Irradiated Microparticles Suppress Prostate Cancer by Tumor Microenvironment Reprogramming and Ferroptosis

Irradiated Microparticles Suppress Prostate Cancer by Tumor Microenvironment Reprogramming and Ferroptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Irradiated Microparticles Suppress Prostate Cancer by Tumor Microenvironment Reprogramming and Ferroptosis Zihan Deng, Binghui Li, Muyang Yang, Lisen Lu, Xiujuan Shi, Jonathan Lovell, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3911119/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 May, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted 8 You are reading this latest preprint version Abstract Immunogenic cell death (ICD) plays a crucial role in triggering the antitumor immune response in the tumor microenvironment (TME) through the release of damage-associated molecular patterns (DAMPs). Recently, considerable attention has been dedicated to ferroptosis, a type of ICD that is induced by intracellular iron and has been demonstrated to change the immune desert status of the TME. However, there remains significant room for improvement among strategies for inducing high levels of ICD through ferroptosis to fight cancers that are characterized by an immune desert, such as prostate cancer. Radiated tumor cell-derived microparticles (RMPs) are radiotherapy mimetics that have been shown to activate the cGAS-STING pathway, induce tumor cell ferroptosis, and inhibit M2 macrophage polarization. RMPs can also act as carriers of agents with remarkable biocompatibility. In the present study, we designed a therapeutic system wherein the ferroptosis inducer RSL-3 was loaded into RMPs to treat prostate cancer, which is considered a cold tumor, using in vitro and in vivo models involving RM-1 prostate carcinoma cells. Apoptosis inducer CT20 peptide (CT20p) was also added into the RMPs to aggravate ICD. In vitro experiments demonstrated that RSL-3- and CT20p-loaded RMPs (RC@RMPs) led to ferroptosis and apoptosis of RM-1 cells, and CT20p had a synergistic effect on ferroptosis by promoting ROS production and mitochondrial instability. RC@RMPs elevated the dendritic cell (DC) expression of MHCⅡ, CD80, and CD86 and facilitated M1 macrophage polarization. In a syngeneic mouse model of prostate cancer induced by RM-1 cells, RC@RMPs significantly inhibited tumor growth and prolonged survival time via DC activation, macrophage reprogramming, enhancement of CD8 + T cell presence, and proinflammatory cytokine production, without diffusing outside the tumor tissue. Moreover, combination treatment with anti-PD-1 showed improved effectiveness to inhibit RM-1 progression. This method provides a novel strategy for the synergistic enhancement of ICD for prostate cancer immunotherapies. Cancer immunotherapy Microparticle Ferroptosis Immunogenic cell death Tumor microenvironment Macrophage reprogramming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION Prostate cancer is an age-related tumor of the male genitourinary system that has a high incidence in the elderly population. Concurrent with the rapidly aging population, prostate cancer has become the leading male urinary system tumor in the world, and has one of fastest growing incidences among malignant tumors in males in the past decade [ 1 ] . The main treatment for prostate cancers involves prostatectomy combined with endocrine therapy and radiotherapy or chemotherapy. Unfortunately, after a period of treatment, most patients develop castration-resistant prostate cancer (CRPC) that is insensitive to treatment [ 2 , 3 ] . However, as prostate cancer is normally considered as a cold tumor, targeted therapy and immunotherapy often show only limited efficacy in the clinic [ 4 , 5 ] . Therefore, there is an urgent need to explore the mechanism of the occurrence and development of prostate cancer and to develop new therapeutic drugs for prostate cancer. Ferroptosis is a form of immunogenic cell death (ICD) that can enhance the ability of innate immune cells to recognize tumor cells and initiate the adaptive immune response [ 6 ] . Thus, ferroptosis can effectively promote the transformation of cold tumors into hot tumors, ultimately improving the response of cancer patients to immunotherapy [ 7 – 9 ] . SLC7A11 and GPX4 are two key molecules that inhibit ferroptosis, and are highly expressed in prostate cancer and CRPC [ 10 ] . Association studies have shown that nine genes associated with ferroptosis are closely related to the prognosis of patients with prostate cancer. Conversely, the CEMIP, HSPB1, and PANX2 genes, which interfere with the process of ferroptosis, can effectively promote the survival of prostate cancer cells, suggesting that ferroptosis-related genes may be prognostic biomarkers and potential drug targets for patients with prostate cancer [ 11 – 13 ] . Related studies have also suggested that the ferroptosis of neutrophils may promote the occurrence and development of tumors [ 14 ] ; however, whether this phenomenon exists in prostate cancer requires further investigation. Moreover, the heterogeneity of prostate cancer often results in hyposusceptibility to ferroptosis, and the agents that trigger ferroptosis can also induce the death of T cells and non-tumor tissue damage, which limits the therapeutic effect of ferroptosis-inducing agents for cancer treatment [ 15 ] . Therefore, it is necessary to develop novel strategies to enhance ferroptosis with improved specificity to target tumor cells. The tumor immunosuppressive microenvironment is the main cause of clinical prostate cancer recurrence and immunotherapy failure, among which myeloid-derived suppressor cells (MDSCs), inhibitory neutrophils, regulatory dendritic cell (DC), and tumor-promoting M2 macrophages are the main causes of tumor immunosuppressive microenvironment formation [ 16 – 20 ] . It has been reported that commonly used radiotherapy techniques can improve the tumor microenvironment (TME) to some extent [ 21 , 22 ] . However, radiotherapy treatment for prostate cancer commonly results in unavoidable adverse side effects, including lower urinary tract symptoms, intestinal complications, erectile dysfunction, and myelosuppression, which limits the application of radiotherapy in patients [ 23 ] . In the previous study, we reported that radiated tumor cell-derived microparticles (RMPs) are the main medium of the bystander effect induced by radiotherapy [ 24 ] . To some extent, the RMPs act as mimetics of radiotherapy, inducing tumor cell ferroptosis and the reprogramming of tumor-promoting M2 macrophages, which may activate type I interferon signaling through the cGAS-STING pathway [ 24 ] . As RMPs originate from the tumor tissue itself, they have an innate ability to target tumor cells. Microparticles have been proven as good carrier of agents for cancer therapies [ 25 , 26 ] . Our previous studies showed that RMPs loaded with agents and adjuvants can inhibit progression of lung cancer and its brain metastasis [ 27 – 29 ] , suggesting that RMPs derived from prostate cancer may a good drug carrier for the treatment of advanced prostate cancer. To sum up, this study used RM-1 prostate cancer cells as a tumor model, and extracted the RMPs of the tumor cells to be repurposed as drug carriers that encapsulate RSL-3, a ferroptosis inducer targeting GPX-4 proteins, which are a key inhibitor of ferroptosis [ 30 ] . In addition, mitochondrial targeting peptide CT20p (peptide sequence: VTIFVAGVLTASLTIWKKMG, an inducer of apoptosis), which can induce multimodel death in tumor cells, was also loaded into RMPs. The CT20p peptide is the C-terminal of the pro-apoptotic protein Bax, which can regulate the activity of Chaperonin-Containing TCP protein in prostate cancer, resulting in mitochondrial instability and cytoskeletal disruption to promote the effective killing of tumor cells [ 31 ] . The results of this study showed that RMPs encapsulating RSL-3 and CT20p (RC@RMPs) retain the characteristics of RMPs and effectively target and kill tumor cells in vitro and in vivo . As an apoptosis inducer, CT20p altered the mitochondrial membrane potential and aggravated ferroptosis by increasing ROS production. Furthermore, RC@RMPs could activate DC cells and reprogram macrophage polarization. RC@RMPs enhanced both adaptive immunity via CD8 + T cells and innate immunity to effectively kill prostate cancer cells. Together, these data provide proof-of-concept for the use of RMP carriers in the treatment of prostate cancer. 2. METHODS Materials The medium of cell culture was purchased from Gibco Life Technologies, Inc. (Grand Island, NY, USA), including Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium. Fetal Bovine Serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co., Ltd. (Huzhou, China). Plasmocin was bought from InvivoGene (Toulouse, France) and penicillin/streptomycin was obtained from Biosharp (Hefei, China). Sterile 1×phosphate buffered saline was purchased from Gibco Life Technologies, Inc. (Grand Island, NY, USA). All the cytokines were purchased from Biolegend (San Diego, CA, USA), containing granulocyte-macrophage colony-stimulating factor macrophage colony-stimulating factor, interleukin-4, interleukin-13, lipopolysaccharide and interferon-γ. RSL3 was bought from Selleck (Houston, TX, USA). CT20p peptide was bought from BankPeptide Inc. (Hefei, China). Sucrose for electroporation buffer was gained from Sinopharm (China). Acetonitrile, methanol and chloroform for High Performance Liquid Chromatography (HPLC) were all purchased from Thermo Fisher Scientific (Waltham, MA, USA) and their purity was more than 99%. The fluorescence dye DiO, DiD and Rhodamine were obtained from Yeasen (Shanghai, China). H2DCFDA and PKH26 were bought from MedChemExpress (NJ, USA). FITC-Liperfluo was obtained from Dojindo (Japan) and Phen Green SK diacetate was bought from GLPBIO (USA). Radioimmunoprecipitation assay buffer and the inhibitors of protease and phosphatase were obtained from Beyotime (Shanghai, China). For western blot, primary antibodies STING, p-STING, NFκB, p-NFκB and Laminin B1 were purchased from ABclonal ( Boston, MA, USA) and GAPDH, CD63, CD81 and Alix were obtained from Proteintech Group, Inc. (Chicago, IL, USA). Secondary antibodies goat anti-mouse IgG H&L-HRP conjugated and goat anti-rabbit IgG H&L-HRP conjugated were bought from Abcam (Cambridge, UK). Collagenase IV and hyaluronidase were purchased from Biosharp (Hefei, China). All the antibodies for flow cytometry and immunocyte depletion were bought from Biolegend (San Diego, CA, USA). Clodronate liposomes were purchased from FormuMax (Silicon Valley, CA, USA). PD-1 mAb for treatment was obtained from BioXell (Italy). Cells culture All the murine cell lines were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China), including prostate cancer cell line (RM-1), Lewis lung carcinoma cell (LLC), mammary cancer cell line (4T1), colon adenocarcinoma cell line (MC38), B16F10 melanoma cells, GL261 glioma cells, DC line (DC2.4) and monocyte cell line (RAW264.7). All the cell lines were treated with 25 µg mL − 1 Plasmocin for at least two weeks and were mycoplasma - negative as determined by MycoProbe Mycoplasma Detection Kit (R&D Systems, Minneapolis, MN, USA). RM-1, 4T1 and LLC cells were cultured in DMEM while other cells were maintained in RPMI 1640 medium. Bone marrow-derived dendritic cells and macrophages (BMDCs & BMDMs) from C57BL/6J mice were generated as previous descriptions in RPMI 1640 medium [ 32 , 33 ] . All the mediums were added with 10% (v/v) FBS and 100 µg mL − 1 penicillin/streptomycin. Preparation of RMPs In 10-cm cell culture dishes, 6×10 6 RM-1 cells were planted and irradiated with a single dose of 20 Gy by 6-MV x-rays (CHIRAD 225). Next, the medium of irradiated cells was renewed by 20 mL DMEM completed medium which its microparticles had been removed via centrifugation. 72 hours later, the medium was collected, and cell debris were removed by 1000 g for 10 min and 14,000 g for 2 min. RMPs were gained from the supernatant via 14,000 g for further 60 min at 4 ℃ and washed with sterile 1×PBS for 2 times. At last, the RMPs were resuspended with 1×PBS for subsequent experiments. RMPs encapsulated with RSL-3 and CT20p via electroporation RSL3 was dissolved in DMSO to a concentration of 10 mg mL − 1 . CT20 peptide (CT20p) was diluted by ultrapure water. RSL3 or CT20p was mixed with RMPs in a 1:2 ratio of mass in 400 mM sucrose solution. By an electroporation system (GenePulser Xcell, BioRad, USA), 400 µL mixture was electroporated in 0.2 cm cuvettes via exponential pulse (voltage: 500 V; capacitance: 125 µF). High Performance Liquid Chromatography (HPLC) HPLC analysis was conducted using a LC-2030C Plus instrument (Shimadzu, Japan). The separation was implemented with a ShimNex C18 chromatographic column (4.6×250 mm, 5 µm, 100 A, Shimadzu, Japan). Three times volume of acetonitrile was mixed with RMPs and then added chloroform (1:2, v/v). After vortexed, the mixture was centrifuged at 10,000 g for 5 min and the lower layer was extracted for measurement. As the standard solution, 10 mg RSL3 was dissolved in acetonitrile and chloroform in a ratio the same as RMPs. All the samples were filtered through a 0.45 µm polytetrafluoroethylene filter. The components were separated and eluted by mobile phase eluted (A: methanol; B: acetonitrile) in the column at 25 ℃ with a flow rate of 1 mL min − 1 . An ultraviolet wavelength (254 nm) was selected for the detection of RSL3. Quantification of RMPs The quantification of RMPs were determined by their protein concentrations. Radioimmunoprecipitation assay buffer were applied to lyse RMPs at 4°C for half an hour. Then, the lysis was centrifuged at 12,000 g for 30 min at 4°C and the supernatant was transferred into a new centrifuge tube for protein concentration measurement by BCA Protein Assay Kit (Thermo Fisher Scientific). Characterization of RMPs Size and Transmission electron microscopy (TEM) Determination One milliliter of 30 ng mL − 1 RMPs were taken for the measurement of the particle size and polydispersity index by Malvern laser particle size analyzer (Zetasizer Nano ZSP). For further identification of the sizes and morphology of RMPs were washed by ddH 2 O, deposited on copper mesh and then observed by TEM (HT7700-SS/FEI Tecnai G20 TWIN). Cell viability measurement All the cells to evaluate viability were planted into 96-well plates (5000 cells per well). After 24 hours growing, distinct RMPs were treated with the cells for further 48 hours. A cell counting kit-8 (CCK-8) assay kit (Meilunbio, Dalian, Chian) was used to measure cell viability. In vitro cellular uptake assay To evaluate the cellular uptake of RMPs, different cell lines were planted into 6-well plates and incubated with DiO pre-dyed MPs for 3 or 6 hours. The cells were collected, washed by PBS and analyzed through flow cytometry (Beckman CytoFLEX S, USA). Identification of the colocalization of CT20p and mitochondrion RMPs with CT20p-Rhodamine B and Rhodamine B were added into the cells which were sequently incubated with 100 nM MitoTracker Green® FM at 25 ℃ for 30 minutes. Confocal laser scanning microscopy (Carl Zeiss LSM710) was used to observe the colocalization of CT20p and MitoTracker Green® FM (standing for mitochondrion) whose Pearson correlation coefficient was calculated by image J software. Analysis of cell apoptosis and ferroptosis Cells were cultured in 24-well plates (30,000 cells per well) and then incubated with MPs, RSL3@MPs, CT20p@MPs and RC@RMPs for 24 hours. The cells were harvested for relevant measurement. Apoptosis was evaluated by Annexin V-Alexa Fluor 488/7-AAD apoptosis detection kit (yeasen, China). The protocol complied with the instruction of the kit. To determine mitochondrial membrane potential, JC-1 assay kit (yeasen, China) was applied. The method to dye and measure cell apoptosis and mitochondrial membrane potential obeyed the instruction of the kits. To evaluate total ROS, lipid ROS and Fe 2+ level, the cells were respectively dyed with H2DCFDA (10 µM), FITC-Liperfluo (5 µM) and Phen Green SK diacetate (10 µM) in 1 mL PBS for 30 min at 37°C in a cell culture incubator. Wased by PBS twice, the cells were resuspended with 200 µL PBS and analyzed via flow cytometry. Western blotting All the MPs and cells that be detected were lysed by RIPA buffer with the inhibitor of protease and phosphatase at 4°C for 30 minutes, and then centrifuged at at 12,000 g for 30 min at 4°C. The mass of the sample loading was adjusted to the same according to their protein concentrations that were detected by BCA Protein Assay Kit. The samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane after boiled for 5 minutes. The membranes block by 5% not-fat milk at room temperature for 1 hour and incubated with related primary antibodies at 4°C overnight. With serveral wash by Tris-buffered saline with 0.05% Tween-20, secondary antibodies incubated with the membranes at room temperature for 1 hour. NcmECL Ultra (P10100, NCM Biotech) was applied for chemiluminescent exposure of the blot. Mice Male C57BL/6J mice (aged 6–8 week, weighted 18–20 g) were purchased from SHULAIBAO Biotech. All mice were kept in micro-isolator cages, and the experimental protocols were approved by the Hubei Provincial Animal Care and Use Committee and were in compliance with the experimental guidelines of the Animal Experimentation Ethics Committee of Huazhong Agricultural University. In vivo cellular internalization assay To identify RMPs uptake by cells in tumor in vivo , we intratumorally injected 100 µL PKH26 marked RMPs to mice with RM-1 tumor burden. 24 hours later, the mice were sacrificed and the tumors were digested into single cell for flow cytometry analysis before they were incubated with antibodies of CD45 (clone S18009F), CD3 (clone 17A2), B220 (clone RA3-6B2), CD11b (clone M1/70), Ly6G (clone S19018G), F480 (clone BM8), CD11c (clone N418), MHCII (clone M5/114.15.2) and NK1.1 (clone PK136). Besides, some tumor tissues were fixed, dehydrated and sectioned into frozen sections which were going to stain by related antibodies and observed via confocal laser scanning microscopy. To image the distribution of RMPs, RC@RMPs were stained with DiD, the 540/20 nm excitation filter and 620/20 nm emission filter were used and the exposure time was 15 s. Subcutaneously implanted prostate tumor model and treatment with RMPs RM-1 tumor cells (1×10 6 cells in 100 µL PBS) were subcutaneously implanted into right back. Five days after tumor inoculation, mice with uniform tumor volume were randomly divided into 7 groups including control group, PD-1 mAb group, RMPs group, RSL3@RMPs group, CT20p@RMPs group, RC@RMPs group and RC@RMPs combined with PD-1 mAb group, and were treated with corresponding RMPs (intratumoral injection with 100 µg in 100 µL PBS) and PD-1 mAb (10 mg/kg, intraperitoneal injection) at 6, 8, 10, 12 and 14 days after grouping. Vernier caliper was applied to measure the length ( L ) and width ( W ) of subcutaneous tumors every other day. The volume of tumor was calculated by the the formula V = ( L × W 2 ) / 2. Mice were sacrificed when the tumor volume reached 1000 mm 3 . Detection of immunocytes in tumor and draining lymph nodes (dLNs) RM-1 tumors from mice were digested into single cell by cutting into small pieces and incubating with Collagenase IV (0.32 mg mL − 1 ) and hyaluronidase (0.5 mg mL − 1 ) for 1 hours at 37°C. The tumor cells were filtered through 70 µm cell strainer after lysis of RBCs. All the samples were blocked Fc receptors followed by incubating with detection antibodies containing CD3 (clone 17A2), CD8 (clone SK1), CD69 (clone H1.2F3), CD4 (clone GK1.5), PD1 (clone 29F.1A12), TOX (clone 6E6D03), TCF1 (clone 7F11A10), CD11c (clone N418), CD11b (clone M1/70), CD86 (clone GL-1), MHCII (clone M5/114.15.2), CD44 (clone IM7), CD62L (clone MEL-14) and Zombie NIR™, and then measured via flow cytometry. Cytokines detection RM-1 tumors from mice were weighted and grinded into homogenate. The supernatant was collected by 6000 g centrifugation for 20 minutes at 4 ℃. The LEGENDplex Mouse Cytokine Release Syndrome Panel (13-plex) with VBottom Plate (purchased from Biolegend) was used for cytokine detection. Immune cell depletion T helper cells and CTLs were depleted by CD4 (clone GK1.5) and CD8 (clone 2.43) antibodies respectively. One day before treatment, 200 µg antibodies were intraperitoneal injected into mice for 5 times at 2-day intervals. Macrophages were depleted by clodronate liposomes. One day before treatment, 200 µL clodronate liposomes were intravenously injected into mice for 5 times at 3-day intervals. Neutrophils were depleted by Ly6G (clone 1A8) antibody. One day before treatment, 200 µg antibodies were intraperitoneal injected into mice for 5 times at 2-day intervals. Statistical analysis All the data were analyzed by Prism software (GraphPad Prism 6.0 software). The log-rank (Mantel-Cox) test was applied to compare survival rates between groups.Kaplan-Meier analysis was used to analyze tumor growth, and comparisons of three or more groups were calculated by one-way analysis of variance (ANOVA). Two-tailed unpaired t test or the Mann-Whitney U test was performed to determine the significance of two groups. P values of < 0.05 were determined statistically significant. Data are presented as means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001 and ns stands for no significant. 3. RESULTS 3.1 Prostate cancer gene expression patterns and immunological correlation of ferroptosis pathway factors To investigate the relationship between ferroptosis-related genes and prostate cancer, we downloaded data for a total of 88 ferroptosis-related genes from a published dataset [ 34 ] to analyse the differentially expressed genes in prostate cancer versus normal samples (|log2FC| >= 1, p < 0.05). We obtained a total of 8 ferroptosis-related genes (“SLC7A11”, “CBS”, “ALOX15”, “DPP4”, “SLC39A8”, “TP53”, and “GPX4”) in prostate cancer and paired normal samples (Fig. 1 A). The TME of prostate cancer is relatively devoid of immune infiltration compared to other malignancies. Therefore, we reanalyzed published single-cell data on prostate cancer to explore the infiltration of immune cells in prostate cancer tumors. The results showed that the vast majority of cells in prostate cancer were epithelial cells, and that any immune cells present were predominantly exhausted CD8 + T cells, confirming the immune desert phenotype of prostate cancer (Fig. 1 B). 28 immune cell infiltration scores were evaluated in prostate cancer according to single sample gene set enrichment analysis (ssGSEA), and the correlations between these 8 ferroptosis-related genes and immune cell infiltration scores were calculated. The results showed that the expression of these genes was significantly correlated with immune infiltration (Fig. 1 C). Because the GPX4 gene was significantly highly expressed in prostate cancer (p < 0.05) and associated with infiltrated immunocytes as shown above, we further explored the relationship between GPX4 and prostate cancer. We investigated the expression of the GPX4 gene in different cancers, and found that GPX4 was highly expressed in the vast majority of moderate cancers, including prostate cancer (Fig. 1 D). Surprisingly, however, there was no significant correlation between GPX4 expression and tumor immune infiltration scores in prostate cancer (Fig. 1 E). In summary, we identified several vital ferroptosis pathway factors that are highly expressed in prostate cancer and correlated with immune cell infiltration, suggesting that they may be good targets to induce ferroptosis to improve the immune desert status in prostate cancer. 3.2 Preparation and characterization of RC@RMPs GPX4 is a peroxidase involved lipid metabolism that is vital for inhibiting ferroptosis [ 35 ] . GPX4 is correlated with the infiltration of various immune cells in prostate cancer, as indicated by the above bioinformatics analysis. The inhibition of GPX4 is considered a potential strategy for initiating ferroptosis [ 36 ] . RMPs, which are derived from radiotherapy-treated cells, are carriers of large quantities of DAMPs and have been shown to induce tumor cell death via ferroptosis [ 24 ] . We loaded the GPX-4 inhibitor RSL3 into RMPs to investigate the potential for a synergistic effect of GPX-4 inhibition and RMPs on ferroptosis to treat prostate cancer. Additionally, the C-terminal of the pro-apoptotic protein Bax, the CT20p peptide, which induces mitochondrial damage, was also added into RMPs to intensify ICD for an enhanced anti-tumor immune response. The combined system (RC@RMPs) was constructed by obtaining and centrifigating the supernatant of irridiated RM-1 tumor cells loaded with CT20p and RSL3 through electroporation, as described in the experimental section and Fig. 2 A. As the quantity of active agents in RMPs differed based on electroporation parameters and the ratio of RMPs to agents, we tried several different conditions for electroporation. Based on High Performance Liquid Chromatography (HPLC) analysis, the highest quantity of RSL3 in RMPs was achieved using following electroporation parameters: 500 V voltage, 125 µF capacitance, and exponential decay wave mode, when 100 µg RSL3 was mixed with 100 µg RMPs (Fig. 2 B and Figure S1 ). Maintaining the above electroporation conditions, we further optimized the mass of RSL3 mixed with 100 µg RMPs, and found that the RSL3 concentration in the RMPs as the highest when 150 µg RSL3 was used (Fig. 2 C). Characterization of zeta potential (Fig. 2 D) and size (Fig. 2 E) showed no significant differences among RMPs, RSL3@RMPs, CT20p@RMPs, and RC@RMPs. Transmission electron microscopy (TEM) indicated that RMPs and RC@RMPs had a regular spherical morphology (Fig. 2 F). Therefore, the loading of RSL3 and CT20p agents did not influence the structure of the RMPs. Western blot analysis demonstrated all the RMPs were rich in extracellular vesicle–associated proteins such as CD63 and CD81, whose expression was not influenced by encapsulation of CT20p and RSL3 (Fig. 2 G). Thus, the RMPs that we extracted had similar composition as extracellular vesicles. 3.3 RC@RMPs can kill RM-1 cells by causing ferroptosis and apoptosis To identify whether RMPs can be ingested by tumor cells, 20 µg mL − 1 DiO pre-labeled RMPs were incubated with some murine tumor cell lines for 3 or 6 hours. We detected a high level of DiO fluorescence intensity in RM-1 cells, indicating that RM-1 cells can effectively take up RMPs (Fig. 3 A). We then evaluated the toxicity of different RMPs to RM-1 tumor cells. RC@RMPs were the most effective at eliminating RM-1 cells with the lowest IC (50) values (4.474 µg mL − 1 vs 53.13 µg mL − 1 for RMPs, 5.467 µg mL − 1 for RSL3@RMPs and 47.43 µg mL − 1 for CT20p@RMPs) (Fig. 3 B). RSL3 is considered to induce ferroptosis by increasing ROS production and mitochondrial dysfunction. CT20p peptides were added into RMPs to synergistically promote ferroptosis by mitochondrial damage. RMPs with CT20p-Rhodamine B and Rhodamine B (control) were incubated with RM-1 to identify the subcellular localization of CT20p. Confocal microscopy showed that CT20p-Rhodamine B colocalized with mitochondria as identified by staining with MitoTracker Green® FM, which was confirmed by Pearson correlation coefficient analysis (Fig. 3 C). Therefore, RMPs loaded with CT20p were targeted to the mitochondria. Flow cytometry analysis revealed that RC@RMPs in large part caused RM-1 cells to proceed to late apotosis, which was difficult to recover from (Fig. 3 D). Only RSL3-loaded RMPs resulted in significantly less cell death than RC@RMPs; CT20p peptides-loaded RMPs were able to induce early apoptosis by producing damaged mitochondria, though this may be reversed by mitophagy (Fig. 3 D). Mechanistically, the toxicity of RC@RMPs was primarily related to the induction of ferroptosis, as indicated by increased total ROS production, increased lipid peroxidation, increased Fe 2+ levels, and decreased mitochondrial membrane potential, which occurred to a greater extent in cells treated with RC@RMPs compared to other RMPs (Fig. 3 E-H). ROS measurement based on H2DCFDA mean fluorescence intensity (MFI) was significantly higher in cells treated with RSL3@RMPs or CT20p@RMPs compared to RMPs, suggesting that both RSL3 and CT20p were able to elevate ROS production. RC@RMP-treated cells showed the highest ROS levels, demonstrating a synergism of RSL3 and CT20p (Fig. 3 E). Meanwhile, there was greater green fluorescence in CT20p@RMP-treated cells compared with the RMPs group, confirming that CT20p played a key role in effects on mitochondria (Fig. 3 F). Overall, the combination of RSL3 and CT20p appears to exert strong synergy to trigger ferroptosis and may augment the subsequent immune response. 3.4 RC@RMPs activate DCs and regulate macrophage polarization As Antigen Presenting Cells (APCs), DCs are vital for initiating anti-tumor immunity. In the TME, they capture and process tumor antigens then present these antigens to tumor-specific T cells, which then recognize and eliminate tumor cells [ 37 ] . Macrophages are also APCs and function to activate T cells. However, tumor-associated macrophages (TAMs) always act as promoters of tumor progression by secreting anti-inflammatory cytokines such as IL-10 and TGF-β [ 20 , 38 ] . Therefore, we measured the direct influence of RC@RMPs on APCs to evaluate the effect that RC@RMPs may have on reshaping the immunological environment of the tumor. DiO pre-labeled RMPs and RC@RMPs at different concentrations were incubated with DC2.4 and RAW264.7 cells. We found that the quantities of RMPs and RC@RMPs taken by APCs increased in a dose-depenent manner (Figure S2 ). CCK8 cell toxicity assays showed that the cell growth of DCs treated with RMPs remained above 50% when the concentration of RMPs was less than 25 µg mL − 1 , indicating that DCs were insensitive to CT20p- and RSL3-loaded RMPs (Figure S3 A). Moreover, flow cytometry analysis showed that incubation with RC@RMPs could boost DC activation by significantly increasing the expression of CD80, CD86, and MHCⅡ, compared to control groups and RMPs loaded with a single agent (Fig. 3 I). RMPs encapsulating DAMP-like DNA fragments generated by radiation may trigger the cGAS-STING pathway. Therefore, we performed Western blots to measure the expression of proteins related to cGAS-STING activation, including pSTING and pNF-κB (p65), in DC2.4 cells treated with different RMPs. The phosphorylation of STING and NF-κB were eleveated when the cells were incubated with RMPs, RMPs loaded either RSL3 or CT20p, or RMPs loaded with both agents, compared to the control group (Figure S4 ), indicating that RSL3 and CT20p loading does not influence RMP-induced activation of the cGAS-STING pathway. In contrast to DCs, RSL3-loaded RMPs showed relatively higher toxicity to macrophages (IC (50) values: 13.1 µg mL − 1 for RSL3@RMPs and 9.08 µg mL − 1 for RC@RMPs). Macrophages treated with RC@RMPs expressed high levels of CD86, suggesting that RC@RMPs contributed to the M1 polarization of macrophages (Fig. 3 J). All RMPs reduced CD206 expression, with no significant differences among RMP-treated groups, demonstrating that RSL3 and CT20p loading maintains the ability of RMPs to inhibit macrophage polarization to M2(Figure S5 ). Together, these findings indicated that RC@RMPs can directly promote inflammation by activating DCs and promoting M1 macrophage polarization. 3.5 RC@RMPs can be taken up by tumor cells and immunocytes in the TME in vivo To explore the tissue distribution of RMPs in vivo , we performed intratumoral injections of 100 µg PKH26-labeled RMPs (100 µL) into mice previously implanted with RM-1 cells. 24 hours later, the mice were sacrificed and the tumor and organs including the heart, liver, spleen, lung, kidney, brain, and dLNs were observed using the IVIS Spectrum Imaging System. The distribution of RMPs was limited to the tumor tissue, as shown in Figure S6 A. Then, we dispersed the tumor to generate a single cell suspension for flow cytometry analysis. There was no significant differences in the percentages of PKH26-positive tumor cells, T cells, B cells, neutrophils, DCs, or M2 macrophages between mice injected with RMPs and those given RC@RMPs (Fig. 4 A). Tumor sections were stained using antibodies targeting neutrophils, DCs, and macrophages. The results confirmed that RMPs could be taken up by the relevant immune cells (Fig. 4 B and Figure S6 B). 3.6 RC@RMPs reshape the tumor immune microenvironment and the combination of RC@RMPs and anti-PD-1 mAb shows synergistic antitumor activity In order to evaluate the therapeutic effect of RC@RMPs on prostate cancer, we treated subcutaneously implanted RM-1 tumors with control, RMPs, RSL3@RMPs, CT20p@RMPs, and RC@RMPs according to the treatment scheme shown in Fig. 5 A. RC@RMP therapy significantly reduced the growth of RM-1 tumors and prolonged survival time compared with mice treated with control, RMPs, RSL3@RMPs, or CT20p@RMPs (Fig. 5 B-C). The median survival time of RC@RMP-treated mice was the longest (39 days) among all groups, although all mice reached ethical endpoint by 62 days after tumor inoculation (Fig. 5 C). To clarify the effects of RC@RMP treatment on the immune microenvironment, immunocytes in the tumor were measured by flow cytometry. We found a significant increase in the number of neutrophils, CD86 + MHCⅡ + DCs, total CD8 + T cells, IFN-γ + CD8 + T cells, and memory CD8 + T cells, and a decrease in the number of M2 macrophages compared with the groups given control and RMPs (Fig. 5 D-K). The level of related cytokines were also measured; only IL-6 was found to be significantly elevated by RC@RMPs compared with the control and RMP groups (Figure S8 G). PD-1 is a negative regulator of the immune system that prevents overactivity and subsequent cytokine release syndrome. The expression of PD-1 is enhanced upon T cell activation, forming a negative feedback loop. Results showed that RC@RMPs significant promoted PD-1 expression, further indicating an uptick in T cell activity. However, RMPs also increased the expression of PD-L1 on macrophages, which can induce programmed cell death in T cells with high PD-1 expression. Therefore, we undertook a combined therapy strategy using an anti-PD-1 antibody and RC@RMPs to antagonize the immunosuppressive effect of the PD-1/PD-L1 pathway during RC@RMP treatment. The combination treatment promoted the inhibition of RM-1 growth (Fig. 5 B) and resulted in a 50% survival rate 62 days after tumor inoculation (Fig. 5 C). The combination therapy also significantly enhanced the presence of CD86 + MHCⅡ + DCs, the proportion of CD8 + T cells among CD3 + T cells, IFN-γ + CD8 + T cells, and TCF-1 + CD8 + T cells, while decreasing the number of M2 macrophages, compared with monotherapy treatment with only RC@RMPs. Furthermore, proinflammatory cytokines including CXCL9, TNF-α, CCL4, and CCL3 were also upregulated in the tumor following the combined therapy, further demonstrating the change in the immune environment (Figure S8 A-I). To identify the key immune subsets affected by our treatment, sections of tumor were stained for CD3, Ly6G, F4/80, and CD206 to observe the presence of T cells, neutrophils, and M2 macrophages. Histological analysis showed that RC@RMPs markedly increased T cell infiltration (Figure S9 A) and decreased the number of M2 macrophages present (Fig. 6 A). To directly assess the role of infiltrating immunocytes, we depleted T helper cells, cytotoxic T cells, neutrophils, and macrophages using CD4 mAb, CD8 mAb, Ly6G mAb, and clodronate liposomes, respectively (Fig. 6 B). These targeted cell subsets were rapidly depleted in peripheral blood within 24 hours, which was confirmed by flow cytometry (Figure S10 ). We observed that the depletion of CD8 + T cells and macrophages impaired the efficacy of RC@RMP treatment (Fig. 6 C), suggesting that these two cell subsets are the main targets of RC@RMPs. Finally, we tested for possible toxicity following RC@RMP treatment. All mice in treated groups showed similar levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CREA) as the control group (Figure S11 A). No abnormalities were observed in the heart, liver, spleen, lung, and kidney after the different RMP therapies, as determined by histopathological examination (Figure S11 B). Together, these results indicated that RC@RMPs successfully remodeled the immune desert environment of RM-1 tumors, and PD-1 blockade enhanced the effectiveness of this immune enhancement and tumor cell killing. 4. DISCUSSION Although ferroptosis is a potent trigger of the innate immune system, there remain significant obstacles for treatment strategies that focus on inducing ferroptosis. Many ferroptosis-inducing agents have limited efficacy against cold tumors due to their short half life, hyposusceptibility of the tumor to ferroptosis, and toxicity to normal cells. Therefore, a major challenge for the clinical application of ferroptosis inducers is to determine strategies to improve the potency and tumor-targeting capabilities of ferroptosis-inducing agents. As one of the most crucial organelles in cell, mitochondria are rich in metabolism-related molecules which can trigger ICD [ 39 ] . Despite some controversy, targeting mitochondria to stimulate the release of DAMPs has been shown to cause ferroptosis via the release of ROS and free iron [ 40 , 41 ] . Recent evidence indicates that radiation triggers ferroptosis and increases the susceptibility of cancer cells to ferroptosis [ 42 , 43 ] . Mechanistically, radiotherapy impairs lipid metabolism through the promotion of ROS production and ACSL4 expression, and downregulation of SLC7A11, all of which mediate ferroptosis [44–46] . As radiotherapy derivatives, RMPs induce tumor cell ferroptosis and reprogram macrophage polarization through DAMPs generated by radiation, as previously demonstrated [ 24 ] . Furthermore, RMPs are microparticles that can act as carriers for active agents, with good stability and biocompatibility. As they are generated from tumor cells, RMPs are also able to target the tumor and act as a source of tumor antigens to APCs. Thus, the combination of RMPs and RSL3 may provide a new strategy to more effectively induce tumor cell ferroptosis and thus stimulate innate immunity. In the study, we designed a combined system (RC@RMPs) of RMPs loaded with the ferroptosis inducer RSL3 and apoptosis inducer CT20p. This system has some distinct advantages. (i) Firstly, the system demonstrates strong synergy to induce tumor cytotoxicity: RC@RMPs were significantly more effective at killing RM-1 cells through inducing apoptosis and ferroptosis, compared to RMPs with a single agent (Fig. 3 A-H). We first discovered that CT20p enhanced ferroptosis through the production of ROS and disruption of the mitochondrial membrane potential (Fig. 3 E-H). This peptide has been shown to target mitochondria and induce mitochondrial membrane hyperpolarization, which impairs the distribution and movement of mitochondria [ 31 ] . Consequently, mitochondrial metabolism is compromised and ROS release occurs, inducing apoptosis and accelerating the ferroptosis process. Nevertheless, the complete mechanism of the synergy through which RSL3, CT20p, and RMPs promote ferroptosis requires further elucidation. We note that as we used relatively low doses of RMPs compared to previous literature [ 24 ] , the RMPs alone did not initiate a significant ferroptosis effect, although DAMPs in the RMPs triggered innate immunity. (ii) Secondly, RC@RMPs caused DC activation and M1 macrophage polarization. ROS production is essential in tumor therapy, for it did not increases ICD in tumor cells but also reprograms the phenotypes of DCs. The highest expression levels of B7 molecules in DCs was achieved by RC@RMP treatment (Fig. 3 I), which was associated with increased ROS levels that may have activated the CD80/CD86 promoters via the release of Ca 2+ and expression of positive transcription elongation factor b (P-TEFb) [ 47 ] . Moreover, DAMPs in RMPs can trigger the activation of the cGAS-STING pathway to facilitate the expression of type Ι interferon, and cause the autoactivation of APCs (Figure S5 ). As our results have shown, all the RMPs promoted the phosphorylation of key components of the cGAS-STING pathway in DC2.4 cells (Figure S5 ), suggesting that the synergistic activity of RSL3 and CT20p in RMPs were able to mobilize a more intense innate immune response. M1 macrophage polarization is essential in antitumor immunity, as demonstrated in Fig. 6 C. ROS may not be the main factor that promotes M1 macrophage polarization, as CD86 expression was not differentially expressed when comparing groups given RMPs and RSL3@RMPs, even though RSL3@RMPs produced much more ROS than RMPs (Fig. 3 E and J). Macrophages in mice treated with CT20p@RMPs showed a stronger tendency towards M1 phenotypes, implying that CT20p promoted M1 polarization, possibly by influencing mitochondrial metabolism in macrophages. (iii) Thirdly, RC@RMPs were capable of targeting tumor sites without spreading throughout other organs, which is likely due to the nature of RMPs, which are derived from tumor cells. Our data confirmed that the RMPs did not distribute into other organs and tissues when injected intratumorally (Figure S6 A). Notably, the greatest RMP uptake was observed in macrophages within the tumor (Fig. 4 A), indicating the importance of reprogramming macrophage polarization. The tumor-targeting of RMPs is critical for minimizing side effects by allowing appropriate dose reduction without compromising therapeutic effect. (iv) Fourthly, the combination therapy with anti-PD-1 mAb demonstrates a highly promising treatment modality. High PD-1 expression is a characteristic of activated CD8 + T cells and a vital checkpoint for immunosuppression by binding with PD-L1. RC@RMP treatment significantly enhanced the percentage of PD-1 + CD8 + T cells, which may limit the therapeutic effect. Addition of anti-PD-1 mAb to increase the presence of inflammatory immunocytes and cytokines in the TME successfully inhibited tumor growth and prolonged the survival time of mice burdened with RM-1 tumors. (v) Finally, RMPs show good biocompatibility and safety for use as cancer vaccines (Fig. 5 ). Our results showed no obvious damage in the major organs after treatment with RMPs (Figure S11 ). To sum up, RC@RMPs specifically targeted tumor sites where they mediated tumor cell apoptosis and synergistically enhanced the ferroptosis of tumor cells, while remodeling the tumor immune microenvironment of prostate cancer by DC activation and M1 macrophage polarization. There remain some limitations in our system. The toxicity of the RC@RMPs was not entirely specific to prostate cancer cells, leading to the death of macrophages and limiting the inflammatory effects of M1 macrophages. Moreover, the precise mechanism by which the components of RC@RMPs synergistically induce M1 macrophage polarization is not yet fully characterized. Further efforts should seek to screen novel genes that specifically contribute to the ferroptosis of prostate cancer cells and elucidate the relationship between macrophage polarization, changes in mitochondrial functionality, and ferroptosis, to support the development of more efficient and accurate therapies for prostate cancer. Data statement Sample sizes were predetermined based on previous experience using at minimum three groups of mice, and all experiments were replicated at least twice to confirm findings. Statistical analyses were conducted with a two-tailed unpaired t-test or one-way ANOVA as described below. Mice were randomly assigned to treatment groups, and where possible, treatment groups were blinded until statistical analysis. No animals or potential outliers were excluded from the data sets presented in this study. The data used and analyzed during the current study are available from the corresponding author on reasonable request. Abbreviations CRPC: castration-resistant prostate cancer; ICD: Immunogenic cell death; TME: Tumor microenvironment; DAMPs: Damage-associated molecular patterns; RMPs: Radiated tumor cell-derived microparticles; CT20p: CT20 peptide; RC@RMPs: RSL-3- and CT20p-loaded RMPs; DC: Dendritic cell; MDSCs: Myeloid-derived suppressor cells; HPLC: High Performance Liquid Chromatography; dLNs: Draining lymph nodes; TEM: Transmission electron microscopy; MFI: Mean fluorescence intensity; APCs: Antigen Presenting Cells; TAMs: Tumor-associated macrophages; ALT: alanine transaminase; AST: aspartate transaminase; BUN: blood urea nitrogen; CREA: creatinine. Declarations Data availability The authors declare that all data supporting the results of this study are available in the paper and supplementary information. Source data are provided in this paper. Acknowledgements We thank all members of the laboratory for their kindness and help. This study was supported by grants from the National Natural Science Foundation of China (No. 82102900, 82022040, 82272851), The China postdoctoral Science Foundation (2023M731223, 2023T160253). We thank the Optical Bioimaging Core Facility of WNLO-HUST for the support in data acquisition. Competing interests The authors declare no competing interest. References S. Sandhu, C. M. Moore, E. Chiong, H. Beltran, R. G. Bristow, S. G. Williams. Prostate cancer. The Lancet. 2021 ; 398 :1075. R. J. Rebello, C. Oing, K. E. Knudsen, S. Loeb, D. C. Johnson, R. E. Reiter, S. Gillessen, T. Van der Kwast, R. G. Bristow. Prostate cancer. Nature Reviews Disease Primers. 2021 ; 7 . M. Cai, X.-L. Song, X.-A. Li, M. Chen, J. Guo, D.-H. Yang, Z. Chen, S.-C. Zhao. Current therapy and drug resistance in metastatic castration-resistant prostate cancer. Drug Resistance Updates. 2023 ; 68 . K. D. Runcie, M. C. Dallos. Prostate Cancer Immunotherapy—Finally in From the Cold? Current Oncology Reports. 2021 ; 23 . H. Liang, Y. Liu, J. Guo, M. Dou, X. Zhang, L. Hu, J. Chen. Progression in immunotherapy for advanced prostate cancer. Frontiers in Oncology. 2023 ; 13 . X. Chen, R. Kang, G. Kroemer, D. Tang. Broadening horizons: the role of ferroptosis in cancer. Nature Reviews Clinical Oncology. 2021 ; 18 :280. W. Gao, X. Wang, Y. Zhou, X. Wang, Y. Yu. Autophagy, ferroptosis, pyroptosis, and necroptosis in tumor immunotherapy. Signal Transduction and Targeted Therapy. 2022 ; 7 . K. Hänggi, B. Ruffell. Cell death, therapeutics, and the immune response in cancer. Trends in Cancer. 2023 ; 9 :381. R. Tang, J. Xu, B. Zhang, J. Liu, C. Liang, J. Hua, Q. Meng, X. Yu, S. Shi. Ferroptosis, necroptosis, and pyroptosis in anticancer immunity. Journal of Hematology & Oncology. 2020 ; 13 . A. Ghoochani, E.-C. Hsu, M. Aslan, M. A. Rice, H. M. Nguyen, J. D. Brooks, E. Corey, R. Paulmurugan, T. Stoyanova. Ferroptosis Inducers Are a Novel Therapeutic Approach for Advanced Prostate Cancer. Cancer Research. 2021 ; 81 :1583. B. Liu, X. Li, D. Wang, Y. Yu, D. Lu, L. Chen, F. Lv, Y. Li, L. Cheng, Y. Song, Y. Xing. CEMIP promotes extracellular matrix‐detached prostate cancer cell survival by inhibiting ferroptosis. Cancer Science. 2022 ; 113 :2056. S. Zhao, P. Li, W. Wu, Q. Wang, B. Qian, X. Li, M. Shen. Roles of ferroptosis in urologic malignancies. Cancer Cell International. 2021 ; 21 . D. Liao, G. Yang, Y. Yang, X. Tang, H. Huang, J. Shao, Q. Pan. Identification of Pannexin 2 as a Novel Marker Correlating with Ferroptosis and Malignant Phenotypes of Prostate Cancer Cells. OncoTargets and Therapy. 2020 ; Volume 13 :4411. R. Kim, A. Hashimoto, N. Markosyan, V. A. Tyurin, Y. Y. Tyurina, G. Kar, S. Fu, M. Sehgal, L. Garcia-Gerique, A. Kossenkov, B. A. Gebregziabher, J. W. Tobias, K. Hicks, R. A. Halpin, N. Cvetesic, H. Deng, L. Donthireddy, A. Greenberg, B. Nam, R. H. Vonderheide, Y. Nefedova, V. E. Kagan, D. I. Gabrilovich. Ferroptosis of tumour neutrophils causes immune suppression in cancer. Nature. 2022 ; 612 :338. H. Xu, D. Ye, M. Ren, H. Zhang, F. Bi. Ferroptosis in the tumor microenvironment: perspectives for immunotherapy. Trends in Molecular Medicine. 2021 ; 27 :856. W. Wu, X. a. Wang, W. Le, C. Lu, H. Li, Y. Zhu, X. Chen, W. An, C. Xu, Q. Wu, L. Wang. Immune microenvironment infiltration landscape and immune-related subtypes in prostate cancer. Frontiers in Immunology. 2023 ; 13 . S. S. Nair, R. Weil, Z. Dovey, A. Davis, A. K. Tewari. The Tumor Microenvironment and Immunotherapy in Prostate and Bladder Cancer. Urologic Clinics of North America. 2020 ; 47 :e17. K. Li, H. Shi, B. Zhang, X. Ou, Q. Ma, Y. Chen, P. Shu, D. Li, Y. Wang. Myeloid-derived suppressor cells as immunosuppressive regulators and therapeutic targets in cancer. Signal Transduction and Targeted Therapy. 2021 ; 6 . M. A. Giese, L. E. Hind, A. Huttenlocher. Neutrophil plasticity in the tumor microenvironment. Blood. 2019 ; 133 :2159. Y. Pan, Y. Yu, X. Wang, T. Zhang. Tumor-Associated Macrophages in Tumor Immunity. Frontiers in Immunology. 2020 ; 11 . M. McLaughlin, E. C. Patin, M. Pedersen, A. Wilkins, M. T. Dillon, A. A. Melcher, K. J. Harrington. Inflammatory microenvironment remodelling by tumour cells after radiotherapy. Nature Reviews Cancer. 2020 ; 20 :203. J. Peng, X. Yin, W. Yun, X. Meng, Z. Huang. Radiotherapy-induced tumor physical microenvironment remodeling to overcome immunotherapy resistance. Cancer Letters. 2023 ; 559 . J. Murgić. Recent Advances in Radiotherapy Modalities for Prostate Cancer. Acta Clinica Croatica. 2022 . C. Wan, Y. Sun, Y. Tian, L. Lu, X. Dai, J. Meng, J. Huang, Q. He, B. Wu, Z. Zhang, K. Jiang, D. Hu, G. Wu, J. F. Lovell, H. Jin, K. Yang. Irradiated tumor cell–derived microparticles mediate tumor eradication via cell killing and immune reprogramming. Science Advances. 2020 ; 6 . Y. Ding, Y. Wang, Q. Hu. Recent advances in overcoming barriers to cell‐based delivery systems for cancer immunotherapy. Exploration. 2022 ; 2 . N. Desai, P. Katare, V. Makwana, S. Salave, L. K. Vora, J. Giri. Tumor-derived systems as novel biomedical tools—turning the enemy into an ally. Biomaterials Research. 2023 ; 27 . L. Lu, H. Zi, J. Zhou, J. Huang, Z. Deng, Z. Tang, L. Li, X. Shi, P. C. Lo, J. F. Lovell, D. Deng, C. Wan, H. Jin. Engineered Microparticles for Treatment of Murine Brain Metastasis by Reprograming Tumor Microenvironment and Inhibiting MAPK Pathway. Advanced Science. 2023 ; 10 . Z. Tuo, Q. He, Z. Zhang, Y. Wang, J. Sun, Q. Wei, Y. Hu, J. F. Lovell, H. Jin, K. Yang. Irradiation conditioning of adjuvanted, autologous cancer cell membrane nanoparticle vaccines. Chemical Engineering Journal. 2022 ; 433 . Z. Liao, J. Huang, P.-C. Lo, J. F. Lovell, H. Jin, K. Yang. Self-adjuvanting cancer nanovaccines. Journal of Nanobiotechnology. 2022 ; 20 . Y. Liu, Y. Wan, Y. Jiang, L. Zhang, W. Cheng. GPX4: The hub of lipid oxidation, ferroptosis, disease and treatment. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. 2023 ; 1878 . M. W. Lee, R. Bassiouni, N. A. Sparrow, A. Iketani, R. J. Boohaker, C. Moskowitz, P. Vishnubhotla, A. S. Khaled, J. Oyer, A. Copik, C. Fernandez-Valle, J. M. Perez, A. R. Khaled. The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption. Cell Death & Disease. 2014 ; 5 :e1249. A. Porgador, E. Gilboa. Bone marrow-generated dendritic cells pulsed with a class I-restricted peptide are potent inducers of cytotoxic T lymphocytes. The Journal of experimental medicine. 1995 ; 182 :255. J. Weischenfeldt, B. Porse. Bone Marrow-Derived Macrophages (BMM): Isolation and Applications. Cold Spring Harbor Protocols. 2008 ; 2008 . Y. Zou, J. Xie, S. Zheng, W. Liu, Y. Tang, W. Tian, X. Deng, L. Wu, Y. Zhang, C.-W. Wong, D. Tan, Q. Liu, X. Xie. Leveraging diverse cell-death patterns to predict the prognosis and drug sensitivity of triple-negative breast cancer patients after surgery. International Journal of Surgery. 2022 ; 107 . T. M. Seibt, B. Proneth, M. Conrad. Role of GPX4 in ferroptosis and its pharmacological implication. Free Radical Biology and Medicine. 2019 ; 133 :144. F.-J. Li, H.-Z. Long, Z.-W. Zhou, H.-Y. Luo, S.-G. Xu, L.-C. Gao. System Xc−/GSH/GPX4 axis: An important antioxidant system for the ferroptosis in drug-resistant solid tumor therapy. Frontiers in Pharmacology. 2022 ; 13 . T. L. Murphy, K. M. Murphy. Dendritic cells in cancer immunology. Cellular & Molecular Immunology. 2021 ; 19 :3. U. Basak, T. Sarkar, S. Mukherjee, S. Chakraborty, A. Dutta, S. Dutta, D. Nayak, S. Kaushik, T. Das, G. Sa. Tumor-associated macrophages: an effective player of the tumor microenvironment. Frontiers in Immunology. 2023 ; 14 . F. J. Bock, S. W. G. Tait. Mitochondria as multifaceted regulators of cell death. Nature Reviews Molecular Cell Biology. 2019 ; 21 :85. Y.-Q. Wang, S.-Y. Chang, Q. Wu, Y.-J. Gou, L. Jia, Y.-M. Cui, P. Yu, Z.-H. Shi, W.-S. Wu, G. Gao, Y.-Z. Chang. The Protective Role of Mitochondrial Ferritin on Erastin-Induced Ferroptosis. Frontiers in Aging Neuroscience. 2016 ; 8 . X. Fang, H. Wang, D. Han, E. Xie, X. Yang, J. Wei, S. Gu, F. Gao, N. Zhu, X. Yin, Q. Cheng, P. Zhang, W. Dai, J. Chen, F. Yang, H.-T. Yang, A. Linkermann, W. Gu, J. Min, F. Wang. Ferroptosis as a target for protection against cardiomyopathy. Proceedings of the National Academy of Sciences. 2019 ; 116 :2672. J. G. Berdeja, D. Madduri, S. Z. Usmani, A. Jakubowiak, M. Agha, A. D. Cohen, A. K. Stewart, P. Hari, M. Htut, A. Lesokhin, A. Deol, N. C. Munshi, E. O'Donnell, D. Avigan, I. Singh, E. Zudaire, T.-M. Yeh, A. J. Allred, Y. Olyslager, A. Banerjee, C. C. Jackson, J. D. Goldberg, J. M. Schecter, W. Deraedt, S. H. Zhuang, J. Infante, D. Geng, X. Wu, M. J. Carrasco-Alfonso, M. Akram, F. Hossain, S. Rizvi, F. Fan, Y. Lin, T. Martin, S. Jagannath. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): a phase 1b/2 open-label study. The Lancet. 2021 ; 398 :314. J. Su, C. Bian, Z. Zheng, H. Wang, L. Meng, Y. Xin, X. Jiang. Cooperation effects of radiation and ferroptosis on tumor suppression and radiation injury. Frontiers in Cell and Developmental Biology. 2022 ; 10 . G. Lei, Y. Zhang, P. Koppula, X. Liu, J. Zhang, S. H. Lin, J. A. Ajani, Q. Xiao, Z. Liao, H. Wang, B. Gan. The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression. Cell Research. 2020 ; 30 :146. G. Lei, C. Mao, Y. Yan, L. Zhuang, B. Gan. Ferroptosis, radiotherapy, and combination therapeutic strategies. Protein & Cell. 2021 ; 12 :836. X. Lang, M. D. Green, W. Wang, J. Yu, J. E. Choi, L. Jiang, P. Liao, J. Zhou, Q. Zhang, A. Dow, A. L. Saripalli, I. Kryczek, S. Wei, W. Szeliga, L. Vatan, E. M. Stone, G. Georgiou, M. Cieslik, D. R. Wahl, M. A. Morgan, A. M. Chinnaiyan, T. S. Lawrence, W. Zou. Radiotherapy and Immunotherapy Promote Tumoral Lipid Oxidation and Ferroptosis via Synergistic Repression of SLC7A11. Cancer Discovery. 2019 ; 9 :1673. D. L. Medina, S. Di Paola, I. Peluso, A. Armani, D. De Stefani, R. Venditti, S. Montefusco, A. Scotto-Rosato, C. Prezioso, A. Forrester, C. Settembre, W. Wang, Q. Gao, H. Xu, M. Sandri, R. Rizzuto, M. A. De Matteis, A. Ballabio. Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. Nature Cell Biology. 2015 ; 17 :288. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.tif Supplymentmaterials.docx Cite Share Download PDF Status: Published Journal Publication published 05 May, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 07 Mar, 2024 Reviews received at journal 22 Feb, 2024 Reviewers agreed at journal 16 Feb, 2024 Reviewers agreed at journal 08 Feb, 2024 Reviewers invited by journal 08 Feb, 2024 Editor assigned by journal 03 Feb, 2024 Submission checks completed at journal 03 Feb, 2024 First submitted to journal 30 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3911119","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":270822267,"identity":"7ccdb5be-db19-4b02-b6c1-04d64e4ab7fc","order_by":0,"name":"Zihan Deng","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Zihan","middleName":"","lastName":"Deng","suffix":""},{"id":270822268,"identity":"fda9abd4-cc82-481f-891c-a2c7ee6bfa22","order_by":1,"name":"Binghui Li","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Binghui","middleName":"","lastName":"Li","suffix":""},{"id":270822269,"identity":"70404e45-6838-4ee0-a652-39af34935102","order_by":2,"name":"Muyang Yang","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Muyang","middleName":"","lastName":"Yang","suffix":""},{"id":270822270,"identity":"29b3b4d1-566d-4d12-911f-27fb7cfbe3a9","order_by":3,"name":"Lisen Lu","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lisen","middleName":"","lastName":"Lu","suffix":""},{"id":270822271,"identity":"6f18b95f-f0b4-41ea-975c-d053f515db25","order_by":4,"name":"Xiujuan Shi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiujuan","middleName":"","lastName":"Shi","suffix":""},{"id":270822272,"identity":"01d2eb16-b6a0-4786-ac6f-89267c6da900","order_by":5,"name":"Jonathan Lovell","email":"","orcid":"","institution":"University at Buffalo, State University of New York","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Lovell","suffix":""},{"id":270822273,"identity":"1b8b97c6-c69f-4a35-bbb9-5ece17bc86df","order_by":6,"name":"Xiantao Zeng","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Xiantao","middleName":"","lastName":"Zeng","suffix":""},{"id":270822274,"identity":"7f5823df-bab7-4b63-afb4-72341c365460","order_by":7,"name":"Weidong Hu","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Weidong","middleName":"","lastName":"Hu","suffix":""},{"id":270822275,"identity":"1d451c06-f8f1-46a1-a9fd-845bffff9f32","order_by":8,"name":"Honglin Jin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYBACxmYGxsdQtgHRWpiNgbQE8VqAgE2aNC3M7bzHqgvb6uoY2Ju3STDU3CHGYXxpt2e2HZZg4DlWJsFw7BkxWnjMbvO2HZBgkMgxk2BsOEyclmLetjoJBvk3JGhh5m1jBtrCQ7wWY2mec4cl23jSii0SjhGhxbD/jOFnnrI6fn72wxtvfKghRksDlMEGIhIIa2BgkCdG0SgYBaNgFIxwAADJlS1M+dPxLgAAAABJRU5ErkJggg==","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Honglin","middleName":"","lastName":"Jin","suffix":""}],"badges":[],"createdAt":"2024-01-30 16:06:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3911119/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3911119/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-024-02496-3","type":"published","date":"2024-05-05T19:58:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50729379,"identity":"b038dd2b-10c2-4782-be50-0b2d9e4f823b","added_by":"auto","created_at":"2024-02-06 12:29:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":796579,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMany key factors of ferroptosis significantly increased in tumor tissue and correlated with infiltration of different immune cells in patients with prostate cancer. \u003c/strong\u003e(A) Difference of ferroptosis pathway factors expression between carcinoma and paracancerous tissue of prostate cancer. (B) The clusters of single cell transcriptome analysis of immunocytes in prostate cancer. (C) Correlation between ferroptosis pathway factors and distinct immune cells in microenvironment of prostate cancer. (D) The expression of GPX4 in pan-cancers. (E) Correlation between GPX4 expression and immune score in prostate cancer. Spearman correlation analysis was used to determine significant differences by P-value. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3911119/v1/11d150f92cb813d44f573064.png"},{"id":50729913,"identity":"1b4289c1-6e85-45a8-8fec-2f00455df511","added_by":"auto","created_at":"2024-02-06 12:37:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":862828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation and characterization of RC@RMPs. \u003c/strong\u003e(A) Schematic of RC@RMP preparation. (B) Determination of the most appropriate electroporation condition to load RSL3 in RMPs as much as possible. 100 μg RSL3 and 100 μg RMPs were mixed in the system. (C) Determination of the most appropriate ratio of RSL3 and RMPs to load RSL3 in RMPs as much as possible. The mass of RMPs in the system were set to 100 μg. (D) Zeta potential and (E) size of RMPs, RSL3@ RMPs, CT20p@RMPs and RC@RMPs measured by Malvern laser particle size analyzer. (F) TEM image of RMPs and RC@RMPs. (G) The expression of CD63, CD81 and Alix of RMPs, RSL3@ RMPs, CT20p@RMPs and RC@RMPs analyzed by Western blot. P-values were calculated by one-way analysis of variance (ANOVA). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3911119/v1/037f1d4d1cea48331a6d8f21.png"},{"id":50729375,"identity":"542fe804-6e75-431b-ba1a-3863e8cad3da","added_by":"auto","created_at":"2024-02-06 12:29:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1809401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRC@RMPs induce intense ferroptosis of RM-1 tumor cell, activate DCs and promote M1 macrophages polarization.\u003c/strong\u003e (A) Different tumor cells’ ability to ingest RMPs. RM-1, LLC, 4T1, B16F10, MC38 and GL261 tumor cells were detected mean fluorescence intensity (MFI) in FL1 channel 3 hours and 6 hours after incubated. (B) Relative cell growth of RM-1 cells respectively incubating RMPs, RSL3@ RMPs, CT20p@RMPs and RC@RMPs for 24 hours. (C) The colocalization of CT20p and mitochondrion through Confocal laser scanning microscopy. 100 μg CT20p-Rhodamine B was capsulated into 100 μg RMPs by electroporation. As control, Rhodamine B with equal amount of substance as CT20p-Rhodamine B was loaded into 100 μg RMPs. 20 μg mL\u003csup\u003e−1 \u003c/sup\u003eCT20p-Rhodamine B-RMPs and Rhodamine B-RMPs were added into the cells was observed via Confocal microscopy and Pearson correlation coefficient was calculated by image J software. (D) The rates of RM-1 apoptosis induced by different agents and RMPs. RM-1 were respectively incubated with PBS (control), 5 μg mL\u003csup\u003e−1\u003c/sup\u003e RSL3, 20 μg mL\u003csup\u003e−1 \u003c/sup\u003edifferent RMPs containing RMPs, RSL3@RMPs, CT20p@RMPs and RC@RMPs. Annexin V-Alexa Fluor 488/7-AAD apoptosis detection kit were used to evaluate the percentages of apoptosis RM-1 24 hours after different agents and RMPs incubation. Annexin V\u003csup\u003e+\u003c/sup\u003e and 7-AAD\u003csup\u003e-\u003c/sup\u003e stands for early apoptosis, while the cells with double positive are considered as late apoptosis. (E) ROS levels detected through H2DCFDA probe quantified by flow cytometer. The RM-1 cells in (D) were dyed with H2DCFDA which can react with ROS and emit fluorescence around 520 nm. (F) The evaluation of mitochondrial membrane potential of RM-1 cells in (D) with JC-1 assay kit. The wavelength of JC-1 emission changes from 590 nm to 529 nm when mitochondrial membrane potential decreases. The MFI of FL1 channel reflects mitochondrion damage degree. (G) FITC-Liperfluo (5 μM) and Phen Green SK diacetate (PGSK) (10 μM) were incubated with the cells in (D) and measured MFI of FITC. (I) The expression of CD80, CD86 and MHCⅡ of DC2.4 cells and (J) CD86 of RAW264.7 incubating RMPs, RSL3@ RMPs, CT20p@RMPs and RC@RMPs for 24 hours. P-values were calculated by one-way analysis of variance (ANOVA). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3911119/v1/57da2cd8c49419e996367d89.png"},{"id":50729378,"identity":"1f58faf3-8c33-4716-9024-e23b815382de","added_by":"auto","created_at":"2024-02-06 12:29:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2725255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRC@RMPs accumulated in tumor cells and different subsets of immunocytes in tumor tissue \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) Quantization of RMPs and RC@RMPs accumulation in tumor cells distinct immunocytes in tumor. 100 μg PKH26 pre-dyed RMPs and RC@RMPs were intratumorally injected and the mice were sacrificed 24 hour after treatment. MFI of PKH26 was detected by flow cytometer and percentages of PKH26 positive immunocytes were calculated. (B) Representative immunofluorescence images showing RMPs internalization of F4/80\u003csup\u003e+\u003c/sup\u003e macrophage and CD11c\u003csup\u003e+\u003c/sup\u003e DCs in tumor tissue 24 hour after treatment.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3911119/v1/0c2268f1571965d12f221e4c.png"},{"id":50729377,"identity":"477c0c1e-4e07-4cc1-a60a-fa47dadb4a92","added_by":"auto","created_at":"2024-02-06 12:29:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":751662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRC@RMPs treatment inhibited the growth of RM-1 tumor by remodeling infiltration of immunocytes, and the effect can be strengthened via combination with anti-PD-1 mAb. \u003c/strong\u003e(A) Schematic of subcutaneously implanted RM-1 tumor treatment by intratumoral injection of RMPs, RSL3@ RMPs, CT20p@RMPs and RC@RMPs in a dose of 100 μg per mouse one time. Anti-PD-1 mAb was intraperitoneal injected (10 mg kg\u003csup\u003e-1\u003c/sup\u003e) in corresponding groups. (B) The curve of tumor growth by measuring tumor volumes every 2 days. (C) Survival curve of RM-1 burden mice in all groups. (D-L) The changes of immunocytes in tumor after treatment were analyzed by flow cytometer. The log-rank (Mantel-Cox) test was applied to compare survival rates between groups. P-values of other experiments were calculated by one-way analysis of variance (ANOVA). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3911119/v1/1556a4af842b4e3574a3234f.png"},{"id":50729380,"identity":"5c4bfa1f-2f31-4781-a3e9-288f8a66d6bb","added_by":"auto","created_at":"2024-02-06 12:29:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2500830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe anti-tumor effect of RC@RMPs depends on CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells and macrophages. \u003c/strong\u003e(A) Representative immunofluorescence images showing the number of M2 macrophage in tumor tissue. (B) Schematic of subcutaneously implanted RM-1 tumor treatment by RC@RMPs with depletion of CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, neutrophils and macrophages. (C) The curve of tumor growth by measuring tumor volumes of mice every 2 days. P-values were calculated by one-way analysis of variance (ANOVA). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ns stands for on significance.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3911119/v1/b29abcb4400e5592d820d1e5.png"},{"id":56043026,"identity":"ae56b6ac-9dc5-4565-8d79-835b37c7862f","added_by":"auto","created_at":"2024-05-07 20:09:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4694839,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3911119/v1/9f403aa2-6d61-494b-b9e4-de928bc3ac82.pdf"},{"id":50729382,"identity":"f430e692-c491-4394-98fc-ef17d221001e","added_by":"auto","created_at":"2024-02-06 12:29:44","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6830950,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-3911119/v1/b5284f93b89b41487c93625e.tif"},{"id":50729383,"identity":"1b1ee68e-7b4e-44a7-aace-043d9e57b74d","added_by":"auto","created_at":"2024-02-06 12:29:44","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6920630,"visible":true,"origin":"","legend":"","description":"","filename":"Supplymentmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3911119/v1/25d92c7f564ecb63369cf850.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Irradiated Microparticles Suppress Prostate Cancer by Tumor Microenvironment Reprogramming and Ferroptosis","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eProstate cancer is an age-related tumor of the male genitourinary system that has a high incidence in the elderly population. Concurrent with the rapidly aging population, prostate cancer has become the leading male urinary system tumor in the world, and has one of fastest growing incidences among malignant tumors in males in the past decade\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The main treatment for prostate cancers involves prostatectomy combined with endocrine therapy and radiotherapy or chemotherapy. Unfortunately, after a period of treatment, most patients develop castration-resistant prostate cancer (CRPC) that is insensitive to treatment\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. However, as prostate cancer is normally considered as a cold tumor, targeted therapy and immunotherapy often show only limited efficacy in the clinic\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, there is an urgent need to explore the mechanism of the occurrence and development of prostate cancer and to develop new therapeutic drugs for prostate cancer.\u003c/p\u003e \u003cp\u003eFerroptosis is a form of immunogenic cell death (ICD) that can enhance the ability of innate immune cells to recognize tumor cells and initiate the adaptive immune response\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Thus, ferroptosis can effectively promote the transformation of cold tumors into hot tumors, ultimately improving the response of cancer patients to immunotherapy\u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. SLC7A11 and GPX4 are two key molecules that inhibit ferroptosis, and are highly expressed in prostate cancer and CRPC\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Association studies have shown that nine genes associated with ferroptosis are closely related to the prognosis of patients with prostate cancer. Conversely, the CEMIP, HSPB1, and PANX2 genes, which interfere with the process of ferroptosis, can effectively promote the survival of prostate cancer cells, suggesting that ferroptosis-related genes may be prognostic biomarkers and potential drug targets for patients with prostate cancer\u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Related studies have also suggested that the ferroptosis of neutrophils may promote the occurrence and development of tumors\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e; however, whether this phenomenon exists in prostate cancer requires further investigation. Moreover, the heterogeneity of prostate cancer often results in hyposusceptibility to ferroptosis, and the agents that trigger ferroptosis can also induce the death of T cells and non-tumor tissue damage, which limits the therapeutic effect of ferroptosis-inducing agents for cancer treatment\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is necessary to develop novel strategies to enhance ferroptosis with improved specificity to target tumor cells.\u003c/p\u003e \u003cp\u003eThe tumor immunosuppressive microenvironment is the main cause of clinical prostate cancer recurrence and immunotherapy failure, among which myeloid-derived suppressor cells (MDSCs), inhibitory neutrophils, regulatory dendritic cell (DC), and tumor-promoting M2 macrophages are the main causes of tumor immunosuppressive microenvironment formation\u003csup\u003e[\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. It has been reported that commonly used radiotherapy techniques can improve the tumor microenvironment (TME) to some extent\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. However, radiotherapy treatment for prostate cancer commonly results in unavoidable adverse side effects, including lower urinary tract symptoms, intestinal complications, erectile dysfunction, and myelosuppression, which limits the application of radiotherapy in patients\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In the previous study, we reported that radiated tumor cell-derived microparticles (RMPs) are the main medium of the bystander effect induced by radiotherapy\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. To some extent, the RMPs act as mimetics of radiotherapy, inducing tumor cell ferroptosis and the reprogramming of tumor-promoting M2 macrophages, which may activate type I interferon signaling through the cGAS-STING pathway\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. As RMPs originate from the tumor tissue itself, they have an innate ability to target tumor cells. Microparticles have been proven as good carrier of agents for cancer therapies\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Our previous studies showed that RMPs loaded with agents and adjuvants can inhibit progression of lung cancer and its brain metastasis\u003csup\u003e[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR29\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, suggesting that RMPs derived from prostate cancer may a good drug carrier for the treatment of advanced prostate cancer.\u003c/p\u003e \u003cp\u003eTo sum up, this study used RM-1 prostate cancer cells as a tumor model, and extracted the RMPs of the tumor cells to be repurposed as drug carriers that encapsulate RSL-3, a ferroptosis inducer targeting GPX-4 proteins, which are a key inhibitor of ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. In addition, mitochondrial targeting peptide CT20p (peptide sequence: VTIFVAGVLTASLTIWKKMG, an inducer of apoptosis), which can induce multimodel death in tumor cells, was also loaded into RMPs. The CT20p peptide is the C-terminal of the pro-apoptotic protein Bax, which can regulate the activity of Chaperonin-Containing TCP protein in prostate cancer, resulting in mitochondrial instability and cytoskeletal disruption to promote the effective killing of tumor cells\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. The results of this study showed that RMPs encapsulating RSL-3 and CT20p (RC@RMPs) retain the characteristics of RMPs and effectively target and kill tumor cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. As an apoptosis inducer, CT20p altered the mitochondrial membrane potential and aggravated ferroptosis by increasing ROS production. Furthermore, RC@RMPs could activate DC cells and reprogram macrophage polarization. RC@RMPs enhanced both adaptive immunity via CD8\u003csup\u003e+\u003c/sup\u003e T cells and innate immunity to effectively kill prostate cancer cells. Together, these data provide proof-of-concept for the use of RMP carriers in the treatment of prostate cancer.\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cp\u003e \u003cb\u003eMaterials\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe medium of cell culture was purchased from Gibco Life Technologies, Inc. (Grand Island, NY, USA), including Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium. Fetal Bovine Serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co., Ltd. (Huzhou, China). Plasmocin was bought from InvivoGene (Toulouse, France) and penicillin/streptomycin was obtained from Biosharp (Hefei, China). Sterile 1\u0026times;phosphate buffered saline was purchased from Gibco Life Technologies, Inc. (Grand Island, NY, USA). All the cytokines were purchased from Biolegend (San Diego, CA, USA), containing granulocyte-macrophage colony-stimulating factor macrophage colony-stimulating factor, interleukin-4, interleukin-13, lipopolysaccharide and interferon-γ. RSL3 was bought from Selleck (Houston, TX, USA). CT20p peptide was bought from BankPeptide Inc. (Hefei, China). Sucrose for electroporation buffer was gained from Sinopharm (China). Acetonitrile, methanol and chloroform for High Performance Liquid Chromatography (HPLC) were all purchased from Thermo Fisher Scientific (Waltham, MA, USA) and their purity was more than 99%. The fluorescence dye DiO, DiD and Rhodamine were obtained from Yeasen (Shanghai, China). H2DCFDA and PKH26 were bought from MedChemExpress (NJ, USA). FITC-Liperfluo was obtained from Dojindo (Japan) and Phen Green SK diacetate was bought from GLPBIO (USA). Radioimmunoprecipitation assay buffer and the inhibitors of protease and phosphatase were obtained from Beyotime (Shanghai, China). For western blot, primary antibodies STING, p-STING, NFκB, p-NFκB and Laminin B1 were purchased from \u003cem\u003eABclonal (\u003c/em\u003eBoston, MA, USA) and GAPDH, CD63, CD81 and Alix were obtained from Proteintech Group, Inc. (Chicago, IL, USA). Secondary antibodies goat anti-mouse IgG H\u0026amp;L-HRP conjugated and goat anti-rabbit IgG H\u0026amp;L-HRP conjugated were bought from Abcam (Cambridge, UK). Collagenase IV and hyaluronidase were purchased from Biosharp (Hefei, China). All the antibodies for flow cytometry and immunocyte depletion were bought from Biolegend (San Diego, CA, USA). Clodronate liposomes were purchased from FormuMax (Silicon Valley, CA, USA). PD-1 mAb for treatment was obtained from BioXell (Italy).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCells culture\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll the murine cell lines were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China), including prostate cancer cell line (RM-1), Lewis lung carcinoma cell (LLC), mammary cancer cell line (4T1), \u003cem\u003ecolon adenocarcinoma cell\u003c/em\u003e line (MC38), B16F10 melanoma cells, GL261 glioma cells, DC line (DC2.4) and monocyte cell line (RAW264.7). All the cell lines were treated with 25 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Plasmocin for at least two weeks and were mycoplasma\u003csup\u003e-\u003c/sup\u003enegative as determined by MycoProbe Mycoplasma Detection Kit (R\u0026amp;D Systems, Minneapolis, MN, USA). RM-1, 4T1 and LLC cells were cultured in DMEM while other cells were maintained in RPMI 1640 medium. Bone marrow-derived dendritic cells and macrophages (BMDCs \u0026amp; BMDMs) from C57BL/6J mice were generated as previous descriptions in RPMI 1640 medium\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. All the mediums were added with 10% (v/v) FBS and 100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e penicillin/streptomycin.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of RMPs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn 10-cm cell culture dishes, 6\u0026times;10\u003csup\u003e6\u003c/sup\u003e RM-1 cells were planted and irradiated with a single dose of 20 Gy by 6-MV x-rays (CHIRAD 225). Next, the medium of irradiated cells was renewed by 20 mL DMEM completed medium which its microparticles had been removed via centrifugation. 72 hours later, the medium was collected, and cell debris were removed by 1000 \u003cem\u003eg\u003c/em\u003e for 10 min and 14,000 \u003cem\u003eg\u003c/em\u003e for 2 min. RMPs were gained from the supernatant via 14,000 \u003cem\u003eg\u003c/em\u003e for further 60 min at 4 ℃ and washed with sterile 1\u0026times;PBS for 2 times. At last, the RMPs were resuspended with 1\u0026times;PBS for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRMPs encapsulated with RSL-3 and CT20p via electroporation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRSL3 was dissolved in DMSO to a concentration of 10 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. CT20 peptide (CT20p) was diluted by ultrapure water. RSL3 or CT20p was mixed with RMPs in a 1:2 ratio of mass in 400 mM sucrose solution. By an electroporation system (GenePulser Xcell, BioRad, USA), 400 \u0026micro;L mixture was electroporated in 0.2 cm cuvettes via exponential pulse (voltage: 500 V; capacitance: 125 \u0026micro;F).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHigh Performance Liquid Chromatography (HPLC)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHPLC analysis was conducted using a LC-2030C Plus instrument (Shimadzu, Japan). The separation was implemented with a ShimNex C18 chromatographic column (4.6\u0026times;250 mm, 5 \u0026micro;m, 100 A, Shimadzu, Japan). Three times volume of acetonitrile was mixed with RMPs and then added chloroform (1:2, v/v). After vortexed, the mixture was centrifuged at 10,000 \u003cem\u003eg\u003c/em\u003e for 5 min and the lower layer was extracted for measurement. As the standard solution, 10 mg RSL3 was dissolved in acetonitrile and chloroform in a ratio the same as RMPs. All the samples were filtered through a 0.45 \u0026micro;m polytetrafluoroethylene filter. The components were separated and eluted by mobile phase eluted (A: methanol; B: acetonitrile) in the column at 25 ℃ with a flow rate of 1 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. An ultraviolet wavelength (254 nm) was selected for the detection of RSL3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantification of RMPs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe quantification of RMPs were determined by their protein concentrations. Radioimmunoprecipitation assay buffer were applied to lyse RMPs at 4\u0026deg;C for half an hour. Then, the lysis was centrifuged at 12,000 \u003cem\u003eg\u003c/em\u003e for 30 min at 4\u0026deg;C and the supernatant was transferred into a new centrifuge tube for protein concentration measurement by BCA Protein Assay Kit (Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization of RMPs Size and Transmission electron microscopy (TEM) Determination\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOne milliliter of 30 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e RMPs were taken for the measurement of the particle size and polydispersity index by Malvern laser particle size analyzer (Zetasizer Nano ZSP). For further identification of the sizes and morphology of RMPs were washed by ddH\u003csub\u003e2\u003c/sub\u003eO, deposited on copper mesh and then observed by TEM (HT7700-SS/FEI Tecnai G20 TWIN).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability measurement\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll the cells to evaluate viability were planted into 96-well plates (5000 cells per well). After 24 hours growing, distinct RMPs were treated with the cells for further 48 hours. A cell counting kit-8 (CCK-8) assay kit (Meilunbio, Dalian, Chian) was used to measure cell viability.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro cellular uptake assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the cellular uptake of RMPs, different cell lines were planted into 6-well plates and incubated with DiO pre-dyed MPs for 3 or 6 hours. The cells were collected, washed by PBS and analyzed through flow cytometry (Beckman CytoFLEX S, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of the colocalization of CT20p and mitochondrion\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRMPs with CT20p-Rhodamine B and Rhodamine B were added into the cells which were sequently incubated with 100 nM MitoTracker Green\u0026reg; FM at 25 ℃ for 30 minutes. Confocal laser scanning microscopy (Carl Zeiss LSM710) was used to observe the colocalization of CT20p and MitoTracker Green\u0026reg; FM (standing for mitochondrion) whose Pearson correlation coefficient was calculated by image J software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of cell apoptosis and ferroptosis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCells were cultured in 24-well plates (30,000 cells per well) and then incubated with MPs, RSL3@MPs, CT20p@MPs and RC@RMPs for 24 hours. The cells were harvested for relevant measurement. Apoptosis was evaluated by Annexin V-Alexa Fluor 488/7-AAD apoptosis detection kit (yeasen, China). The protocol complied with the instruction of the kit. To determine mitochondrial membrane potential, JC-1 assay kit (yeasen, China) was applied. The method to dye and measure cell apoptosis and mitochondrial membrane potential obeyed the instruction of the kits. To evaluate total ROS, lipid ROS and Fe\u003csup\u003e2+\u003c/sup\u003e level, the cells were respectively dyed with H2DCFDA (10 \u0026micro;M), FITC-Liperfluo (5 \u0026micro;M) and Phen Green SK diacetate (10 \u0026micro;M) in 1 mL PBS for 30 min at 37\u0026deg;C in a cell culture incubator. Wased by PBS twice, the cells were resuspended with 200 \u0026micro;L PBS and analyzed via flow cytometry.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blotting\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll the MPs and cells that be detected were lysed by RIPA buffer with the inhibitor of protease and phosphatase at 4\u0026deg;C for 30 minutes, and then centrifuged at at 12,000 \u003cem\u003eg\u003c/em\u003e for 30 min at 4\u0026deg;C. The mass of the sample loading was adjusted to the same according to their protein concentrations that were detected by BCA Protein Assay Kit. The samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane after boiled for 5 minutes. The membranes block by 5% not-fat milk at room temperature for 1 hour and incubated with related primary antibodies at 4\u0026deg;C overnight. With serveral wash by Tris-buffered saline with 0.05% Tween-20, secondary antibodies incubated with the membranes at room temperature for 1 hour. NcmECL Ultra (P10100, NCM Biotech) was applied for chemiluminescent exposure of the blot.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMale C57BL/6J mice (aged 6\u0026ndash;8 week, weighted 18\u0026ndash;20 g) were purchased from SHULAIBAO Biotech. All mice were kept in micro-isolator cages, and the experimental protocols were approved by the Hubei Provincial Animal Care and Use Committee and were in compliance with the experimental guidelines of the Animal Experimentation Ethics Committee of Huazhong Agricultural University.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo cellular internalization assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo identify RMPs uptake by cells in tumor \u003cem\u003ein vivo\u003c/em\u003e, we intratumorally injected 100 \u0026micro;L PKH26 marked RMPs to mice with RM-1 tumor burden. 24 hours later, the mice were sacrificed and the tumors were digested into single cell for flow cytometry analysis before they were incubated with antibodies of CD45 (clone S18009F), CD3 (clone 17A2), B220 (clone RA3-6B2), CD11b (clone M1/70), Ly6G (clone S19018G), F480 (clone BM8), CD11c (clone N418), MHCII (clone M5/114.15.2) and NK1.1 (clone PK136). Besides, some tumor tissues were fixed, dehydrated and sectioned into frozen sections which were going to stain by related antibodies and observed via confocal laser scanning microscopy. To image the distribution of RMPs, RC@RMPs were stained with DiD, the 540/20 nm excitation filter and 620/20 nm emission filter were used and the exposure time was 15 s.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSubcutaneously implanted prostate tumor model and treatment with RMPs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRM-1 tumor cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells in 100 \u0026micro;L PBS) were subcutaneously implanted into right back. Five days after tumor inoculation, mice with uniform tumor volume were randomly divided into 7 groups including control group, PD-1 mAb group, RMPs group, RSL3@RMPs group, CT20p@RMPs group, RC@RMPs group and RC@RMPs combined with PD-1 mAb group, and were treated with corresponding RMPs (intratumoral injection with 100 \u0026micro;g in 100 \u0026micro;L PBS) and PD-1 mAb (10 mg/kg, intraperitoneal injection) at 6, 8, 10, 12 and 14 days after grouping. Vernier caliper was applied to measure the length (\u003cem\u003eL\u003c/em\u003e) and width (\u003cem\u003eW\u003c/em\u003e) of subcutaneous tumors every other day. The volume of tumor was calculated by the the formula \u003cem\u003eV\u003c/em\u003e = (\u003cem\u003eL\u003c/em\u003e \u0026times; \u003cem\u003eW\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e) / 2. Mice were sacrificed when the tumor volume reached 1000 mm\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of immunocytes in tumor and draining lymph nodes (dLNs)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRM-1 tumors from mice were digested into single cell by cutting into small pieces and incubating with Collagenase IV (0.32 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and hyaluronidase (0.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 1 hours at 37\u0026deg;C. The tumor cells were filtered through 70 \u0026micro;m cell strainer after lysis of RBCs. All the samples were blocked Fc receptors followed by incubating with detection antibodies containing CD3 (clone 17A2), CD8 (clone SK1), CD69 (clone H1.2F3), CD4 (clone GK1.5), PD1 (clone 29F.1A12), TOX (clone 6E6D03), TCF1 (clone 7F11A10), CD11c (clone N418), CD11b (clone M1/70), CD86 (clone GL-1), MHCII (clone M5/114.15.2), CD44 (clone IM7), CD62L (clone MEL-14) and Zombie NIR\u0026trade;, and then measured via flow cytometry.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytokines detection\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRM-1 tumors from mice were weighted and grinded into homogenate. The supernatant was collected by 6000 \u003cem\u003eg\u003c/em\u003e centrifugation for 20 minutes at 4 ℃. The LEGENDplex Mouse Cytokine Release Syndrome Panel (13-plex) with VBottom Plate (purchased from Biolegend) was used for cytokine detection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmune cell depletion\u003c/b\u003e \u003c/p\u003e \u003cp\u003eT helper cells and CTLs were depleted by CD4 (clone GK1.5) and CD8 (clone 2.43) antibodies respectively. One day before treatment, 200 \u0026micro;g antibodies were intraperitoneal injected into mice for 5 times at 2-day intervals. Macrophages were depleted by clodronate liposomes. One day before treatment, 200 \u0026micro;L clodronate liposomes were intravenously injected into mice for 5 times at 3-day intervals. Neutrophils were depleted by Ly6G (clone 1A8) antibody. One day before treatment, 200 \u0026micro;g antibodies were intraperitoneal injected into mice for 5 times at 2-day intervals.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll the data were analyzed by Prism software (GraphPad Prism 6.0 software). The log-rank (Mantel-Cox) test was applied to compare survival rates between groups.Kaplan-Meier analysis was used to analyze tumor growth, and comparisons of three or more groups were calculated by one-way analysis of variance (ANOVA). Two-tailed unpaired t test or the Mann-Whitney \u003cem\u003eU\u003c/em\u003e test was performed to determine the significance of two groups. \u003cem\u003eP\u003c/em\u003e values of \u0026lt;\u0026thinsp;0.05 were determined statistically significant. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and ns stands for no significant.\u003c/p\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Prostate cancer gene expression patterns and immunological correlation of ferroptosis pathway factors\u003c/h2\u003e \u003cp\u003eTo investigate the relationship between ferroptosis-related genes and prostate cancer, we downloaded data for a total of 88 ferroptosis-related genes from a published dataset\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e to analyse the differentially expressed genes in prostate cancer versus normal samples (|log2FC| \u0026gt;= 1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). We obtained a total of 8 ferroptosis-related genes (\u0026ldquo;SLC7A11\u0026rdquo;, \u0026ldquo;CBS\u0026rdquo;, \u0026ldquo;ALOX15\u0026rdquo;, \u0026ldquo;DPP4\u0026rdquo;, \u0026ldquo;SLC39A8\u0026rdquo;, \u0026ldquo;TP53\u0026rdquo;, and \u0026ldquo;GPX4\u0026rdquo;) in prostate cancer and paired normal samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The TME of prostate cancer is relatively devoid of immune infiltration compared to other malignancies. Therefore, we reanalyzed published single-cell data on prostate cancer to explore the infiltration of immune cells in prostate cancer tumors. The results showed that the vast majority of cells in prostate cancer were epithelial cells, and that any immune cells present were predominantly exhausted CD8\u003csup\u003e+\u003c/sup\u003e T cells, confirming the immune desert phenotype of prostate cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). 28 immune cell infiltration scores were evaluated in prostate cancer according to single sample gene set enrichment analysis (ssGSEA), and the correlations between these 8 ferroptosis-related genes and immune cell infiltration scores were calculated. The results showed that the expression of these genes was significantly correlated with immune infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Because the GPX4 gene was significantly highly expressed in prostate cancer (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and associated with infiltrated immunocytes as shown above, we further explored the relationship between GPX4 and prostate cancer. We investigated the expression of the GPX4 gene in different cancers, and found that GPX4 was highly expressed in the vast majority of moderate cancers, including prostate cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Surprisingly, however, there was no significant correlation between GPX4 expression and tumor immune infiltration scores in prostate cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In summary, we identified several vital ferroptosis pathway factors that are highly expressed in prostate cancer and correlated with immune cell infiltration, suggesting that they may be good targets to induce ferroptosis to improve the immune desert status in prostate cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Preparation and characterization of RC@RMPs\u003c/h2\u003e \u003cp\u003eGPX4 is a peroxidase involved lipid metabolism that is vital for inhibiting ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. GPX4 is correlated with the infiltration of various immune cells in prostate cancer, as indicated by the above bioinformatics analysis. The inhibition of GPX4 is considered a potential strategy for initiating ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. RMPs, which are derived from radiotherapy-treated cells, are carriers of large quantities of DAMPs and have been shown to induce tumor cell death via ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. We loaded the GPX-4 inhibitor RSL3 into RMPs to investigate the potential for a synergistic effect of GPX-4 inhibition and RMPs on ferroptosis to treat prostate cancer. Additionally, the C-terminal of the pro-apoptotic protein Bax, the CT20p peptide, which induces mitochondrial damage, was also added into RMPs to intensify ICD for an enhanced anti-tumor immune response. The combined system (RC@RMPs) was constructed by obtaining and centrifigating the supernatant of irridiated RM-1 tumor cells loaded with CT20p and RSL3 through electroporation, as described in the experimental section and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. As the quantity of active agents in RMPs differed based on electroporation parameters and the ratio of RMPs to agents, we tried several different conditions for electroporation. Based on High Performance Liquid Chromatography (HPLC) analysis, the highest quantity of RSL3 in RMPs was achieved using following electroporation parameters: 500 V voltage, 125 \u0026micro;F capacitance, and exponential decay wave mode, when 100 \u0026micro;g RSL3 was mixed with 100 \u0026micro;g RMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Maintaining the above electroporation conditions, we further optimized the mass of RSL3 mixed with 100 \u0026micro;g RMPs, and found that the RSL3 concentration in the RMPs as the highest when 150 \u0026micro;g RSL3 was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Characterization of zeta potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and size (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) showed no significant differences among RMPs, RSL3@RMPs, CT20p@RMPs, and RC@RMPs. Transmission electron microscopy (TEM) indicated that RMPs and RC@RMPs had a regular spherical morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Therefore, the loading of RSL3 and CT20p agents did not influence the structure of the RMPs. Western blot analysis demonstrated all the RMPs were rich in extracellular vesicle\u0026ndash;associated proteins such as CD63 and CD81, whose expression was not influenced by encapsulation of CT20p and RSL3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Thus, the RMPs that we extracted had similar composition as extracellular vesicles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.3 RC@RMPs can kill RM-1 cells by causing ferroptosis and apoptosis\u003c/h2\u003e \u003cp\u003eTo identify whether RMPs can be ingested by tumor cells, 20 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DiO pre-labeled RMPs were incubated with some murine tumor cell lines for 3 or 6 hours. We detected a high level of DiO fluorescence intensity in RM-1 cells, indicating that RM-1 cells can effectively take up RMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We then evaluated the toxicity of different RMPs to RM-1 tumor cells. RC@RMPs were the most effective at eliminating RM-1 cells with the lowest IC (50) values (4.474 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e vs 53.13 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for RMPs, 5.467 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for RSL3@RMPs and 47.43 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CT20p@RMPs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). RSL3 is considered to induce ferroptosis by increasing ROS production and mitochondrial dysfunction. CT20p peptides were added into RMPs to synergistically promote ferroptosis by mitochondrial damage. RMPs with CT20p-Rhodamine B and Rhodamine B (control) were incubated with RM-1 to identify the subcellular localization of CT20p. Confocal microscopy showed that CT20p-Rhodamine B colocalized with mitochondria as identified by staining with MitoTracker Green\u0026reg; FM, which was confirmed by Pearson correlation coefficient analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Therefore, RMPs loaded with CT20p were targeted to the mitochondria. Flow cytometry analysis revealed that RC@RMPs in large part caused RM-1 cells to proceed to late apotosis, which was difficult to recover from (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Only RSL3-loaded RMPs resulted in significantly less cell death than RC@RMPs; CT20p peptides-loaded RMPs were able to induce early apoptosis by producing damaged mitochondria, though this may be reversed by mitophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Mechanistically, the toxicity of RC@RMPs was primarily related to the induction of ferroptosis, as indicated by increased total ROS production, increased lipid peroxidation, increased Fe\u003csup\u003e2+\u003c/sup\u003e levels, and decreased mitochondrial membrane potential, which occurred to a greater extent in cells treated with RC@RMPs compared to other RMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H). ROS measurement based on H2DCFDA mean fluorescence intensity (MFI) was significantly higher in cells treated with RSL3@RMPs or CT20p@RMPs compared to RMPs, suggesting that both RSL3 and CT20p were able to elevate ROS production. RC@RMP-treated cells showed the highest ROS levels, demonstrating a synergism of RSL3 and CT20p (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Meanwhile, there was greater green fluorescence in CT20p@RMP-treated cells compared with the RMPs group, confirming that CT20p played a key role in effects on mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Overall, the combination of RSL3 and CT20p appears to exert strong synergy to trigger ferroptosis and may augment the subsequent immune response.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.4 RC@RMPs activate DCs and regulate macrophage polarization\u003c/h2\u003e \u003cp\u003eAs Antigen Presenting Cells (APCs), DCs are vital for initiating anti-tumor immunity. In the TME, they capture and process tumor antigens then present these antigens to tumor-specific T cells, which then recognize and eliminate tumor cells\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Macrophages are also APCs and function to activate T cells. However, tumor-associated macrophages (TAMs) always act as promoters of tumor progression by secreting anti-inflammatory cytokines such as IL-10 and TGF-β\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Therefore, we measured the direct influence of RC@RMPs on APCs to evaluate the effect that RC@RMPs may have on reshaping the immunological environment of the tumor. DiO pre-labeled RMPs and RC@RMPs at different concentrations were incubated with DC2.4 and RAW264.7 cells. We found that the quantities of RMPs and RC@RMPs taken by APCs increased in a dose-depenent manner (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). CCK8 cell toxicity assays showed that the cell growth of DCs treated with RMPs remained above 50% when the concentration of RMPs was less than 25 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating that DCs were insensitive to CT20p- and RSL3-loaded RMPs (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). Moreover, flow cytometry analysis showed that incubation with RC@RMPs could boost DC activation by significantly increasing the expression of CD80, CD86, and MHCⅡ, compared to control groups and RMPs loaded with a single agent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). RMPs encapsulating DAMP-like DNA fragments generated by radiation may trigger the cGAS-STING pathway. Therefore, we performed Western blots to measure the expression of proteins related to cGAS-STING activation, including pSTING and pNF-κB (p65), in DC2.4 cells treated with different RMPs. The phosphorylation of STING and NF-κB were eleveated when the cells were incubated with RMPs, RMPs loaded either RSL3 or CT20p, or RMPs loaded with both agents, compared to the control group (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), indicating that RSL3 and CT20p loading does not influence RMP-induced activation of the cGAS-STING pathway. In contrast to DCs, RSL3-loaded RMPs showed relatively higher toxicity to macrophages (IC (50) values: 13.1 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for RSL3@RMPs and 9.08 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for RC@RMPs). Macrophages treated with RC@RMPs expressed high levels of CD86, suggesting that RC@RMPs contributed to the M1 polarization of macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). All RMPs reduced CD206 expression, with no significant differences among RMP-treated groups, demonstrating that RSL3 and CT20p loading maintains the ability of RMPs to inhibit macrophage polarization to M2(Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Together, these findings indicated that RC@RMPs can directly promote inflammation by activating DCs and promoting M1 macrophage polarization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.5 RC@RMPs can be taken up by tumor cells and immunocytes in the TME \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo explore the tissue distribution of RMPs \u003cem\u003ein vivo\u003c/em\u003e, we performed intratumoral injections of 100 \u0026micro;g PKH26-labeled RMPs (100 \u0026micro;L) into mice previously implanted with RM-1 cells. 24 hours later, the mice were sacrificed and the tumor and organs including the heart, liver, spleen, lung, kidney, brain, and dLNs were observed using the IVIS Spectrum Imaging System. The distribution of RMPs was limited to the tumor tissue, as shown in Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA. Then, we dispersed the tumor to generate a single cell suspension for flow cytometry analysis. There was no significant differences in the percentages of PKH26-positive tumor cells, T cells, B cells, neutrophils, DCs, or M2 macrophages between mice injected with RMPs and those given RC@RMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Tumor sections were stained using antibodies targeting neutrophils, DCs, and macrophages. The results confirmed that RMPs could be taken up by the relevant immune cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6 RC@RMPs reshape the tumor immune microenvironment and the combination of RC@RMPs and anti-PD-1 mAb shows synergistic antitumor activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003e In order to evaluate the therapeutic effect of RC@RMPs on prostate cancer, we treated subcutaneously implanted RM-1 tumors with control, RMPs, RSL3@RMPs, CT20p@RMPs, and RC@RMPs according to the treatment scheme shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. RC@RMP therapy significantly reduced the growth of RM-1 tumors and prolonged survival time compared with mice treated with control, RMPs, RSL3@RMPs, or CT20p@RMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). The median survival time of RC@RMP-treated mice was the longest (39 days) among all groups, although all mice reached ethical endpoint by 62 days after tumor inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). To clarify the effects of RC@RMP treatment on the immune microenvironment, immunocytes in the tumor were measured by flow cytometry. We found a significant increase in the number of neutrophils, CD86\u003csup\u003e+\u003c/sup\u003e MHCⅡ\u003csup\u003e+\u003c/sup\u003e DCs, total CD8\u003csup\u003e+\u003c/sup\u003e T cells, IFN-γ\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells, and memory CD8\u003csup\u003e+\u003c/sup\u003e T cells, and a decrease in the number of M2 macrophages compared with the groups given control and RMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-K). The level of related cytokines were also measured; only IL-6 was found to be significantly elevated by RC@RMPs compared with the control and RMP groups (Figure \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003eG). PD-1 is a negative regulator of the immune system that prevents overactivity and subsequent cytokine release syndrome. The expression of PD-1 is enhanced upon T cell activation, forming a negative feedback loop. Results showed that RC@RMPs significant promoted PD-1 expression, further indicating an uptick in T cell activity. However, RMPs also increased the expression of PD-L1 on macrophages, which can induce programmed cell death in T cells with high PD-1 expression. Therefore, we undertook a combined therapy strategy using an anti-PD-1 antibody and RC@RMPs to antagonize the immunosuppressive effect of the PD-1/PD-L1 pathway during RC@RMP treatment. The combination treatment promoted the inhibition of RM-1 growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and resulted in a 50% survival rate 62 days after tumor inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The combination therapy also significantly enhanced the presence of CD86\u003csup\u003e+\u003c/sup\u003e MHCⅡ\u003csup\u003e+\u003c/sup\u003e DCs, the proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells among CD3\u003csup\u003e+\u003c/sup\u003e T cells, IFN-γ\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells, and TCF-1\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells, while decreasing the number of M2 macrophages, compared with monotherapy treatment with only RC@RMPs. Furthermore, proinflammatory cytokines including CXCL9, TNF-α, CCL4, and CCL3 were also upregulated in the tumor following the combined therapy, further demonstrating the change in the immune environment (Figure \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003eA-I). To identify the key immune subsets affected by our treatment, sections of tumor were stained for CD3, Ly6G, F4/80, and CD206 to observe the presence of T cells, neutrophils, and M2 macrophages. Histological analysis showed that RC@RMPs markedly increased T cell infiltration (Figure \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003eA) and decreased the number of M2 macrophages present (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To directly assess the role of infiltrating immunocytes, we depleted T helper cells, cytotoxic T cells, neutrophils, and macrophages using CD4 mAb, CD8 mAb, Ly6G mAb, and clodronate liposomes, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These targeted cell subsets were rapidly depleted in peripheral blood within 24 hours, which was confirmed by flow cytometry (Figure \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e). We observed that the depletion of CD8\u003csup\u003e+\u003c/sup\u003e T cells and macrophages impaired the efficacy of RC@RMP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), suggesting that these two cell subsets are the main targets of RC@RMPs. Finally, we tested for possible toxicity following RC@RMP treatment. All mice in treated groups showed similar levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CREA) as the control group (Figure \u003cspan refid=\"MOESM11\" class=\"InternalRef\"\u003eS11\u003c/span\u003eA). No abnormalities were observed in the heart, liver, spleen, lung, and kidney after the different RMP therapies, as determined by histopathological examination (Figure \u003cspan refid=\"MOESM11\" class=\"InternalRef\"\u003eS11\u003c/span\u003eB). Together, these results indicated that RC@RMPs successfully remodeled the immune desert environment of RM-1 tumors, and PD-1 blockade enhanced the effectiveness of this immune enhancement and tumor cell killing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eAlthough ferroptosis is a potent trigger of the innate immune system, there remain significant obstacles for treatment strategies that focus on inducing ferroptosis. Many ferroptosis-inducing agents have limited efficacy against cold tumors due to their short half life, hyposusceptibility of the tumor to ferroptosis, and toxicity to normal cells. Therefore, a major challenge for the clinical application of ferroptosis inducers is to determine strategies to improve the potency and tumor-targeting capabilities of ferroptosis-inducing agents. As one of the most crucial organelles in cell, mitochondria are rich in metabolism-related molecules which can trigger ICD\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Despite some controversy, targeting mitochondria to stimulate the release of DAMPs has been shown to cause ferroptosis via the release of ROS and free iron\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Recent evidence indicates that radiation triggers ferroptosis and increases the susceptibility of cancer cells to ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Mechanistically, radiotherapy impairs lipid metabolism through the promotion of ROS production and ACSL4 expression, and downregulation of SLC7A11, all of which mediate ferroptosis\u003csup\u003e[44\u0026ndash;46]\u003c/sup\u003e. As radiotherapy derivatives, RMPs induce tumor cell ferroptosis and reprogram macrophage polarization through DAMPs generated by radiation, as previously demonstrated\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Furthermore, RMPs are microparticles that can act as carriers for active agents, with good stability and biocompatibility. As they are generated from tumor cells, RMPs are also able to target the tumor and act as a source of tumor antigens to APCs. Thus, the combination of RMPs and RSL3 may provide a new strategy to more effectively induce tumor cell ferroptosis and thus stimulate innate immunity.\u003c/p\u003e \u003cp\u003eIn the study, we designed a combined system (RC@RMPs) of RMPs loaded with the ferroptosis inducer RSL3 and apoptosis inducer CT20p. This system has some distinct advantages. (i) Firstly, the system demonstrates strong synergy to induce tumor cytotoxicity: RC@RMPs were significantly more effective at killing RM-1 cells through inducing apoptosis and ferroptosis, compared to RMPs with a single agent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-H). We first discovered that CT20p enhanced ferroptosis through the production of ROS and disruption of the mitochondrial membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H). This peptide has been shown to target mitochondria and induce mitochondrial membrane hyperpolarization, which impairs the distribution and movement of mitochondria\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Consequently, mitochondrial metabolism is compromised and ROS release occurs, inducing apoptosis and accelerating the ferroptosis process. Nevertheless, the complete mechanism of the synergy through which RSL3, CT20p, and RMPs promote ferroptosis requires further elucidation. We note that as we used relatively low doses of RMPs compared to previous literature\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, the RMPs alone did not initiate a significant ferroptosis effect, although DAMPs in the RMPs triggered innate immunity. (ii) Secondly, RC@RMPs caused DC activation and M1 macrophage polarization. ROS production is essential in tumor therapy, for it did not increases ICD in tumor cells but also reprograms the phenotypes of DCs. The highest expression levels of B7 molecules in DCs was achieved by RC@RMP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), which was associated with increased ROS levels that may have activated the \u003cem\u003eCD80/CD86\u003c/em\u003e promoters via the release of Ca\u003csup\u003e2+\u003c/sup\u003e and expression of positive transcription elongation factor b (P-TEFb)\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Moreover, DAMPs in RMPs can trigger the activation of the cGAS-STING pathway to facilitate the expression of type Ι interferon, and cause the autoactivation of APCs (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). As our results have shown, all the RMPs promoted the phosphorylation of key components of the cGAS-STING pathway in DC2.4 cells (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e), suggesting that the synergistic activity of RSL3 and CT20p in RMPs were able to mobilize a more intense innate immune response. M1 macrophage polarization is essential in antitumor immunity, as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC. ROS may not be the main factor that promotes M1 macrophage polarization, as CD86 expression was not differentially expressed when comparing groups given RMPs and RSL3@RMPs, even though RSL3@RMPs produced much more ROS than RMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and J). Macrophages in mice treated with CT20p@RMPs showed a stronger tendency towards M1 phenotypes, implying that CT20p promoted M1 polarization, possibly by influencing mitochondrial metabolism in macrophages. (iii) Thirdly, RC@RMPs were capable of targeting tumor sites without spreading throughout other organs, which is likely due to the nature of RMPs, which are derived from tumor cells. Our data confirmed that the RMPs did not distribute into other organs and tissues when injected intratumorally (Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA). Notably, the greatest RMP uptake was observed in macrophages within the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), indicating the importance of reprogramming macrophage polarization. The tumor-targeting of RMPs is critical for minimizing side effects by allowing appropriate dose reduction without compromising therapeutic effect. (iv) Fourthly, the combination therapy with anti-PD-1 mAb demonstrates a highly promising treatment modality. High PD-1 expression is a characteristic of activated CD8\u003csup\u003e+\u003c/sup\u003e T cells and a vital checkpoint for immunosuppression by binding with PD-L1. RC@RMP treatment significantly enhanced the percentage of PD-1\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells, which may limit the therapeutic effect. Addition of anti-PD-1 mAb to increase the presence of inflammatory immunocytes and cytokines in the TME successfully inhibited tumor growth and prolonged the survival time of mice burdened with RM-1 tumors. (v) Finally, RMPs show good biocompatibility and safety for use as cancer vaccines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Our results showed no obvious damage in the major organs after treatment with RMPs (Figure \u003cspan refid=\"MOESM11\" class=\"InternalRef\"\u003eS11\u003c/span\u003e). To sum up, RC@RMPs specifically targeted tumor sites where they mediated tumor cell apoptosis and synergistically enhanced the ferroptosis of tumor cells, while remodeling the tumor immune microenvironment of prostate cancer by DC activation and M1 macrophage polarization.\u003c/p\u003e \u003cp\u003eThere remain some limitations in our system. The toxicity of the RC@RMPs was not entirely specific to prostate cancer cells, leading to the death of macrophages and limiting the inflammatory effects of M1 macrophages. Moreover, the precise mechanism by which the components of RC@RMPs synergistically induce M1 macrophage polarization is not yet fully characterized. Further efforts should seek to screen novel genes that specifically contribute to the ferroptosis of prostate cancer cells and elucidate the relationship between macrophage polarization, changes in mitochondrial functionality, and ferroptosis, to support the development of more efficient and accurate therapies for prostate cancer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData statement\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSample sizes were predetermined based on previous experience using at minimum three groups of mice, and all experiments were replicated at least twice to confirm findings. Statistical analyses were conducted with a two-tailed unpaired t-test or one-way ANOVA as described below. Mice were randomly assigned to treatment groups, and where possible, treatment groups were blinded until statistical analysis. No animals or potential outliers were excluded from the data sets presented in this study. The data used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCRPC: castration-resistant prostate cancer; ICD: Immunogenic cell death; TME: Tumor microenvironment; DAMPs: Damage-associated molecular patterns; RMPs: Radiated tumor cell-derived microparticles; CT20p: CT20 peptide; RC@RMPs: RSL-3- and CT20p-loaded RMPs; DC: Dendritic cell; MDSCs: Myeloid-derived suppressor cells; HPLC: High Performance Liquid Chromatography; dLNs: Draining lymph nodes; TEM: Transmission electron microscopy; MFI: Mean fluorescence intensity; APCs: Antigen Presenting Cells; TAMs: Tumor-associated macrophages; ALT: alanine transaminase; AST: aspartate transaminase; BUN: blood urea nitrogen; CREA: creatinine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that all data supporting the results of this study are available in the paper and supplementary information. Source data are provided in this paper.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all members of the laboratory for their kindness and help. This study was supported\u0026nbsp;by grants from the National Natural Science Foundation of China (No. 82102900, 82022040, 82272851), The China postdoctoral Science Foundation (2023M731223, 2023T160253). We thank the Optical Bioimaging Core Facility of WNLO-HUST for the support in data acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting interests \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eS. Sandhu, C. M. Moore, E. Chiong, H. Beltran, R. G. Bristow, S. G. Williams. Prostate cancer. \u003cem\u003eThe Lancet. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e398\u003c/em\u003e:1075.\u003c/li\u003e\n\u003cli\u003eR. J. Rebello, C. Oing, K. E. Knudsen, S. Loeb, D. C. Johnson, R. E. Reiter, S. Gillessen, T. Van der Kwast, R. G. Bristow. Prostate cancer. \u003cem\u003eNature Reviews Disease Primers. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e7\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eM. Cai, X.-L. Song, X.-A. Li, M. Chen, J. Guo, D.-H. Yang, Z. Chen, S.-C. Zhao. Current therapy and drug resistance in metastatic castration-resistant prostate cancer. \u003cem\u003eDrug Resistance Updates. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e;\u003cem\u003e68\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eK. D. Runcie, M. C. Dallos. Prostate Cancer Immunotherapy\u0026mdash;Finally in From the Cold? \u003cem\u003eCurrent Oncology Reports. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e23\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eH. Liang, Y. Liu, J. Guo, M. Dou, X. Zhang, L. Hu, J. Chen. Progression in immunotherapy for advanced prostate cancer. \u003cem\u003eFrontiers in Oncology. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e;\u003cem\u003e13\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eX. Chen, R. Kang, G. Kroemer, D. Tang. Broadening horizons: the role of ferroptosis in cancer. \u003cem\u003eNature Reviews Clinical Oncology. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e18\u003c/em\u003e:280.\u003c/li\u003e\n\u003cli\u003eW. Gao, X. Wang, Y. Zhou, X. Wang, Y. Yu. Autophagy, ferroptosis, pyroptosis, and necroptosis in tumor immunotherapy. \u003cem\u003eSignal Transduction and Targeted Therapy. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e;\u003cem\u003e7\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eK. H\u0026auml;nggi, B. Ruffell. Cell death, therapeutics, and the immune response in cancer. \u003cem\u003eTrends in Cancer. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e;\u003cem\u003e9\u003c/em\u003e:381.\u003c/li\u003e\n\u003cli\u003eR. Tang, J. Xu, B. Zhang, J. Liu, C. Liang, J. Hua, Q. Meng, X. Yu, S. Shi. Ferroptosis, necroptosis, and pyroptosis in anticancer immunity. \u003cem\u003eJournal of Hematology \u0026amp; Oncology. \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e;\u003cem\u003e13\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eA. Ghoochani, E.-C. Hsu, M. Aslan, M. A. Rice, H. M. Nguyen, J. D. Brooks, E. Corey, R. Paulmurugan, T. Stoyanova. Ferroptosis Inducers Are a Novel Therapeutic Approach for Advanced Prostate Cancer. \u003cem\u003eCancer Research. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e81\u003c/em\u003e:1583.\u003c/li\u003e\n\u003cli\u003eB. Liu, X. Li, D. Wang, Y. Yu, D. Lu, L. Chen, F. Lv, Y. Li, L. Cheng, Y. Song, Y. Xing. CEMIP promotes extracellular matrix‐detached prostate cancer cell survival by inhibiting ferroptosis. \u003cem\u003eCancer Science. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e;\u003cem\u003e113\u003c/em\u003e:2056.\u003c/li\u003e\n\u003cli\u003eS. Zhao, P. Li, W. Wu, Q. Wang, B. Qian, X. Li, M. Shen. Roles of ferroptosis in urologic malignancies. \u003cem\u003eCancer Cell International. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e21\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eD. Liao, G. Yang, Y. Yang, X. Tang, H. Huang, J. Shao, Q. Pan. \u0026lt;p\u0026gt;Identification of Pannexin 2 as a Novel Marker Correlating with Ferroptosis and Malignant Phenotypes of Prostate Cancer Cells\u0026lt;/p\u0026gt;. \u003cem\u003eOncoTargets and Therapy. \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e;\u003cem\u003eVolume 13\u003c/em\u003e:4411.\u003c/li\u003e\n\u003cli\u003eR. Kim, A. Hashimoto, N. Markosyan, V. A. Tyurin, Y. Y. Tyurina, G. Kar, S. Fu, M. Sehgal, L. Garcia-Gerique, A. Kossenkov, B. A. Gebregziabher, J. W. Tobias, K. Hicks, R. A. Halpin, N. Cvetesic, H. Deng, L. Donthireddy, A. Greenberg, B. Nam, R. H. Vonderheide, Y. Nefedova, V. E. Kagan, D. I. Gabrilovich. Ferroptosis of tumour neutrophils causes immune suppression in cancer. \u003cem\u003eNature. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e;\u003cem\u003e612\u003c/em\u003e:338.\u003c/li\u003e\n\u003cli\u003eH. Xu, D. Ye, M. Ren, H. Zhang, F. Bi. Ferroptosis in the tumor microenvironment: perspectives for immunotherapy. \u003cem\u003eTrends in Molecular Medicine. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e27\u003c/em\u003e:856.\u003c/li\u003e\n\u003cli\u003eW. Wu, X. a. Wang, W. Le, C. Lu, H. Li, Y. Zhu, X. Chen, W. An, C. Xu, Q. Wu, L. Wang. Immune microenvironment infiltration landscape and immune-related subtypes in prostate cancer. \u003cem\u003eFrontiers in Immunology. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e;\u003cem\u003e13\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eS. S. Nair, R. Weil, Z. Dovey, A. Davis, A. K. Tewari. The Tumor Microenvironment and Immunotherapy in Prostate and Bladder Cancer. \u003cem\u003eUrologic Clinics of North America. \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e;\u003cem\u003e47\u003c/em\u003e:e17.\u003c/li\u003e\n\u003cli\u003eK. Li, H. Shi, B. Zhang, X. Ou, Q. Ma, Y. Chen, P. Shu, D. Li, Y. Wang. Myeloid-derived suppressor cells as immunosuppressive regulators and therapeutic targets in cancer. \u003cem\u003eSignal Transduction and Targeted Therapy. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e6\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eM. A. Giese, L. E. Hind, A. Huttenlocher. Neutrophil plasticity in the tumor microenvironment. \u003cem\u003eBlood. \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e;\u003cem\u003e133\u003c/em\u003e:2159.\u003c/li\u003e\n\u003cli\u003eY. Pan, Y. Yu, X. Wang, T. Zhang. Tumor-Associated Macrophages in Tumor Immunity. \u003cem\u003eFrontiers in Immunology. \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e;\u003cem\u003e11\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eM. McLaughlin, E. C. Patin, M. Pedersen, A. Wilkins, M. T. Dillon, A. A. Melcher, K. J. Harrington. Inflammatory microenvironment remodelling by tumour cells after radiotherapy. \u003cem\u003eNature Reviews Cancer. \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e;\u003cem\u003e20\u003c/em\u003e:203.\u003c/li\u003e\n\u003cli\u003eJ. Peng, X. Yin, W. Yun, X. Meng, Z. Huang. Radiotherapy-induced tumor physical microenvironment remodeling to overcome immunotherapy resistance. \u003cem\u003eCancer Letters. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e;\u003cem\u003e559\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eJ. Murgić. Recent Advances in Radiotherapy Modalities for Prostate Cancer. \u003cem\u003eActa Clinica Croatica. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eC. Wan, Y. Sun, Y. Tian, L. Lu, X. Dai, J. Meng, J. Huang, Q. He, B. Wu, Z. Zhang, K. Jiang, D. Hu, G. Wu, J. F. Lovell, H. Jin, K. Yang. Irradiated tumor cell\u0026ndash;derived microparticles mediate tumor eradication via cell killing and immune reprogramming. \u003cem\u003eScience Advances. \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e;\u003cem\u003e6\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eY. Ding, Y. Wang, Q. Hu. Recent advances in overcoming barriers to cell‐based delivery systems for cancer immunotherapy. \u003cem\u003eExploration. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e;\u003cem\u003e2\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eN. Desai, P. Katare, V. Makwana, S. Salave, L. K. Vora, J. Giri. Tumor-derived systems as novel biomedical tools\u0026mdash;turning the enemy into an ally. \u003cem\u003eBiomaterials Research. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e;\u003cem\u003e27\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eL. Lu, H. Zi, J. Zhou, J. Huang, Z. Deng, Z. Tang, L. Li, X. Shi, P. C. Lo, J. F. Lovell, D. Deng, C. Wan, H. Jin. Engineered Microparticles for Treatment of Murine Brain Metastasis by Reprograming Tumor Microenvironment and Inhibiting MAPK Pathway. \u003cem\u003eAdvanced Science. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e;\u003cem\u003e10\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eZ. Tuo, Q. He, Z. Zhang, Y. Wang, J. Sun, Q. Wei, Y. Hu, J. F. Lovell, H. Jin, K. Yang. Irradiation conditioning of adjuvanted, autologous cancer cell membrane nanoparticle vaccines. \u003cem\u003eChemical Engineering Journal. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e;\u003cem\u003e433\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eZ. Liao, J. Huang, P.-C. Lo, J. F. Lovell, H. Jin, K. Yang. Self-adjuvanting cancer nanovaccines. \u003cem\u003eJournal of Nanobiotechnology. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e;\u003cem\u003e20\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eY. Liu, Y. Wan, Y. Jiang, L. Zhang, W. Cheng. GPX4: The hub of lipid oxidation, ferroptosis, disease and treatment. \u003cem\u003eBiochimica et Biophysica Acta (BBA) - Reviews on Cancer. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e;\u003cem\u003e1878\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eM. W. Lee, R. Bassiouni, N. A. Sparrow, A. Iketani, R. J. Boohaker, C. Moskowitz, P. Vishnubhotla, A. S. Khaled, J. Oyer, A. Copik, C. Fernandez-Valle, J. M. Perez, A. R. Khaled. The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption. \u003cem\u003eCell Death \u0026amp; Disease. \u003c/em\u003e\u003cstrong\u003e2014\u003c/strong\u003e;\u003cem\u003e5\u003c/em\u003e:e1249.\u003c/li\u003e\n\u003cli\u003eA. Porgador, E. Gilboa. Bone marrow-generated dendritic cells pulsed with a class I-restricted peptide are potent inducers of cytotoxic T lymphocytes. \u003cem\u003eThe Journal of experimental medicine. \u003c/em\u003e\u003cstrong\u003e1995\u003c/strong\u003e;\u003cem\u003e182\u003c/em\u003e:255.\u003c/li\u003e\n\u003cli\u003eJ. Weischenfeldt, B. Porse. Bone Marrow-Derived Macrophages (BMM): Isolation and Applications. \u003cem\u003eCold Spring Harbor Protocols. \u003c/em\u003e\u003cstrong\u003e2008\u003c/strong\u003e;\u003cem\u003e2008\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eY. Zou, J. Xie, S. Zheng, W. Liu, Y. Tang, W. Tian, X. Deng, L. Wu, Y. Zhang, C.-W. Wong, D. Tan, Q. Liu, X. Xie. Leveraging diverse cell-death patterns to predict the prognosis and drug sensitivity of triple-negative breast cancer patients after surgery. \u003cem\u003eInternational Journal of Surgery. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e;\u003cem\u003e107\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eT. M. Seibt, B. Proneth, M. Conrad. Role of GPX4 in ferroptosis and its pharmacological implication. \u003cem\u003eFree Radical Biology and Medicine. \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e;\u003cem\u003e133\u003c/em\u003e:144.\u003c/li\u003e\n\u003cli\u003eF.-J. Li, H.-Z. Long, Z.-W. Zhou, H.-Y. Luo, S.-G. Xu, L.-C. Gao. System Xc\u0026minus;/GSH/GPX4 axis: An important antioxidant system for the ferroptosis in drug-resistant solid tumor therapy. \u003cem\u003eFrontiers in Pharmacology. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e;\u003cem\u003e13\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eT. L. Murphy, K. M. Murphy. Dendritic cells in cancer immunology. \u003cem\u003eCellular \u0026amp; Molecular Immunology. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e19\u003c/em\u003e:3.\u003c/li\u003e\n\u003cli\u003eU. Basak, T. Sarkar, S. Mukherjee, S. Chakraborty, A. Dutta, S. Dutta, D. Nayak, S. Kaushik, T. Das, G. Sa. Tumor-associated macrophages: an effective player of the tumor microenvironment. \u003cem\u003eFrontiers in Immunology. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e;\u003cem\u003e14\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eF. J. Bock, S. W. G. Tait. Mitochondria as multifaceted regulators of cell death. \u003cem\u003eNature Reviews Molecular Cell Biology. \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e;\u003cem\u003e21\u003c/em\u003e:85.\u003c/li\u003e\n\u003cli\u003eY.-Q. Wang, S.-Y. Chang, Q. Wu, Y.-J. Gou, L. Jia, Y.-M. Cui, P. Yu, Z.-H. Shi, W.-S. Wu, G. Gao, Y.-Z. Chang. The Protective Role of Mitochondrial Ferritin on Erastin-Induced Ferroptosis. \u003cem\u003eFrontiers in Aging Neuroscience. \u003c/em\u003e\u003cstrong\u003e2016\u003c/strong\u003e;\u003cem\u003e8\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eX. Fang, H. Wang, D. Han, E. Xie, X. Yang, J. Wei, S. Gu, F. Gao, N. Zhu, X. Yin, Q. Cheng, P. Zhang, W. Dai, J. Chen, F. Yang, H.-T. Yang, A. Linkermann, W. Gu, J. Min, F. Wang. Ferroptosis as a target for protection against cardiomyopathy. \u003cem\u003eProceedings of the National Academy of Sciences. \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e;\u003cem\u003e116\u003c/em\u003e:2672.\u003c/li\u003e\n\u003cli\u003eJ. G. Berdeja, D. Madduri, S. Z. Usmani, A. Jakubowiak, M. Agha, A. D. Cohen, A. K. Stewart, P. Hari, M. Htut, A. Lesokhin, A. Deol, N. C. Munshi, E. O\u0026apos;Donnell, D. Avigan, I. Singh, E. Zudaire, T.-M. Yeh, A. J. Allred, Y. Olyslager, A. Banerjee, C. C. Jackson, J. D. Goldberg, J. M. Schecter, W. Deraedt, S. H. Zhuang, J. Infante, D. Geng, X. Wu, M. J. Carrasco-Alfonso, M. Akram, F. Hossain, S. Rizvi, F. Fan, Y. Lin, T. Martin, S. Jagannath. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): a phase 1b/2 open-label study. \u003cem\u003eThe Lancet. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e398\u003c/em\u003e:314.\u003c/li\u003e\n\u003cli\u003eJ. Su, C. Bian, Z. Zheng, H. Wang, L. Meng, Y. Xin, X. Jiang. Cooperation effects of radiation and ferroptosis on tumor suppression and radiation injury. \u003cem\u003eFrontiers in Cell and Developmental Biology. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e;\u003cem\u003e10\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eG. Lei, Y. Zhang, P. Koppula, X. Liu, J. Zhang, S. H. Lin, J. A. Ajani, Q. Xiao, Z. Liao, H. Wang, B. Gan. The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression. \u003cem\u003eCell Research. \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e;\u003cem\u003e30\u003c/em\u003e:146.\u003c/li\u003e\n\u003cli\u003eG. Lei, C. Mao, Y. Yan, L. Zhuang, B. Gan. Ferroptosis, radiotherapy, and combination therapeutic strategies. \u003cem\u003eProtein \u0026amp; Cell. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e;\u003cem\u003e12\u003c/em\u003e:836.\u003c/li\u003e\n\u003cli\u003eX. Lang, M. D. Green, W. Wang, J. Yu, J. E. Choi, L. Jiang, P. Liao, J. Zhou, Q. Zhang, A. Dow, A. L. Saripalli, I. Kryczek, S. Wei, W. Szeliga, L. Vatan, E. M. Stone, G. Georgiou, M. Cieslik, D. R. Wahl, M. A. Morgan, A. M. Chinnaiyan, T. S. Lawrence, W. Zou. Radiotherapy and Immunotherapy Promote Tumoral Lipid Oxidation and Ferroptosis via Synergistic Repression of SLC7A11. \u003cem\u003eCancer Discovery. \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e;\u003cem\u003e9\u003c/em\u003e:1673.\u003c/li\u003e\n\u003cli\u003eD. L. Medina, S. Di Paola, I. Peluso, A. Armani, D. De Stefani, R. Venditti, S. Montefusco, A. Scotto-Rosato, C. Prezioso, A. Forrester, C. Settembre, W. Wang, Q. Gao, H. Xu, M. Sandri, R. Rizzuto, M. A. De Matteis, A. Ballabio. Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. \u003cem\u003eNature Cell Biology. \u003c/em\u003e\u003cstrong\u003e2015\u003c/strong\u003e;\u003cem\u003e17\u003c/em\u003e:288.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cancer immunotherapy, Microparticle, Ferroptosis, Immunogenic cell death, Tumor microenvironment, Macrophage reprogramming","lastPublishedDoi":"10.21203/rs.3.rs-3911119/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3911119/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImmunogenic cell death (ICD) plays a crucial role in triggering the antitumor immune response in the tumor microenvironment (TME) through the release of damage-associated molecular patterns (DAMPs). Recently, considerable attention has been dedicated to ferroptosis, a type of ICD that is induced by intracellular iron and has been demonstrated to change the immune desert status of the TME. However, there remains significant room for improvement among strategies for inducing high levels of ICD through ferroptosis to fight cancers that are characterized by an immune desert, such as prostate cancer. Radiated tumor cell-derived microparticles (RMPs) are radiotherapy mimetics that have been shown to activate the cGAS-STING pathway, induce tumor cell ferroptosis, and inhibit M2 macrophage polarization. RMPs can also act as carriers of agents with remarkable biocompatibility. In the present study, we designed a therapeutic system wherein the ferroptosis inducer RSL-3 was loaded into RMPs to treat prostate cancer, which is considered a cold tumor, using in vitro and in vivo models involving RM-1 prostate carcinoma cells. Apoptosis inducer CT20 peptide (CT20p) was also added into the RMPs to aggravate ICD. In vitro experiments demonstrated that RSL-3- and CT20p-loaded RMPs (RC@RMPs) led to ferroptosis and apoptosis of RM-1 cells, and CT20p had a synergistic effect on ferroptosis by promoting ROS production and mitochondrial instability. RC@RMPs elevated the dendritic cell (DC) expression of MHCⅡ, CD80, and CD86 and facilitated M1 macrophage polarization. In a syngeneic mouse model of prostate cancer induced by RM-1 cells, RC@RMPs significantly inhibited tumor growth and prolonged survival time via DC activation, macrophage reprogramming, enhancement of CD8\u003csup\u003e+ \u003c/sup\u003eT cell presence, and proinflammatory cytokine production, without diffusing outside the tumor tissue. Moreover, combination treatment with anti-PD-1 showed improved effectiveness to inhibit RM-1 progression. This method provides a novel strategy for the synergistic enhancement of ICD for prostate cancer immunotherapies.\u003c/p\u003e","manuscriptTitle":"Irradiated Microparticles Suppress Prostate Cancer by Tumor Microenvironment Reprogramming and Ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-06 12:29:38","doi":"10.21203/rs.3.rs-3911119/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-08T01:28:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-23T04:29:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"065b4646-c429-47af-9b87-b9036a40cede","date":"2024-02-17T00:43:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c92cc0af-9ba8-4470-b555-e041e85bbf1b","date":"2024-02-09T00:25:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-08T23:36:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-03T15:19:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-03T15:19:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-01-30T15:49:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cfe5817e-256f-446b-990c-bd28a0f24b48","owner":[],"postedDate":"February 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-07T20:07:18+00:00","versionOfRecord":{"articleIdentity":"rs-3911119","link":"https://doi.org/10.1186/s12951-024-02496-3","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2024-05-05 19:58:22","publishedOnDateReadable":"May 5th, 2024"},"versionCreatedAt":"2024-02-06 12:29:38","video":"","vorDoi":"10.1186/s12951-024-02496-3","vorDoiUrl":"https://doi.org/10.1186/s12951-024-02496-3","workflowStages":[]},"version":"v1","identity":"rs-3911119","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3911119","identity":"rs-3911119","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}