A prodrug hydrogel with tumor microenvironment and near-infrared light dual-responsive action for synergistic cancer immunotherapy

Open AccessCCS ChemistryRESEARCH ARTICLE1 Feb 2022Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy Shaorui Jia†, Zhiyuan Gao†, Zelin Wu, Heqi Gao, He Wang, Hanlin Ou and Dan Ding Shaorui Jia† Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 , Zhiyuan Gao† Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 , Zelin Wu Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 , Heqi Gao Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 , He Wang Department of Urology, First Affiliated Hospital of Soochow University, Suzhou 215006 , Hanlin Ou Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 and Dan Ding *Corresponding author: E-mail Address: [email protected] Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 https://doi.org/10.31635/ccschem.021.202101458 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The combination of immunogenic cell death (ICD) induction and immune checkpoint blockade has emerged as a major direction of cancer immunotherapy. Among currently available ICD inducers, sonosensitizers that produce reactive oxygen species (ROS) under an external trigger to evoke ICD of tumor cells have shown great promise. However, a highly efficient sonosensitizer-based ICD inducer with an aggregation-induced emission (AIE) characteristic has yet to be developed. Herein, a novel AIE sonosensitizer with a twisted molecular structure, very small energy gap between the singlet and triplet excited states (ΔEST), and efficient ROS generation ability, which can serve as an effective ICD inducer, is reported for sonodynamic processes in cancer immunotherapy. Furthermore, an AIE sonosensitizer-based nanosystem with surface modification of anti-PD-L1 peptide is constructed for boosting antitumor immunotherapy. In this system, AIE sonosensitizer-mediated sonodynamic therapy can successfully convert a hypoimmunogenic cold tumor to a hot one and further facilitate the multivalent blocking of programed death ligand (PD-L1) by anti-PD-L1 peptides. Such an advanced nanosystem could effectively initiate the activation of antitumoral immune reactions and modulation of an immunosuppressive microenvironment, contributing to systemic antitumor effects to further inhibit the growth of distant tumors. Download figure Download PowerPoint Introduction Antitumor immunotherapy, which boosts specific cytotoxic T cells to eliminate tumor cells, is recognized as an effective cancer treatment strategy.1 Currently, inducing immunogenic cell death (ICD) of tumor cells is an effective strategy for enhancing recruitment and infiltration of specific cytotoxic T cells into solid tumors (e.g., triple negative breast cancer), converting a cold tumor, with a paucity of T cell infiltration, to hot.2–4 During ICD of tumors, tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) are generated, featuring surface-exposed calreticulin (ecto-CRT), adenosine triphosphate (ATP) secretion, and release of high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70).5,6 DAMPs act as a vital signal and a natural adjuvant to stimulate the presentation of TAA by antigen-presenting cells (APCs) (such as dendritic cells (DCs)) to T cells, which lead to the further activation of tumor-specific T cell-mediated immune response.7,8 Because of the pivotal role of ICD in antitumor immunotherapy, the development of highly effective ICD inducers with few side effects has attracted great interest during the past decade.5,9 Among currently available ICD inducers, photosensitizers evoke ICD of tumor cells by producing reactive oxygen species (ROS) upon light irradiation, which has showed conspicuous spatiotemporal precision and excellent biocompatibility.10 Our previous work has demonstrated that an aggregation-induced emission (AIE) photosensitizer with high ROS production capacity can massively induce sufficient ICD to evoke antitumor immunity.11,12 However, the limited penetration depth of light is a major challenge for the application of photosensitizer-based photodynamic therapy (PDT) in vivo.13 Sonodynamic therapy (SDT), derived from PDT, which utilizes ultrasound (US) to activate the sonosensitizer to produce ROS, enables deep tissue penetration, provides a more effective and safer strategy for the treatment of deep tumors, and shows unique advantages and great potential in antitumor immunotherapy.14–16 The mechanism of SDT is a complex, combinational output of different mechanisms.17–19 Among them, sonoluminescence was proven important to generate ROS.15,20 Sonoluminescence refers to the light emitted by the collapse of a bubble that is generated by US irradiation, which can activate the energy-matching photoactive sonosensitizers and generate the same photochemical reaction like PDT.21,22 For traditional organic photosensitizers with planar molecular structure, strong intermolecular interactions (e.g., π–π stacking) facilitate a nonradiative pathway of excited states, and the aggregation of photosensitizers within nanoparticles (NPs) causes quenching of the emission and ROS generation.23,24 At present, many organic photosensitizers that can be used as sonosensitizers to produce ROS, kill tumor cells, and induce ICD in tumor cells have been reported in SDT.25–27 Similar to photosensitizers, the aggregation-caused quenching (ACQ) effect also occurs when they are used as sonosensitizers in the aggregate state.28,29 Fortunately, sonosensitizers with AIE properties provide a good solution for the ACQ problem.30,31 AIE sonosensitizers have peripheral intramolecular motion units (e.g., rotating benzene rings) and a three-dimensional (3D) molecular structure.32,33 The 3D molecular structure significantly reduces the intermolecular interactions in NPs and aggregates. Meanwhile, the steric hindrance restricts the molecular motion in the excited state, which allows as much absorbed excitation energy as possible to be used in fluorescence emission or ROS generation.34,35 To the best of our knowledge, however, the ICD effect of AIE in SDT has never been studied. The reason may be the lack of appropriate strategies and AIE sonosensitizers with high efficiency. The application of SDT in immune system activation also faces challenges. Therefore, the design of an efficient AIE sonosensitizer is urgently needed to explore whether AIE can build an effective platform for sonosensitivity-based ICD inducers. Additionally, after successfully eliciting antitumor immune response by the induction of ICD, tumors may express autoprotective checkpoint molecules such as programmed cell death-ligand 1 (PD-L1) to bind with programmed death-1 (PD-1) on activated T cells, escaping attack from the immune system.36–38 Thus, an immune checkpoint blockade (ICB), such as the PD-1/PD-L1 blockade, has been employed to block PD-1/PD-L1 interaction to improve the tumor cell killing effect of T cells, thereby enhancing the inhibition efficacy against ICD-induced hot tumors.39–43 Although ICB holds great promise in cancer treatment, its therapeutic effect is largely limited by the insensitive response and insufficient T cells in cold tumors.44,45 Moreover, the synergistic anticancer efficacy of the integrated ICD induction and ICB is superior to monotherapy with only one of them.37,46,47DPPA (CNYSKPTDRQYHF, D-type PD-L1 peptide antagonists) can effectively block the PD-1/PD-L1 pathway, offering salient advantages such as higher stability, lower cost, and easier modification as compared with clinical anti-PD-L1 monoclonal antibodies.48,49 Consequently, for tremendously amplifying the effect of antitumor immunotherapy, the strategy of combing anti-PD-L1 peptide and ICD inducer in a single nanosystem is valuable and highly desirable but currently rare.50,51 Herein, we report sonosensitized AIE ICD inducer-based dots modified with anti-PD-L1 peptides on the surface for considerable improvement of antitumor immunotherapy outcomes. We designed and synthesized three new AIE luminogens (AIEgens): diphenylamino (DPA)-tetraphenylethylene (TPE)-(4-styryl-cyano)pyridinium salt (SCP), triphenylamine (TPA)-Ph-SCP, and TPA-2Ph-SCP). They have D–π–A structure and abundant intramolecular movement unit. Compared with TPA-Ph-SCP and TPA-2Ph-SCP, DPA-TPE-SCP has a more twisted molecular structure and very small ΔEST, which lead to stronger AIE activity, weaker intermolecular interactions of aggregates, and more efficient ROS generation. By comparison, DPA-TPE-SCP is not only the best photosensitizer, but also the best sonosensitizer. AIE sonosensitizers under US excitation showed superior ICD induction capability when compared with the reported sonosensitizing ICD inducer hematoporphyrin monomethyl ether (HMME), and successfully transformed a cold tumor into a hot one that was sensitized to PD-L1 blocking in vivo. Subsequently, surface modification of sonosensitized AIE dots with anti-PD-L1 peptides endowed them complementary advantages. In vivo experiments demonstrate that such function-cooperative dots significantly enhanced immune responses and relieved immune suppression, triggering a systemic antitumor immune therapeutic effect by virtue of the excellent ICD induction and multivalent PD-L1 blockade. Thus, this work not only provides a molecular guideline to design advanced sonosensitizer-based ICD inducers but also introduces new insights into the combination of ICD induction and ICB for synergistic antitumor immunotherapy (Scheme 1). Scheme 1 | Synthesis of AIE-dots-DPPA and the mechanism of antitumor immune responses induced by AIE-dots-DPPA-mediated sonodynamic therapy. Download figure Download PowerPoint Experimental Methods Computational method Structures of TPA-Ph-SCP, TPA-2Ph-SCP, and DPA-TPE-SCP were optimized in the water phase with the B3LYP method and 6-311G (d, p) basis set, Gaussian 09 program. Energy levels of S1–S6 and T1–T6 were calculated by the vertical excitation of the above optimized structures, with the same method of B3LYP/6-311G (d, p). Preparation of nano-sonosensitizer Nano-sonosensitizer was formulated by a nanoprecipitation approach using an amphiphilic co-polymer, DSPE-PEG2000. In brief, sonosensitizer (1 mg) and DSPE-PEG2000 (3 mg) were dissolved in 1 mL of tetrahydrofuran (THF); the solution was added dropwise into 9 mL of water undergoing sonication (3 min) by a microtip probe sonicator (XL2000, Misonix). An air pump was subsequently used to volatilize THF in the mixture to obtain a sonosensitizer-encapsulated dot aqueous solution. Then, the sonosensitizer-encapsulated dot solution was purified by 5000 rpm ultrafiltration for 10 min and filtration using a 0.45 μm syringe driven filter. Preparation of DPPA-conjugated dots DPPA peptides were modified on the AIE dots. First, DPPA peptides were added to the dots suspension, and then the suspension was stirred for 12 h to couple the thiol group of the peptide with the maleimide group of PEG2000 via the click reaction. Peptides that were not conjugated to the dots were then removed by centrifugation. ROS detection in 4T1 cells The 4T1 cancer cells were incubated in special confocal chambers with each nano-sonosensitizer (10 μg/mL of DPA-TPE-SCP dots, 10 μg/mL of HMME NPs) for 4 h at 37 °C. Subsequently, the cells were washed with 1× phosphate-buffered saline (PBS) three times, and then incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA; 20 μM) in FBS free culture medium. The above-mentioned process was performed in the dark. Then, under US exposure (1 MHz, 50% duty cycle, 0.5 W/cm2, 3 min), the cells were imaged with confocal laser scanning microscopy (CLSM) for DCF detection (Ex: 488 nm, Em: 530 ± 20 nm). Ecto-CRT staining in 4T1 cancer cells After the 4T1 cancer cells were incubated with each ICD inducer (10 μg/mL of DPA-TPE-SCP dots, 10 μg/mL of HMME NPs) for 4 h at 37 °C, the cells were washed and irradiated by US for 3 min (1 MHz, 50% duty cycle, 0.5 W/cm2). After 12 h, the cells were washed by precooling 1× saline, fixed with 4% paraformaldehyde on ice for 20 min, and then successively incubated with anticalreticulin antibody (ab2907, 1:200 dilution with 1× PBS) for 2 h and stained with Alexa Fluor 633-conjugated secondary antibody (1:200 dilution with 1× PBS) for another 2 h. The ecto-CRT expression of each ICD inducer-treated cancer cell was visualized by the ecto-CRT immunofluorescence using CLSM with the excitation at 633 nm and signal acquisition in the range from 640 to 670 nm. Detection of extracellular ATP, HMGB1, and HSP70 The 4T1 cancer cells (1 × 105 cells mL−1) were cultured in a black 12-well plate. After adherence, the 4T1 cancers cells were incubated with 10 μg/mL of DPA-TPE-SCP dots and 10 μg/mL of HMME NPs for 4 h at 37 °C, respectively. Next, the treated cells were washed and irradiated by US for 3 min (1 MHz, 50% duty cycle, 0.5 W/cm2). After 12 h, the supernatants were collected and then subjected to centrifugation using 12,000 rpm for 10 min at 4 °C, which was followed by addition of protease and phosphatase inhibitors into the supernatants. Furthermore, the cancer cells in each group were harvested and lysed, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Finally, the extracellular levels of HMGB1 and HSP70 were analyzed by western blot with the anti-HMGB1 antibody (1:500, Abcam, ab79823) and anti-HSP70 antibody (1∶1000, Abcam, ab181606), respectively. The level of secreted ATP was quantitatively determined by ATP Bioluminescent Assay Kit per manufacturer's instruction. In vivo SDT treatment 4T1 cells (1 × 106) dispersed in 50 μL of 1× PBS were injected into the second breast fat pad on the right side of each BALB/c mouse. The mice bearing 4T1 tumors were randomly divided into four groups when their tumors reached about 50 mm3. The AIE dots were intravenously injected into the mice three times on day 2, 4 and 6, respectively (once a day for three days (2, 4, and 6)). The tumors were exposed to US irradiation (1.0 MHz, 50% duty cycle, 1.5 W/cm2, 5 min) at 6-h post each injection. The tumor volume was measured by a caliper and determined with the following formula: Volume = Width2 × Length/2. The mice with tumors reaching 1500 mm3 were euthanized due to the standard animal protocol in this work. To establish a bilateral 4T1 orthotopic tumor model, 4T1 cells (1 × 106) were injected into the second breast fat pad on the right side of each female BALB/c mouse as primary tumor. Four days later, 4T1 cells (2 × 105) were injected into the second breast fat pad on the left side of mice as a distant tumor. The mice bearing ∼50 mm3 of primary tumors were randomly divided into four groups. The AIE dots were intravenously injected into the mice three times on day 2, 4 and 6, respectively (once a day for three days (2, 4, and 6)). At 6 h after each injection, the tumors were exposed to US irradiation for 5 min (1.0 MHz, 50% duty cycle, 1.5 W/cm2). The tumor volume was measured by a caliper and determined with the following formula: Volume = Width2 × Length/2. Flow cytometry analysis At designated time points, lymph nodes and tumors were harvested and homogenized to single-cell suspensions. After that, cell suspensions of lymph nodes were co-stained with anti-CD11c-FITC, anti-CD86-APC, and anti-CD80-PE for flow cytometry analysis of DCs. Single-cell suspensions in tumors were co-stained with different antibodies for flow cytometry analysis of CD8+ T cells (anti-CD3-FITC, anti-CD8-PE, or anti-CD8-APC), PD-L1+ tumor cells (anti-CD45-PE and anti-PD-L1-APC), Treg cells (anti-CD3-FITC, anti-CD4-APC, anti-CD25-PerCP/Cyanine5.5, and anti-Foxp3-PE), and IFN-γ+ of CD8+ T cells (anti-CD3-FITC, anti-CD8-APC, and anti-IFN-γ-PE). Statistical analysis Quantitative data are shown as mean ± standard deviation (SD). All the experiments were repeated at least three times. Statistical comparisons were made by ANOVA analysis and two-sample Student's t-test. P value < 0.05 was considered statistically significant. Results and Discussion Design, synthesis, and characterization of AIEgens Based on our design strategy, the phenylethylene core was used as a π-linker, and compared to triphenylethylene and diphenylethylene, TPE is an excellent AIE skeleton. The D–π–A structure was constructed using a DPA block as an electronic donor (D) and the pyridinium as an electronic acceptor (A). The compounds DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP were designed and synthesized according to the synthetic route shown in Figure 1a. Compound 1 was prepared through a Horner–Wadsworth–Emmons reaction. After quenching with dimethylformamide (DMF), compound 1 containing bromine was functionalized to afford the corresponding aldehyde derivatives, and DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP were obtained from the Knoevenagel condensation of compound 2 and SCP under basic conditions (for detailed synthetic routes of all compounds see Supporting Information Scheme S1). The intermediates were characterized by 1H NMR, and DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP were characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS) ( Supporting Information Figures S1–S17). Figure 1 | (a) Synthetic route to DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP. (b) Plot of ln(A0/A) against light exposure time, where A0 and A are the ABDA absorbance (378 nm) before and after irradiation, respectively. (c) Jablonski diagrams displaying the photophysical properties of the DPA-TPE-SCP. (d) HOMO–LUMO distributions by DFT calculations of DPA-TPE-SCP. (e) Energy levels of S1–S6 and T1–T6 calculated by the vertical excitation of the optimized structures in (f). (f) The dihedral angles of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP by DFT calculations. Download figure Download PowerPoint Photophysical properties The absorption and photoluminescence (PL) spectra of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP are shown in Supporting Information Figure S18. The conjugated structure of TPA-Ph-SCP, TPA-2Ph-SCP, and DPA-TPE-SCP becomes more and more distorted, so that their absorption peaks show hypochromatic shifts at 447, 382, and 350 nm; gradual red-shifting maximum emission wavelengths are located at 628, 630, and 638 nm. The PL quantum yields (QYs) of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP in the solid state were 12.4%, 7.9%, and 2.6%, respectively. When a poor solvent (toluene) was gradually added to the good solution [dimethyl sulfoxide (DMSO)], aggregate formation occurred, and the PL intensities of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP were significantly intensified, which prove that DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP have an obvious AIE feature. Notably the DPA-TPE-SCP showed the best AIE property when comparing the PL intensity of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP in a DMSO/toluene mixture with 99% toluene fraction to that in pure DMSO ( Supporting Information Figure S18). Because high ROS generation capacity is a prerequisite for an ICD inducer, we next studied and compared the ROS production of DPA-TPE-SCP, TPA-Ph-SCP, and in a 10 μM) under light irradiation (10 with as the ROS shown in Figure the ABDA of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP were and that DPA-TPE-SCP is an mechanism Based on the Jablonski the fixed absorption energy excited by light is through three fluorescence through followed by production of ROS or and nonradiative one or energy as much absorbed energy as possible on the is an effective to the efficacy of an To the mechanism of the superior ROS production of DPA-TPE-SCP compared to and TPA-Ph-SCP, calculations were The on electronic structures in the state that DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP a with the molecular on the and the molecular located on the and Supporting Information Figure in Figure TPA-Ph-SCP has a gap between the singlet and triplet excited of However, using the TPE as the π-linker, DPA-TPE-SCP has a very small value of to the highly ΔEST, was which for the superior ROS generation capability of DPA-TPE-SCP. shown in Figure the optimized structures calculated by DFT that compared with and TPA-Ph-SCP, DPA-TPE-SCP has a much molecular a TPE group used as a π-linker, the structure becomes more For the same dihedral between and to and in DPA-TPE-SCP from the of groups ( Supporting Information Figure The more twisted molecular structure the intermolecular interactions such as π–π and nonradiative in the or solid state, the absorbed excitation energy flow to the Therefore, successfully DPA-TPE-SCP showed higher fluorescence and ROS generation and TPA-Ph-SCP is for DPA-TPE-SCP, TPA-2Ph-SCP, and Such excellent of DPA-TPE-SCP is to our molecular design structure, of intramolecular motion units on π-linker, 3D twisted molecular which the process and the ROS generation efficiency. Preparation of AIE dots and detection of ROS in dots The AIE dots were prepared by the method The amphiphilic was used to the microscopy that DPA-TPE-SCP dots with of about nm, respectively dot has on the ROS generation of DPA-TPE-SCP, as by the same ABDA of DPA-TPE-SCP (10 μM) solution and DPA-TPE-SCP dots (10 on upon light ( Supporting Information Figure shown in Figure the reported dots and dots with excellent ROS production and the available sonosensitized compound HMME NPs were used to the ROS production of DPA-TPE-SCP dots dots and TPA-Ph-SCP dots under US irradiation (1 MHz, 50% duty cycle, 1.5 W/cm2). DPA-TPE-SCP dots showed higher ROS generation the sonosensitizers The of ROS production the ROS that the mechanism of DPA-TPE-SCP dots producing ROS may be activated by Next, was used to the species of The was with water and DPA-TPE-SCP dots, followed by US DPA-TPE-SCP dots showed an obvious For was used as the under detection and the that the dots could also generate post US irradiation Consequently, DPA-TPE-SCP dots were then as AIE dots for the following sonosensitized ICD induction Figure 2 | (a) The of sonosensitized AIE dots. (b) and of DPA-TPE-SCP dots. (c) The structure of and (d) Plot of ln(A0/A) against ultrasound (US) exposure time, where A0 and A are the ABDA absorbance (378 nm) before and after irradiation, respectively. (e) generation by DPA-TPE-SCP dots and water with US using (f) generation by DPA-TPE-SCP dots and water with US using Download figure Download PowerPoint In SDT effect and ICD induction by AIE dots has been that SDT can evoke ICD of cancer cells by producing First, of DPA-TPE-SCP dots by 4T1 breast cancer cells within 4 h was analyzed by flow cytometry ( Supporting Information Figure Next, the ROS generation level in 4T1 cells was by of by ROS to the upon US irradiation for 3 min (1 MHz, 0.5 the CLSM of the 4T1 cells treated with DPA-TPE-SCP dots showed the fluorescence and Supporting Information Figure the fluorescence intensity of dots is higher that of NPs ( Supporting Information Figure the excellent ROS production capacity of DPA-TPE-SCP dots. Then, was used to the therapeutic effect of SDT on 4T1 cells in and The of DPA-TPE-SCP dots in 4T1 cells was at different US the great of DPA-TPE-SCP dots. After exposure to US for 3 min (1 MHz, 0.5 the of 4T1 cells as the of DPA-TPE-SCP dots Furthermore, under different US irradiation time, the AIE 4T1 cancer cells more cell that of HMME All demonstrate that DPA-TPE-SCP dots can produce highly efficient ROS in 4T1 cells, a superior SDT effect by DPA-TPE-SCP dots in Figure 3 | (a) CLSM the ROS levels in 4T1 cancer cells after was used as the ROS 50 (b) Cell of 4T1 cells incubated with different of DPA-TPE-SCP dots with or US (c) Cell of 4T1 cells after with DPA-TPE-SCP dots and HMME followed by US irradiation with different (d) CLSM of ecto-CRT on 4T1 cells surface The cell were stained by 50 (e) Quantitative of ATP in the supernatants of 4T1 cells after different (f) the protein levels of HMGB1 and HSP70 in the 4T1 cell

BackgroundPT-112 is a novel immunogenic small molecule1 under Phase II clinical development for cancer therapy.2–8 Besides mediating cytostatic and cytotoxic effects in numerous human and mouse cancer cells, PT-112 elicits...

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Induction of immunogenic cell death (ICD) in tumors can enhance antitumor immunity and modulate immunosuppression in the tumor microenvironment (TME). In the current study, we investigated the effect of silibinin, a natural compound with anticancer activity, and its polymer-based nanoformulations on the induction of apoptosis and ICD in cancer cells. Free and nanoparticulate silibinin were evaluated for their growth-inhibitory effects using an MTT assay. Annexin V/PI staining was used to analyze apoptosis. Calreticulin (CRT) expression was measured by flow cytometry. Western blotting was conducted to examine the levels of elf2α, which plays a role in the ICD pathway. The HSP90 and ATP levels were determined using specific detection kits. Compared to the free drug, silibinin-loaded nanocarriers significantly increased the induction of apoptosis and ICD in B16F10 cells. ICD induction was characterized by significantly increased levels of ICD biomarkers, including CRT, HSP90, and ATP. We also observed an increased expression of p-elf-2α/ elf-2α in B16F10 cells treated with silibinin-loaded micelles compared to cells that received free silibinin. Our findings showed that the encapsulation of silibinin in polymeric nanocarriers can potentiate the effects of this drug on the induction of apoptosis and ICD in B16F10 melanoma cells.

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Most photosensitizers face enormous challenges in tumor hypoxia, the redox microenvironment, and low immune efficacies for reactive oxygen species (ROS). Herein, dye SQ-580 was constructed by coupling the electron-donating indole and thiophenazine-thiophene with the electron-withdrawing dicyanovinyl squaraine. It exhibited a high generation of •OH and O2•- by decreasing ΔES1T2 and acted as an excellent type I photosensitizer for conquering tumor hypoxia. The nanoplatform involving SQ-580, MnO2, and a targeting peptide CREKA was constructed and targeted breast tumor. In the tumor microenvironment, MnO2 reacted with high-expressed GSH and produced Mn2+, which catalyzed H2O2 to decompose into •OH and induced chemodynamic therapy (CDT). The reduction of GSH inhibited the consumption of SQ-580 and maintained its high photodynamic therapy (PDT) efficacy. GSH depletion and ROS resulted in cell ferroptosis. Under the synergy of ferroptosis and ROS, Mn2+ amplified immunogenic cell death (ICD). In the mouse models, SQ-580@MnO2 NPs showed NIRF/MR imaging-guided tumor targeting, effectively inhibited the growth of the primary and distant tumors, and amplified PDT and immune efficacies in the synergy of PDT, CDT, ferroptosis, and ICD. This study provides an effective strategy to design excellent type I photosensitizers and amplify the PDT and ICD efficacies utilizing valence metals and the tumor microenvironment.

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The clinical treatment of metastatic spinal tumor remains a huge challenge owing to the intrinsic limitations of the existing methods. Programmed cell death protein 1 (PD1)/programmed cell death ligand 1 (PD-L1) pathway blockade has been explored as a promising immunotherapeutic strategy; however, their inhibition has a low response rate, leading to the minimal cytotoxic T cell infiltration. To ameliorate the immunosuppressive microenvironment of intractable tumor and further boost the efficacy of immunotherapy, we report an all-round mesoporous nanocarrier composed of an upconverting nanoparticle core and a large-pore mesoporous silica shell (UCMS) that is simultaneously loaded with photosensitizer molecules, the IDO-derived peptide vaccine AL-9, and PD-L1 inhibitor. The IDO-derived peptide can be recognized by the dendritic cells and presented to CD8+ cytotoxic T cells, thereby enhancing the immune response and promoting the killing of the IDO-expressed tumor cells. Meanwhile, the near-infrared (NIR) activated photodynamic therapy (PDT) could induce immunogenic cell death (ICD), which promotes the effector T-cell infiltration. By combining the PDT-elicited ICD, peptide vaccine and immune checkpoint blockade, the designed UCMS@Pep-aPDL1 successfully potentiated local and systemic antitumor immunity and reduced the progression of metastatic foci, demonstrating a synergistic strategy for cancer immunotherapy.

The past decade has witnessed major breakthroughs in cancer immunotherapy. This development has been largely motivated by cancer cell evasion of immunological control and consequent tumor resistance to conventional therapies....

Advances in Immunogenic Cell Death for Cancer Immunotherapy

The Editorial on the Research Topic Immunogenic Cell Death in Cancer: From Benchside Research to Bedside Reality Immunogenic cell death (ICD) has emerged as a cornerstone of therapy-induced antitumor immunity (1–3). ICD is distinguished by spatiotemporally defined emission of danger signals or damage-associated molecular patterns (DAMPs) that elevate the immunogenic potential of dying cells [Garg et al.; (4)]. The important role played by DAMPs in immunity, tissue remodeling, and inflammation is discussed in details by Venereau et al. (Marco E. Bianchi lab). Most potent ICD inducers, characterized so far, elicit danger signaling through oxidative-endoplasmic reticulum stress (5). Several ICD inducers have been characterized, e.g., some chemotherapies, some physicochemical therapies (e.g., radiotherapy or photodynamic therapy/PDT), and oncolytic viruses (2, 6). Here, radiotherapy is among the first recognized immunogenic therapies [on account of “abscopal-effect” (7)]. The immunogenic potential of radiotherapy and possibilities for its combination with immune checkpoint blockers is discussed by Derer et al. (Udo S. Gaipl lab). It is noteworthy that ICD can also be achieved by various “smart” combinatorial strategies – an important point for clinically applied non-ICD inducers, discussed in details by Bezu et al. (Guido Kroemer lab). Several lines of experimental evidence have established the validity of ICD. However, the overreliance on usage of prophylactic vaccination in transplantable (heterotopic) tumor models has attracted some criticism (8). While these criticisms are valid, the field is already moving toward tumors produced orthotopically (curative/therapeutic) or in genetically engineered mouse models (GEMM) (at least for few ICD inducers, e.g., hypericin-PDT, Newcastle disease virotherapy and anthracyclines) (9–12). Moreover, the clinical existence of ICD has been proven through retrospective analysis involving cancer patient’s survival/therapy-responsiveness data (13–17). These observations have encouraged the increased usage of ICD-associated DAMPs as predictive/prognostic biomarkers – a point discussed in detail by Fucikova et al. (Radek Spisek lab). The promising results generated by systemically administered ICD inducers have also paved way for application of ICD-based dendritic cell (DC) vaccines (12). This important development has been discussed from the preclinical/clinical vantage points of various solid tumors by Vandenberk et al. (Stefaan W. van Gool lab) and lymphoma by Zappasodi et al. (Massimo Di Nicola lab). In the latter case, it is clear that the field is moving toward chimeric antigen receptor (CAR)-T cell’s application, and it will be interesting to see its combination with ICD in near future. Nevertheless, the insurmountable complexity of cancer makes it inevitable that in certain contexts, ICD may fail. This failure may stem from various factors, e.g., tumor heterogeneity (8), MHC-level heterogeneity (12), pre-established niches enriched in immunosuppressive factors or immune-checkpoints (1), stem cell-based immune-evasion (12), low mutational load, inactivating mutations/polymorphisms in certain immune-receptors (1), general ablation of danger signaling (14), and other genetic or even epigenetic causes. Several of these pro-cancerous immune-evasive mechanisms and immunotherapeutic strategies required for overcoming them are discussed in detail by Kersten et al. (Karin E. de Visser lab). The strategies for targeting epigenetic processes to improve immunotherapy are further discussed by Wachowska et al. (Jakub Golab lab). We believe that the valuable contributions of key researchers/clinicians toward this research topic/special edition have largely fulfilled its primary aim, i.e., to foster a critical discussion on experimental and clinical relevance of ICD. In fact, to further summarize and organize the fields of ICD and DAMPs, we have produced a multi-author consensus paper within this research topic that attempts to classify DAMPs and ICD inducers with an eye on translational potential of ICD (Garg et al.). This classification paper brings together >50 authors from the fields of ICD and DAMPs, and tries to reach a comprehensive accord on various terminologies related to DAMPs/ICD, the historical background of these concepts, ICD classification system (Type I vs. Type II inducers), and the relevant preclinical/clinical criteria crucial for the field(s) (Garg et al.). We hope that this consensus paper will be a useful literature resource for various researchers/clinicians. These contributions, while summarizing the status quo, have also exposed a set of major questions and challenges that still need to be addressed.

A multifunctional nanosystem catalyzed by cascading natural glucose oxidase and Fe3O4 nanozymes for synergistic chemodynamic and photodynamic cancer therapy

The clinical potency of anti-programmed death-ligand 1 (PD-L1) therapy in metastatic triple-negative breast cancer (TNBC) is modest primarily because of the intrinsic low immunogenicity and an immunosuppressive tumor microenvironment (TME). Photodynamic therapy (PDT), an inducer of immunogenic cell death (ICD), has the potential to enhance antitumor immune response and improve PD-L1 blockade efficacy. DTP, a novel photosensitizer developed previously, has demonstrated potent ROS-dependent photocytotoxicity, yet its immunomodulatory effects remain unexplored. This study investigated the induction of ICD and dendritic cell (DC) maturation following DTP-PDT in vivo and in vitro. A bilateral TNBC model was developed to assess the efficacy of DTP-PDT combined with α-PD-L1 therapy on untreated distant tumors and to explore its potential immunological mechanisms. The results showed that DTP-PDT effectively induced ICD, demonstrated by calreticulin membrane exposure, high mobility group box 1 protein release, and increased secretion of interferon-γ and tumor necrosis factor-α, resulting in DC maturation. The combination of DTP-PDT and α-PD-L1 significantly inhibited distant tumor growth. This effect was associated with increased CD8+ and CD4+ T cells infiltration, and reduced numbers of regulatory T cells, in the distant tumor and spleen. In conclusion, DTP-PDT enhanced TNBC sensitivity to α-PD-L1 by inducing ICD, and its combination withα-PD-L1 could remodel the immunosuppressive TME and enhance systemic immunity, resulting in a therapeutic effect against distant metastasis. This study provides experimental validation for a combined strategy of DTP-PDT and α-PD-L1, proposing a potential therapeutic approach for metastatic TNBC.

Oxygen Nanoshuttles for Tumor Oxygenation and Enhanced Cancer Treatment