Nanotechnology-Assisted Immunogenic Cell Death for Effective Cancer Immunotherapy

Tumor vaccines have been used to treat cancer. How to efficiently induce tumor-associated antigens (TAAs) secretion with host immune system activation is a key issue in achieving high antitumor immunity. Immunogenic cell death (ICD) is a process in which tumor cells upon an external stimulus change from non-immunogenic to immunogenic, leading to enhanced antitumor immune responses. The immune properties of ICD are damage-associated molecular patterns and TAA secretion, which can further promote dendritic cell maturation and antigen presentation to T cells for adaptive immune response provocation. In this review, we mainly summarize the latest studies focusing on nanotechnology-mediated ICD for effective cancer immunotherapy as well as point out the challenges.


Introduction
Cancer immunotherapy is emerging as a promising treatment strategy for many cancer types [1][2][3].It mainly depends on activating host immune response to kill tumor cells, which is in contrast to traditional therapeutic approaches.Immunotherapy approaches include immunogenic cell death (ICD) inducement, cancer vaccination, and adoptive T cell therapy [4][5][6].ICD is used to elicit host immune responses as a result of dying tumor cells becoming immunogenic due to external death-promoting stimuli [7].During this process, damage-associated molecular patterns (DAMPs) and tumor-associated antigens (TAAs) are released and subsequently captured by macrophages and dendritic cells (DCs) [8,9].After processing, the antigen is presented to adaptive immune cells to activate host-specific immune response.DAMPs include adenosine triphosphate (ATP) as the "find me" signal, calreticulin (CRT) as the "eat me" signal, and high mobility group box 1 (HMGB1) for DC activation.In addition, cytokines (e.g., tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)) are released during tumor ICD accompanied by a strong inflammatory response [10].Nowadays, a series of tumor therapies, such as radiotherapy (RT), photodynamic therapy (PDT), hyperthermia therapy (HT), chemodynamic therapy (CDT), and chemotherapy, can be used in ICD-mediated cancer immunotherapy.
Nanomedicines have long circulation time, good biocompatibility, and low side effects in vivo compared with free drugs, thus generally being exploited in antitumor treatment [11,12].In addition, nanoparticles can also accumulate into tumors through the enhanced permeability and retention effect [13][14][15].Surface modification of nanoparticles is able to increase their cellular internalization, which is a prerequisite for triggering ICD [16,17].Moreover, drug loading content can also be improved via electrostatic interaction between nanoparticles and drugs with controlled release when the responsive groups encounter an internal or external stimulus.Thus, nanomedicines have huge advantages in drug delivery with robust antitumor efficacy during cancer immunotherapy [18][19][20].The general nanocarriers include polymeric/inorganic nanoparticles, biomimetic nanosystems, nanogels, covalent organic frameworks, nanocomplexes, etc. [21][22][23][24][25]. Herein, we mainly discuss the nanotechnology-assisted therapeutic strategies for tumor ICD based on RT, PDT, HT, CDT, and chemotherapy (Figure 1).

Radiotherapy
Radiotherapy is a powerful therapeutic modality of cancer which can kill tumor cells through direct DNA damage and indirect reactive oxygen species (ROS) damage without tissue penetration limitation [6,[26][27][28].Recently, it has been reported that RT can induce ICD with TAA and DAMP release, thereby triggering DC maturation and eliciting antitumor immune responses [29][30][31][32][33][34][35][36][37][38].It is widely reported that the coordinated high-order metal elements in nanocrystals can induce tumor ICD via radiotherapy.For example, Huang et al. reported bi-functional coordination nanorod (ZGd-NR) self-assembly from zoledronic acid and Gd 3+ (gadolinium).ZGd-NRs efficiently deposited X-rays and generated hydroxyl radicals, thereby inducing an effective ICD [28].In addition, ZGd-NRs depleted tumor-associated macrophages (TAMs) and reprogrammed the immunosuppressive microenvironment.In another report, Deng et al. synthesized manganese dioxide (MnO 2 ) nanoparticles loaded with programmed death-ligand 1 antibody (aPD-L1) (αPDL1@MnO 2 ) by biomineralization [33].After αPDL1@MnO 2 accumulated in the tumor site, it induced ICD under X-ray irradiation.Simultaneously, MnO 2 catalyzed hydrogen peroxide (H 2 O 2 ) to produce oxygen with tumor hypoxia alleviation and radiotherapy efficacy enhancement, and the released Mn 2+ activated the cyclic GMP-AMP (cGAS)-stimulator of interferon Vaccines 2023, 11, 1440 3 of 22 genes (STING) pathway and triggered tumor-specific immunity.The released αPD-L1 blocked the immune checkpoint, resulting in the reinvigoration of exhausted CD8 + T cells.αPDL1@MnO 2 inhibited tumor growth as well as metastasis by reprogramming the immunosuppressive microenvironment.Choi et al. synthesized radiosensitive snowflake-like Au nanocarriers (S-AuNC) with aPD-L1 encapsulation [39].Under X-ray irradiation, S-AuNC nanoparticles produced ROS, promoted endoplasmic reticulum stress, and induced ICD.The released aPD-L1 revealed a synergistic antitumor efficacy and minimized systemic immune-related adverse effects.Li et al. developed a multifunctional MOF (metal-organic framework) structure (named TZM) using Ta (tantalum) and Zr (zirconium) as metal nodes and tetrakis (4-carboxyphenyl)porphyrin (TCPP) as a photosensitizer ligand [35].TZM preferentially accumulated at tumor sites, absorbed X-ray energy, and effectively generated hydroxyl radicals.The Ta-Zr co-doping design promoted TZM to transfer energy to the photosensitizer TCPP to sensitize the production of singlet oxygen ( 1 O 2 ) for radiodynamic therapy (RDT).Further investigation indicated that TZM-mediated radio-/radiodynamic therapy with tumor ICD promoted DC maturation and up-regulated PD-L1 expression through the cGAS-STING pathway with antitumor immune response amplification.In addition, combination with chemoimmunotherapy is a general strategy during radiotherapy.Lu et al. constructed a mesoporous upconversion nanophosphor (UCNP) based on bismuth (Bi) with doxorubicin (DOX) encapsulation for both radiotherapy-and chemotherapy-mediated ICD as well as TAM polarization [40].In vivo studies indicated that the nanoplatform facilitated abscopal radioimmune efficacy and notably suppressed lung tumor metastasis.In another report, Dong et al. prepared mesoporous organosilica nanoparticles (MONs) decorated with cancer cell membranes which contained X-ray and ROS dual-responsive diselenide bonds and were loaded with DOX for a synergistic ICD inducement [41].Remarkably, the DOX-loaded MONs revealed impressive tumor growth inhibition and metastasis with negligible side effects.
To resolve the hypoxic-environment-mediated radiation resistance and high-radiationdose-mediated safety issues, Chao Ji et al. designed polyvinylpyrrolidone (PVP)-coated tantalum (Ta) nanoparticles (Ta@PVP NPs) to improve blood flow via exploiting photothermal therapy (PTT) and enhance radiotherapy sensitivity via increasing intratumoral oxygen levels (Figure 2a) [42].Ta@PVP NPs displayed a high antitumor efficacy in both in situ and metastatic breast tumors through radiosensitization.The real-time computed tomography (CT) imaging was monitored for the theranostic combination.As shown in Figure 2b, Ta@PVP NPs revealed a stronger CT signal than the clinical contrast agent Ioversol.When Ta@PVP NPs underwent X-Ray and NIR, the highest CTL percentage was detected, showing that RT and PTT enhanced antitumor immune responses.Liu et al. reported a poly(lactic-co-glycolic) acid (PLGA) nanoplatform with hydrophilic catalase (Cat) encapsulation in the core and the hydrophobic immune adjuvant imiquimod (R837) in the shell [43].Owing to the ability of Cat to decompose hydrogen peroxide into oxygen, the PLGA nanoplatform alleviated hypoxia for potent radiotherapy-mediated ICD with tumor-associated antigen release.In the presence of adjuvant R837, the nanoplatform inhibited both primary as well as re-challenged tumor growth after combination with anticytotoxic T-lymphocyte associated protein 4 (CTLA4).Sun et al. used quantum dots with a near-infrared IIb window to deliver catalase, which also reversed hypoxic environments for enhanced radiotherapy and robust antitumor immune response [44].However, the safety issues of the quantum dot nanoplatform, which may restrict its further application, should not be ignored [45].
The low oxygen content in the tumor microenvironment is adverse to PDT, resulting in reduced therapeutic effects.To address this issue, Huang et al. designed a nanoplatform CaO 2 @CuS-MnO 2 @HA (termed CCMH; CaO 2 : calcium peroxide; CuS: copper sulphide; MnO 2 : manganese dioxide; HA: hyaluronic acid) containing multiple ICD inducers (Figure 2d) [51].In the acidic lysosome, CaO 2 decomposed into Ca 2+ and oxygen, resulting in calcium overload with mitochondrial damage and ICD inducement.CaO 2 nanoparticles reacted with water to produce large amounts of oxygen that enhanced PDT efficacy.In addition, CuS nanoparticles produced 1 O 2 under 1064 nm irradiation, which also induced ICD.The authors claimed that the nanoplatform increased the expression of endoplasmic reticulum stress proteins BiP (Binding protein for immunoglobulins)/GRp78 (Glucose-regulated protein of 78 kDa), CHOP (C/EBP homologous protein)/DDIT3 (DNA Damage Inducible Transcript 3), and caspase-12 and promoted DAMP production.CaO 2 @CuS-MnO 2 @HA also caused TAM polarization via escaped oxidation-damaged mitochondrial DNA (ox-mtDNA) to activate the innate immune system.Although PDT triggers ROS generation, it also causes immunosuppressive cytokine release.In order to reverse the immunosuppressive effects, more and more researchers combine PDT with tumor microenvironment modulation for a better antitumor efficacy [56].For example, Chen et al. synthesized a photodynamic immunomodulator, ICy-NLG, which was formed by covalently binding the near-infrared photosensitizer ICy-NH 2 to the immune checkpoint inhibitor NLG919 via the disulfide bond [54].After the disulfide bond cleavage by the intracellular GSH, the activity of the photosensitizer ICy was restored with tumor ICD, and the released NLG919 blocked indoleamine 2,3-dioxygenase (IDO-1) function with regulatory T cell inhibition.The synergistic use of photosensitizers and immune checkpoint inhibitors induced potent immune responses with a promising abscopal effect.Mai et al. constructed a carrier-free nanoparticle C9SN self-assembly from the photosensitizer Ce6 and the glutaminase inhibitor compound 968 (C968) [57].Upon laser irradiation, C9SN produced a large number of ROS resulting in tumor ICD.Moreover, compound 968 inhibited glutamine metabolism, thereby inhibiting GSH production and disrupting REDOX homeostasis.Photodynamic therapy can also combine with chemoimmunotherapy for synergistic antitumor immunity.For example, Kim et al. fabricated a cathepsin B-responsive prodrug nanoplatform when DOX and the photosensitizer verteporfin (VPF) were conjugated via a cleavable linker Phe-Arg-ArgGly (FRRG) to form VPF-FRRG-DOX [58].The authors found that only in the cancer cells with high expression of cathepsin-B, VPF and DOX were released for synergistic ICD with photo-/chemotherapy under laser irradiation.The prodrug platform displayed a strong antitumor activity as well as a durable immune memory response.
Tumor-specific immunity activation requires antigen-presenting cell (APC) maturation, tumor antigen presentation, and co-stimulatory signal activation.However, mature APCs have a short survival time in vivo, and the immunosuppressive microenvironment is another fact not to be ignored.To solve the above problems, Sun et al. designed a photodynamic biomimetic nanosystem when a super artificial dendritic cell (saDC) was constructed with CD86 and anti-LAG3 expression on a cell membrane to target 4T1 cells.In short, the polymer nanoskeleton loaded with an aggregation-induced emission (AIE) active photosensitizer, which was named Fs, was coated by an saDC membrane to form the biomimetic nanosystem saDC@Fs-NP (Figure 3a) [59].In vivo results showed that, under laser irradiation, saDC@Fs-NPs reduced the proportion of bone-marrow-derived suppressor cells (MDSCs) with the immunosuppressive TME modulation, promoted the transformation of a "cold tumor" into a "hot one", and thus enhanced the antitumor immune response.This nanosystem facilitated DC maturation, enhanced CTL infiltration, and reversed T cell exhaustion, which provides a new idea for the combination of PDT and immunotherapy.Chen et al. constructed size-transformable artificial antigen presenting cells (aAPCs) to combine photodynamic immunotherapy for an amplified antitumor immune response [5].The aAPCs decorated with anti-CD28 and peptide major histocompatibility complex (MHC) displayed a size increment and prolonged retention time in tumor tissue when they encountered the preactivated T cells by PDT.This study opens a new approach to treat malignant tumors via aAPCs.Tang et al. utilized dendritic cell (DC) membrane loading with photosensitizer aggregation-induced emission to fabricate biomimetic nanosystems (DC@AIEdots) [60].They found that DC@AIEdots, on the one hand, accumulated in the tumor tissue for PDT-mediated ICD, and also hitchhiked the endogenous T cell triggering for antitumor immunity.Tumor-specific immunity activation requires antigen-presenting cell (APC) maturation, tumor antigen presentation, and co-stimulatory signal activation.However, mature APCs have a short survival time in vivo, and the immunosuppressive microenvironment is another fact not to be ignored.To solve the above problems, Sun et al. designed a photodynamic biomimetic nanosystem when a super artificial dendritic cell (saDC) was constructed with CD86 and anti-LAG3 expression on a cell membrane to target 4T1 cells.In short, the polymer nanoskeleton loaded with an aggregation-induced emission (AIE) active photosensitizer, which was named Fs, was coated by an saDC membrane to form the biomimetic nanosystem saDC@Fs-NP (Figure 3a) [59].In vivo results showed that, under laser irradiation, saDC@Fs-NPs reduced the proportion of bone-marrow-derived suppressor cells (MDSCs) with the immunosuppressive TME modulation, promoted the transformation of a "cold tumor" into a "hot one", and thus enhanced the antitumor immune response.This nanosystem facilitated DC maturation, enhanced CTL infiltration, and reversed T cell exhaustion, which provides a new idea for the combination of PDT and immunotherapy.Chen et al. constructed size-transformable artificial antigen presenting cells (aAPCs) to combine photodynamic immunotherapy for an amplified antitumor immune

Hyperthermia Therapy
Hyperthermic therapy (HT) refers to heating tissues to 39-45 • C, which induces endoplasmic reticulum stress through unfolded protein responses, upregulates heat shock proteins on a cell membrane surface, and releases TAAs, causing tumor ICD [71,72].Photothermal therapy (PTT) is one type of HT, which means that a laser irradiates a tumor area, effectively converting light energy into heat energy through photothermal conversion agents [73].Jingchao Li et al. used NIR-II semiconductor polymer nanoparticles (SPNs) and the toll-like receptor (TLR) agonist resiquimod (R848) to form a core, the surface of which was coated by a heat-responsive lipid shell to construct a multifunctional nanoplatform, SPN II R (Figure 3b) [61].Under NIR II irradiation, SPN II R elicited PTT, which increased the local temperature of the tumor and induced ICD with DAMP and TAA release.In subcuta-neous 4T1 tumor-bearing BALB/c mice, SPN II R-mediated photothermal immunotherapy not only enhanced the inhibitory effect on primary and distal tumor growth, but also significantly suppressed lung metastasis.In addition, the study not only achieved primary tumor ablation and ICD induction, but also achieved an in situ R848 release and remolded the tumor microenvironment.
Local thermal therapy (e.g., PTT and magnetic thermal therapy) has superior advantages, including negligible invasiveness and low side effects [71,[74][75][76][77]. Multiple nanoformulations (e.g., inorganic nanoparticles, semiconductor polymeric nanoplatforms, and organic and inorganic hybrid nanomaterials) can be applied in thermal therapy with negligible toxicological issues which mainly achieve high tumor accumulation via the enhanced permeability and retention effect [61,[78][79][80].Li et al. developed a thermally responsive copolymer micelle PP IR780 -ZMS-containing manganese zinc sulfide nanoparticle (ZMS) and a near-infrared dye IR780 [79].Under NIR light irradiation, IR780 underwent photothermal conversion, thereby allowing PP IR780 -ZMSs to release ZMSs for metal immunotherapy.The released ZMSs produced a large amount of Mn 2+ with hydroxyl radical generation and ICD inducement.Simultaneously, Mn 2+ activated the cGAS-STING pathway to produce type I interferon and enhance tumor ICD.The results showed that PP IR780 -ZMS elicited potent antitumor immune responses with lung tumor metastasis inhibition.Linlin Zhang et al. prepared a magnetic nanoplatform where the water-soluble 2, 2 -azo compound (2-aminopropane) dihydrochloric acid (AAPH) was encapsulated into magnetic iron oxide nanocrystals (IONCs) to form IONC-AAPH for photothermal and magnetic thermal therapy-mediated ICD [81].In the presence of an alternating magnetic field, the heat induced by the azo compound facilitated carbon-centric radical generation (Figure 4a).Both magnetic heating and free radicals caused MC-38 tumor cell death, leading to DAMP release, DC maturation, CTL infiltration, and primary and distal tumor growth inhibition.
In addition, microwave ablation (MWA), which exploits polar molecules to cause strong oscillations under microwave irradiation, followed by heat generation with local thermal ablation of tumors, can also be used in tumor treatment.Yujie Zhu et al. designed a Ca 2+ -surplus alginate (ALG) saline gel which could increase heating efficiency and restrict the heating region under microwave irradiation [82].As a highly effective microwave sensitizer, Ca 2+ enhanced microwave heating in a concentration-dependent manner, leading to local thermal ablation and a large production of TAAs and DAMPs (Figure 4b).In addition, the synergistic effect of an extracellular high concentration of Ca 2+ and hyperthermia induced ICD with intracellular Ca 2+ homeostasis disruption.The results showed that ALG-Ca-assisted MWA treatment could effectively activate DCs in lymph nodes.Simultaneously, mice with ALG-Ca treatment revealed a high ratio of CD3 + CD8 + to regulatory T cells (Tregs), indicating that the host antitumor immunity was activated.Du et al. built a thermal-responsive micellar nanoplatform that separately delivered adenosine receptor antagonist and doxorubicin (DOX) for both hyperthermia/chemotherapy-mediated ICD and immunosuppressive tumor microenvironment modulation under local microwave irradiation [83].The E-selectin modification on the nanoplatform surface facilitated adherence to leukocytes for a higher tumor accumulation.
By changing the 3D structures of constituent units in covalent organic frameworks (COFs), ROS are also able to be generated.Liang Zhang et al. used a COF platform to construct a three-dimensional structure of triphenylamine for ICD-mediated antitumor immunity activation [96].The covalent linkage of COFs facilitated the generation and diffusion of ROS with endoplasmic reticulum stress for further tumor ICD with enhanced CD3 + CD8 + T cell infiltration.After combination with anti-CD47, both the primary and distal 4T1 tumor growth were notably inhibited.
In addition, glucose depletion in the tumor microenvironment (TME) is also an effective tumor treatment approach after combination with CDT.Zhaoming Fu et al. fabricated a folate-modified metal-phenolic network nanosystem (F-MGC) with glucose oxidase (GOx) and chlorogenic acid (CHA) co-encapsulation [84,88].Due to the catalytic activity of GOx and Fe 3+ , F-MGCs effectively consumed glucose in the TME and generated cytotoxic hydroxyl radicals for chemokinetic therapy accompanied by ICD.In vivo studies showed that CHA facilitated M2 phenotype TAM polarization into M1 type, remodeled the immunosuppressive TME, and activated the immune system.This nanoplatform exhibited an excellent biocompatibility and effectively inhibited tumor growth and metastasis.Li et al. developed ROS-responsive nanocomplexes that generated ROS via Fe 3+ -mediated Fenton reaction, delivering αPD-L1 and GOx (Figure 6a) [84].At the tumor site, GOx that was released from the nanocomposites generated H 2 O 2 through the oxidation of glucose, and Fe 3+ induced •OH generation (Figure 6b,c) for CDT-mediated ICD (Figure 6d).The released αPD-L1 blocked PD-1/PDL1 interaction.Moreover, the released GOx increased the •OH production efficiency with the "cold" tumor microenvironment transformation into a "hot" one.Nanocomposites induced favorable systemic immune responses and effectively inhibited both primary and distant tumor growth in a bilateral 4T1 tumor model in mice.By changing the 3D structures of constituent units in covalent organic frameworks (COFs), ROS are also able to be generated.Liang Zhang et al. used a COF platform to construct a three-dimensional structure of triphenylamine for ICD-mediated antitumor immunity activation [96].The covalent linkage of COFs facilitated the generation and diffusion of ROS with endoplasmic reticulum stress for further tumor ICD with enhanced CD3 + CD8 + T cell infiltration.After combination with anti-CD47, both the primary and distal 4T1 tumor growth were notably inhibited.
In addition, glucose depletion in the tumor microenvironment (TME) is also an effective tumor treatment approach after combination with CDT.Zhaoming Fu et al. fabricated a folate-modified metal-phenolic network nanosystem (F-MGC) with glucose oxidase (GOx) and chlorogenic acid (CHA) co-encapsulation [84,88].Due to the catalytic activity GOx that was released from the nanocomposites generated H2O2 through the oxidation of glucose, and Fe 3+ induced •OH generation (Figure 6b,c) for CDT-mediated ICD (Figure 6d).The released αPD-L1 blocked PD-1/PDL1 interaction.Moreover, the released GOx increased the •OH production efficiency with the "cold" tumor microenvironment transformation into a "hot" one.Nanocomposites induced favorable systemic immune responses and effectively inhibited both primary and distant tumor growth in a bilateral 4T1 tumor model in mice.

Chemotherapy
Chemotherapeutic drugs (e.g., adriamycin, cisplatin, oxaliplatin, and doxorubicin) can cause tumor ICD when injected at a low dose (Table 2) [97][98][99].However, in vivo low tumor accumulation as well as side effects are obstacles to the free drugs, which can be better resolved by nanotechnology [100].

Chemotherapy
Chemotherapeutic drugs (e.g., adriamycin, cisplatin, oxaliplatin, and doxorubicin) can cause tumor ICD when injected at a low dose (Table 2) [97][98][99].However, in vivo low tumor accumulation as well as side effects are obstacles to the free drugs, which can be better resolved by nanotechnology [100].
Platinum (Pt) complexes are the most commonly used chemotherapeutic drugs approved in cancer treatment, while their clinical application is limited due to poor pharmacokinetics, severe side effects, and drug resistance.Jiajia Xiang et al. constructed tannic acid-Pt nanocomplex (PTI) nanoparticles, which produced tumor immunogenicity by inducing ICD, simultaneously improving antitumor efficacy after combination with immune checkpoint blockade (ICB) therapy [101].Nanomedicine PTI was prepared in two steps.1, 2-diaminocyclohexane-platinum (II) (DACHPt) was first complexed with natural polyphenols to form nanoparticles, which were then linked with iron (III) by metalcatecholamine coordination bonds.PTI significantly promoted the expression of CRT on cell membranes, which revealed significantly higher exposure than that in other groups, proving its effective induction of ICD in vivo.The infiltration of CTL into the tumor was significantly increased after PTI treatment, which was 1.8-fold higher than that in the oxaliplatin group, and the secretion of IFN-γ and TNF-α was promoted.PTI significantly inhibited tumor growth, and the tumor inhibition rate was significantly higher than that in the control group (83%).In addition, PTI plus αPD-L1 significantly prolonged the survival rate of the mice, which was 100% at 44 days after inoculation, while that for PTI-alone treatment was only 37.5%.Doxorubicin (DOX) is a classic ICD inducer, while the level of ICD induced by DOX alone is not enough to elicit a strong immune response.Pengkai Wu et al. developed a novel carrier-free nanoparticle that was able to deliver DOX, cytolytic peptide melittin (MPI), and an anti-TOX small-gap delivery RNA (siTOX) (Figure 7a) [102].TOX is the abbreviation of thymocyte selection-associated high-mobility group box protein.pH-sensitive fluorinated prodrugs, FDOX (FD) and FMPI (FM), were synthesized to prepare FD/FM NPs through hydrophobic interactions and fluorine interactions.FD/FM@siTOX NPs were prepared by electrostatic interaction between FD/FM NPs and siTOX NPs.CD8 + T cell infiltration increased due to the combination of DOX and MPI with ICD enhancement.Mechanically, siTOX reduced TOX expression, which acted as a "defense" signal to reinvigorate CD8 + T cells.The author claimed that the constructed multifunctional nanosystem transformed a "cold" tumor into a "hot" one with effective antitumor immune responses during hepatoma treatment.Triple-negative breast cancer (TNBC) is prone to metastasis, possessing a cold immune microenvironment and poor prognosis [103,104].Chemotherapy drugs are poorly targeted and have strong systemic toxicity.α5β1 integrin is over-expressed on breast cancer cells and plays an important role in tumor progression and metastasis.Xinyun Qiu et al. improved the targetability of nanocrystals by using a clinical Ac-PhScNK-NH 2 (ATN) peptide to target α5β1 integrin [98].α5β1 integrin-targeting micellar paclitaxel (ATN-MPTX) was constructed via using a reduction-sensitive biodegradable micelle as a carrier.ATN-MPTX induced DC maturation by PTX-mediated ICD.After combination with a STING agonist cyclic dinucleotide (CDN), ATN-MPTX significantly suppressed tumor growth and lung metastasis.Thus, compared with traditional chemotherapy, nanotechnology-assisted chemotherapeutics can achieve enhanced antitumor efficacy and decreased side effects via eliciting tumor ICD.They can also be combined with other approaches, including ICB and gene silencing, to achieve a better antitumor activity.
The STING pathway is an important component of innate immunity and triggers productions of multiple immune stimulatory molecules, which can drive DC maturation, TAM polarization, and T cell priming, leading to immune-mediated tumor elimination.In addition, tumor ICD can also be induced by STING [114][115][116][117][118]. Lingxiao Zhang et al. constructed a Zn 2+ layered zinc dihydroxide (Zn-LDH) nanoparticle, which consumed H + with a large amount of Zn 2+ release, accompanied by activation of the cGAS-STING pathway with tumor ICD [105].Zn-LDH effectively destroyed lysosomes and blocked autophagy.In vivo results showed that Zn-LDH notably inhibited postoperative tumor growth and lung metastasis (Figure 7b).
In addition to the STING pathway, recent studies have found that pyroptosis can also induce ICD.Pyroptosis is an inflammatory form of programmed cell death characterized by cell swelling, giant bubble production, plasma membrane poring, chromatin fragmentation, and cytoplasmic proinflammatory cytokine extravasation.This process can enhance tumor ICD with TAA and DAMP release and promote tumor immunotherapy efficacy [63,119,120].Lei Li et al. constructed carrier-free nanoplatform A-C/NPs with cytarabine (Ara-C) and chlorin e6 (Ce6) encapsulation [63].A-C/NPs triggered GSDME-mediated pyroptosis in a controlled manner through ROS accumulation.The study showed that both Ce6 and A-C/NPs were able to trigger pyroptosis under near-infrared laser irradiation, and A-C/NPs had more swollen cells and more interleukin-1beta (IL-1β), IL-18, and NOD-like receptor family pyrin domain containing 3 (NLRP3) release than Ce6.A-C/NPs under laser irradiation effectively activated the systemic immune response in vivo.Seven days after surgical removal of the primary tumor, mice were subcutaneously inoculated with 4T1-Luc on the contralateral side, and A-C/NPs were found to effectively amplify an antigenspecific antitumor immune response and induce a long-lasting immune response.In another example, Li et al. developed an aggregation-induced emission photosensitive dimer that selectively targeted tumors, inducing ICD due to photodynamic and photothermal therapy [121].Simultaneously, the ROS generation during PDT led to GSDME-mediated pyroptosis for an enhanced ICD with antitumor immunoefficacy.
Ferroptosis is a form of regulated cell death characterized by iron-dependent accumulation of lipid peroxides to lethal levels [122].Ferroptosis has been found to effectively inhibit tumor growth accompanied by ICD [123][124][125][126]. Yaqian Du et al. reported a potent ROS production through a multifunctional nanoplatform, simultaneously leading to a depletion of glutathione peroxidase 4 (GPX4) and glutathione (GSH), which in total enhanced the occurrence of tumor ferroptosis and triggered tumor ICD [127].The nanoplatform consisted of copper and iron silicate mesoporous hollow nanospheres, gold nanoparticles, and immune adjuvant R848.Fe 3+ and Cu 2+ could react with GSH to form glutathione disulfide (GSSG), leading to GSH depletion and GPX4 expression decrement.In addition, the formed Fe 2+ could generate •OH via Fenton reaction, thus causing ferroptosis.It mainly activated host immune responses by causing ICD, increasing DC maturation and T-cell infiltration.The nanoplatform demonstrated a satisfactory efficacy in eliminating both primary and metastatic tumors.Yang et al. constructed a biomimetic magnetic nanoplatform with a glutamate-cystine antiporter system X c − inhibitor sulfasalazine (SAS) encapsulation in the mesoporous Fe 3 O 4 nanoparticle and platelet membrane decoration on the surface [128].They found that the nanoplatform resulted in ferroptosis due to Fe 3 O 4 -mediated LPO accumulation and SAS-mediated system X c − inhibition, which also generated immunogenicity and sensitized immune checkpoint blockade therapy.
Autophagy means that eukaryotic cells remove dysfunctional or unnecessary components to keep cellular homeostasis and manage metabolism [113,129,130].It has a close relationship with tumor ICD when both autophagy inhibition and over-activation will lead to ICD.For example, Li et al. reported semiconducting polymer nanocomplexes with the autophagy inhibitor chloroquine (CQ) and IDO inhibitor NLG919 co-encapsulation [131].CQ suppressed the degradation of intracellular damaged proteins or organelle resulting in PDT-mediated ICD enhancement, which then combined with NLG919-mediated immunometabolism suppression achieving a potent antitumor immunity.In another report, Wu et al. developed a nanomicelle dissolving microneedle (DMN) patch that could deliver the photosensitizer IR780 for PDT-mediated ICD and deliver CQ for autophagy inhibitionmediated damage-associated molecular pattern release [132].The author also found that CQ facilitated TAM autophagy inhibition with M1 phenotype polarization for TME modulation.As for "over-activated" autophagy-mediated ICD, He et al. prepared a smart nanomicelle with oxaliplatin delivery resulting in ICD simultaneously accompanied by autophagy that cleaved the autophagy-responsive bond, leading to STF-62247 release [133].As the autophagy inducer, STF-62247 caused autophagic death of tumor cells and facilitated tumor-associated antigen processing, thereby eliciting antitumor immune response.
Remarkably, in our previous report, we found that nanocarriers with tertiary amine and mercaptan structure also induced ICD selectively in B16F10 cells [127].This finding opens a new avenue to exploiting nanomaterials for ICD-mediated cancer immunotherapy.

Conclusions
Immunotherapy has promising potential as a treatment strategy for many cancers.It mainly depends on activating the host immune system to attack pathological tissue.This review mainly summarized different nanotechnologies (e.g., RT, PDT, HT, CDT, and chemotherapy) mediating ICD for cancer immunotherapy.The detailed mechanisms and latest research advances for each approach were introduced.Simultaneously, the key issues, including hypoxia, tumor-associated macrophages, indoleamine 2,3-dioxygenase, as well as PD-1/PD-L1-mediated immunosuppression, were discussed in this review.The detailed strategies, including combination with TME modulation, cancer vaccination, ICB, and starvation therapy, were presented.In addition, the in vivo short survival time issue of DC was also mentioned, which was able to be resolved by artificial DC construction.We then introduced the relatively new aspects that STING, pyroptosis, ferroptosis, and nanocarriers could also lead to ICD, providing more strategies to potentiate ICD-mediated antitumor immune responses.
Even though nanomedicine-based tumor ICD has shown great promise in preclinical studies, there still exist some challenges.First, nanomedicines may have off-target effects.Second, some nanomaterials may be difficult to metabolize, resulting in long-term toxicity.Although biomimetic nanomaterials have unique advantages, they may have immunogenicity issues.Finally, exploring combinational therapies for synergistic effects is a likely necessity if dramatic improvements in patient survival rates are desired.Nanomedicines display huge superiority compared with free ICD inducers, including good in vivo stability, high tumor accumulation, controlled drug release, achievable combination therapy, and TME modulation.However, some nanomedicines may need complicated preparations and encounter problems in large-scale production.To achieve successful implementation in the clinic, the above issues should be resolved.In addition, the insurmountable gap between animals and humans may increase the clinical translation difficulty of nanomedicines when they display superior antitumor efficacy in cells/animals while being probably far less effective in humans.The simple design and good biodegradability of nanoparticles are essential for clinical translation.

Figure 1 .
Figure 1.ICD can be induced by a series of treatment modalities, including RT, PDT, HT, chemotherapy.RT kills tumor cell by direct DNA or indirect ROS damage.PDT is ind cascade of ROS reactions.HT means heating tissues to a relatively high temperature with The dying tumor cells expose CRT and release DAMPs (e.g., ATP and HMGB1) which c and activate DC to enhance host immune response.ICD: immunogenic cell death; RT: radi PDT: photodynamic therapy; HT: hyperthermia therapy; CDT: chemodynamic therapy damage-associated molecular patterns; DC: dendritic cell; ROS: reactive oxygen species; O 1 O2: singlet oxygen; CRT: Calreticulin; HMGB1: High Mobility Group Box 1; ATP: aden phosphate; •OH: hydroxyl radical; STING: Cyclic GMP-AMP (cGAS)-stimulator of genes.

Figure 1 .
Figure 1.ICD can be induced by a series of treatment modalities, including RT, PDT, HT, CDT, and chemotherapy.RT kills tumor cell by direct DNA or indirect ROS damage.PDT is induced by a cascade of ROS reactions.HT means heating tissues to a relatively high temperature with ER stress.The dying tumor cells expose CRT and release DAMPs (e.g., ATP and HMGB1) which can recruit and activate DC to enhance host immune response.ICD: immunogenic cell death; RT: radiotherapy; PDT: photodynamic therapy; HT: hyperthermia therapy; CDT: chemodynamic therapy; DAMPs: damageassociated molecular patterns; DC: dendritic cell; ROS: reactive oxygen species; O 2 : oxygen; 1 O 2 : singlet oxygen; CRT: Calreticulin; HMGB1: High Mobility Group Box 1; ATP: adenosine triphosphate; •OH: hydroxyl radical; STING: Cyclic GMP-AMP (cGAS)-stimulator of interferon genes.

Figure 6 .
Figure 6.(a) Schematic illustration for the preparation of P/G@EF-TKNPs and the mechanism of in vivo combination therapy of CDT and immune checkpoint blockade (ICB).P/G: PDL1/GOx; EF: oligomerized (−)-epigallocatechin-3-O-gallate, Fe 3+ ; TKNP: thioketal bond-linked nanoparticles.(b) Fluorescence images and (c) flow cytometry analysis of 4T1 cells after incubation for 24 h at the GOx-equivalent concentration of 100 mU mL −1 using the ROS fluorescence probe DCFH-DA.(d) Flow cytometric examination of CRT exposure on the surface of 4T1 tumor cells after various treatments [84].Copyright 2022, American Chemical Society.

Figure 6 .
Figure 6.(a) Schematic illustration for the preparation of P/G@EF-TKNPs and the mechanism of in vivo combination therapy of CDT and immune checkpoint blockade (ICB).P/G: PDL1/GOx; EF: oligomerized (−)-epigallocatechin-3-O-gallate, Fe 3+ ; TKNP: thioketal bond-linked nanoparticles.(b) Fluorescence images and (c) flow cytometry analysis of 4T1 cells after incubation for 24 h at the GOx-equivalent concentration of 100 mU mL −1 using the ROS fluorescence probe DCFH-DA.(d) Flow cytometric examination of CRT exposure on the surface of 4T1 tumor cells after various treatments [84].Copyright 2022, American Chemical Society.

Table 1 .
Summary of PDT-induced ICD to enhance cancer immune efficacy.

Table 2 .
Summary of nanoparticle-loaded chemotherapy-induced ICD to enhance cancer immune efficacy.