and systemic antitumor immune effects. Therefore, OV must be nonvirulent while retaining its immunostimulatory properties.¹³¹ Talimogene laherparepvec (T-VEC) was the first OV based on type 1 modified herpes simplex virus (HSV) and the first authorized viral therapy for unresectable melanoma. The following mechanisms mediate the antitumor responses of OV: (1) OV triggers ICD in cancer cells, resulting in PAMP, DAMP, and TAA production, in addition to its cytotoxic effects on tumor cells by apoptosis, pyroptosis, and necroptosis^131,132; (2) OV-release molecule treatment recruits and activates APCs to the virus-infected site. In addition, BATF3⁺ DCs engulf soluble tumor antigens, migrate to dLNs, and activate adaptive immunity^18,133; (3) enhanced antigen processing and presentation by the release of chemokines and interferons upregulates MHC class I molecules, thereby promoting antigen-specific CD8⁺ T cell recruitment for cytotoxicity toward tumor cells¹³⁴; (4) OV hinders tumor angiogenesis through endothelial cell activation¹³⁵; (5) OV produces ECM degraders and triggers CAFs lysis to inhibit ECM production from inducing stroma degradation, allowing T cell infiltration¹³⁶; and (6) immune checkpoint molecules are also upregulated in tumor cells, increasing sensitivity to ICIs.¹³⁷

A phase II study assessed the regression of lesions in patients with melanoma after OV therapy and found that OV-injected lesions with OV had more effector CD8⁺ T cells but fewer Tregs and MDSCs.¹³⁸ Thirteen patients with primary cutaneous B cell lymphoma (pCBCL) received T-VEC therapy. The results revealed that the virus was found only in the injected lesions and not in the non-injected lesions, indicating that the abscopal effect is mediated by the OV immune response rather than by the direct viral killing of tumors. In addition, type 1 interferon gene expression increased rapidly after injection; infiltration of activated NK cells, monocytes, and polyclonal CD8 T cells was enhanced, while Tregs infiltration decreased.¹³⁹

Immunostimulatory monoclonal antibodies

Some immunostimulatory monoclonal antibodies (mAbs) have been found to have dose-limiting toxicities, which limits the use of optimal doses. Intratumoral administration may improve the therapeutic index while decreasing immune-related adverse events (irAEs) and systemic toxicity.⁷⁹

Intratumoral ICIs cause mAb accumulation in the TME and dLNs. Targeted delivery of ICIs to dLNs enhanced the antitumor immune response compared with systemic ICI therapy. Furthermore, when the mAb dose was reduced, such effects persisted and were equivalent to those obtained by intratumoral administration. These results suggest that intratumoral ICIs boost immune responses by enhancing immunomodulation within dLNs.¹⁴⁰ Several preclinical studies have revealed the use of intratumoral CTLA-4 mAb delivery with potent antitumor activity.^141,142 In a phase I study of 12 patients with unresectable melanoma who were administered intratumoral ipilimumab and IL-2, seven patients achieved complete response at the treated lesion, and eight of nine patients with multiple tumors had a local or abscopal effect.¹⁴³ The combination of CTLA-4 and Toll-like receptor (TLR)-9 reversed immunotherapy resistance and triggered a systemic antitumor immune response.¹⁴⁴

Chimeric antigen receptor T cell therapy

Chimeric antigen receptors (CARs) are recombinant receptors that regulate T cells to recognize and target tumor cells with specific surface molecules.¹⁴⁵ Several clinical trials involving CAR-T targeting CD19 and B cell maturation antigens have yielded remarkable outcomes.^146,147 Compared with TILs, CAR-T cells are not limited by MHC molecules and can trigger immune effects through additional co-stimulatory domains, improving antigen recognition and altering “cold” tumor TME with recruited TILs.¹⁴⁵ However, CAR-T therapy has only been used in hematological malignancy and showed limited efficacy in solid tumors.¹⁴⁸ Furthermore, TME largely influenced the therapeutic effect of CAR-T cells because it inhibits CAR-T cells transport to a specific site, interferes with CAR-T cell metabolism, and builds an immunosuppressive environment that leads to T cell failure.

Locoregional CAR-T cell administration circumvents T cell homing barriers and reduces off-target tumor toxicity, as demonstrated in solid tumors, including the liver breast, brain, and pleura.^{148,149,150,151,152,153,154,155,156} Locoregionally delivered CAR-T cells have the potential to expand and immigrate to other tumor sites, improving the immune response, which may be more resource-intensive than other locoregional therapies.¹⁵⁷ Intratumoral administration of HER2-targeted CAR-T cells in mammary tumor mice induces mammary cancer in mice, which spreads to distant tumor sites and secondary lymphoid.¹⁵⁸ A patient with recurrent multifocal glioblastoma showed regression of all spinal and intracranial tumors through CAR-T intracranial delivery, with increased cytokines and immune cells in the cerebrospinal fluid.¹⁴⁹ A study demonstrated that local intraventricular and intratumoral administration of HER2 CAR-T cells, rather than intravenous administration, resulted in 100% survival even after tumor rechallenge in a human xenograft model of breast cancer metastasis to the brain.¹⁵⁹ A phase 1 study of repeated local delivery of HER2-CAR-T for recurrent and/or refractory central nervous system tumors showed an activated local immune response and was well tolerated.¹⁵³ Local delivery of B7-H3-targeted CAR-T cells triggered enhanced antitumor effect in xenograft mouse models of cerebral atypical teratoid/rhabdoid tumors.¹⁶⁰ Malignant pleural mesothelin is a poor prognostic cancer with a median overall survival of less than 1 year.¹⁶¹ FAP-targeted CAR-T cells were conducted in three patients with pleural mesothelioma, showing the feasibility of local delivery in organ-specific tumors. Two of three patients were alive during the follow-up of 18 months.¹⁵⁵ Adusumilli et al. conducted intrapleural administration of mesothelin-targeted CAR-T cell therapy combined with pembrolizumab for malignant pleural disease. The median overall survival was 23.8 months and the 1-year survival rate was 83%.¹⁵⁴ A phase 1b study of anti-CEA CAR-T hepatic artery infusions and selected internal radiation therapy in liver metastases patients suggested effective and safe results.¹⁵⁶

However, because numerous metastatic solid tumors are ineligible for locoregional delivery, engineered CAR-T trafficking to the desired site is in progress. Numerous intratumoral immunotherapy agents are currently being studied, including pattern recognition receptor (PRR) agonists, cytokines, DNAs, RNAs, pathogens, and biomaterials.⁷⁹

Combination therapy: Locoregional therapy and immunotherapy

Locoregional therapy has been shown to improve TME modulation favorably. However, the immune response triggered by locoregional therapy is relatively weak and transient. The abscopal effect induced by locoregional therapy has only been observed in a few cases. Meanwhile, the recurrence rate of HCC after RFA ranges from 50% to 70% in HCC.⁷⁶ Therefore, it is necessary to explore the use of systemic adjuvant therapies. Locoregional therapy improved immunotherapeutic efficacy by remodeling TME.⁷⁷ For example, OV induces a pro-inflammatory TME, contributing to tumors that are more susceptible to immunotherapy. In addition, several locoregional therapies upregulate immune checkpoint molecules, making them an appealing strategy for combining with ICIs. Considering that, most immunotherapies act in a T cell-dependent manner, tumors that lack T cells present low immunogenicity and cannot benefit from immunotherapy. Locoregional therapy increases intratumoral T cell infiltration, allowing for the use of pre-existing tumor-specific T cells in immunotherapy.¹⁶² It may synergize with immunotherapy to boost the antitumor immune response. Numerous studies on combining immunotherapy and locoregional therapies have been conducted based on promising preclinical and clinical results.

A preclinical study in a prostate cancer mouse model demonstrated that cryoablation combined with CTLA-4 blockade increased infiltration of CD4⁺ and CD8⁺ T cells, with a higher T effector/Tregs ratio in the treated tumors compared with monotherapy and prevented distant secondary tumor growth.¹⁶³ A pilot study in patients with early-stage breast cancer showed that cryo-ipilimumab combination therapy increased systemically peripheral Th1-type cytokines, activated (ICOS⁺) proliferative T cells, and intratumoral proliferating T effector/Tregs ratio.¹⁶⁴ In addition, cryo-ipilimumab combination therapy increased the diversity of the T cell clonal repertoire, which was absent in the ipilimumab monotherapy group.¹⁶⁵ Another pilot study explored cryoablation combined with tremelimumab in patients with metastatic renal cell carcinoma, with immunoassays showing increased T cell infiltration and TLS in clear cell patients.¹⁶⁶

In a colorectal mouse model, combining PD-1 inhibitors with RFA enhances tumor-specific T cell activity and increases the effector T cells/Tregs ratio in distant tumors, prolonging survival.¹⁶⁷ In patients with advanced HCC, ablation (RFA or cryoablation) combined with tremelimumab resulted in a 26% overall response rate with an intratumoral CD8⁺ T cell concentration.¹⁶⁸ Moreover, a cytosine-phosphate-guanine (CpG) TLR 9 agonist enhances RFA-induced CD4⁺ T cell-mediated responses by increasing the immunogenicity of CD11b⁺ F4/80 + MHC-II + macrophages and CD11c⁺ CD103⁺ DCs.¹⁶⁹ Another study attempted to vaccinate patients with heated tumor lysate-pulsed DCs and observed an improvement in immunogenic efficacy by RFA.¹⁷⁰

Combined therapy with ICIs and MWA showed suboptimal therapeutic effects compared with other thermal ablation modalities. Tremelimumab combined with MWA in patients with refractory biliary tract cancer showed limited activation of intratumoral CD8⁺ and CD4⁺ T cells and increased activation of circulating HLA-DR⁺ CD8⁺ T cells. TCR-β screening also demonstrated TCR repertoire expansion; however, this was not statistically significant.¹⁷¹

IRE enhances PD-1 efficacy in pancreatic cancer and melanoma mouse models, promoting proliferating CD8⁺ T cell infiltration by activating DCs and IRE-induced alleviation of immunosuppressive components in stroma.¹¹⁰ Burbach et al. reported that IRE combined with CTLA-4 stimulated antigen-specific CD8⁺ T cells in a mouse prostate cancer model in non-lymphoid tissues, the blood, and tumors. Increased tumor-specific tissue-resident memory T cells are prominent in tumor remission and antitumor immunity.¹⁷² White et al. revealed that IRE, when combined with a PD-1 inhibitor and TLR 7 agonist, improved treatment outcomes by increasing cDC1s, inhibiting primary pancreatic tumor growth, and inducing distant tumor regression.¹⁷³ IRE combined with a PD-1 inhibitor and TLR 3/9 agonists modulated the immune cell repertoire, decreased Tregs, immune-tolerant M2 macrophages, MDSCs, and increased CD8⁺ T cells, immunogenic M1 macrophages, CD169⁺ macrophages, and cDC1s. In addition, IRE reverses the immunosuppressive TME in primary lesions and distant untreated tumors.¹⁷⁴ IRE also enhanced DC-vaccine therapy efficacy by increasing intratumoral CD8⁺ T cells and GZMB⁺ cells in a pancreatic cancer mouse model.¹⁷⁵

M-HIFU combined with ICIs increased intratumoral CD8⁺ and CD4⁺ T cells while decreasing circulating IL-10 in an immunologically “cold” neuroblastoma murine model. In addition, an abscopal effect was observed in the combination group.¹²⁴ Osada et al. demonstrated that PD-L1 blockade combined with M-HIFU remodeled the TME by repolarizing TAM to the immunostimulatory M1 phenotype and enhanced T cells.¹²⁶

OV therapy has been shown to improve the effectiveness of CTLA-4 inhibitors in a melanoma mouse model.^176,177 A phase II trial comparing ipilimumab with T-VEC to ipilimumab alone in advanced melanoma discovered a significant improvement in overall response rates (39% vs. 18%).¹⁷⁸ Another phase 1b study investigated T-VEC combined with pembrolizumab in patients with advanced melanoma with a response rate of 62%. Responders to the combination treatment showed increased IFN-γ gene expression and CD8⁺ T cell infiltration.¹⁷⁹ The potential synergistic effects of OV and ablation have been reported in some studies. Combining IRE and therapy and Alphavirus M1 therapy transformed immune-silent pancreatic cancer into immune-inflamed tumors with T cell activation.¹⁸⁰ Another study revealed that G47Δ (HSV type 1 oncolytic) enhanced the RFA efficacy through a CD8⁺ T cell-mediated immune response that was further enhanced by ICIs.¹⁸¹ (Figure 3 and Table 1).

Figure 3.

Immunomodulatory effect by locoregional therapy combined with immunotherapy

(1) Locoregional therapy avoids total denaturation of tumor antigens and enhances the release of tumor-specific antigens. (2) The release of DAMPs recruits numerous immune cells. (3) Modulating the tumor stroma with increased MVD and vessel permeability, alleviating hypoxia, and softening the extracellular matrix (ECM) through decreased secretion of FAP-α, HABP1, and LOX by CAFs, hindering the construction of the fibrotic network and restoring the infiltration of drugs and immune cells. (4) Activation and maturation of DCs with increases in CD80/86, CD40, MHC-II, and CCR7 molecules. (5) Cytotoxic T cells increased with enhanced secretion of IFN-γ, GZMB, and perforin. (6) Robust expansion of intratumoral T cell clones. (7) Promotion of CD4⁺ T cells differentiating into Th1 and cytotoxic T cells. (8) Generation of protective tumor-specific Trms, enhancing local immunosurveillance. (9) Immunosuppressive populations were reduced, including Tregs and MDSCs, and anti-inflammatory M2 macrophages polarized to pro-inflammatory M1 macrophages, alleviating an immunosuppressive TME. (10) Inhibitory checkpoint molecules, such as PD-1 and LAG-3 on T cells and PD-L1 on tumor cells, were upregulated, indicating the necessity of combination with ICIs. CAFs, cancer-associated fibroblasts; FAPα+, fibroblast activation protein alpha-positive; HABP1, hyaluronic acid-binding protein 1; LOX, lysyl oxidase; MVD, microvessel density; IFN-γ, interferon-gamma; GZMB, granzyme B; Trm, tissue-resident memory T cells.

Table 1.

Immunomodulatory effect of locoregional therapy

Feature	Locoregional therapy	Immunomodulatory effect	Ref
Heat-based approach	RFA	(1) PD-1/PD-L1, LAG-3 expression ↑	Fei et al., Castelletti et al.11,161
		(2) tumor-specific T cells responses ↑	Mizukoshi et al.93
		(3) functional DCs and T cells in distant tumors ↑	Fei et al.11
		(4) Tregs, TAMs, and TANs in distant tumors ↓	Fei et al.11
		(5) cytokine secretion ↑	Biondetti et al.75
	MWA	(1) T cells, cytotoxic NK cells ↑	Yu et al., Dong et al.98,99
		(2) lower cytokines and HSP70 than RFA or cryoablation	Chesney et al., Ribas et al.178,179
		(3) similar pro-inflammatory cytokines with resection	Sun et al.180
	Cryoablation	(1) PD-1/PD-L1 expression ↑	Zeng et al.91
		(2) T cells, TAMs, and TANs ↑ CD8:FOXP3 ratio ↑	Biondetti et al.75
		(3) expansion of T cell clones ↑	Kato et al.88
		(4) cytokine secretion ↑	Biondetti et al.75
Integrity-preserved approaches	IRE	(1) T cells, DC cells ↑	Zhao et al., Dai et al.110,111
		(2) activation and maturation of DCs ↑	Zhao et al.110
		(3) PD-1+ T cells, Tregs ↓	Dai et al.111
		(4) permeability of vessels ↑	Zhao et al.110
		(5) soften extracellular matrix	Zhao et al.110
	M-HIFU	(1) activated cytotoxic T cells and NK cells↑	Lu et al.; Eranki et al.; Abe et al.122,124,126
		(2) M1 macrophage polarization ↑	Abe et al.126
		(3) DC maturation ↑	Abe et al.126
		(4) Tregs ↓	Eranki et al.124

The up arrows represent upregulation and down arrows represent downregulation.

New challenges and dilemmas

Optimal locoregional therapy for boosting immune responses

Locoregional therapies enable the immune system to exert antitumor effects by promoting innate and adaptive immune responses. However, the optimal locoregional therapy strategy for stimulating immune response remains unknown. Optimal locoregional strategies and combination therapies to augment antitumor responses have been investigated as part of ongoing research efforts. Shao et al. explored the immunomodulatory effects of different physical therapies in vitro. This study found that cryotherapy produced the most natural and non-denatured proteins. IRE released the most tumor antigens simultaneously, followed by cryotherapy and thermal therapy. Furthermore, it was significantly more effective than cryotherapy and thermal therapy in stimulating T cell activation. These results indicated that while all locoregional therapy approaches can destroy cells, there are significant differences in the “quantity” and “quality” of tumor antigen released, leading to differences in the ability to prime in the immune system.¹⁰⁸

White et al. reported that IRE induced more T cell and macrophage infiltration within the first 24 h than cryoablation in a murine pancreatic cancer model.¹⁰⁹ Compared with RFA, IRE preserved the permeability of vessels within the ablated area, promoted macrophage and neutrophil infiltration, and triggered a more substantial abscopal effect that persisted in CD3⁺ cell infiltration after liver ablation in a xenograft HCC tumor model.¹⁸² A clinical observational study in patients with HCC found a transient increase in macrophage migration inhibitory factor (MIF) after IRE compared with RFA. MIF attracts immune cells from the bloodstream to the ablated area, inhibits migration, maintains cell viability, and enhances inflammatory activity.¹⁸³ MWA appeared to elicit a minor immune response. Compared with RFA or cryoablation, MWA produced lower levels of inflammatory cytokines and HSP70 in preclinical models.^184,185 Another animal study found that pro-inflammatory cytokine levels after MWA were comparable to those in the surgical resection group.¹⁸⁶

Another preclinical study found that M-HIFU was superior to radiotherapy or thermal ablation at stimulating antitumor immune effects. M-HIFU caused an increase in intratumoral immune cell infiltration, such as DCs, macrophages, NK cells, and neutrophils, especially IFN-γ⁺CD8⁺ T cells. Furthermore, M-HIFU promoted immune responses at untreated tumor sites while suppressing lung metastases in melanoma and hepatocellular carcinoma murine models. One possible explanation reason for the superior immunity induction role of M-HIFU is that it differs from thermal ablation, which results in heat-based denaturation and necrosis, M-HIFU treatment preserves the integrity of subcellular components through cell membrane disruption. The tumor antigen peptides in the intact nonviable tumor homogenate preserved immunogenicity following M-HIFU treatment. In addition, the immunostimulatory effects of M-HIFU enhanced the efficacy of ICIs, which may play a role in the induction of sterile inflammation.^123,127 Disruption of the cell membrane by preserving the integrity of subcellular components, such as that induced by IRE or M-HIFU, has better immunogenic or pro-inflammatory effects on tumor antigen release than heat-based denaturation and necrosis. Physical locoregional therapy methods may have greater clinical potential because it is easier to expand to various types of solid tumors. In contrast, targeted vaccines generally act against specific tumor types. To date, no direct comparison of different combination strategies has been conducted. Given that the integrity of subcellular components boosts the immune response, locoregional therapy with better tumor antigen preservation may be the most effective combination therapy. However, only data from randomized studies will reveal the optimal combination strategy.

Timing of locoregional therapy

Following local treatment, the optimal timing of locoregional therapy and immunotherapy, either concomitant or immunotherapy, has not been established. Numerous clinical trials are ongoing to investigate locoregional therapy combined with immunotherapy; however, few have incorporated the sequence or timing of therapies.¹⁸⁷ Preclinical and clinical studies have shown that local treatment triggers the rapid release of DAMPs, a key immunostimulatory signal of ICD, which induces DC maturation and immigration, ultimately promoting ICI efficacy. It has been reported that ICD-inducing chemotherapy followed by ICI treatment altered the tumor into a “hot” tumor with abundant T cell infiltration (high T effector/Treg cell ratio), enhancing ICI therapeutic effects.¹⁸⁸ Moreover, the expansion of specific T cells and alleviation of immunosuppressive cells occurred within days and tapered off over time following locoregional therapy, creating a window for incorporating immunotherapy.¹¹² After oncolytic therapy, the expression of CTLA-4 and PD-1 was regulated time-dependent, and similar results were found in RFA treatment, indicating the importance of the schedule in ICI treatment after locoregional therapy.^11,167,189

However, Seremet et al. reported that two patients with melanoma who were unresponsive to ICIs showed complete disease regression and disappearance of non-injected lesions following T-VEC intratumoral therapy, indicating that prior ICI treatment enhances the effectiveness of locoregional therapy.¹⁹⁰ Furthermore, a preclinical study revealed that immunotherapy before thermal ablation resulted in a superior antitumor immune response with fewer macrophages and MDSC and increased IFN-γ producing CD8⁺T cells than concomitant or delayed immunotherapy. This phenomenon may be explained by mechanical changes in the TME and inflammation-mediated changes in the immune phenotype following thermal ablation, thus limiting its efficacy.¹⁹¹ Given that the immunomodulatory effects caused by locoregional therapy are short-lived, the timing sequence of combined immunotherapy requires further study to optimize treatment.

Control of immunomodulatory effect by locoregional therapy

How can we precisely control tumor locoregional therapy for its immunomodulatory effect? There is currently no expert consensus on performing locoregional therapies to achieve optimal efficacy; hence, further research is required. (1) Due to the transient and relatively weak antitumor response elicited by locoregional therapy, repeated and multiple sessions may produce a more potent effect by maintaining stable antigen release. Furthermore, tumor heterogeneity often hinders immunotherapy; thus, sustained antigen exposure from primary or distant lesions can theoretically enhance immune responses and even generate de novo responses. Fractionated radiotherapy is reported to be more effective than single-dose applications.¹⁹² (2) It is worth noting that selecting appropriate lesions, including the anatomic location, visibility, practicality, and safety, for locoregional therapy is also crucial and should be carefully considered. Lesion priority rules are also important. However, whether a specific tumor location (lung metastasis versus liver metastasis) triggers a better immune response is unknown. Evaluation of the status of the targeted lesion (progressive or stable) would be valuable for understanding the consequences. Generally, investigators select the most convenient lesions. (3) Locoregional therapy is recommended to achieve a complete radiological response. Incomplete local therapy reduces the tumor burden to some extent and leads to the release of tumor antigens; nevertheless, it has been reported that incomplete tumor destruction by RFA leads to high infiltration of MDSCs, accelerates tumor progression by inhibiting adaptive immunity, and hinders the efficacy of PD-1 immunotherapy. Furthermore, studies have reported that CCR2 antagonists attenuate tumor progression and reverse resistance to ICIs after incomplete RFA. Nevertheless, this treatment approach is insufficient to eliminate residual tumors and prevent distal metastasis.⁹⁶ (4) Technique parameters contributed to immunomodulatory results. For cryoablation, parameters applied during treatment, including duration, minimum temperature, and freeze/thaw cycles, can influence the extent of apoptosis and necrosis in the target lesion. Furthermore, the immunosuppressant is induced by the parameters determined in the intensity of immune stimulation and the balance between the immunostimulant and the large cryoablation volume.¹⁹³ In contrast, smaller cryoablation volumes serve as antitumor immunity stimulants and have been shown to prolong overall survival.¹⁹⁴ (5) In addition, single-lesion ablation is reported to be superior to multi-lesion ablation in reducing the total number of metastases.¹⁹⁴

Assessment of immune effect by locoregional therapy

Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1) and immunotherapeutic trial guidelines (iRECIST), designed for systemic treatment, are unsuitable for locoregional therapy.¹⁹⁵ The modified RECIST (mRECIST) has been widely used to treat intrahepatic lesions with locoregional therapy.¹⁹⁶ Nevertheless, mRECIST is ineligible for atypical immune responses, such as the increased lesion diameter, which may be interpreted as “progression” caused by locoregional immune activity.¹⁹⁷ A guideline for intratumoral RESIST (itRECIST) was recently developed to capture the response of both treated and untreated lesions to predict the benefit of locoregional therapy better.¹⁹⁵

Considering that the immune effect occurs before the ultimate treatment response, the surveillance of dynamic immune changes in lesions treated with locoregional therapy will provide valuable information about the antitumor immune response. Numerous immune effect assessments have been performed to capture the molecular activity of locoregional therapy or the underlying immune response. In contrast to clinical treatment assessment, immune effect assessment focuses more on the immune landscape. The TME undergoes highly dynamic alterations during tumor progression and locoregional therapy. In addition, immune classification of tumors based on Immunescore can guide treatment selection.¹⁶² The biopsy is often used to monitor treatment response; however, it provides limited information. It is not representative of the entire tumor landscape because of substantial immune heterogeneity within each metastasis. Liquid biopsy is a less invasive method that provides a clue for assessing response to therapy; however, peripheral blood does not fully represent alterations in the TME. Therefore, several imaging techniques have been developed for this purpose. Immuno-positron emission tomography (Immuno-PET) is a molecular imaging approach that combines the excellent affinity and specificity of mAbs for cell surface markers with the ultra-high sensitivity of PET.¹⁹⁸ Radiolabeled antibodies targeting lineage-defining molecules such as CD3, CD4, and CD8, and activation markers such as HLA-DR and ICOS can be administered longitudinally and serially at various time points to assess T cell responses and evaluate the efficacy of immunotherapy.^{199,200,201,202} Several preclinical studies have applied immuno-PET to monitor different immunotherapies, including tumor vaccination and ICIs.²⁰²

Profiling the hallmark of TME associated with tumor progression and recurrence by drawing the gene expression landscape of immune cells was first described by Bindea et al.²⁰³ Since then, multiple tools such as CIBERSORT, TIMER, and ImmuneCellAI have been used to estimate the abundance of intratumoral immune cells from gene expression data in bulk RNA-seq and microarray data from deconvolution methods.¹⁶² However, there are limitations, as assigning transcripts to specific cell types intrinsically is inherently unreasonable.¹⁶² In addition, transcriptional information could not reflect rigorous post-translational control of proteins such as PD-1.^3,204 However, with the development of novel high-throughput single-cell multi-omics platforms, such as CyTOF and scRNA-seq, it is now possible to examine cellular phenotypes, functional characteristics, and dynamic changes in the TME to create networks of cell-cell interactions that straighten the landscape of cancer development and metastasis. Understanding the spatial interactions between different cells further advances our understanding of the complex TME. For instance, patients with Merkel cell carcinoma with PD-L1⁺ tumors show high heterogeneity of treatment effects on immunotherapy. Quantification analysis showed that responders had higher infiltration of PD-1⁺ cells adjacent to PD-L1⁺ cells within 20 μm than non-responders.²⁰⁵ These techniques will help decipher the complex TME and interaction effects between combination therapeutic strategies and uncover antitumor immune mechanisms.

Conclusion and prospects

Locoregional therapy is widely used in the treatment of various solid tumors and has the advantages of minimal invasiveness, high reproducibility, and high tumor destruction efficiency. Current research recognizes the superiority of locoregional therapy in remodeling the tumor immune microenvironment, including creating endogenous immune vaccines, stimulating antitumor immunity, attracting tumoral infiltration of cytotoxic T cells, and reversing the immunosuppressive TME. In addition, numerous studies have demonstrated that combining locoregional therapy with various immunotherapy regimens has a positive therapeutic impact and promising applications.

Precision and personalized cancer therapy is being increasingly advocated. The lack of a thorough understanding of the interaction between locoregional therapy and immunotherapy impedes this approach. The individual approach and understanding of TME profiling in each patient will form the basis for combined therapy. It is essential to identify the key characteristics of the TME to provide adequate information for subsequent treatment. Given that T cells play a crucial role in the antitumor immune response, the pre-existing T cell scenery and immune TME should be meticulously assessed to distinguish specific cases. It is reasonable to assert that more strategies are required for “cold” tumors. Based on current research, immunotherapy treatment will maximize therapeutic efficacy. With the approval of more agents designed to modulate different T targets in the future and the determination of optimal locoregional therapy, these optimal combination therapies would favorably target the features of individual immune scenery. Moreover, the TME component will change during treatment, and understanding and unraveling the dynamic interaction with TME after locoregional therapy will provide important insights into exploring tumor-targeted combination strategies to improve the efficacy of immunotherapy.