An endoplasmic reticulum-targeting iridium(iii) complex induces immunogenic cell death in melanoma cells and enhances anti-PD-1 immunotherapy by remodeling tumor microenvironment

{"full_text": " INORGANIC CHEMISTRY\n FRONTIERS\n\n View Article Online\n RESEARCH ARTICLE View Journal\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n An endoplasmic reticulum-targeting iridium(III)\n Cite this: DOI: 10.1039/d3qi00841j\n complex induces immunogenic cell death in\n melanoma cells and enhances anti-PD-1\n immunotherapy by remodeling tumor\n microenvironment\u2020\n Yi Rong,a Zhongxian Fan,b Zhijie Yu,a Li Wei,b Han Shen,a Huaiyi Huang, b\n\n Xiaojuan Hao,a,c Zizhuo Zhaod and Jinquan Wang *a\n\n One of the major challenges for immune checkpoint blockades (ICBs) lies in melanoma with limited T cell\n responses or immunologically \u201ccold\u201d tumors. Inspired by the immunogenicity of immunogenic cell death\n (ICD) that renders tumor cells sensitive to ICBs, in the present study, an endoplasmic reticulum (ER) tar-\n geting iridium(III) metal complex (IrC) was investigated as an ICD inducer. It was found that the IrC-treated\n tumor cells showed the hallmarks of ICD, including cell surface exposure of the endoplasmic reticulum\n protein calreticulin (CRT), secretion of high mobility group box 1 protein (HMGB1), and release of adeno-\n sine triphosphate (ATP). The vaccination of syngeneic immunocompetent mice with IrC-treated dying\n cells resulted in anti-tumor immunity with CD8+ T cell response, Foxp3+ T cell depletion, and memory\n immunity e\ufb00ect. Furthermore, tumor-bearing mice treated with IrC + anti-PD-1 combination therapy\n Received 6th June 2023, showed proin\ufb02ammatory cytokines secretion, increased dendritic cells (DCs) activation, and CD8+ T cell\n Accepted 17th July 2023\n in\ufb01ltration within the tumor, indicating that the combinatorial therapy reconstructs the tumor microenvi-\n DOI: 10.1039/d3qi00841j ronment (TME) and converts an immune \u201ccold\u201d tumor into a \u201chot\u201d one. This work provides a promising\n rsc.li/frontiers-inorganic strategy for cancer chemo-immunotherapy.\n\n\n\n\n Introduction rates of patients.3 The ICBs represented by anti-PD-1 anti-\n bodies (nivolumab and pembrolizumab) and anti-PD-L1 anti-\n Melanoma is the most dangerous type of skin cancer with the bodies (durvalumab and atezolizumab) are approved by the\n worst prognosis and the highest mortality rate.1 The tra- FDA of the United States and led to remarkable survival\n ditional treatments for melanoma include surgical resection, benefits in melanoma patients.4 However, adverse side e\ufb00ects\n chemotherapy, and radiotherapy, which have limited e\ufb03cacy and drug resistance are the main barriers for therapeutic e\ufb00ec-\n and are often accompanied with severe side e\ufb00ects.2 In recent tiveness. In particular, only 20\u201330% of clinical patients are\n years, new generation therapeutic drugs such as small mole- sensitive to ICBs immunotherapy, which severely limits their\n cule kinase targeting inhibitors and ICBs have been developed clinical use.5 Advances in knowledge about the pathophysiolo-\n and contributed to promote the quality of life and survival gical mechanisms of melanoma have revealed that the low\n response rate of melanoma to immunotherapy was due to the\n host immunosuppressive state.6 There was a lack of cytotoxic T\n a\n Guangdong Provincial Key Laboratory of Advanced Drug Delivery, School of lymphocytes (CTLs) in the tumor tissue of non-sensitive\n Bioscience and Biopharmaceutics, Guangdong Pharmaceutical University,\n patients, which showed immunosuppressive tumor microenvi-\n Guangzhou, 510006, P. R. China. E-mail: wangjinquan@gdpu.edu.cn\n b\n School of Pharmaceutical Science (Shenzhen), Shen Zhen Campus of Sun Yat-Sen ronment (TME), so-called the \u201ccold\u201d tumor.7 Therefore, how to\n University, Shenzhen, 518107, P. R. China activate the immune system of non-sensitive patients and turn\n \u201ccold\u201d tumors into \u201chot\u201d ones by remodeling the TME is the\n c\n Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang\n 325001, P. R. China key to improve the therapeutic e\ufb00ect of ICBs on melanoma.\n d\n Department of Ultrasound, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University,\n Recent studies suggest that some chemotherapeutic anti-\n Guangzhou, 510275, P. R. China\n \u2020 Electronic supplementary information (ESI) available. See DOI: https://doi.org/ tumor drugs, such as oxaliplatin,8 doxorubicin,9 and cyclopho-\n 10.1039/d3qi00841j sphamide,10 can induce ICD of tumor cells, which may trigger\n\n\n This journal is \u00a9 the Partner Organisations 2023 Inorg. Chem. Front.\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n robust antitumor immune responses.11 Tumor cells under- with the trifluoromethyl group showed potent cytotoxicity to\n going ICD release damage-associated molecular patterns cancer cells. Notably, IrC was mainly accumulated in the ER\n (DAMPs), which include cell surface exposure of CRT, secretion and induces ER stress. Inspired by these finding, we evaluate\n of ATP, and release of HMGB1.12 These DAMPs could activate the anticancer e\ufb03cacy and mechanism of IrC as an ICD\n immune system, increase the infiltration of CTLs into tumor inducer in the present study.\n tissue, transform \u201ccold\u201d tumor into \u201chot\u201d one, and thus In this work, we demonstrated that IrC could induce ICD\n enhance the e\ufb03cacy of ICBs for immunotherapy.13 Growing through ER stress pathway in melanoma cells. In addition to\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n evidence indicates that the immunomodulatory ability of che- the ROS generation, IrC treatment leads to T cell infiltration,\n motherapeutic drugs may be more important than their cyto- DCs maturation, and proinflammatory cytokines secretion\n toxicity, and the e\ufb00ect of ICD in vivo is independent of the involved with antitumor immunity activation. This promising\n cytotoxicity of chemotherapeutic drugs themselves.14 ICD induction strategy significantly converted tumor cells into\n Chemotherapeutic drugs with ICD e\ufb00ect can activate the anti- an endogenous vaccine and boosted the e\ufb00ects of anti-PD-1\n tumor immunity of the body and enhance the immunothera- (Scheme 1). These results provide a novel insight into the\n peutic e\ufb00ect of ICBs while reducing the toxicity of chemothera- mechanisms of iridium-based complex induced immunogeni-\n peutic drugs.15 Therefore, \u201cchemo-immunotherapy\u201d may city and establish a strategy to enhance immunotherapy by\n become the most promising modality for the treatment of combining metal-based anticancer agent with ICBs.\n melanoma.16\n The traditional understanding of classical metallic anti-\n cancer drugs represented by cisplatin was that it would induce Results and discussion\n immunosuppression of the body.17 However, accumulated evi-\n The photophysical properties of IrC in solution\n dence revealed that some metallic anticancer drugs such as\n oxaliplatin can evoke ICD and induce systemic anticancer The fluorinated tridentate ligand and IrC were prepared follow-\n immune-stimulation for tumor prevention.18 In addition, a ing our previous reported methods with minor modification\n large number of metal-based complexes including platinum(IV) (Fig. S1 and S2\u2020).44 According to the lipo-hydro partition coe\ufb03-\n complexes,19\u201324 ruthenium(II) complexes,25\u201328 iridium(III) cient results, IrC exhibited positive log P value (+1.45 \u00b1 0.08)\n complexes,29\u201334 copper(II) complexes,35 and gold which may enhance cell uptake e\ufb03ciency. IrC exhibited strong\n complexes 36,37\n are being tested as potential ICD inducers. absorption at 250\u2013270 nm, the luminescence emission\n Unfortunately, few of these metal-based ICD inducers have maxima of IrC was located at 615 nm (Fig. 1A). Upon exci-\n entered human clinical trials.17,38 Up to date, oxaliplatin is the tation, IrC showed more intense phosphorescence signal\n only metal-based anticancer drug known to induce ICD that (\u03bbex/em = 405/615 nm) in hydrophobic solvent dichloromethane\n has been used in the clinic.39 Therefore, it is necessary to (DCM) than in H2O. In addition, it is found that the emission\n design and screen novel anticancer metal complexes that can intensity of IrC in the presence of human serum albumin\n act as ICD inducers for immunotherapies of neoplastic (HSA) was enhanced significantly compared to that measured\n disease. Recently, Chao et al. reported that an Ir(III) complex in H2O (Fig. 1B). In addition, the stability of IrC in FBS was\n (Ir1) containing a bis(2-chloroethyl)-azane moiety acted as a assessed by HPLC, and no changes of IrC were observed after\n potential ICD inducer in non-small cell lung cancer (NSCLC). being incubated with FBS for 48 h (Fig. S3\u2020). These results\n Ir1 is the first iridium-based complex that can develop an demonstrated that the introduction of fluorine atom could\n immunomodulatory response by ICD.30 However, the synergis- enhance the interaction of protein and the hydrophobic micro-\n tic therapy in combination with ICBs has not yet been investi- environment such that the protein pocket will enhance the\n gated. Therefore, we propose that the combination of iridium- phosphorescent intensity of IrC.\n based ICD inducer and anti-PD-1 therapy may increase the\n immunogenicity of melanoma and enhance anti-PD-1 immu- IrC induces cytotoxicity, cell apoptosis, ROS and its cellular\n notherapy by reprograming its \u201ccold\u201d TME. localization in melanoma cells\n Fluorination is an important strategy for structure-based We examined the in vitro cytotoxic activity of IrC against\n medicinal chemistry and comprises a substantial proportion human melanoma A375 cells by MTT assay. As shown in\n of commercially available drugs such as Efavirenz, Fig. 2A, IrC displayed a high cytotoxicity towards the A375\n Gemcitabine, and others.40,41 Fluorinated compounds can cancer cells, with the half inhibitory concentrations (IC50)\n modulate the physicochemical and pharmacokinetic pro- values of 4.3 \u03bcM and 2.6 \u03bcM for 24 h and 48 h, respectively. In\n perties of drugs via improving the bioavailability, enhancing addition, CDDP (IC50 = 31.2 \u03bcM and 16.3 \u03bcM for 24 h and\n the selectivity and binding a\ufb03nity to cellular targets, and 48 h, respectively) was less e\ufb00ective than IrC under the same\n increasing the metabolic stability of drugs.42,43 However, condition, indicating that IrC has a better chemotherapeutic\n fluorinated metallic anticancer complex has been seldom profile than cisplatin.\n reported. Previously, our group have designed and synthesized To determine whether IrC and CDDP causes cell death by\n four novel Ir(III) complexes (Ir1\u2013Ir4). Ir1\u2013Ir3 showed photo- apoptosis, annexin V/PI co-staining assay was carried out. The\n induced catalytic anticancer activity.44 Interestingly, we annexin V positive staining in the cell surface is a hallmark of\n recently found that Ir4 (termed IrC in this work) coordinated early-phase apoptosis. In combination with the membrane-\n\n\n Inorg. Chem. Front. This journal is \u00a9 the Partner Organisations 2023\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n Scheme 1 Schematic illustration of IrC-induced ICD for sensitizing melanoma to PD-1 checkpoint blockade immunotherapy.\n\n\n\n\n by colocalization assay with ER-Tracker Green (ERTG), Mito-\n Tracker Green (MTG), and Lyso-Tracker Green (LTG) probe in\n A375 cells. The co-stain patterns of IrC matched poorly with\n those of LTG and MTG. Whereas, an excellent superimposition\n between the signals of IrC (red) and ERTG (green) was\n observed in the overlap images, with a Pearson correlation\n coe\ufb03cient of 0.86, indicating that IrC selectively localized in\n the ER (Fig. 2D). In addition, the cellular uptake of IrC was\n Fig. 1 Photophysical properties of IrC in solution. (A) UV-Vis and phos- quantified by ICP-MS. The results indicated that IrC preferen-\n phorescence spectra of IrC (50 \u03bcM) in H2O and DCM. (B)\n Phosphorescence spectra of IrC (50 \u03bcM) in the absence and presence of\n tially accumulated in the ER of A375 cells, which is consistent\n HSA (50 \u03bcM) in H2O, \u03bbex = 405 nm. with the results of the colocalization assay (Fig. 2C).\n It has been reported that some iridium(III) complexes could\n induce excessive reactive oxygen species (ROS) in tumor cells,\n which would lead to apoptosis.30 Therefore, we investigated\n impermeable DNA stain PI, one can use a flow cytometer to whether IrC could increase the ROS level in A375 cells using a\n distinguish viable cells and apoptosis cells.45 The results ROS probe (DCFH-DA). The strong green fluorescence signals\n showed an increased population in the AV+PI\u2212 and AV+PI+ were observed in the cells treated with IrC, indicating the gene-\n regions after being treated with IrC or CDDP for 24 h. Due to ration of ROS. On the contrary, the control cells that were incu-\n that cells were treated with the dose at the IC50 of IrC (4 \u00b5M) bated with PBS showed no fluorescence. The cells treated with\n or CDDP (30 \u03bcM), similar apoptotic rates were obtained CDDP showed a weak green fluorescence, indicating that\n (Fig. 2B). The results of annexin V/PI co-staining assay CDDP could induce a small amount of ROS in A375 cells\n suggested that the toxicity of IrC and CDDP is attributed to (Fig. 2E). In addition, an increase of ROS was detected by flow\n cell apoptosis. cytometry after IrC-treatment, which is consistent with the\n In order to further investigate the mechanism of the cellu- results observed by CLSM (Fig. 2F). These data suggested that\n lar toxicity of IrC, the intracellular distribution of IrC was the toxicity of IrC likely results from the intracellular ROS\n observed under a confocal laser scanning microscope (CLSM) generation.\n\n\n This journal is \u00a9 the Partner Organisations 2023 Inorg. Chem. Front.\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n Fig. 2 IrC induces cytotoxicity, cell apoptosis, and ROS, and its cellular localization in A375 melanoma cells. (A) Half inhibitory concentrations (IC50)\n of IrC and CDDP in A375 cells. (B) Apoptosis determined by \ufb02ow cytometry with annexin V/PI co-staining assay. (C) Cellular uptake of IrC in the\n nucleus, lysosomes, mitochondria and ER of A375 cells was quanti\ufb01ed by ICP-MS. (D) Co-localization of IrC in A375 cells by CLSM. MTG: Mito-\n Tracker\u2122 Green, ERTG: ER-Tracker\u2122 Green, LTG: Lyso-Tracker\u2122 Green. (E) IrC and CDDP induces ROS generation in the A375 cells detected by\n CLSM. (F) Detection of ROS by \ufb02ow cytometry. Scale bar: 50 \u00b5m.\n\n\n\n\n IrC induces ICD in melanoma cells through ER stress\n The subcellular target determines the initial interaction after\n the drug was up taken by cells. Due to the fact that IrC prefer-\n entially accumulated in the ER of the cells, and induced ROS\n in ER, we wonder if IrC could damage the ER and initiate ER\n stress. Thus, two ER stress related proteins, p-eIF2\u03b1 and CHOP\n were examined by western blot. The results showed that\n p-eIF2\u03b1 and CHOP were up-regulated after the cells were\n treated with IrC. In contrast, only slight up-regulation of\n p-eIF2\u03b1 and CHOP was observed in the CDDP treated cells\n (Fig. 3A). These data indicated that IrC could induce ER stress,\n which is a critical factor in the signaling cascades leading to\n ICD. Furthermore, the hallmarks of ICD, namely DAMPs,\n including CRT, HMGB1 and ATP were investigated. The extra-\n cellular HMGB1 in the medium supernatant was detected by\n western blot. The release of HMGB1 from A375 cells treated\n with IrC (4 \u03bcM) was increased greatly, about 5 times as high as\n that in the CDDP treated cells (Fig. 3B). The surface exposure\n of CRT was detected by immunofluorescence and observed by\n CLSM. CRT on the cell surface of A375 cells was identified by\n green fluorescence after being treated with IrC (4 \u03bcM) for 24 h.\n However, only very weak fluorescence signal was detected in\n CDDP-treated A375 cells, which suggested the failure of CDDP\n to induce ICD in melanoma cells (Fig. 3C). In addition, an Fig. 3 Hallmarks of ER stress and ICD in A375 cells treated with IrC\n earlier time point of 12 h was performed to quantify the CRT (4 \u03bcM) or CDDP (30 \u03bcM). (A) The expression of p-eIF2\u03b1 and CHOP. (B)\n The extracellular HMGB1 in the medium supernatant was detected by\n exposure by flow cytometry after being treated with IrC (4 \u03bcM)\n western blot. (C) CLSM images of CRT on cell surface. Scale bars:\n or CDDP (30 \u03bcM). The IrC group (24.00%) showed significant 50 \u00b5m. (D) The quantitative evaluation of CRT exposure was detected by\n increase of CRT positive cells compared to the CDDP group \ufb02ow cytometry. (E) The extracellular release of ATP after IrC and CDDP\n (6.49%) and the control group (1.05%), which is in accordance treatment. *** = P < 0.001, compared with the control group.\n\n\n\n Inorg. Chem. Front. This journal is \u00a9 the Partner Organisations 2023\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n with the results obtained by CLSM (Fig. 3D). ATP is an impor- these assays to examine ICD related DAMPs can only identify\n tant molecule that is secreted by cells during the process of IrC as an ICD inducer in vitro. To further explore the possibility\n ICD.46 Thus, the extracellular release of ATP from A375 cells that IrC promotes ICD in mice models, we also applied the\n was measured by a commercial ELISA detection kit. The ATP same set of experiments to the murine origin melanoma cells\n secretion from A375 cells after being treated with IrC (4 \u03bcM) B16-F10 (Fig. S5\u2013S7\u2020). Encouragingly, the data suggested that\n was 4.6 times as high as that of the control cells. In compari- IrC induced ICD in B16-F10 cells with similar results to A375\n son, no significant di\ufb00erence of ATP secretion was observed cells. Therefore, we next performed in vivo experiments to\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n between CDDP (30 \u03bcM) treated cells and control cells (Fig. 3E). examine the immune activation e\ufb00ect of IrC using B16-F10\n To approximate the strength of the immune response of IrC, cells.\n we compared and quantified the immunogenic cell death-\n related hallmarks to a known ICD inducer, oxaliplatin. The\n A375 cells were treated with IrC (6 \u03bcM) or oxaliplatin (70 \u03bcM) IrC induces ICD in a syngeneic mouse model\n for 12 hours at the IC50, respectively (Fig. S4A\u2020). The ATP The gold standard for evaluating the ability of a chemical\n secretion of IrC group was 3.5 times as high as that of the agent as a bona fide ICD inducer requires vaccination experi-\n control group, and the oxaliplatin group was 3.7 times as high ment with an immunocompetent mouse model.17 Therefore,\n as that of the control group (Fig. S4B\u2020). Additionally, the quan- we evaluated the ICD e\ufb00ect of IrC in a syngeneic mouse model\n titative analysis of western blot showed that the release of by vaccination experiment using immunocompetent C57BL/\n HMGB1 increased significantly in the IrC group (9.2 times) 6 mice.30 The mice of IrC group and CDDP group were injected\n and oxaliplatin group (9.4 times) compared to the control with the dying B16-F10 tumor cells treated with IrC or CDDP,\n group (Fig. S4C\u2020). In comparison, the percentage of CRT posi- separately. The mice of the control group were injected with\n tive cells in the IrC group (26.2%) and oxaliplatin group the B16-F10 cells treated with the solvent. A week later, all the\n (24.2%) showed significant increase compared the control mice from these three groups were rechallenged by injecting\n group (0.87%), which is in accordance with the results the same type of live tumor cells (Fig. 4A). Tumor volume was\n obtained by CLSM (Fig. S4D and E\u2020). Collectively, the results monitored for 15 days, as shown in Fig. 4B\u2013D. Vaccination\n of the ATP secretion, HMGB1 release, and CRT translocation with cells treated with IrC showed pronounced tumor rejection\n demonstrated that IrC could induce e\ufb00ective ICD in the A375 in immunized mice of IrC group at the end of vaccination\n cells in vitro. Notably, IrC and oxaliplatin have a similar capa- experiment on the day 15. However, no statistical di\ufb00erence of\n bility to induce ICD. Accumulating evidence revealed that tumor inhibition was observed between CDDP and control\n these signaling molecules known as DAMPs could potentiate group. Histological examination (H&E) of the tumors that were\n systemic antitumor immune responses. collected from the mice confirmed these results. Tumor\n For example, ATP released by tumor cells acts as a \u201cfind tissues from IrC group displayed severe alterations, including\n me\u201d signal, which enhances the tumor recognition ability of nucleus fragmentation and breakdown of cytoplasmic struc-\n immune cells.47 CRT, as the \u201ceat me\u201d signal, can enhance anti- ture, which indicated necrosis or apoptosis of the tumor cells.\n tumor immunity,48 and HMGB1, as a binder to immune cells, However, slightly damage was observed in the tumors of CDDP\n can promote the recovery of immune surveillance.49 However, group (Fig. 4E).\n\n\n\n\n Fig. 4 B16-F10 melanoma tumor vaccine experiment in C57BL/6 mice. (A) Schematic of the ICD vaccine experiment. (B) Representative photo-\n graphs in vaccinated mice following di\ufb00erent treatments. (C) Individual tumor growth curves of the rechallenged mice with di\ufb00erent vaccination. (D)\n Average tumor growth curves of the rechallenged mice with di\ufb00erent vaccination. (E) H&E staining results of tumor slices collected from mice in\n each group. *** = P < 0.001, compared with the control group.\n\n\n\n This journal is \u00a9 the Partner Organisations 2023 Inorg. Chem. Front.\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n In addition, the body weight of mice in these three groups\n showed no significant di\ufb00erence throughout the experiments\n (Fig. S8\u2020). The H&E staining of the main organs (heart, liver,\n spleen, lungs, kidneys) showed no significant histological\n damage in all treated groups (Fig. S9\u2020). Taken together, these\n results demonstrate that IrC could promote ICD induction in\n syngeneic mouse model in vivo.\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n ICD e\ufb00ect induced by IrC could induce systemic immune\n stimulation\n Encouraged by the robust local antitumor immune responses\n in the vaccination experiment, we next investigated if the ICD\n e\ufb00ect induced by IrC could induce systemic immune stimu-\n lation. The tumor-draining lymph nodes (TDLNs) and tumors\n were retrieved from the mice with vaccination experiment and\n the immune cells were isolated and analyzed by flow\n cytometry.\n As illustrated in the Fig. 5A and B, the IrC group (29.1%)\n showed significant increase of the matured DCs\n (CD80+CD86+) in TDLNs compared to the CDDP group\n (12.0%) and the control group (17.6%) with statistical di\ufb00er-\n ence. These results suggested that IrC could promote DCs\n maturation in the lymph nodes, which play a key role in initi-\n ating and regulating the innate and adaptive immunity. In\n addition, the presence of CD8+ T cells, termed as cytotoxic T\n lymphocytes (CTLs) in the tumor tissue of the mice were Fig. 5 The ICD e\ufb00ect induced by IrC induced systemic immune stimu-\n lation in the mice in the vaccine experiment. (A) The presence and the\n quantified by flow cytometry. The proportion of CD8+ T cells\n quanti\ufb01cation (B) of mature DC induced by ICD e\ufb00ect in tumor-draining\n in the IrC group (25.8%) was much higher than that in the lymph nodes of the mice. (C) The presence and the quanti\ufb01cation (D) of\n CDDP group (17.2%) and the control group (12.8%) (Fig. 5C CD8+ T cells in the tumors of the mice. (E) Immuno\ufb02uorescence images\n and D). Furthermore, immunofluorescence analysis showed (red: CD8+; blue: Hoechst 33324) and the quanti\ufb01cation analysis (F) of\n the most enrichment of CD8+ T cells in the tumor tissue of the number of CD8+ T cells. (G) Immuno\ufb02uorescence images (green:\n Foxp3+; blue: Hoechst 33324) and the quanti\ufb01cation analysis (H) of the\n IrC group (Fig. 5E and F), indicating the e\ufb00ective infiltration\n number of Foxp3+ cells. Concentration of the TNF-\u03b1 (I), IFN-\u03b3 (J), and\n of CTLs into the tumors. The immunosuppressive cells, IL-10 (K) in the serum of the mice in di\ufb00erent groups. Data from 3 bio-\n Foxp3+ T cells in the tumor tissue of the mice were also deter- logically independent samples for B, D, F, and H. Scale bars: 100 \u03bcm.\n mined. As shown in Fig. 5G and H, the number of Foxp3+ T Data from 4 biologically independent samples for H, I, and J. ** = p <\n cells in the IrC group was much less than that of CDDP 0.01; *** = p < 0.001, compared with the control group.\n group and control group. To further determine the systemic\n immune response, we evaluated the cellular cytokines in the\n serum of the mice in di\ufb00erent groups. The levels of proin-\n flammatory cytokines (TNF-\u03b1 and IFN-\u03b3) in serum of IrC PBMC and spleen from the mice at the end of vaccination\n group were significantly higher than that of CDDP group and experiment. The antitumor capacity of the immune memory\n control group (Fig. 5I and J). The concentration of anti- cells was evaluated by co-culturing the PBMC with B16-F10\n inflammatory cytokine IL-10 of IrC group was lower than that cells in vitro. The cytotoxic e\ufb00ect of PBMC on B16-F10 cells was\n of CDDP group and control group (Fig. 5K). Collectively, these determined by calcein acetoxymethyl (Calcein-AM) staining\n data demonstrated that the ICD e\ufb00ect induced by IrC could assay. The intracellular nonfluorescent Calcein-AM can be con-\n induce systemic immune stimulation by regulating the verted into the intensely green fluorescent calcein by the ester-\n immune cells and immune molecules. ase in living cells, so the cell viability could be quantified by\n the fluorescence intensity of the target cells.51 The PBMC from\n the IrC group exhibited much higher relative cytotoxicity\n IrC elicits antitumor immunological memory against tumor cells than that of CDDP group and the control\n In general, the central memory T cells (TCM) provide immune group (Fig. 6A). The flow cytometric analysis indicated that the\n protections after antigen stimulation, whereas the e\ufb00ector proportions of TEM and TCM in the PBMC of IrC group showed\n memory T cells (TEM) induce immediate protections by produ- markedly increase compared with CDDP group and control\n cing proinflammatory cytokines, such as IFN-\u03b3.50 In order to group (Fig. 6B and C). In addition, the percentages of TEM and\n evaluate whether the IrC-induced systemic immune responses TCM cells in the spleens showed similar results as that in the\n were durable, we further analyzed the TCM and TEM in the PBMC (Fig. S10\u2020). These data demonstrated the immune\n\n\n Inorg. Chem. Front. This journal is \u00a9 the Partner Organisations 2023\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n Fig. 6 IrC triggered immunological memory in the vaccinated C57BL/6 mice. (A) The cytotoxic e\ufb00ect of PBMC on B16-F10 cells was quanti\ufb01ed by\n Calcein-AM staining assay. (B) Proportions of TEM cells (CD3+CD8+CD44+CD62L\u2212) in the PBMC. (C) Proportions of TCM cells\n (CD3+CD8+CD44+CD62L+) in the PBMC. (D) PBMC (Hoechst) co-culture with B16-F10 cells (Calcein-AM) observed by CLSM (scale bar = 50 \u00b5m) and\n SEM (scale bar = 5 \u00b5m). * = p < 0.05; ** = p < 0.01; compared with the control group.\n\n\n\n\n memory e\ufb00ect in the mice of IrC group. The B16-F10 cells were IrC or PD-1 treatment alone, and CDDP + PD-1 groups,\n stained by Calcein-AM and PBMC were stained by Hoechst induced a statistical tumor growth delay versus control group.\n 33324, and observed under CLSM. After being co-cultured for These three groups showed similar inhibition of tumor\n 4 h, the number of PBMC around the tumor cells in the IrC growth. Moreover, compared with the control group, IrC +\n group showed significant increase. However, only few numbers PD-1 group showed the most significant tumor inhibitory\n of PBMC were observed around the target cells in the CDDP e\ufb00ect during the 12 days observation period (Fig. 7A and B).\n group and control group. In addition, scanning electron Histological examination of the tumors slices from di\ufb00erent\n microscopy (SEM) was used to observe the cell interactions, groups confirmed these results. The tumor tissues of the\n most PBMC tightly bound to the tumor cells in the IrC group, CDDP group were partially damaged. In addition, the tumor\n which is in accordance with the results obtained by CLSM tissues of the IrC or PD-1 treatment alone, and CDDP +\n (Fig. 6D). These results of the co-culture experiments not only PD-1 groups showed moderated damage. Moreover, a large\n support the vaccination experiment, but also confirm that the proportion of the tumor tissues was noted to have displayed\n memory immunity e\ufb00ect induced by IrC is involved in antitu- shrunken cells without nucleus in the IrC + PD-1 group, which\n mor immune responses. reflects severe necrosis or apoptosis of the tumor cells\n (Fig. 7D). Furthermore, in corresponding to H&E results, the\n most amount of green fluorescence signals of the TUNEL assay\n IrC enhances anti-PD-1 immunotherapy in vivo were observed in the IrC + PD-1 group, indicating the highest\n Recent studies revealed that some tumors show striking fea- proportion of apoptotic cells in the tumor tissues (Fig. 7E). In\n tures of CTLs absence, which are known as immunologically addition, signs of toxicity, such as emaciation and decreased\n \u201ccold\u201d, and do not respond to ICBs.52 However, our results body weight were observed in the mice of CDDP and CDDP +\n indicated that IrC may convert the immunosuppressive \u201ccold\u201d PD-1 groups (Fig. 7C). Additionally, histopathology examin-\n tumors into immune-responsive \u201chot\u201d ones and enhance ation showed proximal tubular cell degeneration and kidney\n tumor response to PD-1 checkpoint blockade immunotherapy. glomerulus atrophy in these two groups (Fig. 7F), which was\n Therefore, we further examined whether IrC could boost the inconsistent with the previous report.53 On the contrary, no\n anti-tumor immunity of PD-1 inhibitors using a poorly immu- body weight loss and other severe side e\ufb00ects were observed in\n nogenic B16-F10 melanoma model in vivo. CDDP was selected the mice of IrC, PD-1, and IrC + PD-1 groups. The histological\n as a control agent because it does not have ICD-inducing analysis of the other main organs (liver, heart, spleen and\n ability in B16-F10 melanoma. The results showed that the lungs) showed no significant pathological lesion in all treated\n CDDP treatment alone only slightly suppressed tumor growth. groups (Fig. S11\u2020). In addition, the hemolytic analysis\n\n\n This journal is \u00a9 the Partner Organisations 2023 Inorg. Chem. Front.\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n Fig. 7 The therapeutic e\ufb00ect on B16-F10 tumor bearing mouse model with di\ufb00erent treatment strategies. (A) Individual tumor growth curves of the\n mice. (B) Average tumor growth curves of the mice. (C) Average body weight of the mice in each group after removing the tumor. (D) H&E staining\n results of tumor slices collected from the mice. (E) TUNEL staining (green: TUNEL positive; blue: DAPI) results of tumor slices collected from mice.\n (F) H&E staining results of kidney slices collected from the mice in each group. Scale bars: 100 \u00b5m. Data represent mean \u00b1 SEM (n = 6), * = p < 0.05,\n ** = p < 0.01; *** = p < 0.001, compared with the control group.\n\n\n\n showed that the incubation of red blood cells (RBCs) with IrC IrC reconstructs the TME immune phenotype of \u201ccold\u201d tumor\n did not cause any hemolysis at the treatment concentration into \u201chot\u201d one\n (Fig. S12\u2020), confirming the good hemocompatibility of the IrC.\n Collectively, IrC could enhance the anti-tumor immunity of To further confirm if IrC could remodeling the TME of the\n PD-1 in B16-F10 tumor-bearing C57BL/6 mice and possesses poorly immunogenic melanoma model, we exanimated the\n relatively high biosafety in vivo. CTLs (CD8+ T cells) and the immunosuppressive cells (Foxp3+\n\n\n\n\n Fig. 8 Assessment of the immune status of the tumor-bearing mice with di\ufb00erent treatment. (A) Immuno\ufb02uorescence images (red: CD8+; blue:\n DAPI) of CD8+ T cells in the tumor of the mice in each group. (B) Immuno\ufb02uorescence images (green: Foxp3+; blue: DAPI) of Foxp3+ cells in the\n tumor of the mice in each group. (C) Photographs of the spleens retrieved from the mice in each group. (D) The proportion of CD8+/Foxp3+ in the\n tumor tissues of the mice in each group. Concentration of the TNF-\u03b1 (E), IFN-\u03b3 (F), and IL-10 (G) in the serum of the mice in di\ufb00erent group. Data\n from 4 biologically independent samples. Scale bars: 100 \u03bcm. ** = p < 0.01; *** = p < 0.001, compared with the control group.\n\n\n\n Inorg. Chem. Front. This journal is \u00a9 the Partner Organisations 2023\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n T cells) in the tumor tissues of the mice in di\ufb00erent groups. As provide a rationale for combining metal drug chemotherapy\n shown in Fig. 8A, there were almost no CD8+ expressing CTLs with immunotherapy in clinics.\n infiltrated in the tumors of the control group, which was a\n symbol of \u201ccold\u201d tumors. However, the red fluorescence\n signals of CD8+ T cells were observed the most in the IrC + Experimental section\n PD-1 group, indicating the highest level of CD8+ positive cells\n Materials and general instrumentation\n infiltration in the tumor tissues and the ability of IrC to\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n enhance the local immune responses. Whereas, few CD8+ T Iridium complex (IrC) [Ir( ppy)2(L)]Cl, where ppy = 2-phenylpyr-\n cells were observed in the tumors of CDDP and CDDP + idine, L = 4\u2032-(3,5-bis(trifluoromethyl)phenyl)-2,2\u2032:6\u2032,2\u2033-terpyri-\n PD-1 groups, possibly due to the insu\ufb03cient ICD-inducing dine was prepared and characterized according to our previous\n ability of CDDP. The abundance of Foxp3+ T cells represent the report.44 All reagents and solvents from commercial sources\n suppression of immune responses in the tumor and the CD8+/ were of analytical grade unless otherwise noted. Iridium(III)\n Foxp3+ ratio is a critical parameter to evaluate the antitumor chloride trihydrate was purchased from Kunming Institute of\n immune responses.52 As expected, only few green fluorescence Precious Metal. Cisplatin (CDDP), oxaliplatin, 3-(4,5-dimethyl-\n Foxp3+ signals were observed in the tumor tissues from the IrC thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst\n + PD-1 group (Fig. 8B). Furthermore, the IrC + PD-1 group 33342 were purchased from Sigma-Aldrich (USA). Mito-\n showed the highest proportion of CD8+/Foxp3+ in all the tracker\u2122 Green, Lyso-Tracker\u2122 Green, ER-Tracker\u2122 Green,\n treated groups (Fig. 8D). These data indicated that IrC were purchased from Thermo (USA). Reactive oxygen species\n mediated ICD combined with PD-1 therapy significantly assay kit and calcein acetoxymethyl (Calcein-AM) cell viability\n reduced tumor-associated immunosuppression. Notably, the assay kit were purchased from Beyotime (China). Calreticulin\n B16-F10 tumor bearing mice of the IrC treated group showed (D3E6) XP\u00ae Rabbit mAb (Alexa Fluor\u00ae 488 Conjugate) was pur-\n swollen spleen, indicating that the IrC-induced tumor ablation chased from CST (USA). ENLITEN\u00ae ATP assay system bio-\n in vivo is a proinflammatory process, which was inconsistent luminescence detection kit was purchased from Promega\n with the recent report.54 Interestingly, the IrC + PD-1 treatment (USA). IFN-\u03b3, TNF-\u03b1, and IL-10 ELISA kit were purchased from\n could make the swollen spleen of the mice return to normal Multi-sciences (China). \u03b1PD-1 antibody (RMP1\u201314) was pur-\n level after treatment, indicating that combination treatment chased from Bioxcell (USA). TUNEL staining kit was purchased\n group showed better body tolerance, which is important for from Biossci (USA). Mouse peripheral blood lymphocyte\n the safe use in the clinic (Fig. 8C). The ELISA assay results (PMBC) isolation kit, mouse tumor infiltrating lymphocyte iso-\n showed that the contents of proinflammatory cytokines IFN- \u03b3 lation kit and heparin sodium salt were purchased from\n and TNF-\u03b1 in the serum were substantially increased in the Solarbio (China). The inductively coupled plasma mass spec-\n mice of IrC or PD-1 treatment alone groups. Moreover, the IrC trometry (ICP-MS) experiments were carried out on an Agilent\n + PD-1 treatment group showed the most significant increase 7700x instrument (Thermo Elemental Co., Ltd).\n compared with the control group (Fig. 8E and F). In addition,\n Characterization of UV-Vis spectroscopy, phosphorescence\n the content of immunosuppressive cytokine IL-10 of all the\n spectroscopy, and log Po/w measurement\n treatment groups was lower than that of control group\n (Fig. 8G). These results demonstrated that the immune system The UV-Vis spectra of IrC (50 \u03bcM) were obtained at room temp-\n of IrC + PD-1 group is e\ufb00ectively activated, and the IrC + PD-1 erature from 750 to 250 nm with a Yoke T3202S UV-Vis spectro-\n treatment could turn the \u201ccold\u201d tumor into a \u201chot\u201d one due to photometer. The phosphorescence emission measurements of\n the induction of inflamed microenvironment. IrC (50 \u03bcM) in diverse solvents and human serum albumin\n (HSA) (50 \u03bcM) were obtained by a Techcomp FL970 fluo-\n rescence spectrophotometer. log Po/w measurement was per-\n formed as followed, IrC was added to pre-saturated water and\n pre-saturated n-octanol solution with the final concentration\n Conclusions of IrC was 100 \u03bcM, then the mixture was shaken for 24 h at\n We successfully demonstrated that IrC could induce ICD in room temperature. The absorbance of the oil/water phase in\n melanoma cells through ER stress pathway. The IrC induced- acetonitrile solution was measured by a UV-Vis spectrophoto-\n ICD e\ufb00ect not only rejected tumor rechallenge by B16- meter after stationary. The partition coe\ufb03cient of IrC was cal-\n F10 melanoma but also generated antitumor memory immune culated from the equation log Po/w = log(Co/Cw), where Co/Cw\n response. Furthermore, the IrC and anti-PD-1 combination represents the concentration of the IrC in the octanol/water\n therapy e\ufb03ciently remodeled the tumor-immunosuppressive phase.\n microenvironment, including more CD8+ T cell infiltration,\n DCs maturation, and the secretion of antitumor associated Stability in FBS\n cytokines, therefore, turning immunosuppressive \u201ccold\u201d tumor The stability test of IrC in fetal bovine serum (FBS) was per-\n to immune-responsive \u201chot\u201d one and potentiating the anti- formed as previously described.30 IrC (10 \u03bcL, 10 \u03bcM, from\n tumor e\ufb00ect of PD-1 inhibitors. In addition, IrC showed no stock solution 1 mM in DMSO) and diazepam (10 \u03bcL, 10 \u03bcM,\n adverse e\ufb00ects in the in vivo experiment. This study will internal reference) were added to FBS (980 \u03bcL) to total volume\n\n\n This journal is \u00a9 the Partner Organisations 2023 Inorg. Chem. Front.\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n of 1000 \u03bcL. The solution was shaken (\u223c300 rpm) and incu- Annexin V/PI co-staining assay was performed by flow cyto-\n bated for 0 or 48 hours at 37 \u00b0C respectively. After that, the metry according to the manufacturer\u2019s protocol.30 Briefly, cells\n samples were added 2 mL of acetonitrile, then the mixture was were seeded in six-well culture plates and incubated with IrC\n centrifuged (\u00d71000g) for 45 min at 4 \u00b0C. The supernatants were (4 \u00b5M) or CDDP (30 \u03bcM) for 24 h at 37 \u00b0C in a 5% CO2 incuba-\n collected and solvent was evaporated. Then the residue was tor. After treatment, the cells were trypsinized, washed twice\n suspended in 200 \u03bcL of ethanol and was filtered. The samples with cold PBS, and resuspended in 100 \u00b5L of 1\u00d7 binding bu\ufb00er\n were analyzed by HPLC-UV with a C18 reverse phase column. (50 mM HEPES/NaOH, pH 7.4, 700 mM NaCl, 12.5 mM CaCl2).\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n Then 5 \u03bcL of annexin V-FITC and 1 \u03bcL of PI were added to the\n Cell lines and cell culture cell suspension. After incubation for 15 min at room tempera-\n A375 human melanoma cells and B16-F10 mouse melanoma ture, another 400 \u00b5L of binding bu\ufb00er was added. Then the\n cells were obtained from the Shanghai Cell Institute (Cell stained samples were examined using the flow cytometer.\n Bank, Sinica Academia Shanghai, China). These two cell lines\n were confirmed based on DNA detection techniques by detect- Intracellular reactive oxygen species (ROS) measurement\n ing of short tandem repeats (STRs). Cells were cultured in\n Accumulation of ROS was detected using the DCFH-DA probe\n DMEM supplemented with 10% fetal bovine serum and peni-\n according to our previously report.55 The cells were treated\n cillin (100 U mL\u22121)/streptomycin (50 U mL\u22121). Cells were used\n with IrC (4 \u03bcM) or CDDP (30 \u03bcM) for 24 h. Then cells were\n when they reached logarithmic growth phase.\n incubated in 10 \u00b5M DCFH-DA for 30 min and washed three\n Cellular localization and distribution times with PBS to remove DCFH-DA. The cells were trypsi-\n nized, washed twice with cold PBS, then the samples were\n Melanoma cells were cultured in a 35 mm2 glass-bottom\n quantified by flow cytometry. In addition, the cells were\n dishes (Corning) and incubated with IrC (4 \u03bcM) for 24 h. A\n seeded in a 35 mm2 glass-bottom dishes for assessment by\n mitochondria-specific green fluorescent probe Mito-Tracker\n CLSM.\n Green (MTG, 5 \u03bcg mL\u22121), or a lysosome-specific green fluo-\n rescent probe Lyso-Traker Green (LTG, 5 \u03bcg mL\u22121), or an endo-\n plasmic reticulum-specific green fluorescent probe ER-Traker ER stress, cell surface CRT, extracellular HMGB1 and ATP\n Green (ERTG, 5 \u03bcg mL\u22121) and nuclei stain probe Hoechst Western blot was performed to detect the ER stress relative\n 33342 (10 \u03bcg mL\u22121) were then added to the dishes and incu- protein phospho-eIF-2\u03b1 ( p-eIF2\u03b1, 38 kDa), C/EBP homologous\n bated for 30 min in the dark. The cells were then washed three protein (CHOP, 27 kDa), and the ICD hallmark HMGB1\n times with PBS before the samples were observed under a con- (30 kDa). Briefly, melanoma cells were treated with IrC (4 \u03bcM)\n focal laser scanning microscope (CLSM, Carl Zeiss 880, or CDDP (30 \u03bcM) for 24 h. Then the cells were collected and\n Germany). ER-Tracker Green (\u03bbex = 488 nm, \u03bbem = 504\u2013511 nm), washed twice with ice-cold PBS and lysed in RIPA bu\ufb00er to\n Lyso-Traker Green (\u03bbex = 488 nm, \u03bbem = 510\u2013540 nm), Mito- extract the total cellular proteins for detecting p-eIF2\u03b1 and\n Tracker Green (\u03bbex = 488 nm, \u03bbem = 510\u2013540 nm) and Hoechst CHOP. In addition, the cell culture supernatants were collected\n 33342 (\u03bbex = 405 nm, \u03bbem = 410\u2013450 nm). and freeze-dried for detecting extracellular HMGB1. The\n Inductively coupled plasma mass spectrometry (ICP-MS) routine western blot procedure was performed as described in\n was employed to quantify the cellular uptake of IrC, as our previous report.56 The antibody HMGB1 (ab79823) was\n described in our previous report.51 A375 cells were incubated obtained from Abcam (Cambridge, United Kingdom). CHOP\n with IrC (4 \u03bcM) for 24 h at 37 \u00b0C. After incubation, the whole (#35744), p-eIF2\u03b1 (#11279), GAPDH (#41549), and secondary\n cell, the mitochondria, endoplasmic reticulum, nuclear and antibody (#13012) were obtained from SAB (USA).\n cytoplasm fractions were obtained separately using mitochon- Immunofluorescence was performed to detect the cell\n drial, endoplasmic reticulum, nuclear and cytoplasm isolation surface CRT. Melanoma cells were seeded in a 35 mm2 glass-\n kits respectively. The extractions were subsequently digested bottom dishes and treated with IrC (4 \u03bcM) or CDDP (30 \u03bcM)\n by 60% HNO3 for 24 h and analyzed by ICP-MS. The data were for 24 h. Then the cells were incubated with Calreticulin\n reported as the mean \u00b1 standard deviation (n = 3) and the (D3E6) XP\u00ae Rabbit mAb (Alexa Fluor\u00ae 488 Conjugate) accord-\n results were reported based on the cell number. ing to the manufacturer\u2019s protocol. After being washed with\n PBS, the Hoechst 33342 was added and incubated for 15 min.\n The cytotoxicity and apoptosis assay After washing with PBS again, the samples were observed\n MTT assay was employed to measure the cytotoxicity of IrC under the CLSM. The excitation wavelength was 488 nm, the\n and CDDP on melanoma cells. Briefly, exponentially grown emission wavelength was 520 \u00b1 20 nm.\n A375 cells were seeded into 96-well culture plates at a density The quantitative evaluation of CRT exposure was detected\n of 1 \u00d7 104 cells per well and incubated at 37 \u00b0C for 24 h. Then by flow cytometry. Melanoma cells were treated with IrC (4 \u03bcM)\n cells were treated with a range of concentrations of IrC or or CDDP (30 \u03bcM) for 12 h. After the incubation, the cells were\n CDDP and incubated for 24 h or 48 h. The half-maximal trypsinized, and washed twice with cold PBS. Then the cells\n inhibitory concentration (IC50) values were determined by a were collected, fixed, and incubated with Calreticulin (D3E6)\n series of the inhibition rate evaluated by the MTT assay as XP\u00ae Rabbit mAb (Alexa Fluor\u00ae 488 Conjugate) according to\n described in our previous report.55 the manufacturer\u2019s protocol.30 Then the stained samples were\n\n\n Inorg. Chem. Front. This journal is \u00a9 the Partner Organisations 2023\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n examined using the flow cytometer, and the binned popu- Hematoxylin\u2013eosin (H&E) and immunofluorescence (IF)\n lations were expressed as % of total cells. staining\n The extracellular ATP was quantified using an ATP detection The tumors and major organs (including kidney, heart, lung,\n kit (ENLITEN\u00ae, Promega). Melanoma cells were treated as liver, and spleen) from the di\ufb00erent groups were collected after\n describe above, then the cell supernatants were transferred to the mice were sacrificed. Parts of the tumors and organs were\n the non-transparent bottomed white-walled 96-well plates. The embedded in para\ufb03n and sliced into 6 \u03bcm sections. For the\n chemiluminescence derived from ATP was measured and cal- morphological analysis, tumors and major organs sections\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n culated on a multifunction full wavelength scanner (Thermo, were stained with H&E by standard procedures including\n USA). depara\ufb03nization and rehydration (xylene, alcohol, water). For\n immunofluorescence assay, the sections of tumor were stained\n Animal models and therapeutic protocols with anti-CD8-FITC (ab217344), anti-Foxp3-FITC (ab215206)\n antibodies (Cambridge, United Kingdom), and images were\n Syngeneic C57BL/6 male mice aged 5 weeks were purchased analyzed by Zeiss Zen (bule edition) software.\n and bred in the experimental animal center, Guangdong\n Pharmaceutical University (GDPU). All experimental protocols Flow cytometric (FACS) analysis and ELISA assays\n received prior approval from the GDPU Animal Care and Use\n Committee (approval no.: gdpuspf 2017460-3). All surgical pro- DCs maturation and T lymphocyte infiltration were assessed\n cedures were performed under sterile condition using by FACS analysis. Distant tumors and tumor draining lymph\n anesthesia. nodes of the mice in the in vivo vaccination experiment were\n For in vivo vaccination experiment, the mice were randomly harvested and digested into single cell suspensions as pre-\n divided into three groups (n = 7), which were named as IrC viously described.57 Lymphocytes were isolated with a lympho-\n group, CDDP group, and control group. B16-F10 cells were cyte isolation kit. The single-cell suspension was stained with\n treated in vitro with, IrC (5 \u03bcM), CDDP (50 \u03bcM) and the solvent antibodies for CD8+ cytotoxic T cells (anti-mouse CD8-FITC,\n separately for 24 h. Then the treated cells were rinsed in PBS anti-mouse CD3-PerCP-Cy5.5) and for DC cells (CD86-PE \u2013\n and injected subcutaneously into the left flank of the mice in Cyanine7, anti-mouse CD80-FITC). All samples were analyzed\n each group. One week later, mice were re-challenged in the with a flow cytometer and FlowJo software.\n right flanks with live B16-F10 cells (2 \u00d7 106 cells per 100 \u03bcL, The quantification of cytokines TNF-\u03b1, IFN-\u03b3, interleukin-10\n sterile PBS/Matrigel, 1 : 1, v : v). Tumor volume (V) was (IL-10) in mice serum was performed by means of enzyme-\n measured every 3 days and calculated as V = (length \u00d7 width2)/ linked immunosorbent assay (ELISA), according to the manu-\n 2. facturer\u2019s instructions.\n For systemic therapy experiments, C57BL/6 male mice were\n injected in the right flank with B16-F10 (2 \u00d7 106 cells per Analysis of memory T cells and their cytotoxicity on B16-F10\n 100 \u03bcL, sterile PBS/Matrigel, 1 : 1, v : v) to establish a xenograft cells\n model. The mice with well grown tumors were randomly For analysis of memory T cells, the spleens of the mice of\n divided into six groups (n = 6): control group (PBS), CDDP di\ufb00erent treatment groups were collected and the single-cell\n group (6 mg kg\u22121), IrC group (5 mg kg\u22121), PD-1 group (10 mg suspensions were prepared as previously described.52 In\n kg\u22121), CDDP + PD-1 group (CDDP, 6 mg kg\u22121 + PD-1, 10 mg addition, the peripheral blood mononuclear cell (PBMC) of the\n kg\u22121), IrC + PD-1 group (IrC, 5 mg kg\u22121, + PD-1, 10 mg kg\u22121). mice of di\ufb00erent groups were collected using mouse PBMC\n Mice then received intraperitoneal injections of the corres- isolation kit at the end of vaccine experiment. The e\ufb00ector\n ponding drugs every three days for 4 times, and the PD-1 anti- memory T cells (TEM, CD3+CD8+CD44+CD62L\u2212) and central\n body was injected 24 hours later after the chemotherapy drugs memory T cells (TCM, CD3+CD8+CD44+CD62L+) were labeled\n were administered. Tumor volume (V) was measured every 3 with corresponding antibodies and analyzed by a flow\n days. cytometer.50\n For analysis the cytotoxicity of memory T cells, B16-F10\n cells were seeded in glass-bottom dishes for 24 h, then stained\n Hemolysis assay by calcein acetoxymethyl (Calcein-AM) for 30 min. PBMC were\n The blood (1 mL) was collected from healthy C57BL/6 mice stained by Hoechst 33324 for 30 min. Then PBMCs from\n under anesthesia. Then the sample was washed twice with di\ufb00erent group were added in the dishes and co-cultured with\n 4 mL normal saline and centrifuged (600g, 5 min). RBC the B16-F10 cells for another 4 h, with the ratio of PBMCs to\n deposit was obtained when the supernatant was colorless and B16-F10 cells being 25 : 1. The tumor cell viability was\n transparent. Then the RBC deposit was diluted with normal measured using the calcein AM cell viability assay kit (beyo-\n saline. Next, various concentrations of IrC (5, 10, and 20 \u03bcM) time, China) according to the manufacturer\u2019s instructions. The\n were added into the RBC suspension. Distilled water and relative cytotoxicity = (1 \u2212 cell viability)%.\n normal saline were used as positive control and negative The in vitro visualization images were obtained under a con-\n control, respectively. The samples were incubated in a 37 \u00b0C focal laser scanning microscope (CLSM, Carl Zeiss 880,\n water bath, and observed 2 h later. Germany). In addition, the samples were also observed by the\n\n\n This journal is \u00a9 the Partner Organisations 2023 Inorg. Chem. Front.\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n scanning electron microscopy (SEM) analysis as described in 2 H. S. Duarte, C. R. P. Veiga, C. P. Veiga, A. J. A. Wainstein\n the previous report.58 Briefly, the co-culture cells were fixed in and A. P. Drummond-Lage, Toxicity profile of treatment\n 2.5% glutaraldehyde ( pH 7.4) for 2 h and postfixed with 1% with PD-1 inhibitors for lung cancer, melanoma and renal\n osmium tetroxide. Then the samples were prepared after de- cell carcinoma: A real-world Brazilian study, Int.\n hydration with sequential washes in ethanol. Afterward, the Immunopharmacol., 2022, 108, 108727.\n samples were sputter-coated with gold and then observed 3 M. K. Callahan and P. B. Chapman, PD-1 or PD-L1 block-\n under a SEM (HITACHI, Japan). ade adds little to combination of BRAF and MEK inhibition\nPublished on 18 July 2023. Downloaded by Sun Yat-Sen (Zhongshan) University on 7/22/2023 2:04:27 AM.\n\n\n\n\n in the treatment of BRAF V600-mutated melanoma, J. Clin.\n Statistics analysis Oncol., 2022, 40, 1393\u20131395.\n Data processing and statistical analysis were performed using 4 L. Zhou, Q. Xu, L. Huang, J. Jin, X. Zuo, Q. Zhang, L. Ye,\n GraphPad prism 8.0. The one-way ANOVA statistical analysis S. Zhu, P. Zhan, J. Ren, T. Lv and Y. Song, Low-dose carbo-\n was used for comparisons between di\ufb00erent groups. *P < 0.05 platin reprograms tumor immune microenvironment\n was considered statistically significant. through STING signaling pathway and synergizes with PD-1\n inhibitors in lung cancer, Cancer Lett., 2021, 500, 163\u2013171.\n 5 R. Mason, L. Au, A. Ingles Garces and J. Larkin, Current\n Author contributions and emerging systemic therapies for cutaneous metastatic\n melanoma, Expert Opin. Pharmacother., 2019, 20, 1135\u2013\n Jinquan Wang and Huaiyi Huang designed all experiments; 1152.\n Zhongxian Fan and Li Wei synthesized and characterized the 6 S. Asiry, G. Kim, P. S. Filippou, L. R. Sanchez,\n iridium complex; Yi Rong, Zhijie Yu, performed the cell and D. Entenberg, D. K. Marks, M. H. Oktay and\n animal experiments; Han Shen and Zizhuo Zhao analyzed the G. S. Karagiannis, The cancer cell dissemination machinery\n data; Jinquan Wang, Huaiyi Huang, and Xiaojuan Hao wrote as an immunosuppressive niche: a new obstacle towards\n the manuscript. All the authors discussed the results and com- the era of cancer immunotherapy, Front. Immunol., 2021,\n mented on and proofread the manuscript. 12, 654877.\n 7 N. E. Donlon, R. Power, C. Hayes, J. V. Reynolds and\n J. Lysaght, Radiotherapy, immunotherapy, and the tumour\n Con\ufb02icts of interest microenvironment: Turning an immunosuppressive milieu\n into a therapeutic opportunity, Cancer Lett., 2021, 502, 84\u2013\n The authors declare no conflicts of interest.\n 96.\n 8 H. Arai, Y. Xiao, F. Loupakis, N. Kawanishi, J. Wang,\n F. Battaglin, S. Soni, W. Zhang, C. Mancao, B. Salhia,\n Acknowledgements S. M. Mumenthaler, D. J. Weisenberger, G. Liang,\n This work was supported by the National Natural Science C. Cremolini, A. Falcone, J. Millstein and H. J. Lenz,\n Foundation of China (no. 22277013, 22277153, 22007104, Immunogenic cell death pathway polymorphisms for pre-\n 21975053), the Yin Ling Experts Projects of Guangdong dicting oxaliplatin e\ufb03cacy in metastatic colorectal cancer,\n Province (no. 2022A1313030043), the Natural Science J. Immunother. Cancer, 2020, 8, e001714.\n Foundation of Guangdong Province (no. 2022A1515012094), 9 F. Huang, J. Lei, Y. Sun, F. Yan, B. Chen, L. Zhang, Z. Lu,\n Guangdong Basic and Applied Basic Research Foundation (no. R. Cao, Y. Lin, C. Wang and G. Tan, Induction of enhanced\n 2021B1515020050), Science, Technology and Innovation immunogenic cell death through ultrasound-controlled\n Commission of Shenzhen Municipality Project (no. release of doxorubicin by liposome-microbubble com-\n JCYJ20190807152616996), and the Characteristic Innovation plexes, OncoImmunology, 2018, 7, e1446720.\n Projects in Ordinary Colleges and Universities of Guangdong 10 B. Du and D. J. Waxman, Medium dose intermittent cyclo-\n Province (no. 2021KTSCX053). phosphamide induces immunogenic cell death and cancer\n cell autonomous type I interferon production in glioma\n models, Cancer Lett., 2020, 470, 170\u2013180.\n References 11 M. Jiang, J. Zeng, L. Zhao, M. Zhang, J. Ma, X. Guan and\n W. Zhang, Chemotherapeutic drug-induced immunogenic\n 1 V. M. Vorwald, D. M. Davis, R. J. Van Gulick, R. J. Torphy, cell death for nanomedicine-based cancer chemo-immu-\n J. 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