Endoplasmic reticulum-targeted iridium(III) photosensitizer induces pyroptosis for augmented tumor immunotherapy.

{"full_text": " Journal of Inorganic Biochemistry 260 (2024) 112695\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Inorganic Biochemistry\n journal homepage: www.elsevier.com/locate/jinorgbio\n\n\n\n\nEndoplasmic reticulum-targeted iridium(III) photosensitizer induces\npyroptosis for augmented tumor immunotherapy\nYun-Shi Zhi a, b, 1 , Tie Chen b, 1 , Bin-Fa Liang b , Shan Jiang a , Da-Hong Yao a , Zhen-Dan He a ,\nChen-Yang Li b, * , Liang He c, * , Zheng-Yin Pan a, *\na\n College of Pharmacy, Shenzhen Technology University, Shenzhen 518118, China.\nb\n School of Pharmacy, Shenzhen University Medical School, Shenzhen University, Shenzhen 518055, China\nc\n Key Laboratory for Biobased Materials and Energy of Ministry of Education, College of Materials and Energy, South China Agricultural University, Guangzhou 510642,\nChina\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: An ideal tumor treatment strategy involves therapeutic approaches that can enhance the immunogenicity of the\nIridium(III) complex tumor microenvironment while simultaneously eliminating the primary tumor. A cholic acid-modified iridium\nPhotodynamic therapy (III) (Ir3) photosensitizer, targeted to the endoplasmic reticulum (ER), has been reported to exhibit potent type I\nImmunotherapy\n and type II photodynamic therapeutic effects against triple-negative breast cancer (MDA-MB-231). This photo-\nPyroptosis\nEndoplasmic reticulum\n sensitizer induces pyroptotic cell death mediated by gasdermin E (GSDME) through photodynamic means and\nAntitumor enhances tumor immunotherapy. Mechanistic studies have revealed that complex Ir3 induces characteristics of\n damage-related molecular patterns (DAMPs) in MDA-MB-231 breast cancer cells under light conditions. These\n include cell-surface calreticulin (CRT) eversion, extracellular high mobility group box 1 (HMGB1) and ATP\n release, accompanied by ER stress and increased reactive oxygen species (ROS). Consequently, complex Ir3\n promotes dendritic cell maturation and antigen presentation under light conditions, fully activates T cell-\n dependent immune response in vivo, and ultimately eliminates distant tumors while destroying primary tu-\n mors. In conclusion, immune regulation and targeted intervention mediated by metal complexes represent a new\n and promising approach to tumor therapy. This provides an effective strategy for the development of combined\n targeted therapy and immunotherapy.\n\n\n\n\n1. Introduction about 20% of patients and most patients do not achieve lasting benefit\n [3]. The tumor microenvironment (TME), especially the tumor immune\n Tumor immunology is an emerging field of \u201cpersonalized\u201d medicine. microenvironment (TIME) lacking T cell inflammation, determines the\nIt is an antitumor response that strengthens the immune system to attack effectiveness of tumor immunotherapy [4\u20136].\nimmune molecules overexpressed by tumor cells or expressed only by Numerous studies have shown that photodynamic therapy (PDT) can\ntumor cells [1]. Many biomolecular or small molecule drugs are used to cause tumor cell death in an immunogenic manner. Furthermore, PDT is\nrebalance the tumor immune response to kill tumors [2]. Immune a non-invasive treatment and has been extensively studied in the\ncheckpoint inhibitors (ICBs) therapies are highly successful in prevent- treatment of localized cancers [7\u20139]. Under laser irradiation focused on\ning tumor-mediated T cell inactivation. However, it is effective in only localized tumors, photosensitizers (PSs) rapidly generate cytotoxic\n\n\n\n Abbreviations: ER, Endoplasmic reticulum; CA, Cholic acid; GSDME, Gasdermin E; DAMPs, Damage-related molecular patterns; CRT, Calreticulin; HMGB1, High\nmobility group box 1; ROS, Reactive oxygen species; ICBs, Immune checkpoint inhibitors; TME, Microenvironment; PDT, Photodynamic therapy; PS, Photosensitizer;\nICD, Immunogenic cell death; GSDMD, Gasdermin D; LAP, Leucine aminopeptidase; MLCT, Metal-ligand charge transfer; ABDA, 9,10-anthracenediyl-bis(methylene)\ndimalonic acid; PCC, Pearson colocalization coefficient; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC50, Half maximal inhibitory con-\ncentration; PI, Phototoxicity index; DCFH-DA, 2\u2032,7\u2032-dichlorodihydrofluorescein diacetate; ERS, ER stress; UPR, Unfolded protein response; ATF4, Activating tran-\nscription factor 4; CHOP, C/EBP-homologous protein; STAT3, Signal transducer and activator of transcription 3; DCs, Dendritic cells; H&E, Hematoxylin and eosin.\n * Corresponding authors.\n E-mail addresses: lcy@szu.edu.cn (C.-Y. Li), heliang@scau.edu.cn (L. He), panzhengyin@sztu.edu.cn (Z.-Y. Pan).\n 1\n These authors made equal contributions to this work.\n\nhttps://doi.org/10.1016/j.jinorgbio.2024.112695\nReceived 9 June 2024; Received in revised form 17 July 2024; Accepted 10 August 2024\nAvailable online 11 August 2024\n0162-0134/\u00a9 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fY.-S. Zhi et al. Journal of Inorganic Biochemistry 260 (2024) 112695\n\n\nreactive oxygen species (ROS) and selectively damage diseased tissues\n[10]. In addition, ROS produced by PSs can not only promote tumor cell\ndeath, but also trigger endoplasmic reticulum (ER) stress and trigger\ncell-surface calreticulin (CRT) protein exposure, thereby inducing\nimmunogenic cell death (ICD) of cancer cells and releasing damage-\nrelated molecular patterns (DAMPs) and pro-inflammatory cytokines\n[11,12].\n ER is an indispensable organelle responsible for protein synthesis,\nintracellular signal transduction, and calcium homeostasis, and plays a\nvital role in the immune system [13]. In addition, subtle disturbances to\nthe ER redox signaling pathway are likely to lead to ER stress, which\ninduces ICD [12,14]. Recently, Chao\u2019s groups reported that ER-targeting\nmetal iridium complexes combined with PDT can enhance ICD15.\n Pyroptosis is a new type of programmed cell death different from\napoptosis, which can cause a strong inflammatory response. It has the\npotential to stimulate antitumor immunity, eliminate primary tumors,\nand inhibit tumor metastasis [16]. Activating Caspase-1/11/4/5 can cut Scheme 1. Structures of Ir1\u2013Ir3.\nGasdermin D (GSDMD) to form GSDMD-N-terminus (GSDMD-NT) and\nGSDMD-C-terminus (GSDMD-CT). GSDMD-CT \u201coligomerizes\u201d to form What\u2019s more, the antitumor immune-therapeutic effect of Ir3 was\nmembrane pores and induce pyroptosis [17]. In addition, activated verified in a mouse tumor model.\nCaspase-3 can also lyse GSDME to form GSDME-NT and GSDME-CT,\nwhich manifests as pyroptosis [18]. This pathway also activates the 2. Results and discussion\ninflammatory response by perforating the cell membrane and releasing\nthe cell contents outward [19]. Overexpression of GSDME has been 2.1. Synthesis and characterization\nshown to promote tumor sensitivity of small molecule-targeted in-\nhibitors [20]. Pyroptosis can trigger ICD and convert \u201ccold\u201d immuno- The synthesis route of complexes Ir1-Ir3 is shown in Scheme S1.\nsuppressive TME into \u201chot\u201d immunogenic TME with extensive tumor Ligand Phen-CA was synthesized as previously reported [37]. The\nlymphocyte infiltration [21]. complexes Ir1-Ir3 were synthesized by conventional methods [38]. The\n Due to the great clinical success of traditional platinum-based drugs, complexes Ir1-Ir3 were characterized by ESI-MS (Fig. S1-S3), 1H NMR\nresearchers have also begun to focus on other metal drugs, such as (Fig. S4-S6), and 13C NMR spectra (Fig. S7-S9). The mass spectrometry\ncyclometalated iridium(III) complexes [22\u201325]. Cyclometalated iridium data and the result of the 1H and 13C NMR tentative assignments\n(III) complexes have superior photophysical properties and anti-cancer confirmed the composition of these complexes [39,40].\nefficacy, which is helpful to study their localization and anti-cancer The UV\u2013Vis absorption spectra of Ir1-Ir3 exhibit a strong intraligand\nmechanism in cells, so as to develop new anti-cancer drugs with diag- transition band at 250\u2013315 nm, and two relatively weak broad bands at\nnostic and therapeutic functions [26\u201328]. ROS produced by PDT has a 315\u2013365 and 365\u2013460 nm attributed to the mixed metal-ligand charge\ncertain killing range [29]. Organelle localization of photosensitizers is transfer ([1]MLCT and [3]MLCT) (Fig. 1A and Fig. S10A-B) [41]. The\nclosely related to therapeutic effect. Therefore, it is of great significance strong absorption properties of the complexes in the visible region\nto find photosensitizers with organelle-targeting functions [30,31]. (400\u2013480 nm) make it a potential visible excitation photosensitizer.\n Most imaging probes are easily removed quickly after entering the Moreover, complexes Ir1-Ir3 exhibit orange-red emission in the region\nliver and are difficult to accumulate fully in the liver in a short period of of about 510\u2013650 nm, which is attributed to the [3]MLCT excited state\ntime, resulting in low signal intensity and sensitivity in the liver [32]. of the complexes (Fig. 1B and Fig. S10C-D).\nCholic acid, a signaling molecule that enters hepatocytes through The ability of Ir1-Ir3 to generate singlet oxygen (1O2) was investi-\n\u201cactive transport\u201d, is involved in many hepatic metabolisms and exhibits gated by using 9,10-anthracenediyl-bis(methylene)dimalonic acid\ninherent hepatocyte targeting [33,34]. Su\u2019s group designed hCy-CA- (ABDA) as a singlet oxygen (1O2) probe. In the presence of Ir1-Ir3 and\nLAP, a leucine aminopeptidase (LAP)-activated fluorescent probe, 425 nm light irradiation, the maximum absorption of ABDA at 380 nm\nwhich greatly improved the targeting ability of hepatocytes by intro- gradually decreases, indicating that the 1O2 is produced (Fig. 1C).\nducing cholic acid groups, showing high selectivity, high sensitivity, and Among the three complexes, Ir2 and Ir3 have higher singlet oxygen\nlow detection limit (0.0067 U\u22c5mL\u2212 1). It has successfully enabled imag- yields of 0.65 and 0.67, respectively, probably due to the larger conju-\ning diagnostics of trace amounts of LAP in vivo [35]. In addition, Andriy gation properties of the ligands pq and pbt.\nMokhir\u2019s group developed an ER-targeted conjugate, a prodrug of cholic\nacid and N-alkylaminoferrocene, which is specifically activated in can- 2.2. Cellular localization\ncer cells with elevated ROS levels [36]. The conjugate induced ER stress\nby accumulating in the ER of cancer cells, and further caused cell ne- Due to the intrinsic phosphorescent properties of Ir3, its sub-\ncrosis by reducing mitochondrial membrane potential and producing organelle localization after entering cells can be explored by laser\nmitochondrial ROS. confocal fluorescence microscopy. Colocalization experiments were\n Therefore, novel cyclometalated iridium(III) complexes Ir1-Ir3 performed in MDA-MB-231 cells by co-staining Ir3 with the probe of ER-\nfunctionalized with cholic acid groups were designed and synthesized Tracker Deep Red. As shown in Fig. 2, the fluorescence of Ir3 (shown in\n(Scheme 1), which have excellent singlet oxygen production capacity. green) in the cells overlaps to a large extent with the fluorescence of ER-\nAll of these complexes showed photodynamic therapeutic activity Tracker Deep Red (shown in red), and the Pearson colocalization coef-\nagainst triple-negative breast cancer, with Ir3 being the most active. ficient (PCC) value is 0.90, indicating that Ir3 predominantly accumu-\nMechanistic studies have found that Ir3 accumulates in the ER, which lates in the ER after entering the cells. Some other lipophilic cationic\npromotes the in situ production of ROS on the ER and triggers ER stress iridium complexes have also been reported in the literature to localize\nunder visible light (425 nm) irradiation. Ir3 exhibits excellent photo- primarily to the ER [42,43]. Thus, Ir3 acts as an ER-targeted PDT\ntoxicity in nanomolar concentration against human triple-negative photosensitizer. The auxiliary ligand of iridium(III) complexes coupled\nbreast cancer cells MDA-MB-231 and mouse breast cancer 4T1 cells. It with some hydrophobic molecules, such as bis(2-chloroethyl)-azane\neffectively initiates GSDME-mediated pyroptosis and triggers ICD. [15], N-phenethylsuccinamide [30], rhodamine [43], etc., can\n\n 2\n\fY.-S. Zhi et al. Journal of Inorganic Biochemistry 260 (2024) 112695\n\n\n\n\nFig. 1. (A) UV/Vis and (B) emission spectra of Ir1\u2013Ir3 (2 \u00d7 10\u2212 5 M) measured in CH3CN at 298 K. (C) Changes in the absorption spectra of ABDA at 380 nm upon\nirradiation at 425 nm in the presence of Ir1\u2013Ir3 in aerated PBS. [Ru(bpy)3]Cl2 was used as a standard.\n\n\n\n\nFig. 2. Confocal co-localization images of MDA-MB-231 cells treated with Ir3 (10 \u03bcM, 4 h; Ex = 405 nm, Em = 550\u2013600 nm) and then stained with ER-Tracker Deep\nRed FM (150 nM, 30 min; Ex = 633 nm, Em = 660\u2013700 nm). The scale bar represents 20 \u03bcm. (For interpretation of the references to colour in this figure legend, the\nreader is referred to the web version of this article.)\n\n\neffectively target the endoplasmic reticulum. Andriy Mokhir\u2019s team re-\n Table 1\nported that cholic acid-modified prodrugs of N-alkylaminoferrocene\n Antiproliferative activities (IC50/\u03bcM) of the compounds in the absence and\ncould well target the endoplasmic reticulum of cells [36]. In addition,\n presence of 425 nm light in MDA-MB-231 and 4T1 cells.\nthe lipophilicity of Ir3 also facilitates targeting the endoplasmic retic-\nulum. Therefore, we believe that the endoplasmic reticulum targeting Cell Lines MDA-MB-231 4T1\n\nproperties of Ir3 may be related to the lipophilicity and the cholic acid Condition Darka Lightb PIc Darka Lightb PIc\ngroup of the complex. Ir1 6.8 \u00b1 0.098 \u00b1 69.0 10.2 \u00b1 2.4 \u00b1 0.3 4.3\n 0.21 0.1 0.4\n Ir2 4.6 \u00b1 0.01 \u00b1 459.2 3.1 \u00b1 0.18 \u00b1 17.3\n2.3. PDT activity\n 0.13 0.04 0.14 0.04\n Ir3 7.6 \u00b1 0.009 \u00b1 838.9 8.3 \u00b1 0.3 \u00b1 24.6\n The photodynamic activity of Ir1\u2013Ir3 on MDA-MB-231 and 4T1 cells 0.18 0.02 0.12 0.03\nwas detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium\n a: Cells were incubated with the indicated compounds in the dark for 48 h.\nbromide (MTT) colorimetric assay. As shown in Table 1, after 15 min\n b: Cells were incubated with the indicated compounds for 12 h in the dark and\nirradiation with 425 nm laser (15 mW/cm2), the half maximum inhib- then irradiated with light at 425 nm.\nitory concentrations (IC50) obtained for Ir2 and Ir3 on MDA-MB-231 c: PI = Phototoxicity Index, PI is the ratio of the IC50 values in the dark to those\nwere 0.010 \u03bcM and 0.009 \u03bcM, respectively, both down to the nano- obtained upon light irradiation.\nmolar level. Moreover, the IC50 values for 4T1 cells upon light irradia-\ntion were 0.18 \u03bcM and 0.34 \u03bcM, respectively, showing significant\n\n 3\n\fY.-S. Zhi et al. Journal of Inorganic Biochemistry 260 (2024) 112695\n\n\nphototoxicity. Among these complexes, Ir3 showed the highest photo- STAT3 decreases significantly with dose dependence, which facilitates\ntoxicity index (PI) against MDA-MB-231 and 4T1, which were 838.89 the transformation of \u201ccold\u201d tumors to \u201chot\u201d tumors and initiates CHOP-\nand 24.63, respectively. These results show that Ir3 exhibits excellent mediated ERS.\nphotodynamic therapeutic activity in vitro.\n 2.6. Pyroptosis induction\n2.4. ROS generation\n Ir3-induced cell death pattern under PDT treatment was studied\n The therapeutic effect of PDT depends on the fact that cancer cells through a variety of experiments. Firstly, it was observed by inverted\nproduce sufficient cytotoxic ROS. Therefore, we used 2\u2032,7\u2032-dichlor- microscopy and transmission electron microscopy that the cells in the\nodihydrofluorescein diacetate (DCFH-DA) as a capture probe for ROS to control group treated by light alone had normal growth, good elonga-\ndetect the ability of Ir3 to induce ROS production in MDA-MB-231 cells tion, and normal morphology. In the Ir3 plus PDT group, the cells were\nunder light conditions. DCFH-DA is a non-fluorescent probe that can rounded and swollen, the intercellular space was significantly enlarged,\npenetrate cell membranes. It is oxidized by the ROS inside the cell, the cell morphology and contour were unclear, and the volume and\nresulting in highly fluorescent DCF. The results of laser confocal imaging density were significantly reduced. In addition, there are many distinct\nshowed that the intracellular fluorescence in the control group was vacuoles released on the cell membrane surface (Fig. 5A and B). These\nweak, while under laser irradiation, the intracellular fluorescence in the morphological changes are typical of pyroptosis.\nIr3-treated group gradually increased with the increase of Ir3 concen- Furthermore, Western blot analysis was used to detect catalytic\ntration (Fig. 3A). Flow cytometry further validated Ir3\u2019s ability to cleavage of GSDME, which is considered one of the important indicators\ninduce cells to produce ROS after laser irradiation (Fig. 3B). In summary, of pyroptosis. As shown in Fig. 5C, the expression of cleaved caspase-3\nIr3 can effectively induce intracellular ROS production under laser and GSDME-N in Ir3-treated cells increased significantly with laser\nirradiation. time-dependence. These results suggest that Ir3-mediated PDT induces\n It is well known that traditional type II PDT relies on the production cells to activate cleaved caspase-3 and cleaved GSDME (GSDME-N),\nof 1O2, and this PDT mode is less effective in the treatment of hypoxic perforating cell membranes and triggering pyroptosis to kill cancer cells.\ntumors, while the type I PDT that induces the production of superoxide\nanion radicals (O\u2022\u22122 ) has advantages in the treatment of hypoxic tumors 2.7. Tumor immunogenic cell death induction\ndue to its reduced dependence on oxygen [44]. We then used the su-\nperoxide anion (O\u2022\u2212 2 ) probe dihydroethidium to detect the production of PDT-induced pyroptosis may cause immunogenic cell death (ICD).\nO\u2022\u2212\n 2 . By confocal microscopy imaging (Fig. 3C), it can be seen that after ICD has three distinct biochemical signatures of DAMPs: HMGB1\nPDT treatment, as the Ir3 concentration (0 \u03bcM, 0.1 \u03bcM, 0.2 \u03bcM, and 0.4 release, etco-CRT exposure, and ATP exosomes. To investigate whether\n\u03bcM) increases, the O\u2022\u2212 2 produced in MDA-MB-231 cells also increase. PDT treatment with Ir3 triggers ICD, we first observed changes in\nThese results indicate that both type I and type II PDT are involved in the intracellular etco-CRT and HMGB1 expression upon 425 nm laser irra-\nmechanism of Ir3-induced cell death. diation after Ir3 (0 \u03bcM, 0.1 \u03bcM, 0.2 \u03bcM, and 0.4 \u03bcM) treatment.\n Immunofluorescence imaging was shown in Fig. 6A. The red fluores-\n2.5. Analysis of the expression of intracellular endoplasmic reticulum- cence (ecto-CRT) in the cells of the control group was negligible under\nassociated proteins dark conditions, but the red fluorescence in the cells of the Ir3 group was\n significantly enhanced by 425 nm laser irradiation (15 mW/cm2, 15\n External stimuli can cause an increase in unfolded and misfolded min), indicating high expression of ecto-CRT in cells after treatment by\nproteins within the ER, and cause ER stress (ERS), which further rapidly Ir3 and PDT. In addition, quantitative analysis of the above results using\nactivates unfolded protein response (UPR) signals in response [28\u201330]. flow cytometry (Fig. 6B) showed a concentration-dependent increase in\nUnder ERS, PERK auto-phosphorylation and subsequent phosphoryla- intracellular immunofluorescence intensity after treatment with Ir3 and\ntion of eIF2\u03b1 restore cellular homeostasis. However, persistent ERS PDT.\npromotes the expression of activating transcription factor 4 (ATF4) [45]. At the same time, as shown in Fig. 6C, with the increase of Ir3\nATF4 is involved in processes such as cell metabolism, nutrient uptake, concentration, the green fluorescence (HMGB1) inside cells gradually\nand antioxidant. ATF6 is a type 2 transmembrane protein in the ER. In decreases under laser irradiation, indicating that HMGB1 migrates from\nERS, ATF6 promotes transcription of UPR-associated genes to eliminate the nucleus to the cytoplasm and exosomes to the extracellular. Next, we\nmisfolded proteins, allowing cells to survive under ERS [46,47]. How- tested the change in extracellular ATP content after treatment with Ir3\never, under long-term ERS, ATF4 and ATF6 can activate downstream (0 \u03bcM, 0.1 \u03bcM, 0.2 \u03bcM, and 0.4 \u03bcM) under light and dark conditions\nproteins such as C/EBP-homologous protein (CHOP), thereby inducing (Fig. 6D). ATP in the extracellular supernatant of cells treated with Ir3\napoptosis [48]. (0.4 \u03bcM) and PDT was significantly increased compared to the control\n In summary, when UPR cannot alleviate ERS, many pro-apoptotic group. In summary, cells treated by Ir3 and PDT have high immuno-\nsignaling pathways, such as CHOP up-regulation, are initiated. After genicity and excellent anti-tumor immunity potential.\nMDA-MB-231 cells were stimulated by Ir3 and laser, a large amount of\nROS was accumulated in the ER, which increased the pressure on the ER. 2.8. In vivo anti-tumor evaluation\nCompared with the control group, the expression of PERK and ATF6\nproteins decreased and the expression of ATF4 and CHOP proteins To investigate the antitumor efficacy and systemic immune response\nincreased significantly with the increase of Ir3 concentration in MDA- of Ir3-mediated PDT in vivo, we chose to construct a BALB/C mouse\nMB-231 cells treated with Ir3 and laser (Fig. 4). The above results model of 4T1 tumors instead of human triple-negative breast cancer\nindicate that Ir3 damages the ER and cause tumor cell death. MDA-MB-231 tumors. Mice were implanted with primary tumors on the\n Signal transducer and activator of transcription 3 (STAT3) is the key right side. After 7 days, when the volume of the primary tumor reached\nmolecule in many oncogenic signaling pathways that can induce 50\u2013100 mm3, mice were implanted with the distal tumor on the left side.\nepithelial-mesenchymal transformation and promote apoptosis [49]. After 1 day, mice were randomly divided into 4 groups. 5 mg/kg Ir3 was\nStudies have pointed out that inhibition of p-STAT3 can promote the administered within the primary tumor, followed by 425 nm PDT\nexpression of CHOP [50]. In addition, STAT3 promotes communication treatment. Distal tumors were left untreated to observe the immune\nbetween tumors and individual immune cell subsets, impeding tumor response. Among them, Ctrl + dark group, Ctrl + light group, and Ir3\ntransformation from \u201ccold\u201d to \u201chot\u201d [51]. Therefore, Ir3 is targeted to group were used as controls.\nthe ER, and after laser stimulation, the expression of intracellular p- As shown in Fig. S11, Ir3 has a good inhibitory effect on 4T1 tumor\n\n 4\n\fY.-S. Zhi et al. Journal of Inorganic Biochemistry 260 (2024) 112695\n\n\n\n\nFig. 3. Detection of cellular ROS levels in MDA-MB-231 cells by (A) confocal microscopy and (B) flow cytometry. Cells were treated with Ir3 (0.1, 0.2, 0.4 \u03bcM) for 12\nh and stained with DCFH-DA (10 \u03bcM) for 20 min. Cells were then irradiated with a green LED light (\u03bbir = 425 nm, 15 mW/cm2) for 15 min. Ex = 488 nm, Em = 520\n\u00b1 20 nm. Scale bar, 20 \u03bcm. (C) Detection of the production of O\u2022\u2212 2 in MDA-MB-231 cells by flow cytometry after Ir3 and PDT treatment. Scale bar: 20 \u03bcm.\nDihydroethidium: Ex = 561 nm, Em = 620 \u00b1 20 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of\nthis article.)\n\n\n\n\n 5\n\fY.-S. Zhi et al. Journal of Inorganic Biochemistry 260 (2024) 112695\n\n\n Ir3 had excellent anti-tumor immune effects.\n Tumor-infiltrated immune cells were analyzed by flow cytometry to\n assess the anti-tumor immune response of mice. Firstly, we assessed the\n degree of maturation of dendritic cells (DCs) in the lymph nodes of tu-\n mors, which is necessary for anti-tumor immunity. As shown in Fig. 7C,\n CD80+ CD86+ dendritic cells in the Ir3 + light group increased signif-\n icantly from 7.83% to 23.6% compared with the Ctrl + light group, Ctrl\n + dark group, and Ir3 group, indicating that the maturity of DCs\n increased after Ir3 and PDT treatment. Secondly, we quantified CD8+\n cytotoxic T cells and CD4+ helper T cells which are indicators of adap-\n tive immune responses in distant tumors. The proportion of tumor-\n infiltrated CD3+ CD4+ and CD3+ CD8+ T lymphocytes in the Ir3 +\n light group were 36.3% and 21.7%, respectively, which were 3\u20137 times\n higher than those in the Ctrl + dark group (11% and 3.76%), the Ctrl +\n light group (0.6% and 5.76%) and the Ir3 group (14.3% and 6.34%)\n (Fig. 7D and E). In summary, cytotoxic T lymphocytes can be induced by\n Ir3 and PDT to activate adaptive anticancer immune responses.\n During Ir3 and PDT treatment in mice, toxic and side effects of the\n Ir3 + light group were evaluated. The body weight of BALB/C mice\n loaded with 4T1 tumors remained stable, and no mice died or lost\n weight significantly (Fig. S12). Hematoxylin and eosin (H&E) staining\n showed that no significant pathological abnormalities were found in the\n major organs (heart, kidney, liver, spleen and lung) of Ir3 + group\n (Fig. S13). In summary, it shows that Ir3 has very low systemic toxicity\nFig. 4. Expression of STAT3, p-STAT3, ATF6, PERK, ATF4 and CHOP proteins. and side effects in vivo after PDT treatment.\nMDA-MB-231 cells were treated with Ir3 (1 \u03bcM, 4 h), irradiated with 425 nm\nlight for indicated time intervals, and further incubated for 4 h.\n 3. Conclusions\n\ngrowth under light (425 nm) irradiation. On the end day of PDT treat- We have developed a novel iridium(III) photosensitizer Ir3 that\nment (day 16), tumor volume was suppressed by approximately 86% in targets the ER and can induce pyroptosis, which can effectively activate\nmice treated with Ir3 and PDT (Fig. 7A and B). At the same time, we the anti-tumor immune response in vivo. Ir3 shows nanomolar photo-\nassessed the immune effect of distal tumors in each group by measuring toxicity (Light IC50 = 9 nM), and the phototoxicity index for MDA-MB-\nthe growth rate of distal tumors under dark conditions. There were no 231 reaches 838.9. Under visible light (425 nm) irradiation, singlet\nsignificant suppressive effects on distal tumors in the Ctrl + light group, oxygen can be effectively generated, and it has high photostability.\nCtrl + dark group, and Ir3 group. Their distant tumors were enlarged to Mechanistic studies have shown that Ir3 causes excessive ROS produc-\nabout 402.82 mm3, 412.82 mm3, and 249.65 mm3, respectively. The tion and ERS in MDA-MB-231 cells under light irradiation. Interestingly,\ngrowth rate of distal tumors in the Ir3 + light group was significantly Ir3 can cause GSDME-mediated pyroptosis to enhance tumor immuno-\ninhibited compared with the control group. The inhibition rate of distal genicity. Ir3 promotes dendritic cell maturation and activates T cell-\ntumors in the Ir3 + light group was as high as about 83%, and its size dependent adaptive immune responses in mice. Ultimately, Ir3 elimi-\nonly increased to about 68.68 mm3. These results showed that Ir3 could nates the distant tumor while destroying the primary tumor. In sum-\neffectively inhibit distant tumors after PDT treatment, indicating that mary, the results suggest that Ir3 is a novel photosensitizer that targets\n\n\n\n\nFig. 5. (A) Inverted microscope and (B) transmission electron microscope images of MDA-MB-231 cells treated with Ir3 (0.4 \u03bcM) and PDT. Black arrows indicate\nareas where cellular vacuoles are released. Scale bar: 20 \u03bcm. (C) Expression of cleaved-caspase 3, GSDME, and GSDME-N proteins in MDA-MB-231 cells treated with\nIr3 (0.4 \u03bcM) and PDT.\n\n 6\n\fY.-S. Zhi et al. Journal of Inorganic Biochemistry 260 (2024) 112695\n\n\n\n\nFig. 6. (A) CLSM images and (B) flow cytometry analysis of ecto-CRT in MDA-MB-231 cells treated with Ir3 (0 \u03bcM, 0.1 \u03bcM, 0.2 \u03bcM, and 0.4 \u03bcM) + PDT (15 mW/cm2,\n15 min). Scale bar: 20 \u03bcm. ecto-CRT: Ex = 633 nm, Em = 650 \u00b1 30 nm; Hoechst 33342: Ex = 405 nm, Em = 430 \u00b1 20 nm. (C) CLSM images of HMGB1 in MDA-MB-\n231 cells treated with Ir3 (0 \u03bcM, 0.1 \u03bcM, 0.2 \u03bcM, and 0.4 \u03bcM) + PDT (15 mW/cm2, 15 min). Scale bar: 20 \u03bcm. HMGB1: Ex = 488 nm, Em = 520 \u00b1 20 nm; Hoechst\n33342: Ex = 405 nm, Em = 430 \u00b1 20 nm. (D) ATP levels in cell culture supernatants after Ir3 and PDT treatment (425 nm, 15 mW/cm2, 10 min). *p < 0.05, **p <\n0.01, ***p < 0.001.\n\n\nthe ER and generates an immunomodulatory response through ICD. This Tie Chen: Writing \u2013 original draft, Investigation, Formal analysis. Bin-\nis a promising photodynamic immunotherapy strategy. Fa Liang: Investigation. Shan Jiang: Investigation. Da-Hong Yao: Re-\n sources. Zhen-Dan He: Resources. Chen-Yang Li: Resources, Funding\nCRediT authorship contribution statement acquisition. Liang He: Writing \u2013 review & editing, Resources, Funding\n acquisition. Zheng-Yin Pan: Writing \u2013 review & editing, Supervision,\n Yun-Shi Zhi: Writing \u2013 original draft, Investigation, Formal analysis. Funding acquisition, Conceptualization.\n\n\n 7\n\fY.-S. Zhi et al. Journal of Inorganic Biochemistry 260 (2024) 112695\n\n\n\n\nFig. 7. In vivo anti-tumor efficacy and anti-tumor immune response. (A) Volume changes of primary and distant tumors: (black dot) Ctrl + dark, (red dot) Ctrl +\nlight, (blue dot) Ir3, (pink dot) Ir3 + light, (n = 3). (B) The photographs of the tumor collected at the end of the therapeutic period. In vivo anti-tumor immune\nresponse. (C) Expression of CD 80+ and CD 86+ quantitatively detected by flow cytometry in vivo (n = 3). (D-E) Populations of CD4+ and CD8+ T cells in distant\ntumors (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, by Student\u2019s two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is\nreferred to the web version of this article.)\n\n\n\n 8\n\fY.-S. Zhi et al. Journal of Inorganic Biochemistry 260 (2024) 112695\n\n\nDeclaration of competing interest [17] X. Liu, Z. Zhang, J. Ruan, Y. Pan, V.G. Magupalli, H. Wu, J. Lieberman,\n Inflammasome-activated gasdermin D causes pyroptosis by forming membrane\n pores, Nature 535 (7610) (2016) 153\u2013158.\n The authors declare that they have no known competing financial [18] Y. Wang, W. Gao, X. Shi, J. Ding, W. Liu, H. He, K. Wang, F. Shao, Chemotherapy\ninterests or personal relationships that could have appeared to influence drugs induce pyroptosis through caspase-3 cleavage of a gasdermin, Nature 547\nthe work reported in this paper. (7661) (2017) 99\u2013103.\n [19] C. Rogers, T. Fernandes-Alnemri, L. Mayes, D. Alnemri, G. Cingolani, E.S. Alnemri,\n Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to\nData availability secondary necrotic/pyroptotic cell death, Nat. Commun. 8 (2017) 14128.\n [20] H. Lu, S. Zhang, J. Wu, M. Chen, M.C. Cai, Y. Fu, W. Li, J. Wang, X. 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He, Ferroptosis\nShenzhen Natural Science Fund (the Stable Support Plan Program Photoinduced by new Cyclometalated iridium(III) complexes and its synergism\n20220811110339002), the Shenzhen Science and Technology Innova- with apoptosis in tumor cell inhibition, Angew. Chem. Int. Ed. 60 (15) (2021)\n 8174\u20138181.\ntion Program (JCYJ20230808105913028), Shenzhen Longgang District [24] W. Lin, Y. Liu, J. Wang, Z. Zhao, K. Lu, H. Meng, R. Luoliu, X. He, J. Shen, Z.\nScience and Technology Innovation Bureau (LGKCYLWS2021000001). W. Mao, W. Xia, Engineered Bacteria labeled with iridium(III) photosensitizers for\n enhanced photodynamic immunotherapy of solid tumors, Angew. Chem. Int. Ed.\n 62 (43) (2023) e202310158, https://doi.org/10.1002/anie.202310158.\nAppendix A. Supplementary data [25] M.M. Wang, F.J. Xu, Y.Y. Su, X.T. Geng, X.L. Qian, Y.Q. Xue, Z.H. Yu Kong, H.\n K. Liu, Z. 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