NK Cell-Derived Extracellular Vesicles Function as Immunomodulators to Regulate Breast Cancer Progression by Activating MHC-I Signalling

NK Cell-Derived Extracellular Vesicles Function as Immunomodulators to Regulate Breast Cancer Progression by Activating MHC-I Signalling | 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 Article NK Cell-Derived Extracellular Vesicles Function as Immunomodulators to Regulate Breast Cancer Progression by Activating MHC-I Signalling Limin Zhang, Yidan Xu, Jing Guo, Xin Lv, Libin Liao, Yajie Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7555077/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Although immune checkpoint inhibitors (ICIs) and adoptive T cell therapy have shown success in cancer treatment, these therapies often have limited efficacy in breast cancer patients because these tumours use various mechanisms to evade immune surveillance and elimination; however, the underlying mechanisms remaining unclear. This study revealed that natural killer (NK) cell-derived extracellular vesicles (NK-EVs) can be internalized by breast cancer cells in vitro and exhibit tumour-homing properties in tumour-bearing mice. Proteomic sequencing revealed that treatment with NK-EVs significantly upregulated the expression of major histocompatibility complex (MHC) class I-related proteins, including antigen processing-related transporters (TAP1 and TAP2), transporter associated with antigen processing binding protein (TAPBP, tapasin), immunoproteasome subunits (PSMB9 and PSMB10), and β2-microglobulin (B2M), in breast cancer cells. These proteins are involved in MHC-I synthesis and endogenous peptide processing/presentation by tumour cells. Moreover, MHC-I expression is essential for antigen presentation and T cell-mediated immune responses. NK-EVs upregulated MHC-I expression on the surfaces of breast cancer cells, thus facilitating the recognition and killing of breast cancer cells by CD8 + T cells. Additionally, an environment with increased MHC-I levels promoted NK cell maturation and enhanced their cytotoxic functions. Finally, in tumour-bearing mice, NK-EVs combined with NK cell therapy inhibited tumour progression and exhibited good safety. These results suggest that similar strategies may be novel therapeutic approaches for treating patients in the clinic. In the future, the combination of NK-EVs with ICIs or adoptive cell therapy may overcome the limitations of currently available breast cancer immunotherapies by performing two functions, namely, directly killing tumour cells and sensitizing immune cells. Biological sciences/Cancer Biological sciences/Immunology Health sciences/Oncology Breast cancer NK cell-derived extracellular vesicles MHC class I Antigen presentation CD8+T cell Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Breast cancer is one of the most common malignant tumours in women worldwide, and an increasing incidence has been observed among younger populations 1 . Although advances in breast cancer screening, diagnosis, and treatment strategies have led to decreases in mortality rates, some patients still fail to achieve ideal outcomes 2 3 . For example, endocrine therapy is the only treatment for patients with luminal-A type breast cancer (hormone receptor-positive). However, some tumours develop acquired resistance, thereby rendering endocrine therapy ineffective. Moreover, triple-negative breast cancer (TNBC) lacks clear therapeutic targets, and chemotherapy is the main treatment for TNBC patients 4 – 6 . However, only approximately 20% of is a common occurrence and is partly due to the active downregulation of cell surface major histocompatibility complex (MHC)-I expression during tumour immune escape 7 8 . TNBC patients exhibit intratumorally heterogeneity in regional MHC-I expression 9 . In mouse models, MHC-I deficiency abrogates antitumour immunity and the response to immune checkpoint inhibitors (ICIs), whereas intratumorally MHC-I heterogeneity leads to increased natural killer (NK) cell infiltration in an IFN-γ-dependent manner. Spatial technology has revealed that MHC-I heterogeneity is associated with clinical resistance to anti-programmed cell death ligand 1 (PD-L1) therapy and an increased NK: T cell ratio in human breast tumours. Additionally, tumours with heterogeneous MHC-I require NKG2A to suppress NK cell function. Combination therapy with anti-NKG2A and anti-PD-L1 restores complete remission in heterogeneous MHC-I mouse models, which depends on the presence of tumour-infiltrating activated NK and CD8 + T cells 9 10 . These results suggest that similar strategies may result in greater benefits for patients in clinical trials. Immune checkpoint inhibitor therapy aims to utilize the full repertoire of T cells that are capable of destroying malignant cells, although the relevant T cells and ligand peptides are often undefined 11 . Increasing evidence indicates that successful immunotherapy with ICIs depends on sustained expression of MHC class I molecules by tumour cells 12 . Tumour-specific MHC-I (tsMHC-I) antigen presentation can be disrupted by multiple mechanisms, including immune editing or selection against neoantigens, the loss of MHC-I allelic diversity, or mutations that suppress interferon-sensing pathways and subsequent MHC-I upregulation 13 – 15 . In breast cancer, the somatic genetic loss of HLA loci or epigenetic silencing via promoter hypermethylation has been observed 16 . In recent years, adoptive immunotherapy has gained widespread attention for its promising potential in the treatment of solid tumours 17 ; however, its application is limited by factors such as the poor tumour homing ability of immune cells, the inability to penetrate biological barriers, and the tumour microenvironment (TME) 18 . Extracellular vesicles (EVs) are membrane-bound vesicles that are released into the extracellular matrix after the fusion of intracellular multivesicular bodies (MVBs) with the cell membrane 19 . These nanoscale lipid structures (30–150 nm in diameter) encapsulate proteins, mRNAs, miRNAs, and other substances. NK cell-derived vesicles contain various cytotoxic proteins, miRNAs, and cytokines; moreover, they exhibit cell-killing activity similar to that of NK cells 20 . Additionally, NK cell-derived EVs (NK-EVs) have a small particle size and low immunogenicity, and the acidic TME of solid tumours promotes their uptake by tumour cells, thereby enabling NK-EVs to readily enter the TME, kill tumour cells, and exert antitumour effects 21 . For example, NK-EVs mediate the apoptosis of melanoma cells via the activation of the caspase/PARP apoptotic pathway through the perforin and granzyme B enzymes they carry. Additionally, NK-EVs can regulate immune responses while killing tumour cells, reversing tumour-induced immune suppression and restoring the cytotoxicity of other immune cells to synergistically eliminate tumour cells 22 23 . For example, NK-EVs carrying miR-186 can restore NK cell function in the TME to inhibit neuroblastoma growth and migration 24 . Based on these findings, the exploration of the application and mechanism of NK-EVs in breast cancer is very important for identifying new strategies for treating breast cancer patients. Here, we report for the first time that NK-EVs can alter the expression of proteins that are involved in tumour immune response pathways. A previous study revealed that NK-EVs upregulate MHC-I pathway-related proteins, including antigen processing transporters (TAP2), TAP-binding proteins (TAPBPs), immunoproteasomes (PSMB9 and PSMB10), and β2-microglobulin (B2M), in tumours, thereby increasing CD8 + T-cell-mediated killing of tumour cells and increasing NK cell cytotoxic activity. In vitro, the cytotoxic effect of the combination of NK-EVs and NK cells/T cells on breast cancer cells was significantly greater than that of NK cells/T cells alone. Furthermore, in a nude mouse model of subcutaneous TNBC, NK-EVs demonstrated potent tumour-targeting and antitumour effects. Compared with NK cell monotherapy, the NK-EV combined with NK cell treatment regimen more significantly delayed tumour growth without significantly affecting mouse body weights or organ tissues. In summary, NK-EVs can inhibit tumour progression by activating the tumour MHC-I pathway, overcoming the suppressive nature of the TME, and increasing immune responses to tumours. Notably, minimal NK-EV intervention can increase the effectiveness of immune cells in killing tumours, thereby inhibiting tumour immune escape. Our findings provide evidence for the antitumour activity of NK-EVs and highlight their potential for use as an immunotherapeutic strategy for solid tumours, including breast cancer. 2. Methods 2.1 Cell Culture NK Cell Preparation : Healthy donors aged 20–30 years were recruited for this study. Ethical approval was provided by the First Affiliated Hospital of Xinjiang Medical University, and informed consent was obtained from the donors. Within 2 hours of blood collection, cell sorting was performed with PluriSpin Human NK Cell Enrichment (Plurselect, Germany). The PluriSpin suspension (50 µl/ml whole blood) was added to whole blood samples, and the samples were vortexed for 2 seconds, incubated for 15 minutes, diluted with an equal volume of washing buffer, and gently mixed. The diluted samples were overlaid on a density gradient medium (Ficoll, TBD; HY2015, China) and centrifuged at 800 × g for 30 minutes to obtain NK cells (purity of 75–90%). The cells were expanded in Corning KBM 581 Serum-free Cell Medium supplemented with cytokines (CIMI MAX NK KIT, Zhongke Sai'er, China), and an approximately 450-fold expansion was observed after 14 days of in vitro culture. NK cell purity was determined by flow cytometry (Novo Cyte 3000, Agilent Technologies) on days 3, 7, and 14 by using the following antibodies: APC anti-human CD3, PE anti-human CD56 (NCAM), and Pacific Blue™ anti-human CD16 antibodies. Moreover, the following isotype controls were used: APC mouse IgG2a, PE mouse IgG1, and Pacific Blue™ Mouse IgG1 (BioLegend, USA). CD8 + T cell Preparation : Within 2 hours of blood collection from healthy donors, cell sorting was performed with pluriSpin Human CD8 + T Cell Enrichment (Plurselect, Germany). The cells were expanded in Corning KBM 581 Serum-free Cell Medium supplemented with IL-2 (1000 U/ml), and flow cytometry was performed on day 7 with the following antibodies: APC anti-human CD3, PE anti-human CD56 (NCAM), Pacific Blue™ anti-human CD16, and FITC anti-human CD8. The isotype controls that were mentioned above were used (BioLegend, USA). Cell Lines MCF-7 and MDA-MB-231 cells were purchased from Pricella (China), whereas the ECA109, U87, HCT116, and HepG2 cell lines were obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in high-glucose DMEM (DAKEWE) supplemented with 10% foetal bovine serum (FBS; Sigma) and 1% penicillin-streptomycin (P/S; Corning) at 37°C in a 5% CO 2 incubator (HERACELL-240i; Thermo). 2.2 Isolation of NK-EVs NK cell culture supernatants were collected, and NK-EVs were obtained via ultracentrifugation methods that were previously described in the literature 25 . 2.3 Transmission Electron Microscopy (TEM) NK-EVs were resuspended to a concentration of 5×10¹¹ particles/mL. A carbon-coated copper grid (ZK200; Rigorous) was placed grid-side down on a drop of NK-EVs for 10 minutes, and excess EVs were removed by washing with PBS. The grid was fixed on a drop of 2.5% glutaraldehyde (P1126; Solarbio) for 5 minutes, washed 3 times with PBS (3 minutes per wash), stained with a uranyl acetate solution (U25690; ACMEC) for 60 seconds, blotted dry with filter paper, and air-dried at room temperature. TEM images were captured with a JEM-F200 instrument (JEOL) at an acceleration voltage of 80 kV. 2.4 Nanoparticle Tracking Analysis (NTA) The nanoparticle tracking analyser (ZetaView; Particle Metrix) was calibrated, and PBS-diluted samples were injected into the system for measurement. The system utilized laser scattering microscopy to record the Brownian motion trajectories of NK-EVs in solution, and the particle size and EV concentration were calculated with the Stokes-Einstein equation. 2.5 Western Blotting Analysis NK cells and NK-EVs were lysed with RIPA buffer (AR0102-100; BOSTER) supplemented with a protease inhibitor (PMSF) at 4°C for 30 minutes, and the supernatants were collected by centrifugation. Animal tissue proteins were extracted by homogenizing the tumour tissues with RIPA buffer, and the supernatants were collected after centrifugation at 12,000 rpm for 20 minutes. The protein concentrations were determined with a BCA protein assay kit (CoWin Biosciences) and normalized, and then, protein samples were mixed with 4x protein loading buffer (P1017; Solarbio), denatured by heating at 95°C for 10 minutes, and subjected to SDS-PAGE (P1200; Solarbio). After electrophoresis, the proteins were transferred to PVDF membranes (ZSEQ00010; Millipore), which were blocked with 5% BSA (9048-46-8; Solarbio) at room temperature for 1 hour, washed 3 times with TBST (T1082; Solarbio), and incubated with primary antibodies at 4°C overnight. The next day, after the membranes were washed 3 times with TBST, a rabbit anti-mouse HRP-conjugated secondary antibody (AB6721; Abcam) was added, and the samples were incubated at room temperature for 1 hour. ECL reagent (AB133406; Abcam) was added, and immunoblotting was performed with a 4800Muti fully automated chemiluminescence system (Tanon; China). Band intensity was quantified with ImageJ software for differential analysis. Antibodies against the following NK-EVs-specific proteins were used: CD63 (AB134045; Abcam), TSG101 (AB125011; Abcam), Alix (AB275377; Abcam), perforin (AB256453; Abcam), Granzyme B (AB255598; Abcam), PSMB9 (AB242061; Abcam), PSMB10 (38662A12; Invitrogen), TAP1 (38660A50; Invitrogen), TAP2 (AB235110; Abcam), TAPBP (AB288565; Abcam), B2M (AB75853; Abcam), MHC-I (MA35712; Thermo), and p-STAT1 (AF3300; Affinity). 2.6 Assay of NK-EV Uptake by Cells Tumour cells (1000 cells/well) were seeded in laser confocal dishes, cultured at 37°C in 5% CO 2 for 24 hours, and incubated with 10 µL of Dil-stained NK-EVs (22102; AAT Bioquest) for 24 hours. After the supernatants were removed, the cells were fixed with 4% paraformaldehyde (P0099; Beyotime), stained with DAPI (BioLegend; 422801) to detect the nuclei and AlexaFluor®488 (AAT Bioquest; 23115) to detect the cytoplasm, washed 3 times with PBS, and observed under a confocal laser scanning microscope (CLSM). 2.7 Real-Time Cell Analysis (RTCA) The effect of NK-EVs on tumour cell proliferation was dynamically monitored via RTCA (Agilent; China). DMEM was added to the E-Plate16 for background impedance measurement. Tumour cells in the logarithmic phase of growth (7000 cells/well) were seeded in the E-Plate, maintained at room temperature for 15 minutes, and subsequently placed onto an electrified detection platform for real-time dynamic cell proliferation detection. When the cell index reached a value of approximately 1, the corresponding concentrations of NK-EVs, NK cells, or T cells were added to each well, and tumour cell proliferation curves were generated to calculate the inhibition rates at different time points. The following equation was used to calculate the inhibition rate: inhibition rate (%) = [(control cell index - experimental cell index)/control cell index] × 100%. 2.8 CCK-8 Assay To evaluate the effect of NK-EVs on tumour cell viability, an experiment was performed according to the instructions of the CCK-8 assay kit (CA1210; Solarbio). The cells (10,000 cells/well) were seeded in 96-well plates, cultured for 24 hours, and treated with various factors (such as NK-EVs, NK cells, or T cells) for an additional 48 hours, followed by the addition of the CCK-8 reagent. After 4 hours, the absorbance at 450 nm was measured with a microplate reader, and the cell viability (%) was calculated as follows: cell viability = [(experimental well - blank well)/(negative control well - blank well)] × 100%. 2.9 4D-FastDIA Quantitative Proteomics To analyse the signalling pathways that are regulated by NK-EVs in breast cancer cells, breast cancer cells were randomly divided into control and NK-EV groups (n ≥ 3) and cultured in 75-cm² TC-treated flasks (5×10⁶ cells/flask). After 24 hours, NK-EVs (25 µg/mL) were added to the NK-EV group, and an equal volume of PBS was added to the control group. After 48 hours, the cell pellets were collected to extract total proteins, which were separated and enriched by liquid chromatography, enzymatically digested into peptides, and identified by mass spectrometry for protein quantification. Bioinformatics analysis was performed based on the quantitative results to identify the key proteins that are involved in regulating breast cancer cells. 2.10 Animal Experiments For in vivo experiments, female BALB/c nude mice (6–8-weeks-old) were purchased from Vital River Laboratories (Beijing, China) and housed in the Experimental Animal Center of Xinjiang Medical University under specific pathogen-free conditions. All of the procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Xinjiang Medical University and were conducted in accordance with guidelines and policies (ethics approval number: "IACUC-JT-20230420-46"). The animal studies were conducted following ARRIVE guidelines 2.0 (Animal Research: Reporting of In vivo Experiments). All methods were carried out in accordance with relevant guidelines and regulations. For anesthesia, we used isoflurane (5% induction, 2% maintenance); for euthanasia, we adopted intraperitoneal injection of sodium pentobarbital (150 mg/kg). 2.10.1 Nude Mouse Xenograft Subcutaneous Tumour Model MDA-MB-231 cells (1×10⁷) were resuspended in 500 µL of PBS and subcutaneously injected into the right axilla of female BALB/c nude mice (body weight ≈ 15 g). The length and width of the tumours were measured every 3 days, and the subcutaneous tumours were observed to reach a size of approximately 1 cm 3 after ~ 14 days. In accordance with our animal welfare protocol, mice were euthanized if the tumour volume exceeded 1500 mm³ or if signs of distress were observed. The maximum tumour volume allowed in this study was 1500 mm³. The ECA cell model was established following similar protocols. 2.10.2 NK-EV Homing Assay in Tumour-Bearing Nude Mice NK-EVs were stained with Dil (V22887; Thermo Fisher) according to the kit instructions. Tumour-bearing nude mice were intravenously injected with Dil-labelled NK-EVs (500 µg resuspended in 100 µL of PBS). The distribution of fluorescent NK-EVs in the nude mice was detected with an in vivo imaging system (IVIS Spectrum; PerkinElmer) at 4 hours, 8 hours, and 24 hours. The mice were sacrificed at 24 hours, and tumour tissues and major organs (including the heart, liver, spleen, lung, and kidney tissues) were collected to detect the fluorescence intensity of NK-EVs in ex vivo tissues. 2.10.3 Assay of NK-EV-mediated Protein Regulation in Animal Tumour Tissues Tumour-bearing nude mice were randomly divided into 2 groups (n = 6). The experimental group was intravenously injected with NK-EVs (resuspended in 100 µL of PBS), and the control group was injected with an equal volume of PBS. The mice were sacrificed 24 hours later, and tumour tissues were collected. The expression levels of the PSMB9, PSMB10, TAP1, TAP2, TAPBP, B2M, and KRT9 proteins in the tumour tissues were detected by Western blotting and IHC, whereas the MHC-I and STAT1 protein levels were detected by Western blotting. 2.10.4 Assessment of the Therapeutic Efficacy and Safety of NK-EVs Combined with NK Cells in Tumour-Bearing Nude Mice Tumour-bearing nude mice were randomly divided into control, NK, and NK-EV + NK groups (n = 6) and were subjected to a 28-day intravenous injection treatment regimen. Specifically, on days 1, 7, 13, 19, and 25 of treatment, the mice in the NK and NK-EV + NK groups were intravenously injected with NK cells (1×10⁶ cells/mouse), and those in the control group were injected with an equal volume of PBS. On days 4, 10, 16, 22, and 28 of treatment, the mice in the NK-EV + NK group were intravenously injected with NK-EVs (100 µg/mouse), and those in the other two groups were injected with an equal volume of PBS. The mice were sacrificed on day 28. Body weights and tumour volumes were recorded during the experiment. Moreover, heart, liver, spleen, lung, kidney, and tumour tissues were collected, weighed, and photographed. 2.11 Immunohistochemistry (IHC) Paraffin-embedded sections were subjected to IHC. Specifically, the sections were deparaffinized, and antigen retrieval was performed by heating in 0.01 M citrate buffer (pH = 6.0); subsequently, endogenous peroxidase activity was blocked with 3% hydrogen peroxide, after which the sections were blocked with serum, incubated with primary and secondary antibodies in sequence, developed with DAB chromogen (G1212-200T; Servicebio), counterstained with haematoxylin, and mounted. Stained tissue sections were observed under an optical microscope (Fi3; Nikon), and quantitative analysis was performed with ImageJ software to compare the mean densities between the groups. 2.12 H&E Staining Collected organs and tumour tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned with a microtome (RM2016; Leica, Germany). After deparaffinization and rehydration, the sections were stained with haematoxylin (H9627; Sigma) to detect nuclei and eosin (71014544; Sinopharm) to detect cytoplasm, after which they were dehydrated, mounted, and observed under a light microscope. 2.13 Ethics Approval for Human Subjects Research Human Subjects: The study involving human blood samples from healthy donors was approved by the Ethics Review Committee of the First Affiliated Hospital of Xinjiang Medical University. All procedures strictly adhered to the principles of the Declaration of Helsinki (World Medical Association, 2024 revision). Written informed consent was obtained from all participants prior to the study. 2.14 Ethics Approval for Animal Experiments Animal Experiments: All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Xinjiang Medical University (approval number: IACUC-JT-20230420-46) and were conducted in accordance with institutional guidelines and the ARRIVE guidelines 2.0. For anesthesia, isoflurane was used (5% induction, 2% maintenance); for euthanasia, intraperitoneal injection of sodium pentobarbital (150 mg/kg) was adopted. 2.15 Statistical Analysis Statistical analysis was performed with SPSS 26.0 software. Independent sample t tests were used to analyse differences between two groups, and one-way ANOVA was used to analyse differences among three or more groups. The results are presented as the means ± standard deviations. Significance levels are denoted as * p < 0.05, ** p < 0.01, and ns (not significant). The graphs were generated with GraphPad Prism 8.0 software. 3. Results 3.1 Identification of NK-EVs and Their Antitumour Effects NK-EVs were collected by ultracentrifugation (Fig. 1A) . The NTA results revealed that the NK-EVs were physically homogeneous particles with a diameter of approximately 112.1 ± 48.3 nm and a concentration of ~ 3.5×10¹² particles/mL (Fig. 1B) . TEM imaging revealed that the NK-EVs exhibited uniform spherical structures with distinct membrane structures, and they were approximately 110 nm in diameter (Fig. 1C) . Western blotting analysis revealed that the NK-EVs expressed the exosome markers CD63, Alix, and TSG101, as well as the cytotoxic protein markers perforin and granzyme B. Notably, the NK-EVs did not express calnexin, thus confirming the absence of organelle membrane components (Fig. 1D) . Confocal fluorescence microscopy of Dil-labelled NK-EVs revealed their localization in the cytoplasm and around the nuclei of MCF-7 and MDA-MB-231 cells (Fig. 1E) , thereby demonstrating that breast cancer cells can internalize NK-EVs. In addition, endocytosis was also observed in other tumor types (HCT116, ECA109, U87 and HepG2) (SFig. 1A) . The RTCA results revealed that after the addition of NK-EVs at different concentrations, the proliferation of both MCF-7 and MDA-MB-231 cells were inhibited in a dose-dependent manner (Fig. 1F, G) . The inhibition rates of the MCF-7 and MDA-MB-231 cells reached 96.50% and 94.68%, respectively, at 48 hours after NK-EV intervention (Fig. 1H-I) . A CCK-8 assay was similarly performed to determine the effects of different concentrations of NK-EVs on breast cancer cell viability at 48 hours, and the results revealed dose-dependent inhibition of cancer cell viability, thus further verifying that NK-EVs can inhibit tumour cell proliferation and viability (Fig. 1J-K) . 3.2 Quantitative Proteomics Analysis of Breast Cancer Cells (MCF-7) after NK-EV Intervention Quantitative proteomics analysis was performed on MCF-7 cells after treatment with NK-EVs or the PBS control, and 38 differentially expressed proteins (including 30 upregulated and 8 downregulated proteins) were identified (Fig. 2A-B) . GO and KEGG 26 27 enrichment analyses of the differentially expressed proteins (Fig. 2C-D) revealed significant enrichment of interacting proteins in the MHC-I and immune-related pathways. A protein-protein interaction (PPI) network was constructed using the differentially expressed proteins (Fig. 2E) , which revealed that NK-EV intervention significantly upregulated the expression of PSMB9, PSMB10, TAP1, TAP2, TAPBP, B2M, and other proteins that are closely related to intracellular antigen peptide transport and MHC-I expression. Similar were obtained in MDA-MB-231 cells via the same method (SFig. 2) . 3.3 Protein Expression in Breast Cancer Cells after NK-EV Intervention After coculturing MCF-7 and MDA-MB-231 cells with NK-EVs for 24 hours and removing the supernatants, the cells were subjected to Western blotting. The results revealed increased expression of the PSMB9, PSMB10, TAP2, TAPBP, B2M, MHC-I, and STAT1 proteins in MCF-7 cells (Fig. 3A-B) . Similarly, PSMB9, PSMB10, TAP2, and TAPBP protein expression was upregulated in MDA-MB-231 cells (Fig. 3C-D) , which was consistent with the results of 4D-FastDIA quantitative proteomics. 3.4 NK-EV Intervention Increases Immune Cell-Mediated Tumour Growth Inhibition NK-EVs can not only directly induce tumour apoptosis but also promote the function of cytotoxic T lymphocytes through multiple pathways, including the reprogramming of tumour-associated macrophages and upregulation of MHC-I on tumour cells. We first isolated and cultured NK cells and CD8 + T cells from human peripheral blood. Breast cancer cells were seeded in RTCA plates, and the following experimental groups were established: A) the ① PBS, ② NK-EV, ③ CD8 + T cell, and ④ NK-EV + CD8 + T cell groups; and B) the ① PBS, ② NK-EV, ③ NK cell, and ④ NK-EV + NK cell groups. Dynamic curves of cell proliferation were subsequently generated. The results revealed that in both groups, the rates of tumour cell proliferation inhibition in the ④ NK-EV + CD8 + T cell and ④ NK-EV + NK cell groups were more significant (Fig. 4A-H) . Moreover, compared with the tumour cell inhibition rate following NK cell monotherapy, the rate of MDA-MB-231 cell inhibition following NK-EV treatment and NK cell treatment reached 87.93%, which demonstrated a significant difference. Similarly, the CCK-8 results revealed that NK-EVs reduced the viability of MCF-7 and MDA-MB-231 cells after CD8 + T cell or NK cell intervention (Fig. 4I-L) . 3.5 NK-EVs Exhibit Tumour-Homing Properties in Tumour-Bearing Mice Exogenous NK-EVs are reported to exhibit a strong ability to target particular tissues in vivo. An IVIS imaging system was used to monitor intravenously injected Dil-fluorescent dye-labelled NK-EVs in tumour-free mice and in MDA-MB-231 breast cancer tumour-bearing mice (Fig. 5A). IVIS imaging revealed that NK-EVs demonstrated no major aggregation area in control mice, whereas obvious aggregation at the tumour site was observed in breast cancer tumour-bearing mice (Fig. 5B) . The mice were sacrificed 24 hours after NK-EV injection, and the tumour tissues and major organs were collected and subjected to ex vivo fluorescence imaging. The results revealed that NK-EVs mainly accumulated in the livers of control mice, whereas in breast cancer and oesophageal cancer tumour-bearing mice, NK-EVs accumulated in both the tumour and liver, with stronger fluorescence signals being detected in the tumours than in the liver (Fig. 5C). 3.6 Effects of NK-EVs Combined with NK Cells in Model Mice Due to the high immunogenicity of T cells, severe immune reactions may occur during allogeneic infusion. In contrast, NK cells have low immunogenicity, and thus, the use of NK cells can avoid inhibition mediated by inhibitory receptors. Therefore, NK cells were selected for combination therapy with NK-EVs in the in vivo experiments. Previous studies have evaluated the therapeutic effect of NK-EVs in tumours and reported that NK-EVs at concentrations of 200–500 µg exert good antitumour effects in tumour-bearing mice, with some antitumour effects beginning to be observed following the administration of 100 µg of NK-EVs. The treatment protocol is shown in (Fig. 5D) . Tumour growth curves revealed that, compared with NK cell monotherapy, the combined intervention of NK-EVs and NK cells significantly inhibited tumour growth (Fig. 5F-I) . The ex vivo tumour volumes and weights in the NK-EV combined with NK cell treatment group were significantly different from those in the other two groups (Fig. 5E, 5J) . Additionally, H&E staining revealed that, compared with the PBS control and NK cell-treated groups, the NK-EV combined with NK cell treatment group exhibited increased tumour apoptosis in mice (Fig. 5L) . These data demonstrate that the combination of NK-EVs and NK cells significantly inhibits breast cancer tumour growth, with a more potent antitumour effect being observed compared to NK cell monotherapy alone. During the entire treatment process, there was no significant difference observed in body weights among the groups (Fig. 5K) . In all of the treatment groups, no obvious tumour metastasis or liver injury was observed in the liver tissues ex vivo (Fig. S3 A) ; additionally, the organ weights remained within normal ranges (Fig. S3 B-F) , and H&E staining of tissue sections revealed no obvious pathological damage to the liver or other organs among the groups (Fig. S3 G) . In summary, the combination of NK-EVs with NK cell therapy is safe. 3.7 NK-EVs Participate in Regulating the MHC-I Pathway in Tumour-Bearing Nude Mice Nude mouse models of MDA-MB-231 cell-derived tumours were intravenously injected with either PBS control or NK-EVs (500 µg) via the tail vein. The mice were sacrificed 24 hours later, and the tumour tissues were collected. The Western blotting and IHC results revealed increased protein expression of PSMB9, PSMB10, TAP2, TAPBP, and B2M, in the tumour tissues of the NK-EV intervention group (Fig. 6A-D) . 3.8 Expression of MHC-I Pathway-Related Proteins in Breast Cancer Patients Tumour pathological sections from patients with different stages of breast cancer were collected, and IHC staining revealed increased protein expression of PSMB9, PSMB10, TAP1, TAP2, TAPBP, and B2M in stage II breast cancer tumours from patients (Fig. 6D) . 4. Discussion In this study, we obtained high-quality NK-EVs and demonstrated that NK-EVs can be internalized by breast cancer cells. Additionally, the uptake of NK-EVs was similarly observed in glioma cells (U87), oesophageal cancer cells (ECA109), colon cancer cells (HCT116), and liver cancer cells (HepG2), which is consistent with previous findings on NK-EVs in cancer research 20 . These results suggest that our findings may be applicable to multiple solid tumours. In the animal experiments, after the intravenous injection of Dil-NK-EVs into mice with breast cancer, aggregation of fluorescence signals at the tumour site were observed at 4 hours, and the fluorescence signals did not weaken or disappear at 24 hours. These results verify the good tumour-targeting homing ability of NK-EVs, thereby indicating that NK-EVs exhibit great advantages as antitumour drugs or drug delivery systems for future treatments 22 28 . We observed that NK-EVs carry perforin and granzyme B. Additionally, quantitative proteomics sequencing of breast cancer cells after NK-EV treatment revealed significant enrichment of multiple related pathways in the GO analysis, including protein binding, protein activity, enzyme receptor activity, and signal receptor activity (including TRAIL and transmembrane transporter activity) 29 . Natural killer cell-mediated cytotoxicity was also significantly enriched in the GO and KEGG analyses, thereby suggesting that the pathway by which NK-EVs kill breast cancer cells in this study may be similar to that described in previous studies 30 – 32 . Furthermore, cell killing experiments revealed both dose-dependent and time-dependent killing effects of NK-EVs on breast cancer cells. At a concentration of 50 µg/100 µL, tumour cell proliferation was inhibited, and obvious cell death was observed at an effector-target ratio of 200 µg/100 µL cell. After breast cancer cells were treated with NK-EVs at an effector-target ratio lower than the tumour-killing threshold for 24 hours, tumour cells were more susceptible to killing by CD8 + T cells. The protein sequencing results revealed that the upregulated proteins were closely related to antigen peptide presentation by tumour cells and MHC-I expression. MHC-I plays a crucial role in cell-mediated immune responses. Reduced or absent MHC-I expression leads to tumour immune escape, which is primarily due to the lack of tumour antigen presentation for recruiting and activating CD8 + T cells 7 33 . The downregulation of MHC-I molecules has been observed in various tumours. PSMB9 and PSMB10 perform proteolysis in a trypsin-like manner, whereby they cleave endogenous peptides to generate peptides with appropriate anchor residues, which are then transported to the endoplasmic reticulum lumen by the transporters TAP1 and TAP2. TAPBP acts as a bridge for TAP and MHC-I molecules, thereby mediating peptide loading onto the MHC-I structure to form the MHC-I complex 34 35 . The MHC-I complex consists of three components, including the MHC-I heavy chain, B2M, and amino acid peptides derived from endogenous proteins (which are transported to the cell membrane surface by the Golgi apparatus). In this study, our unintended manipulation may have resulted in significant effects due to the exposures of hidden endogenous peptides of breast cancer cells, thus potentially inducing the aggregation and killing functions of CD8 + T cells. Typically, the antigen presentation machinery (APM) can be upregulated by cytokines, such as IFN-γ, that are secreted by activated lymphocytes and NK cells 36 . IFN-γ receptor signalling via the JAK-STAT pathway can induce the expression several APM components, including MHC-I heavy chains and B2M in cis or trans. In this study, both the protein sequencing and Western blotting results revealed increased STAT1 expression. Therefore, after NK-EVs are internalized by tumour cells, it is unknown which component/process leads to STAT1 phosphorylation, and this question requires further detection of NK-EV components and additional experiments for clarification 37 . Finally, we observed that NK cells exhibited an increased ability to kill breast cancer cells that were pretreated with NK-EVs. This effect may be explained by the study of Ardolino M. et al., who suggested that reduced or absent MHC-I expression prevents NK cells from achieving "permission," thereby leading to their inability to express inhibitory receptors such as KIRs, which are necessary for effective tumour cell recognition and killing 38 39 . NK cells infiltrating MHC-I-deficient tumour environments exhibit a hyporesponsive state, whereas those infiltrating MHC-I-sufficient tumour environments exhibit a hyperresponsive state 40 41 . Our results show that NK-EVs increase CD8 + T cell recognition and killing and promote NK cell cytotoxicity by upregulating MHC-I expression in breast cancer cells (Fig. 7). In the animal model, there were no significant effects observed on mouse body weights or organ tissue. Based on the results of staining of clinical samples, high expression of MHC-I pathway-related proteins may indicate a better prognosis for patients with breast cancer, and these proteins may serve as indicators for evaluating breast cancer prognosis. 5. Conclusion In summary, our findings demonstrate that NK-EVs are innovative nanotherapeutic candidates that can be internalized by tumour cells; moreover, they exhibit tumour-homing properties and are safe. To our knowledge, this study is the first to report that NK-EVs can increase tumour antigen presentation and that the combination of NK-EVs with other adoptive immunotherapies or ICIs may be a new strategy for treating breast cancer. In future research, further investigations of NK-EV components, mechanisms of action, and TME changes are crucial. Abbreviations APM Antigen Presentation Machinery B2M β2-Microglobulin CLSM Confocal Laser Scanning Microscope EVs Extracellular Vesicles ICIs Immune Checkpoint Inhibitors IFN-γ Interferon-γ IVIS In Vivo Imaging System MHC Major Histocompatibility Complex NTA Nanoparticle Tracking Analysis NK Natural Killer PD-L1 Programmed Cell Death Ligand 1 PSMB9/PSMB10 Proteasome Subunit Beta Type 9/10 RTCA Real-Time Cell Analysis TAP1/TAP2 Transporter Associated with Antigen Processing 1/2 TAPBP Transporter Associated with Antigen Processing Binding Protein TEM Transmission Electron Microscopy TME Tumour Microenvironment Declarations Acknowledgements None. Author contributions All the authors contributed to the study’s conception and design. Limin Zhang and Yidan Xu planned the experiment, composed the manuscript and managed experimental data. Jing Guo, Hu Li and carried out cytological and molecular biological experiments, analysed data and made charts. Yidan Xu and Jing Guo were responsible for animals breeding performed animal researches and executed data analysis. Xin Lv, Libin Liao, Yajie Zhang, Jiyang Li, Xinxin Gu, Hu Li assisted data analysis with constructive recommendation. Qi Pan and Shengbin Bai gave suggestions on writing and revising the manuscript. All authors have read and approved the final manuscript. Funding This study was supported by the Open Project Fund of the Key Laboratory of Tissue and Cell Engineering of Xinjiang Uygur Autonomous Region, China. (Project name: the Role and Mechanism of Exosomes Derived from Natural Killer Cells in Enhancing Tumour Immune Response by Upregulating the MHC Class Ⅰ Pathway in Triple-negative Breast Cancer, project number: XJXS002). Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate Animal experiments were approved by the Animal Ethics Committee of Medical Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University. (Permit Number: IACUC-JT-20230420-46). The animal studies were conducted according to the ARRIVE guidelines 2.0 (Animal Research: Reporting of In Vivo Experiments). All methods were carried out in accordance with relevant guidelines and regulations. The authors declare that they have not use AI-generated work in this manuscript. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Kim J, Harper A, McCormack V, et al. Global patterns and trends in breast cancer incidence and mortality across 185 countries. Nature medicine 2025;31(4):1154-62. doi: 10.1038/s41591-025-03502-3 [published Online First: 2025/02/25] Xiong X, Zheng LW, Ding Y, et al. Breast cancer: pathogenesis and treatments. 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Scandinavian journal of immunology 2020;92(4):e12938. doi: 10.1111/sji.12938 [published Online First: 2020/07/23] Cózar B, Greppi M, Carpentier S, et al. Tumor-Infiltrating Natural Killer Cells. Cancer discovery 2021;11(1):34-44. doi: 10.1158/2159-8290.Cd-20-0655 [published Online First: 2020/12/06] Additional Declarations No competing interests reported. Supplementary Files OriginalImageforWesternBlot.pptx Tumorsizeimage.pdf fig8.jpg Supplementary Figure 1.Schematic diagram of the NK-EV uptake assay. Nuclei were stained with DAPI (blue), and the cytoplasm was stained with Flour488 (green). Scale bar: 100 μm. fig9.jpg Supplementary Figure 2.Quantitative proteomics analysis of breast cancer cells (MDA-MB-231) after NK-EV intervention. (A) Volcano plot of differentially expressed proteins (fold change > 1.5, p < 0.05). (B) Heatmap of differentially expressed proteins tumour cells. (C-D) GO and KEGG enrichment analyses of differentially expressed proteins, p < 0.05. (E) Protein-protein interaction network of the differentially expressed proteins. BP, biological process; CC, cellular component; MF, molecular function. fig10.jpg Supplementary Figure 3. Safety test of NK-Evs in mice with breast cancer. (A) Photographs of isolated livers. (B) Liver weight. (C) Heart weight. (D) Weight of spleen. (E) Lung weight. (F) Kidney weight. (G) H&E-stained pathological pictures of organs. Scale bars are indicated in the figure. All of the data are presented as the means±SEMs, * p < 0.05, ** p < 0.01. H&E, haematoxylin and eosin. 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23:25:11","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2206839,"visible":true,"origin":"","legend":"","description":"","filename":"Figures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/f2d6b49bc31829da778dc5e6.pdf"},{"id":94048842,"identity":"1f5deb8b-3a7a-49ec-b22a-4da02efcf6ba","added_by":"auto","created_at":"2025-10-21 23:25:11","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":122935,"visible":true,"origin":"","legend":"","description":"","filename":"93b46dfe427d4a5481e13428fa8f44ad1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/04b272041b14cff8ecd94a75.xml"},{"id":94048834,"identity":"4726d1da-3f6f-44b9-b4a3-c57b2a6cfaab","added_by":"auto","created_at":"2025-10-21 23:25:10","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138429,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/46aa5549049d1d500ec87b96.html"},{"id":94048822,"identity":"2658a830-3abe-4c26-b1cd-0dea475065da","added_by":"auto","created_at":"2025-10-21 23:25:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":793715,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation and identification of NK-EVs. (A) Schematic diagram of NK-EVisolation by ultracentrifugation. (B) Size distribution and concentration of NK-EVs as measured by NTA. (C) TEM image of NK-EV morphology. Scale bar: 100 nm. (D) Western blotting analysis of the levels of the exosome markers CD63, Alix, and TSG101, as well as the cytotoxic proteins perforin and granzyme B. β-tubulin served as the internal control. (E) Schematic diagram of the NK-EVuptake assay. Nuclei were stained with DAPI(blue), and the cytoplasm was stained with Flour488(green). Scale bar: 100 μm. (F-G) RTCA of the proliferation curves of MCF-7 (F) and MDA-MB-231 (G) cells treated with different doses of NK-EVs. The arrows indicate the time points of NK-EVintervention. (H-I) RTCA of the rates of MCF-7 (H) and MDA-MB-231 (I) cell inhibition at 24 hours, 48 hours, and 72 hours after intervention with different doses of NK-EVs. (J-K) CCK-8 assay of the viability of MCF-7 (J) and MDA-MB-231 (K) cells at 48 hours after intervention with different doses of NK-EVs. All of the data are presented as the means±SEMs from at least three independent experiments, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. NK-EVs, NK cell-derived extracellular vesicles; TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis.\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/a13d93f49213888ca641c063.jpg"},{"id":94048823,"identity":"814810ff-c30f-4b3c-a480-d9127e47b69b","added_by":"auto","created_at":"2025-10-21 23:25:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":458165,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative proteomics analysis of breast cancer cells (MCF-7) after NK-EVintervention. (A) Volcano plot of differentially expressed proteins (fold change\u0026gt; 1.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). (B) Heatmap of differentially expressed proteins in MCF-7 tumour cells. (C-D) GO and KEGG enrichment analyses of differentially expressed proteins, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. (E) Protein-protein interaction network of the differentially expressed proteins. BP, biological process; CC, cellular component; MF, molecular function.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/8a6a30ae0fa03ebe55b477b2.jpg"},{"id":94049773,"identity":"3185c962-53ad-44df-9a3a-4fb7a573d216","added_by":"auto","created_at":"2025-10-21 23:41:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":845738,"visible":true,"origin":"","legend":"\u003cp\u003eNK-EVs affect the expression of MHC-I pathway-related proteins in breast cancer cells. (A) Western blotting analysis of the levels of MHC-I pathway-related proteins (PSMB9, PSMB10, TAP1, TAP2, TAPBP, B2M, MHC Ⅰ, and p-STAT) in MCF-7 cells. (B) Expression was quantified by ImageJ software and normalized to that of β-tubulin. (C) Western blottinganalysis of MHC-I pathway-related proteins (PSMB9, PSMB10, TAP1, TAP2, TAPBP, B2M, MHC Ⅰ, and p-STAT) in MDA-MB-231 cells. (D) Expression was quantified by ImageJ software and normalized to that of β-tubulin. All of the data are presented as the means±SEMsfrom at least three independent experiments. Compared with untreated MCF-7 and MDA-MB-231 cells, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/9eb47111e351ae7ca0ea6220.jpg"},{"id":94049373,"identity":"0769bb2a-8c32-41c7-ba53-abc073853e36","added_by":"auto","created_at":"2025-10-21 23:33:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":586234,"visible":true,"origin":"","legend":"\u003cp\u003eNK-EVintervention increases the killing of tumour cells by immune cells. In panels A, C, E, and G, the red arrow indicates the addition of10 μg of NK-EVs to each well of the ④ NK-EVs+CD8+T cell group in experiment A and the ④ NK-EV+NK cell group in experiment B. The black arrow indicates (via changing medium in all of the wells) the addition ofCD8+T cells and NK cells to the ④ NK-EVs+CD8+T cell group in experiment A and the ④ NK-EV+NK cell group in experiment B at an effector-target ratio of 1:5. The yellow arrow indicates the addition of10 μg of NK-EVs to each well of the ② NK-EV group in experiments A and B. (A-B) RTCA of cell proliferation curves (A) and inhibition rates at 24 hours, 48 hours, and 72 hours (B) in MCF-7 cells treated with NK-EVs combined with CD8+T cells. (C-D) RTCA of proliferation curves (C) and inhibition rates at 24 hours, 48 hours, and 72 hours (D) in MCF-7 cells treated with NK-EVs combined with NK cells. (E-F) RTCA of cell proliferation curves (E) and inhibition rates at 24 hours, 48 hours, and 72 hours (F) in MDA-MB-231 cells treatedwith NK-EVs combined with CD8+T cells. (G-H) RTCA of proliferation curves (G) and inhibition rates at 24 hours, 48 hours, and 72 hours (H) in MDA-MB-231 cells treated with NK-EVs combined with NK cells. (I) CCK-8 analysis of the viability of MCF-7 cells at 48 hours after intervention with NK-EVs combined with CD8+Tcells. (J) CCK-8 analysis of the viability of MCF-7 cells at 48 hours after intervention with NK-EVs combined with NK cells. (K) CCK-8 analysis of theviability of MDA-MB-231 cells at 48 hours after intervention with NK-EVs combined with CD8+T cells. (L) CCK-8 analysis of theviability of MDA-MB-231 cells at 48 hours after intervention with NK-EVs combined with NK cells. All of the data are presented as the means±SEMsfrom at least three independent experiments, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01. RTCA, real-time cell analysis.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/30632773af8d94b2f7e62aa8.jpg"},{"id":94048833,"identity":"d0c2c3e2-8f9b-498b-9948-c370c12da4e2","added_by":"auto","created_at":"2025-10-21 23:25:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":572944,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo targeting ability and effects of NK-EVs in a subcutaneous xenograft tumour mouse model. (A) Schematic diagram of the experimental protocol for evaluating the tumour-targeting ability of NK-EVs in subcutaneous xenograft tumour model mice. (B-C) IVIS detection of living mice (B), as well as major organs (liver, lung, heart, spleen, and kidney) and ex vivo tumours (C). (D) Schematic diagram of the experimental protocol for evaluating the therapeutic efficacy and safety of NK-EVs combined with NK cells in MDA-MB-231 subcutaneous tumour-bearing mice. (E) Optical images of ex vivo tumour tissues obtained from mice at the end of treatment. (F-I) Tumourgrowth curves of the control (F), NK cell treatment (G), and NK-EV+NK celltreatment (H) groups during treatment, as well as the overall tumourgrowth curve comparison (I). (J) Tumour weights recorded at the end of treatment. (K) Mouse body weights recorded during treatment. (L) Representative histological images of H\u0026amp;E-stained mouse tumour tissue sections. Scale bar: 100 μm. All of the data are presented as the means±SEMs, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. H\u0026amp;E, haematoxylin and eosin.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/976b26fa8d20d971bb94c287.jpg"},{"id":94048835,"identity":"24034897-9f80-4002-a99c-18b0f8a7a049","added_by":"auto","created_at":"2025-10-21 23:25:10","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":749954,"visible":true,"origin":"","legend":"\u003cp\u003eNK-EVs affect the expression of MHC-I-related proteins in breast cancer tumour tissues. (A) Western blotting analysis of the levels of MHC-I pathway-related proteins (PSMB9, PSMB10, TAP1, TAP2, TAPBP, and B2M). (B) Expression was quantified by ImageJ software and normalized to β-tubulin expression. (C) IHC staining of MHC-I pathway-related proteins in the tumour tissues of PBS-treated and NK-EV-treated mouse breast cancer tissues. Scale bar: 200 μm. (D) Quantitative analysis with ImageJsoftware was performed to compare the mean optical density values between groups. (E) IHC staining of MHC-I pathway-related proteins in tumour pathological sections of clinical breast cancer patients.Stage II indicates grade 2 breast cancer pathology, and stage IIIindicates grade 3 breast cancer pathology. Scale bar: 100 μm. All of the data are presented as the means±SEMs, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01. H\u0026amp;E, haematoxylin and eosin.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/b7a9e721617a07c6fe86fa2b.jpg"},{"id":94048836,"identity":"f93ceef9-7e56-408c-8773-dfa316abf60a","added_by":"auto","created_at":"2025-10-21 23:25:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":305060,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of how NK-EVs regulate the tumour MHC-I pathway to increaseimmune cell killing. After NK cell-derived exosomes are captured by tumour cells, cytokines such as IFN-γ are released into the cytoplasm. IFN-γ and TNF-α upregulate the expression of immunoproteasomes (including PSMB9 and PSMB10), thus further influencing antigen processing transporters that are (including TAP1 and TAP2) localized on the endoplasmic reticulum that function to transport peptides that are generated by proteasomal degradation in the cytoplasm and nucleus into the endoplasmic reticulum lumen. Here, the interaction between newly assembled MHC-I molecules and TAP-related transporters is mediated by TAP binding protein (TAPBP). β2-microglobulin (β2M) enables MHC-I molecules to effectively present peptides to immune cells, thereby enhancing NK cell-mediated killing and improving CD8+T cell recognition and killing of tumours. Therefore, the regulatory effect of NK-EVs on the tumour MHC-I pathway promotes the response of immune cells to tumours.\u003c/p\u003e","description":"","filename":"fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/9c3eeec32a71f4aef1685a48.jpg"},{"id":94050815,"identity":"7d798efc-2533-4ade-b6ef-23cde7003afe","added_by":"auto","created_at":"2025-10-21 23:49:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5535507,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/c85ae181-f170-4e00-a0c6-17601ba2ab99.pdf"},{"id":94048839,"identity":"c535a0aa-49ba-4bad-a906-9fe2b4a66aee","added_by":"auto","created_at":"2025-10-21 23:25:11","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16562630,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalImageforWesternBlot.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/9e735eeb13ee67c6269e1583.pptx"},{"id":94049372,"identity":"64d267cd-3d21-4323-b4d1-d43b522f7269","added_by":"auto","created_at":"2025-10-21 23:33:10","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":187734,"visible":true,"origin":"","legend":"","description":"","filename":"Tumorsizeimage.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/3baf25dc832354aeb5610a63.pdf"},{"id":94048827,"identity":"db61bf58-182b-49e9-a702-82264bb32caa","added_by":"auto","created_at":"2025-10-21 23:25:10","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":224215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1.\u003c/strong\u003eSchematic diagram of the NK-EV uptake assay. Nuclei were stained with DAPI (blue), and the cytoplasm was stained with Flour488 (green). Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/ec864ed050c48afd0b719d88.jpg"},{"id":94048825,"identity":"d759ebbc-dbc6-4c4f-afdd-dde8463b7fa3","added_by":"auto","created_at":"2025-10-21 23:25:10","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":474838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2.\u003c/strong\u003eQuantitative proteomics analysis of breast cancer cells (MDA-MB-231) after NK-EV intervention. (A) Volcano plot of differentially expressed proteins (fold change \u0026gt; 1.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). (B) Heatmap of differentially expressed proteins tumour cells. (C-D) GO and KEGG enrichment analyses of differentially expressed proteins, p \u0026lt; 0.05. (E) Protein-protein interaction network of the differentially expressed proteins. BP, biological process; CC, cellular component; MF, molecular function.\u003c/p\u003e","description":"","filename":"fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/c9fb8a98cf5add3abfc09791.jpg"},{"id":94048837,"identity":"7d1d8ef6-1a64-4efd-aba3-a97643bfb306","added_by":"auto","created_at":"2025-10-21 23:25:11","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1143180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. \u003c/strong\u003eSafety test of NK-Evs in mice with breast cancer. (A) Photographs of isolated livers. (B) Liver weight. (C) Heart weight. (D) Weight of spleen. (E) Lung weight. (F) Kidney weight. (G) H\u0026amp;E-stained pathological pictures of organs. Scale bars are indicated in the figure. All of the data are presented as the means±SEMs, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01. H\u0026amp;E, haematoxylin and eosin.\u003c/p\u003e","description":"","filename":"fig10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555077/v1/0d9cb1fb6f591313eb1750e1.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"NK Cell-Derived Extracellular Vesicles Function as Immunomodulators to Regulate Breast Cancer Progression by Activating MHC-I Signalling","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBreast cancer is one of the most common malignant tumours in women worldwide, and an increasing incidence has been observed among younger populations\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Although advances in breast cancer screening, diagnosis, and treatment strategies have led to decreases in mortality rates, some patients still fail to achieve ideal outcomes\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. For example, endocrine therapy is the only treatment for patients with luminal-A type breast cancer (hormone receptor-positive). However, some tumours develop acquired resistance, thereby rendering endocrine therapy ineffective. Moreover, triple-negative breast cancer (TNBC) lacks clear therapeutic targets, and chemotherapy is the main treatment for TNBC patients\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, only approximately 20% of is a common occurrence and is partly due to the active downregulation of cell surface major histocompatibility complex (MHC)-I expression during tumour immune escape\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. TNBC patients exhibit intratumorally heterogeneity in regional MHC-I expression\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In mouse models, MHC-I deficiency abrogates antitumour immunity and the response to immune checkpoint inhibitors (ICIs), whereas intratumorally MHC-I heterogeneity leads to increased natural killer (NK) cell infiltration in an IFN-γ-dependent manner. Spatial technology has revealed that MHC-I heterogeneity is associated with clinical resistance to anti-programmed cell death ligand 1 (PD-L1) therapy and an increased NK: T cell ratio in human breast tumours. Additionally, tumours with heterogeneous MHC-I require NKG2A to suppress NK cell function. Combination therapy with anti-NKG2A and anti-PD-L1 restores complete remission in heterogeneous MHC-I mouse models, which depends on the presence of tumour-infiltrating activated NK and CD8\u003csup\u003e+\u003c/sup\u003eT cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These results suggest that similar strategies may result in greater benefits for patients in clinical trials.\u003c/p\u003e\u003cp\u003eImmune checkpoint inhibitor therapy aims to utilize the full repertoire of T cells that are capable of destroying malignant cells, although the relevant T cells and ligand peptides are often undefined\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Increasing evidence indicates that successful immunotherapy with ICIs depends on sustained expression of MHC class I molecules by tumour cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Tumour-specific MHC-I (tsMHC-I) antigen presentation can be disrupted by multiple mechanisms, including immune editing or selection against neoantigens, the loss of MHC-I allelic diversity, or mutations that suppress interferon-sensing pathways and subsequent MHC-I upregulation\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In breast cancer, the somatic genetic loss of HLA loci or epigenetic silencing via promoter hypermethylation has been observed\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In recent years, adoptive immunotherapy has gained widespread attention for its promising potential in the treatment of solid tumours\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e; however, its application is limited by factors such as the poor tumour homing ability of immune cells, the inability to penetrate biological barriers, and the tumour microenvironment (TME)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eExtracellular vesicles (EVs) are membrane-bound vesicles that are released into the extracellular matrix after the fusion of intracellular multivesicular bodies (MVBs) with the cell membrane\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These nanoscale lipid structures (30\u0026ndash;150 nm in diameter) encapsulate proteins, mRNAs, miRNAs, and other substances. NK cell-derived vesicles contain various cytotoxic proteins, miRNAs, and cytokines; moreover, they exhibit cell-killing activity similar to that of NK cells\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Additionally, NK cell-derived EVs (NK-EVs) have a small particle size and low immunogenicity, and the acidic TME of solid tumours promotes their uptake by tumour cells, thereby enabling NK-EVs to readily enter the TME, kill tumour cells, and exert antitumour effects\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. For example, NK-EVs mediate the apoptosis of melanoma cells via the activation of the caspase/PARP apoptotic pathway through the perforin and granzyme B enzymes they carry. Additionally, NK-EVs can regulate immune responses while killing tumour cells, reversing tumour-induced immune suppression and restoring the cytotoxicity of other immune cells to synergistically eliminate tumour cells\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. For example, NK-EVs carrying miR-186 can restore NK cell function in the TME to inhibit neuroblastoma growth and migration\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Based on these findings, the exploration of the application and mechanism of NK-EVs in breast cancer is very important for identifying new strategies for treating breast cancer patients.\u003c/p\u003e\u003cp\u003eHere, we report for the first time that NK-EVs can alter the expression of proteins that are involved in tumour immune response pathways. A previous study revealed that NK-EVs upregulate MHC-I pathway-related proteins, including antigen processing transporters (TAP2), TAP-binding proteins (TAPBPs), immunoproteasomes (PSMB9 and PSMB10), and β2-microglobulin (B2M), in tumours, thereby increasing CD8\u003csup\u003e+\u003c/sup\u003eT-cell-mediated killing of tumour cells and increasing NK cell cytotoxic activity. In vitro, the cytotoxic effect of the combination of NK-EVs and NK cells/T cells on breast cancer cells was significantly greater than that of NK cells/T cells alone. Furthermore, in a nude mouse model of subcutaneous TNBC, NK-EVs demonstrated potent tumour-targeting and antitumour effects. Compared with NK cell monotherapy, the NK-EV combined with NK cell treatment regimen more significantly delayed tumour growth without significantly affecting mouse body weights or organ tissues. In summary, NK-EVs can inhibit tumour progression by activating the tumour MHC-I pathway, overcoming the suppressive nature of the TME, and increasing immune responses to tumours. Notably, minimal NK-EV intervention can increase the effectiveness of immune cells in killing tumours, thereby inhibiting tumour immune escape. Our findings provide evidence for the antitumour activity of NK-EVs and highlight their potential for use as an immunotherapeutic strategy for solid tumours, including breast cancer.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Cell Culture\u003c/h2\u003e\u003cp\u003e\u003cb\u003eNK Cell Preparation\u003c/b\u003e: Healthy donors aged 20\u0026ndash;30 years were recruited for this study. Ethical approval was provided by the First Affiliated Hospital of Xinjiang Medical University, and informed consent was obtained from the donors. Within 2 hours of blood collection, cell sorting was performed with PluriSpin Human NK Cell Enrichment (Plurselect, Germany). The PluriSpin suspension (50 \u0026micro;l/ml whole blood) was added to whole blood samples, and the samples were vortexed for 2 seconds, incubated for 15 minutes, diluted with an equal volume of washing buffer, and gently mixed. The diluted samples were overlaid on a density gradient medium (Ficoll, TBD; HY2015, China) and centrifuged at 800 \u0026times; g for 30 minutes to obtain NK cells (purity of 75\u0026ndash;90%). The cells were expanded in Corning KBM 581 Serum-free Cell Medium supplemented with cytokines (CIMI MAX NK KIT, Zhongke Sai'er, China), and an approximately 450-fold expansion was observed after 14 days of in vitro culture. NK cell purity was determined by flow cytometry (Novo Cyte 3000, Agilent Technologies) on days 3, 7, and 14 by using the following antibodies: APC anti-human CD3, PE anti-human CD56 (NCAM), and Pacific Blue\u0026trade; anti-human CD16 antibodies. Moreover, the following isotype controls were used: APC mouse IgG2a, PE mouse IgG1, and Pacific Blue\u0026trade; Mouse IgG1 (BioLegend, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCD8\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eT cell Preparation\u003c/b\u003e: Within 2 hours of blood collection from healthy donors, cell sorting was performed with pluriSpin Human CD8\u003csup\u003e+\u003c/sup\u003eT Cell Enrichment (Plurselect, Germany). The cells were expanded in Corning KBM 581 Serum-free Cell Medium supplemented with IL-2 (1000 U/ml), and flow cytometry was performed on day 7 with the following antibodies: APC anti-human CD3, PE anti-human CD56 (NCAM), Pacific Blue\u0026trade; anti-human CD16, and FITC anti-human CD8. The isotype controls that were mentioned above were used (BioLegend, USA).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCell Lines\u003c/strong\u003e\u003cp\u003eMCF-7 and MDA-MB-231 cells were purchased from Pricella (China), whereas the ECA109, U87, HCT116, and HepG2 cell lines were obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in high-glucose DMEM (DAKEWE) supplemented with 10% foetal bovine serum (FBS; Sigma) and 1% penicillin-streptomycin (P/S; Corning) at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator (HERACELL-240i; Thermo).\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Isolation of NK-EVs\u003c/h2\u003e\u003cp\u003eNK cell culture supernatants were collected, and NK-EVs were obtained via ultracentrifugation methods that were previously described in the literature\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Transmission Electron Microscopy (TEM)\u003c/h2\u003e\u003cp\u003eNK-EVs were resuspended to a concentration of 5\u0026times;10\u0026sup1;\u0026sup1; particles/mL. A carbon-coated copper grid (ZK200; Rigorous) was placed grid-side down on a drop of NK-EVs for 10 minutes, and excess EVs were removed by washing with PBS. The grid was fixed on a drop of 2.5% glutaraldehyde (P1126; Solarbio) for 5 minutes, washed 3 times with PBS (3 minutes per wash), stained with a uranyl acetate solution (U25690; ACMEC) for 60 seconds, blotted dry with filter paper, and air-dried at room temperature. TEM images were captured with a JEM-F200 instrument (JEOL) at an acceleration voltage of 80 kV.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Nanoparticle Tracking Analysis (NTA)\u003c/h2\u003e\u003cp\u003eThe nanoparticle tracking analyser (ZetaView; Particle Metrix) was calibrated, and PBS-diluted samples were injected into the system for measurement. The system utilized laser scattering microscopy to record the Brownian motion trajectories of NK-EVs in solution, and the particle size and EV concentration were calculated with the Stokes-Einstein equation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Western Blotting Analysis\u003c/h2\u003e\u003cp\u003eNK cells and NK-EVs were lysed with RIPA buffer (AR0102-100; BOSTER) supplemented with a protease inhibitor (PMSF) at 4\u0026deg;C for 30 minutes, and the supernatants were collected by centrifugation. Animal tissue proteins were extracted by homogenizing the tumour tissues with RIPA buffer, and the supernatants were collected after centrifugation at 12,000 rpm for 20 minutes. The protein concentrations were determined with a BCA protein assay kit (CoWin Biosciences) and normalized, and then, protein samples were mixed with 4x protein loading buffer (P1017; Solarbio), denatured by heating at 95\u0026deg;C for 10 minutes, and subjected to SDS-PAGE (P1200; Solarbio). After electrophoresis, the proteins were transferred to PVDF membranes (ZSEQ00010; Millipore), which were blocked with 5% BSA (9048-46-8; Solarbio) at room temperature for 1 hour, washed 3 times with TBST (T1082; Solarbio), and incubated with primary antibodies at 4\u0026deg;C overnight. The next day, after the membranes were washed 3 times with TBST, a rabbit anti-mouse HRP-conjugated secondary antibody (AB6721; Abcam) was added, and the samples were incubated at room temperature for 1 hour. ECL reagent (AB133406; Abcam) was added, and immunoblotting was performed with a 4800Muti fully automated chemiluminescence system (Tanon; China). Band intensity was quantified with ImageJ software for differential analysis. Antibodies against the following NK-EVs-specific proteins were used: CD63 (AB134045; Abcam), TSG101 (AB125011; Abcam), Alix (AB275377; Abcam), perforin (AB256453; Abcam), Granzyme B (AB255598; Abcam), PSMB9 (AB242061; Abcam), PSMB10 (38662A12; Invitrogen), TAP1 (38660A50; Invitrogen), TAP2 (AB235110; Abcam), TAPBP (AB288565; Abcam), B2M (AB75853; Abcam), MHC-I (MA35712; Thermo), and p-STAT1 (AF3300; Affinity).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Assay of NK-EV Uptake by Cells\u003c/h2\u003e\u003cp\u003eTumour cells (1000 cells/well) were seeded in laser confocal dishes, cultured at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours, and incubated with 10 \u0026micro;L of Dil-stained NK-EVs (22102; AAT Bioquest) for 24 hours. After the supernatants were removed, the cells were fixed with 4% paraformaldehyde (P0099; Beyotime), stained with DAPI (BioLegend; 422801) to detect the nuclei and AlexaFluor\u0026reg;488 (AAT Bioquest; 23115) to detect the cytoplasm, washed 3 times with PBS, and observed under a confocal laser scanning microscope (CLSM).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Real-Time Cell Analysis (RTCA)\u003c/h2\u003e\u003cp\u003eThe effect of NK-EVs on tumour cell proliferation was dynamically monitored via RTCA (Agilent; China). DMEM was added to the E-Plate16 for background impedance measurement. Tumour cells in the logarithmic phase of growth (7000 cells/well) were seeded in the E-Plate, maintained at room temperature for 15 minutes, and subsequently placed onto an electrified detection platform for real-time dynamic cell proliferation detection. When the cell index reached a value of approximately 1, the corresponding concentrations of NK-EVs, NK cells, or T cells were added to each well, and tumour cell proliferation curves were generated to calculate the inhibition rates at different time points.\u003c/p\u003e\u003cp\u003eThe following equation was used to calculate the inhibition rate: inhibition rate (%) = [(control cell index - experimental cell index)/control cell index] \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 CCK-8 Assay\u003c/h2\u003e\u003cp\u003eTo evaluate the effect of NK-EVs on tumour cell viability, an experiment was performed according to the instructions of the CCK-8 assay kit (CA1210; Solarbio). The cells (10,000 cells/well) were seeded in 96-well plates, cultured for 24 hours, and treated with various factors (such as NK-EVs, NK cells, or T cells) for an additional 48 hours, followed by the addition of the CCK-8 reagent. After 4 hours, the absorbance at 450 nm was measured with a microplate reader, and the cell viability (%) was calculated as follows: cell viability = [(experimental well - blank well)/(negative control well - blank well)] \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 4D-FastDIA Quantitative Proteomics\u003c/h2\u003e\u003cp\u003eTo analyse the signalling pathways that are regulated by NK-EVs in breast cancer cells, breast cancer cells were randomly divided into control and NK-EV groups (n\u0026thinsp;\u0026ge;\u0026thinsp;3) and cultured in 75-cm\u0026sup2; TC-treated flasks (5\u0026times;10⁶ cells/flask). After 24 hours, NK-EVs (25 \u0026micro;g/mL) were added to the NK-EV group, and an equal volume of PBS was added to the control group. After 48 hours, the cell pellets were collected to extract total proteins, which were separated and enriched by liquid chromatography, enzymatically digested into peptides, and identified by mass spectrometry for protein quantification. Bioinformatics analysis was performed based on the quantitative results to identify the key proteins that are involved in regulating breast cancer cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Animal Experiments\u003c/h2\u003e\u003cp\u003eFor in vivo experiments, female BALB/c nude mice (6\u0026ndash;8-weeks-old) were purchased from Vital River Laboratories (Beijing, China) and housed in the Experimental Animal Center of Xinjiang Medical University under specific pathogen-free conditions. All of the procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Xinjiang Medical University and were conducted in accordance with guidelines and policies (ethics approval number: \"IACUC-JT-20230420-46\"). The animal studies were conducted following ARRIVE guidelines 2.0 (Animal Research: Reporting of In vivo Experiments). All methods were carried out in accordance with relevant guidelines and regulations. For anesthesia, we used isoflurane (5% induction, 2% maintenance); for euthanasia, we adopted intraperitoneal injection of sodium pentobarbital (150 mg/kg).\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.10.1 Nude Mouse Xenograft Subcutaneous Tumour Model\u003c/h2\u003e\u003cp\u003eMDA-MB-231 cells (1\u0026times;10⁷) were resuspended in 500 \u0026micro;L of PBS and subcutaneously injected into the right axilla of female BALB/c nude mice (body weight\u0026thinsp;\u0026asymp;\u0026thinsp;15 g). The length and width of the tumours were measured every 3 days, and the subcutaneous tumours were observed to reach a size of approximately 1 cm\u003csup\u003e3\u003c/sup\u003e after ~\u0026thinsp;14 days. In accordance with our animal welfare protocol, mice were euthanized if the tumour volume exceeded 1500 mm\u0026sup3; or if signs of distress were observed. The maximum tumour volume allowed in this study was 1500 mm\u0026sup3;. The ECA cell model was established following similar protocols.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.10.2 NK-EV Homing Assay in Tumour-Bearing Nude Mice\u003c/h2\u003e\u003cp\u003eNK-EVs were stained with Dil (V22887; Thermo Fisher) according to the kit instructions. Tumour-bearing nude mice were intravenously injected with Dil-labelled NK-EVs (500 \u0026micro;g resuspended in 100 \u0026micro;L of PBS). The distribution of fluorescent NK-EVs in the nude mice was detected with an in vivo imaging system (IVIS Spectrum; PerkinElmer) at 4 hours, 8 hours, and 24 hours. The mice were sacrificed at 24 hours, and tumour tissues and major organs (including the heart, liver, spleen, lung, and kidney tissues) were collected to detect the fluorescence intensity of NK-EVs in ex vivo tissues.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.10.3 Assay of NK-EV-mediated Protein Regulation in Animal Tumour Tissues\u003c/h2\u003e\u003cp\u003eTumour-bearing nude mice were randomly divided into 2 groups (n\u0026thinsp;=\u0026thinsp;6). The experimental group was intravenously injected with NK-EVs (resuspended in 100 \u0026micro;L of PBS), and the control group was injected with an equal volume of PBS. The mice were sacrificed 24 hours later, and tumour tissues were collected. The expression levels of the PSMB9, PSMB10, TAP1, TAP2, TAPBP, B2M, and KRT9 proteins in the tumour tissues were detected by Western blotting and IHC, whereas the MHC-I and STAT1 protein levels were detected by Western blotting.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.10.4 Assessment of the Therapeutic Efficacy and Safety of NK-EVs Combined with NK Cells in Tumour-Bearing Nude Mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTumour-bearing nude mice were randomly divided into control, NK, and NK-EV\u0026thinsp;+\u0026thinsp;NK groups (n\u0026thinsp;=\u0026thinsp;6) and were subjected to a 28-day intravenous injection treatment regimen. Specifically, on days 1, 7, 13, 19, and 25 of treatment, the mice in the NK and NK-EV\u0026thinsp;+\u0026thinsp;NK groups were intravenously injected with NK cells (1\u0026times;10⁶ cells/mouse), and those in the control group were injected with an equal volume of PBS. On days 4, 10, 16, 22, and 28 of treatment, the mice in the NK-EV\u0026thinsp;+\u0026thinsp;NK group were intravenously injected with NK-EVs (100 \u0026micro;g/mouse), and those in the other two groups were injected with an equal volume of PBS. The mice were sacrificed on day 28. Body weights and tumour volumes were recorded during the experiment. Moreover, heart, liver, spleen, lung, kidney, and tumour tissues were collected, weighed, and photographed.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Immunohistochemistry (IHC)\u003c/h2\u003e\u003cp\u003eParaffin-embedded sections were subjected to IHC. Specifically, the sections were deparaffinized, and antigen retrieval was performed by heating in 0.01 M citrate buffer (pH\u0026thinsp;=\u0026thinsp;6.0); subsequently, endogenous peroxidase activity was blocked with 3% hydrogen peroxide, after which the sections were blocked with serum, incubated with primary and secondary antibodies in sequence, developed with DAB chromogen (G1212-200T; Servicebio), counterstained with haematoxylin, and mounted. Stained tissue sections were observed under an optical microscope (Fi3; Nikon), and quantitative analysis was performed with ImageJ software to compare the mean densities between the groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.12 H\u0026amp;E Staining\u003c/h2\u003e\u003cp\u003eCollected organs and tumour tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned with a microtome (RM2016; Leica, Germany). After deparaffinization and rehydration, the sections were stained with haematoxylin (H9627; Sigma) to detect nuclei and eosin (71014544; Sinopharm) to detect cytoplasm, after which they were dehydrated, mounted, and observed under a light microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Ethics Approval for Human Subjects Research\u003c/h2\u003e\u003cp\u003e Human Subjects: The study involving human blood samples from healthy donors was approved by the Ethics Review Committee of the First Affiliated Hospital of Xinjiang Medical University. All procedures strictly adhered to the principles of the Declaration of Helsinki (World Medical Association, 2024 revision). Written informed consent was obtained from all participants prior to the study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Ethics Approval for Animal Experiments\u003c/h2\u003e\u003cp\u003e Animal Experiments: All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Xinjiang Medical University (approval number: IACUC-JT-20230420-46) and were conducted in accordance with institutional guidelines and the ARRIVE guidelines 2.0. For anesthesia, isoflurane was used (5% induction, 2% maintenance); for euthanasia, intraperitoneal injection of sodium pentobarbital (150 mg/kg) was adopted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Statistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed with SPSS 26.0 software. Independent sample t tests were used to analyse differences between two groups, and one-way ANOVA was used to analyse differences among three or more groups. The results are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations. Significance levels are denoted as *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ns (not significant). The graphs were generated with GraphPad Prism 8.0 software.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Identification of NK-EVs and Their Antitumour Effects\u003c/h2\u003e\u003cp\u003eNK-EVs were collected by ultracentrifugation \u003cb\u003e(Fig.\u0026nbsp;1A)\u003c/b\u003e. The NTA results revealed that the NK-EVs were physically homogeneous particles with a diameter of approximately 112.1\u0026thinsp;\u0026plusmn;\u0026thinsp;48.3 nm and a concentration of ~\u0026thinsp;3.5\u0026times;10\u0026sup1;\u0026sup2; particles/mL \u003cb\u003e(Fig.\u0026nbsp;1B)\u003c/b\u003e. TEM imaging revealed that the NK-EVs exhibited uniform spherical structures with distinct membrane structures, and they were approximately 110 nm in diameter \u003cb\u003e(Fig.\u0026nbsp;1C)\u003c/b\u003e. Western blotting analysis revealed that the NK-EVs expressed the exosome markers CD63, Alix, and TSG101, as well as the cytotoxic protein markers perforin and granzyme B. Notably, the NK-EVs did not express calnexin, thus confirming the absence of organelle membrane components \u003cb\u003e(Fig.\u0026nbsp;1D)\u003c/b\u003e. Confocal fluorescence microscopy of Dil-labelled NK-EVs revealed their localization in the cytoplasm and around the nuclei of MCF-7 and MDA-MB-231 cells \u003cb\u003e(Fig.\u0026nbsp;1E)\u003c/b\u003e, thereby demonstrating that breast cancer cells can internalize NK-EVs. In addition, endocytosis was also observed in other tumor types (HCT116, ECA109, U87 and HepG2) \u003cb\u003e(SFig. 1A)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe RTCA results revealed that after the addition of NK-EVs at different concentrations, the proliferation of both MCF-7 and MDA-MB-231 cells were inhibited in a dose-dependent manner \u003cb\u003e(Fig.\u0026nbsp;1F, G)\u003c/b\u003e. The inhibition rates of the MCF-7 and MDA-MB-231 cells reached 96.50% and 94.68%, respectively, at 48 hours after NK-EV intervention \u003cb\u003e(Fig.\u0026nbsp;1H-I)\u003c/b\u003e. A CCK-8 assay was similarly performed to determine the effects of different concentrations of NK-EVs on breast cancer cell viability at 48 hours, and the results revealed dose-dependent inhibition of cancer cell viability, thus further verifying that NK-EVs can inhibit tumour cell proliferation and viability \u003cb\u003e(Fig.\u0026nbsp;1J-K)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Quantitative Proteomics Analysis of Breast Cancer Cells (MCF-7) after NK-EV Intervention\u003c/h2\u003e\u003cp\u003eQuantitative proteomics analysis was performed on MCF-7 cells after treatment with NK-EVs or the PBS control, and 38 differentially expressed proteins (including 30 upregulated and 8 downregulated proteins) were identified \u003cb\u003e(Fig.\u0026nbsp;2A-B)\u003c/b\u003e. GO and KEGG\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e enrichment analyses of the differentially expressed proteins \u003cb\u003e(Fig.\u0026nbsp;2C-D)\u003c/b\u003e revealed significant enrichment of interacting proteins in the MHC-I and immune-related pathways. A protein-protein interaction (PPI) network was constructed using the differentially expressed proteins \u003cb\u003e(Fig.\u0026nbsp;2E)\u003c/b\u003e, which revealed that NK-EV intervention significantly upregulated the expression of PSMB9, PSMB10, TAP1, TAP2, TAPBP, B2M, and other proteins that are closely related to intracellular antigen peptide transport and MHC-I expression. Similar were obtained in MDA-MB-231 cells via the same method \u003cb\u003e(SFig. 2)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Protein Expression in Breast Cancer Cells after NK-EV Intervention\u003c/h2\u003e\u003cp\u003eAfter coculturing MCF-7 and MDA-MB-231 cells with NK-EVs for 24 hours and removing the supernatants, the cells were subjected to Western blotting. The results revealed increased expression of the PSMB9, PSMB10, TAP2, TAPBP, B2M, MHC-I, and STAT1 proteins in MCF-7 cells \u003cb\u003e(Fig.\u0026nbsp;3A-B)\u003c/b\u003e. Similarly, PSMB9, PSMB10, TAP2, and TAPBP protein expression was upregulated in MDA-MB-231 cells \u003cb\u003e(Fig.\u0026nbsp;3C-D)\u003c/b\u003e, which was consistent with the results of 4D-FastDIA quantitative proteomics.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.4 NK-EV Intervention Increases Immune Cell-Mediated Tumour Growth Inhibition\u003c/h2\u003e\u003cp\u003eNK-EVs can not only directly induce tumour apoptosis but also promote the function of cytotoxic T lymphocytes through multiple pathways, including the reprogramming of tumour-associated macrophages and upregulation of MHC-I on tumour cells. We first isolated and cultured NK cells and CD8\u0026thinsp;+\u0026thinsp;T cells from human peripheral blood. Breast cancer cells were seeded in RTCA plates, and the following experimental groups were established: A) the ① PBS, ② NK-EV, ③ CD8\u0026thinsp;+\u0026thinsp;T cell, and ④ NK-EV\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cell groups; and B) the ① PBS, ② NK-EV, ③ NK cell, and ④ NK-EV\u0026thinsp;+\u0026thinsp;NK cell groups. Dynamic curves of cell proliferation were subsequently generated. The results revealed that in both groups, the rates of tumour cell proliferation inhibition in the ④ NK-EV\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cell and ④ NK-EV\u0026thinsp;+\u0026thinsp;NK cell groups were more significant \u003cb\u003e(Fig.\u0026nbsp;4A-H)\u003c/b\u003e. Moreover, compared with the tumour cell inhibition rate following NK cell monotherapy, the rate of MDA-MB-231 cell inhibition following NK-EV treatment and NK cell treatment reached 87.93%, which demonstrated a significant difference. Similarly, the CCK-8 results revealed that NK-EVs reduced the viability of MCF-7 and MDA-MB-231 cells after CD8\u0026thinsp;+\u0026thinsp;T cell or NK cell intervention \u003cb\u003e(Fig.\u0026nbsp;4I-L)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.5 NK-EVs Exhibit Tumour-Homing Properties in Tumour-Bearing Mice\u003c/h2\u003e\u003cp\u003eExogenous NK-EVs are reported to exhibit a strong ability to target particular tissues in vivo. An IVIS imaging system was used to monitor intravenously injected Dil-fluorescent dye-labelled NK-EVs in tumour-free mice and in MDA-MB-231 breast cancer tumour-bearing mice \u003cb\u003e(Fig.\u0026nbsp;5A).\u003c/b\u003e IVIS imaging revealed that NK-EVs demonstrated no major aggregation area in control mice, whereas obvious aggregation at the tumour site was observed in breast cancer tumour-bearing mice \u003cb\u003e(Fig.\u0026nbsp;5B)\u003c/b\u003e. The mice were sacrificed 24 hours after NK-EV injection, and the tumour tissues and major organs were collected and subjected to ex vivo fluorescence imaging. The results revealed that NK-EVs mainly accumulated in the livers of control mice, whereas in breast cancer and oesophageal cancer tumour-bearing mice, NK-EVs accumulated in both the tumour and liver, with stronger fluorescence signals being detected in the tumours than in the liver \u003cb\u003e(Fig.\u0026nbsp;5C).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Effects of NK-EVs Combined with NK Cells in Model Mice\u003c/h2\u003e\u003cp\u003eDue to the high immunogenicity of T cells, severe immune reactions may occur during allogeneic infusion. In contrast, NK cells have low immunogenicity, and thus, the use of NK cells can avoid inhibition mediated by inhibitory receptors. Therefore, NK cells were selected for combination therapy with NK-EVs in the in vivo experiments. Previous studies have evaluated the therapeutic effect of NK-EVs in tumours and reported that NK-EVs at concentrations of 200\u0026ndash;500 \u0026micro;g exert good antitumour effects in tumour-bearing mice, with some antitumour effects beginning to be observed following the administration of 100 \u0026micro;g of NK-EVs. The treatment protocol is shown in \u003cb\u003e(Fig.\u0026nbsp;5D)\u003c/b\u003e. Tumour growth curves revealed that, compared with NK cell monotherapy, the combined intervention of NK-EVs and NK cells significantly inhibited tumour growth \u003cb\u003e(Fig.\u0026nbsp;5F-I)\u003c/b\u003e. The ex vivo tumour volumes and weights in the NK-EV combined with NK cell treatment group were significantly different from those in the other two groups \u003cb\u003e(Fig.\u0026nbsp;5E, 5J)\u003c/b\u003e. Additionally, H\u0026amp;E staining revealed that, compared with the PBS control and NK cell-treated groups, the NK-EV combined with NK cell treatment group exhibited increased tumour apoptosis in mice \u003cb\u003e(Fig.\u0026nbsp;5L)\u003c/b\u003e. These data demonstrate that the combination of NK-EVs and NK cells significantly inhibits breast cancer tumour growth, with a more potent antitumour effect being observed compared to NK cell monotherapy alone. During the entire treatment process, there was no significant difference observed in body weights among the groups \u003cb\u003e(Fig.\u0026nbsp;5K)\u003c/b\u003e. In all of the treatment groups, no obvious tumour metastasis or liver injury was observed in the liver tissues ex vivo \u003cb\u003e(Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA)\u003c/b\u003e; additionally, the organ weights remained within normal ranges \u003cb\u003e(Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB-F)\u003c/b\u003e, and H\u0026amp;E staining of tissue sections revealed no obvious pathological damage to the liver or other organs among the groups \u003cb\u003e(Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG)\u003c/b\u003e. In summary, the combination of NK-EVs with NK cell therapy is safe.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.7 NK-EVs Participate in Regulating the MHC-I Pathway in Tumour-Bearing Nude Mice\u003c/h2\u003e\u003cp\u003eNude mouse models of MDA-MB-231 cell-derived tumours were intravenously injected with either PBS control or NK-EVs (500 \u0026micro;g) via the tail vein. The mice were sacrificed 24 hours later, and the tumour tissues were collected. The Western blotting and IHC results revealed increased protein expression of PSMB9, PSMB10, TAP2, TAPBP, and B2M, in the tumour tissues of the NK-EV intervention group \u003cb\u003e(Fig.\u0026nbsp;6A-D)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Expression of MHC-I Pathway-Related Proteins in Breast Cancer Patients\u003c/h2\u003e\u003cp\u003eTumour pathological sections from patients with different stages of breast cancer were collected, and IHC staining revealed increased protein expression of PSMB9, PSMB10, TAP1, TAP2, TAPBP, and B2M in stage II breast cancer tumours from patients \u003cb\u003e(Fig.\u0026nbsp;6D)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we obtained high-quality NK-EVs and demonstrated that NK-EVs can be internalized by breast cancer cells. Additionally, the uptake of NK-EVs was similarly observed in glioma cells (U87), oesophageal cancer cells (ECA109), colon cancer cells (HCT116), and liver cancer cells (HepG2), which is consistent with previous findings on NK-EVs in cancer research\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These results suggest that our findings may be applicable to multiple solid tumours. In the animal experiments, after the intravenous injection of Dil-NK-EVs into mice with breast cancer, aggregation of fluorescence signals at the tumour site were observed at 4 hours, and the fluorescence signals did not weaken or disappear at 24 hours. These results verify the good tumour-targeting homing ability of NK-EVs, thereby indicating that NK-EVs exhibit great advantages as antitumour drugs or drug delivery systems for future treatments\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe observed that NK-EVs carry perforin and granzyme B. Additionally, quantitative proteomics sequencing of breast cancer cells after NK-EV treatment revealed significant enrichment of multiple related pathways in the GO analysis, including protein binding, protein activity, enzyme receptor activity, and signal receptor activity (including TRAIL and transmembrane transporter activity)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Natural killer cell-mediated cytotoxicity was also significantly enriched in the GO and KEGG analyses, thereby suggesting that the pathway by which NK-EVs kill breast cancer cells in this study may be similar to that described in previous studies\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Furthermore, cell killing experiments revealed both dose-dependent and time-dependent killing effects of NK-EVs on breast cancer cells. At a concentration of 50 \u0026micro;g/100 \u0026micro;L, tumour cell proliferation was inhibited, and obvious cell death was observed at an effector-target ratio of 200 \u0026micro;g/100 \u0026micro;L cell.\u003c/p\u003e\u003cp\u003eAfter breast cancer cells were treated with NK-EVs at an effector-target ratio lower than the tumour-killing threshold for 24 hours, tumour cells were more susceptible to killing by CD8\u003csup\u003e+\u003c/sup\u003eT cells. The protein sequencing results revealed that the upregulated proteins were closely related to antigen peptide presentation by tumour cells and MHC-I expression. MHC-I plays a crucial role in cell-mediated immune responses. Reduced or absent MHC-I expression leads to tumour immune escape, which is primarily due to the lack of tumour antigen presentation for recruiting and activating CD8\u003csup\u003e+\u003c/sup\u003eT cells\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The downregulation of MHC-I molecules has been observed in various tumours. PSMB9 and PSMB10 perform proteolysis in a trypsin-like manner, whereby they cleave endogenous peptides to generate peptides with appropriate anchor residues, which are then transported to the endoplasmic reticulum lumen by the transporters TAP1 and TAP2. TAPBP acts as a bridge for TAP and MHC-I molecules, thereby mediating peptide loading onto the MHC-I structure to form the MHC-I complex\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The MHC-I complex consists of three components, including the MHC-I heavy chain, B2M, and amino acid peptides derived from endogenous proteins (which are transported to the cell membrane surface by the Golgi apparatus). In this study, our unintended manipulation may have resulted in significant effects due to the exposures of hidden endogenous peptides of breast cancer cells, thus potentially inducing the aggregation and killing functions of CD8\u003csup\u003e+\u003c/sup\u003eT cells.\u003c/p\u003e\u003cp\u003eTypically, the antigen presentation machinery (APM) can be upregulated by cytokines, such as IFN-γ, that are secreted by activated lymphocytes and NK cells\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. IFN-γ receptor signalling via the JAK-STAT pathway can induce the expression several APM components, including MHC-I heavy chains and B2M in cis or trans. In this study, both the protein sequencing and Western blotting results revealed increased STAT1 expression. Therefore, after NK-EVs are internalized by tumour cells, it is unknown which component/process leads to STAT1 phosphorylation, and this question requires further detection of NK-EV components and additional experiments for clarification\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFinally, we observed that NK cells exhibited an increased ability to kill breast cancer cells that were pretreated with NK-EVs. This effect may be explained by the study of Ardolino M. et al., who suggested that reduced or absent MHC-I expression prevents NK cells from achieving \"permission,\" thereby leading to their inability to express inhibitory receptors such as KIRs, which are necessary for effective tumour cell recognition and killing\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. NK cells infiltrating MHC-I-deficient tumour environments exhibit a hyporesponsive state, whereas those infiltrating MHC-I-sufficient tumour environments exhibit a hyperresponsive state\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur results show that NK-EVs increase CD8\u0026thinsp;+\u0026thinsp;T cell recognition and killing and promote NK cell cytotoxicity by upregulating MHC-I expression in breast cancer cells (Fig.\u0026nbsp;7). In the animal model, there were no significant effects observed on mouse body weights or organ tissue. Based on the results of staining of clinical samples, high expression of MHC-I pathway-related proteins may indicate a better prognosis for patients with breast cancer, and these proteins may serve as indicators for evaluating breast cancer prognosis.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, our findings demonstrate that NK-EVs are innovative nanotherapeutic candidates that can be internalized by tumour cells; moreover, they exhibit tumour-homing properties and are safe. To our knowledge, this study is the first to report that NK-EVs can increase tumour antigen presentation and that the combination of NK-EVs with other adoptive immunotherapies or ICIs may be a new strategy for treating breast cancer. In future research, further investigations of NK-EV components, mechanisms of action, and TME changes are crucial.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAPM \u0026nbsp; Antigen Presentation Machinery\u003c/p\u003e\n\u003cp\u003eB2M \u0026nbsp;\u0026nbsp;\u0026beta;2-Microglobulin\u003c/p\u003e\n\u003cp\u003eCLSM \u0026nbsp; \u0026nbsp;Confocal Laser Scanning Microscope\u003c/p\u003e\n\u003cp\u003eEVs\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; Extracellular Vesicles\u003c/p\u003e\n\u003cp\u003eICIs \u0026nbsp; \u0026nbsp;Immune Checkpoint Inhibitors\u003c/p\u003e\n\u003cp\u003eIFN-\u0026gamma;\u0026nbsp; \u0026nbsp;Interferon-\u0026gamma;\u003c/p\u003e\n\u003cp\u003eIVIS \u0026nbsp; \u0026nbsp;In Vivo Imaging System\u003c/p\u003e\n\u003cp\u003eMHC \u0026nbsp; Major Histocompatibility Complex\u003c/p\u003e\n\u003cp\u003eNTA \u0026nbsp; \u0026nbsp;Nanoparticle Tracking Analysis\u003c/p\u003e\n\u003cp\u003eNK \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp;Natural Killer\u003c/p\u003e\n\u003cp\u003ePD-L1 \u0026nbsp; Programmed Cell Death Ligand 1\u003c/p\u003e\n\u003cp\u003ePSMB9/PSMB10 \u0026nbsp; \u0026nbsp;Proteasome Subunit Beta Type 9/10\u003c/p\u003e\n\u003cp\u003eRTCA\u0026nbsp; \u0026nbsp; Real-Time Cell Analysis\u003c/p\u003e\n\u003cp\u003eTAP1/TAP2 \u0026nbsp; Transporter Associated with Antigen Processing 1/2\u003c/p\u003e\n\u003cp\u003eTAPBP\u0026nbsp;\u0026nbsp; Transporter Associated with Antigen Processing Binding Protein\u003c/p\u003e\n\u003cp\u003eTEM\u0026nbsp; \u0026nbsp;\u0026nbsp;Transmission Electron Microscopy\u003c/p\u003e\n\u003cp\u003eTME \u0026nbsp; \u0026nbsp;Tumour Microenvironment\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors contributed to the study\u0026rsquo;s conception and design. Limin Zhang and Yidan Xu planned the experiment, composed the manuscript and managed experimental data. Jing Guo, Hu Li and carried out cytological and molecular biological experiments, analysed data and made charts. Yidan Xu and Jing Guo were responsible for animals breeding performed animal researches and executed data analysis. Xin Lv, Libin Liao, Yajie Zhang, Jiyang Li, Xinxin Gu, Hu Li assisted data analysis with constructive recommendation. Qi Pan and Shengbin Bai gave suggestions on writing and revising the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Open Project Fund of the Key Laboratory of Tissue and Cell Engineering of Xinjiang Uygur Autonomous Region, China. (Project name: the Role and Mechanism of Exosomes Derived from Natural Killer Cells in Enhancing Tumour Immune Response by Upregulating the MHC Class Ⅰ Pathway in Triple-negative Breast Cancer, project number: XJXS002).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were approved by the Animal Ethics Committee of Medical Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University. (Permit Number: IACUC-JT-20230420-46). The animal studies were conducted according to the ARRIVE guidelines 2.0 (Animal Research: Reporting of In Vivo Experiments). All methods were carried out in accordance with relevant guidelines and regulations. The authors declare that they have not use AI-generated work in this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKim J, Harper A, McCormack V, et al. Global patterns and trends in breast cancer incidence and mortality across 185 countries. \u003cem\u003eNature medicine\u003c/em\u003e 2025;31(4):1154-62. doi: 10.1038/s41591-025-03502-3 [published Online First: 2025/02/25]\u003c/li\u003e\n\u003cli\u003eXiong X, Zheng LW, Ding Y, et al. 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Tumor-Infiltrating Natural Killer Cells. \u003cem\u003eCancer discovery\u003c/em\u003e 2021;11(1):34-44. doi: 10.1158/2159-8290.Cd-20-0655 [published Online First: 2020/12/06]\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, NK cell-derived extracellular vesicles, MHC class I, Antigen presentation, CD8+T cell","lastPublishedDoi":"10.21203/rs.3.rs-7555077/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7555077/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough immune checkpoint inhibitors (ICIs) and adoptive T cell therapy have shown success in cancer treatment, these therapies often have limited efficacy in breast cancer patients because these tumours use various mechanisms to evade immune surveillance and elimination; however, the underlying mechanisms remaining unclear. This study revealed that natural killer (NK) cell-derived extracellular vesicles (NK-EVs) can be internalized by breast cancer cells in vitro and exhibit tumour-homing properties in tumour-bearing mice. Proteomic sequencing revealed that treatment with NK-EVs significantly upregulated the expression of major histocompatibility complex (MHC) class I-related proteins, including antigen processing-related transporters (TAP1 and TAP2), transporter associated with antigen processing binding protein (TAPBP, tapasin), immunoproteasome subunits (PSMB9 and PSMB10), and β2-microglobulin (B2M), in breast cancer cells. These proteins are involved in MHC-I synthesis and endogenous peptide processing/presentation by tumour cells. Moreover, MHC-I expression is essential for antigen presentation and T cell-mediated immune responses. NK-EVs upregulated MHC-I expression on the surfaces of breast cancer cells, thus facilitating the recognition and killing of breast cancer cells by CD8\u003csup\u003e+\u003c/sup\u003eT cells. Additionally, an environment with increased MHC-I levels promoted NK cell maturation and enhanced their cytotoxic functions. Finally, in tumour-bearing mice, NK-EVs combined with NK cell therapy inhibited tumour progression and exhibited good safety. These results suggest that similar strategies may be novel therapeutic approaches for treating patients in the clinic. In the future, the combination of NK-EVs with ICIs or adoptive cell therapy may overcome the limitations of currently available breast cancer immunotherapies by performing two functions, namely, directly killing tumour cells and sensitizing immune cells.\u003c/p\u003e","manuscriptTitle":"NK Cell-Derived Extracellular Vesicles Function as Immunomodulators to Regulate Breast Cancer Progression by Activating MHC-I Signalling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-21 23:25:05","doi":"10.21203/rs.3.rs-7555077/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-20T06:23:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-19T18:09:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T12:37:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187056064684277197128625489614737565307","date":"2025-10-10T15:14:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260996028980063550351961418417029564249","date":"2025-10-09T08:53:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-08T14:55:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-03T08:52:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-22T09:05:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-17T09:28:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-17T09:24:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cb8a24dd-f4de-4fa6-bb3e-4e753bed8cdb","owner":[],"postedDate":"October 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":56558402,"name":"Biological sciences/Cancer"},{"id":56558403,"name":"Biological sciences/Immunology"},{"id":56558404,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-11-20T06:38:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-21 23:25:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7555077","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7555077","identity":"rs-7555077","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}