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. 2025 Dec 12;23:765. doi: 10.1186/s12951-025-03842-9

Targeting pre-metastatic niche with inhalable dendritic cell vesicles to prevent breast cancer lung metastasis

Qingqing Leng 1,4, Zhengxin Yu 1,4, Yu Zhao 1,4, Yue Shao 1,4, Qiuyang Jin 1,4, Qiyu Zhao 1,4, Ying Yang 3,4, Mengmeng Ji 3,5, Hua Yang 5, Lili Ren 5, Guang Jia 3,4, Xueyi Wang 2, Jinchao Zhang 3,4,✉, Zhenhua Li 2,✉, Huifang Liu 1,4,✉
PMCID: PMC12699861  PMID: 41388289

Abstract

Distant metastasis remains the primary cause of mortality in breast cancer, yet therapeutic options to prevent or treat metastatic progression are still limited. Emerging evidence suggests that the formation of the pre-metastatic niche (PMN) serves as a pivotal step in the process of breast cancer metastasis. Lung tissue is the major site of breast cancer metastasis with elevated prostaglandin E2 (PGE2) levels, which fosters immunosuppression and promotes niche establishment. Although EP2 and EP4 receptor antagonists have shown promise in counteracting PGE2-driven immunosuppression, their clinical translation is hindered by poor selectivity and bioavailability. To address these limitations, we developed a nanotherapeutic platform using dendritic cell–derived nanovesicles (NVs) engineered with α-lactalbumin (α-LA) and loaded with the EP2 antagonist TG6-10-1 and the EP4 antagonist GW627368, termed L-TG/GW-NVs. L-TG/GW-NVs exploit the homing ability of DC-derived NVs and retain immune-stimulatory molecules, thereby preventing PMN formation by blocking PGE2 signaling and reactivating suppressed dendritic cells and cytotoxic T cells. This synergistic strategy markedly suppressed lung metastasis by disrupting niche formation, enhancing immune activation, and reversing T cell exhaustion. Collectively, our findings establish a novel framework for metastatic breast cancer therapy and provide valuable insights for future translational studies and combinational immunotherapies.

Graphical Abstract

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Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-025-03842-9.

Keywords: Dendritic extracellular vesicle, Pre-metastatic niche, Prostaglandin E2 antagonists, Lung metastasis, Dendritic cell dysfunction

Introduction

In recent years, advancements in immunotherapy, gene therapy, and nanotechnology have brought hope for long-term survival and even potential cures for breast cancer patients [1–3]. However, distant metastasis remains a major challenge. It is a multifaceted process involving remodeling of the tumor microenvironment, colonization by circulating tumor cells, and the induction of angiogenesis [4]. Recent studies further emphasize that the establishment of a pre-metastatic niche (PMN) within target organs represents a critical step in this progression. PMN forms through signals from primary tumors, including exosomes, cytokines (e.g., TGF-β, VEGF), and metabolites (e.g., lactate), which remodel distal immune environments. In the mature PMN, immune cells such as dendritic cells (DCs), T cells, and NK (Natural Killer) cells are suppressed, thereby promoting metastasis [5–7]. Thus, targeting PMN formation represents a promising strategy to inhibit cancer metastasis.

The lung is a primary site for breast cancer metastasis, with about 35.7% of patients developing lung metastases [8]. Elevated prostaglandin E2 (PGE2) levels in the lungs of these patients trigger chronic inflammation [9, 10] and disrupt immune cell function, promoting the formation of a premetastatic niche (PMN) [11–13]. PGE2 exerts its effects through receptors, notably EP2 and EP4, which significantly suppress immune responses [14]. Reducing PGE2’s impact could alleviate its immunosuppressive effects. For example, knocking out PGE2 receptor genes eliminates these effects, but ethical and safety issues render this approach impractical for clinical use. A more viable option involves using specific EP2/EP4 receptor antagonists, though their effectiveness in cancer metastasis is limited by poor drug selectivity and low bioavailability [15–17]. This underscores the critical need for innovative drug delivery systems.

Antigen-presenting cells (APCs), particularly DCs, are crucial for antitumor immunity. In PGE2-rich lung microenvironments, DCs dysfunction occur due to disrupted cAMP-PKA-CREM signaling, which reduces CXCL9 and IL-12 production [18, 19]. This impairs CD8+ cytotoxic T lymphocyte (CTL) activation and weakens immune surveillance of circulating tumor cells (CTCs) [20]. Artificial DC nanovesicles (DC-NVs) mimic natural DC outer vesicles and show strong homing affinity for DCs [21]. Additionally, DC-NVs share the same membrane surface traits as mature DCs, retaining numerous co-stimulatory molecules and MHC-antigen complexes, ensuring their effectiveness as tumor vaccines [22–24]. These characteristics make DC-NVs powerful carriers for immunotherapy.

To tackle these issues, we created lyophilized engineered DC nanovesicles (L-TG/GW-NVs) loaded with α-lactalbumin (α-LA), a triple-negative breast cancer antigen, combined with receptor antagonists TG6-10-1 (TG) and GW627368 (GW). Using a 4T1 breast cancer mouse model, we tested inhaled L-TG/GW-NVs for their ability to prevent lung metastasis. The results demonstrated that this drug delivery system reversed PMN formation in lung metastases by disrupting PGE2-induced immunosuppression through PGE2 antagonists and targeting DCs, thereby restoring their T-cell activation capacity. Additionally, inhalation delivery ensured significant drug accumulation in lung tissues (Fig. 5). In conclusion, L-TG/GW-NVs provide novel insights and critical data for preventing and treating breast cancer lung metastasis, offering substantial research significance (Fig. 1)

Fig. 5.

Fig. 5

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Therapeutic efficacy of L-TG/GW-NVs to suppress postoperative metastasis of TNBC. (A) Schematic representation of a model of lung metastasis from breast cancer. (B) Ex vivo fluorescence imaging. Scale bar, 5 mm. (C) Ex vivo imaging of lung tissue. (D) The representative fields of lung tissues with metastasis areas (blue arrows) from each group. Scale bar, 2 mm. (E) 4T1-GFP immunofluorescence analysis images of the macroscopic lung metastatic nodules. Scale bar, 50 μm. (F) Ki67 immunofluorescence analysis images of the macroscopic lung metastatic nodules. Scale bar, 50 μm

Fig. 1.

Fig. 1

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Schematic Diagram of the Anti-metastasis Effects of L-TG/GW-NVs. L-TG/GW-NVs were obtained through artificial extrusion and ultrasonic drug loading. Inhalation of L-TG/GW-NVs freeze-dried powder by mice effectively alleviated the suppressive effect of PGE2 on DCs in the lungs, activated T-cell-mediated tumor immunity, and prevented the formation of PMN in the lungs

Materials and methods

Cell lines and purification of LA-NVs

The DC2.4 cell line was purchased from iCell Bioscience Incorporated (Shanghai, China). DCs were cultured under an environment of 37 ℃ and 5% CO2 with RPMI 1640 medium. To prepare LA-NVs, DCs were treated with 0.1 mg/mL alpha-lactalbumin (α-LA) for 24 h, then replaced the medium with fresh RPMI 1640. After 24 h, DCs were harvested and washed twice using PBS buffer. The cell suspension was successively passed via extrusion through polycarbonate membrane filters of 10 μm, 5 μm, 1 μm, and 0.4 μm pore sizes (Nuclepore, Whatman Inc., USA), with a mini-extruder (Avanti Polar Lipids, Birmingham, USA) used for the process. The extruded sample (20 mL) was harvested and subjected to centrifugation at 4 ℃: first at 300×g for 10 min, then 3000×g for 10 min, and subsequently 10,000×g for 30 min. The resulting supernatant was concentrated using a 100 KDa ultrafiltration centrifuge tube via centrifugation at 4500×g for 5 min. This concentrated supernatant was centrifuged further via an ultracentrifuge: the conditions were 100,000×g, 4 ℃, and a duration of 70 min [25]. The supernatant was first discarded, after which NVs filtered utilizing a filter with a 0.45 μm pore size. The obtained suspension of LA-NVs was stored at −80 ℃.

Loading of TG and GW

TG (HY-16978, MedChemExpress, CN) and GW (HY-16963, MedChemExpress, CN) were loaded into LA-NVs via ultrasound, yielding L-TG/GW-NVs. The ultrasound parameters were set as follows: amplitude: 30%; 30 s of sonication followed by 30 s of pause, repeated for 6 cycles, totaling 6 min. In brief, the TG and GW both at a concentration of 100 µg/mL were mixed with 100 µg/mL LA-NVs in a 1:1 ratio and placed into an ultrasonic cell disruptor. Following sonication, the mixture underwent incubation at 37 ℃ for 1 h to permit the spontaneous repair of membranes. After that, the L-TG/GW-NVs were subjected to ultracentrifugation (4 ℃, 120000 g, 1 h) to remove the unencapsulated drugs. The precipitate of L-TG/GW-NVs was resuspended in PBS and stored in a −80 ℃ refrigerator for subsequent experiments.

L-TG/GW-NVs powder preparation and room-temperature stability test

The L-TG/GW-NVs were diluted in PBS with 25 mM trehalose (S11052, Shanghai yuanye Bio-Technology, CN) at a 1:8 ratio. Using trehalose as a lyoprotectant could ensure the morphological integrity of NVs and facilitate the formation of inhalable powder [26]. The lyophilized solution was equilibrated at 40 ℃ for 50 min, then pre-frozen at −80 ℃ for 14 h, and then freeze-dried at −60 ℃ and 15 Pa for 48 h to obtain the final product. In order to test the stability of freeze-dried nanoparticles under room-temperature condition, powder with L-TG/GW-NVs were kept in room-temperature conditions over periods of 0, 7, 14, 21, and 28 days. At different points in time, L-TG/GW-NV power was resuspended in PBS, and this suspension was used to determine particle size changes through dynamic light scattering (DLS, ZetaView PMX 110, Particle Metrix, GER).

Characterization of L-TG/GW-NVs

The JEM 1011 microscope (JEOL, Japan) was used to capture transmission electron microscope (TEM) images of NVs. The DLS was applied to monitor the diameter of exosomes. Nanoparticle tracking analysis (NTA, Malvern, UK) was employed to determine the particle concentration and diameter distribution of exosomes. Western Blot (WB) analysis was used to detect the surface marker proteins CD80, CD86 and MHC II on L-TG/GW-NVs.

The attachment of L-TG/GW-NVs to T cell and DCs

T cells or DCs, together with L-TG/GW-NVs, were labeled with the membrane dye DiO (34215-57-1, MeilunBio, CN) and DiD (127274-91-3, MeilunBio, CN). T cells or DCs were co-incubated with L-TG/GW-NVs for 2 h. Laser confocal scanning microscopy (LCSM) was used to analyze the uptake of L-TG/GW-NVs by T cells or DCs.

The activation of T cell in vitro

Lymphocytes isolated from mouse spleens were incubated with either PBS (as a control) or LA-NVs for 24 h in a cell culture incubator. T cells were analyzed by flow cytometry (FCM).

The inhibitory effect of LA-TG/GW-NVs on DC dysfunction in vitro

Co-incubate DCs with PGE2 (900117P, Sigma-Aldrich, CN) at a concentration of 200 ng/mL for 48 h. After replacing the culture medium, DCs incubated with PBS and L-TG/GW-NVs for 24 h. Subsequently, ELISA kits and CCK8 kits were used to perform the necessary assays.

Toxicity evaluation in vitro

The safety of materials at different concentrations was verified using the hemolytic test method. Mouse blood was collected via orbital blood sampling, and blood cells were harvested. Then, 1 mL of L-TG/GW-NVs at concentrations ranging from 10 to 300 µg/mL, 1 mL of pure water, and 1 mL of PBS were respectively mixed thoroughly with 20 µL of blood cells. The mixtures were centrifuged after incubating for 1 h, then photographs were taken to observe the hemolysis.

Lung distribution determination in vivo

L-TG/GW-NVs were labeled with DiD dye and prepared into lyophilized powder under light-proof conditions. After the mice were given inhalation dosing, their lungs were obtained at 0.5 h, 1 h, 2 h, and 3 h to conduct ex vivo fluorescence imaging analysis.

In vivo lung metastasis model

Female BALB/c mice at 4–6 weeks of age were acquired from SPF (Beijing) Biotechnology Co., Ltd. The mice were fed standard rat chow and free to water. 4T1-G cells (1.0 × 106 cells per mouse) were implanted into the third mammary fat pad of female BALB/c mice. Fourteen days later, 4T1-G cells (2.0 × 105 cells per mouse) were intravenously injected into the mice [27].

Efficacy evaluations

The BALB/c mice were randomly allocated into a total of five groups: PBS, TG/GW, NVs, LA-NVs, and L-TG/GW-NVs. For each inhalation administration to mice, 0.35 mg of lyophilized powder was used, with an inhalation cycle of 30 s per round, repeated 6 times. Treatment was initiated on the second day after the establishment of the mouse model of in situ breast cancer. Administer the medicine every two days for a total of five times. After the treatment, the model of lung metastasis of breast cancer was established. 14 days later, the mice were euthanized, with their lungs then collected for subsequent experimental use. Lung metastatic nodule counts in mice across different groups were documented, to assessed breast cancer lung metastasis conditions. Additionally, single-cell suspensions were prepared from a piece of lung tissue. After co-incubating with fluorescently labeled antibodies, FCM was employed to detect the expression of CD3, CD8, CD4, PD-1, and TIM3 proteins on T cell surfaces, thereby analyzing the activation and exhaustion of T cells. A segment of lung tissue was homogenized for assessing the levels of IL-2, IL-6, Gamz-B, TNF-α and IFN-γ using Elisa kit (Proteintech Group, CN). After this, a segment of lung tissue was immersed in 4% paraformaldehyde for fixation. Immunofluorescence was used to detect the infiltration of immune cells in the lungs; for the histological assessment of lung sections, hematoxylin-eosin (H&E) staining was chosen to observe the aggregation of lung nodules.

Toxicity evaluation in vivo

After the experiment, hearts, livers, spleens, and kidneys of mice from different experimental groups were collected, prepared into H&E sections, and their tissue morphology was observed. Moreover, alanine transaminase (ALT), Urea, alkaline phosphatase (ALP), creatinine (CREA) and uric acid (UA) levels of serum were detected.

Statistical analysis

In this study, mean ± standard deviation (SD) is used to express experimental data. To determine significant differences between groups, t-test or one-way analysis of variance (ANOVA) (coupled with multiple comparisons via Fisher’s pairwise comparison) was employed. Statistical analysis was performed with Origin 9.0 (OriginLab, Northampton, Massachusetts, USA) and GraphPad Prism 9.0 (GraphPad Software, San Diego, California, USA).

Results and discussion

Activation of DCs by α-LA

PGE2 primarily acts via cell surface receptors EP2 and EP4. TG and GW, competitive inhibitors of EP2 and EP4 respectively, effectively reduce PGE2’s cellular impact [28]. However, like other small-molecule drugs, their targeting specificity is limited, necessitating carrier systems to improve precision and minimize side effects. Extracellular membrane vesicles are commonly used as drug carriers due to their excellent biocompatibility [29]. Additionally, ultrasound can enhance the loading of small-molecule drugs into extracellular vesicles [30, 31]. Therefore, TG and GW were loaded into LA-NVs using an ultrasonic drug loading method to obtain L-TG/GW-NVs. TEM imaging revealed that NVs, LA-NVs, and L-TG/GW-NVs exhibited similar spherical morphologies with diameters of approximately 150 nm (Fig. 2A). DCs express surface marker proteins CD80, CD86, and MHC II, and prior studies indicate that DC-derived exosomes also carry these proteins [32]. Western blotting confirmed that NVs and LA-NVs contained CD80, CD86, and MHC II, verifying that the extruded NVs retained their immunostimulatory properties (Fig. 2B and C). Nanoparticle Tracking Analyzer (NTA) was used to determine particle sizes of the samples. At the same time, Dynamic Light Scattering (DLS) was also used. The results were approximately 157.4 ± 7.9 nm for NVs, 166.7 ± 4.3 nm for LA-NVs, and 166.5 ± 11.8 nm for L-TG/GW-NVs (Fig. 2D and F), indicating that incorporating TG6-10-1 and GW627368 into LA-NVs did not significantly alter nanovesicle size. Drug loading and encapsulation efficiencies were assessed after co-incubating LA-NVs with varying drug concentrations under ultrasonication, with an optimal drug concentration of 75 µg/mL determined (Fig. 2E and Fig. S1). Hemolysis tests confirmed the biosafety of L-TG/GW-NVs (Fig. S2). DLS analysis of L-TG/GW-NVs stability over different storage times showed a slight size increase, remaining around 170 nm (Fig. 2G).

Fig. 2.

Fig. 2

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Synthesis and characterization of L-TG/GW-NVs. (A) TEM image of NVs, LA-NVs and L-TG/GW-NVs stained with phosphotungstic acid. (B) Western blot analysis of NVs and LA-NVs. (C) Quantitative results of WB images. (D) The size distribution of NVs、LA-NVs and L-TG/GW-NVs by DLS. (E) The loading efficiency of TG/GW among different TG/GW concentrations. (F) The size distribution of NVs、LA-NVs and L-TG/GW-NVs by NTA. (G) Stability studies of L-TG/GW-NVs by DLS. (n = 3, ****p < 0.0001)

Activation of DCs by α-LA

α-Lactalbumin (α-LA) is a key immunodominant protein in triple-negative breast cancer (TNBC), expressed in approximately 70% of cases, making it a promising target for vaccine development [22]. After incubating α-LA with dendritic cells (DCs) for 24 h, tumor antigen-stimulated DCs were obtained (Fig. 3A). Laser confocal scanning microscopy (LCSM) revealed green fluorescence (FITC-α-LA) within DCs, confirming α-LA uptake (Fig. 3B). Flow cytometry (FCM) further validated this uptake (Fig. 3C). Previous studies indicate that DCs, upon taking up tumor antigens, become activated, enter a proliferative state, and increase cytokine secretion [33]. Scanning electron microscopy (SEM) showed morphological changes in α-LA-stimulated DCs, indicating maturation (Fig. 3D). Western blotting (WB) analysis revealed elevated expression of costimulatory molecules CD80 and MHC II on DCs post-α-LA stimulation, suggesting enhanced antigen-presenting capacity (Fig. 3E). The CCK-8 assay demonstrated increased DC proliferation after α-LA treatment (Fig. 3F). FCM analysis confirmed higher expression of CD86 and MHC II on DCs (Fig. 3G and Fig. S3). Additionally, ELISA assays showed significantly elevated levels of IL-6, TNF-α, and IL-12 secreted by DCs following α-LA uptake (Fig. 3H). These findings confirm that α-LA effectively promotes DC maturation and activation. WB analysis of cell vesicles showed that LA-NVs, compared to unstimulated NVs, had higher levels of CD80, CD86, and MHC II (Fig. 2C).

Fig. 3.

Fig. 3

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Activation of DC cells by α-LA. (A) Schematic diagram of DCs activation after uptake of α-LA. (B) LCSM image of DCs after co-incubation with FITC-α-LA. Scale bar, 10 μm. (C) DCs were subjected to FCM analysis after being co-incubated with FITC-α-LA. (D) Morphological examination. Scale bar, 2 μm. (E) Western blot analysis of DCs and LA-DCs. (F) The cell viability of DCs. (G) DCs were subjected to FCM analysis for the expression of CD86 and MHC II after being co-incubated with PBS or α-LA for 48 h. (H) ELISA detected the change in IL-6, TNF-α, and IL-12 by DCs. (n = 3, ***p < 0.001, ****p < 0.0001)

LA-NVs derived from α-LA-stimulated DCs retained costimulatory molecules on their membrane surface and demonstrated enhanced T cell activation capacity. As shown in Fig. S4, expression of T cell markers CD4 and CD8 significantly increased following LA-NVs stimulation. ELISA assays revealed that T cell activation factors IL-2, TNF-α, and Gzms-B were maximally upregulated in the LA-NVs group (Fig. S5). Thus, α-LA stimulation effectively promotes DC maturation and proliferation, and LA-NVs from these DCs retain higher levels of costimulatory molecules, significantly boosting T cell activation compared to plain NVs.

L-TG/GW-NVs Re-activate T cells and DCs

Current research has demonstrated that PGE2 exerts a potent inhibitory effect on T cells and DCs [34, 35]. What’s more, the immunosuppression induced thereby also serves as one of the important causes for the formation of PMN. As we can see from Fig. S6, this effection is mediated through the EP2 and EP4 receptors. The CCK8 results indicated that PGE2 inhibited the cell viability of T cells, and the inhibitory effect becomes stronger with increasing concentrations of PGE2 (Fig. S7). The ELISA results showed that the secretion levels of tumor cell-killing factors, including IFN-γ, Gzms-B, and IL-2, by T-cells decreased under the influence of PGE2, as indicated in Fig. S8. The FCM results showed that under the influence of PGE2, the PD-1 and TIM-3 protein expression of T-cells increased, indicating that the cells entered an exhausted state (Fig. S9). Because the CD80, CD86, and MHC molecules of L-TG/GW-NVs effectively activated T cells by binding to T cells [36, 37]. Therefore, DC-derived L-TG/GW-NVs can target T cells and release PGE2 receptor antagonists (Fig. 4A). The LCSM results showed that L-TG/GW-NVs could effectively bind to T cells (Fig. 4B). T cells were incubated with PGE2 for 48 h, and then different materials were added. The ELISA results showed that L-TG/GW-NVs could effectively restore the ability of T cells to secrete IL-2, IFN-γ and TNF-α (Fig. 4C). Additionally, upon entering an exhausted state, T cells are characterized by elevated surface expression levels of PD-1 and TIM-3 [38, 39]. However, in our experiments, we found that L-TG/GW-NVs can attenuate the expression levels of the exhaustion markers TIM-3 and PD-1 of T cells (Fig. 4D). At the same time, CD4 and CD8 proteins, the activation markers of T cells, were up-regulated by L-TG/GW-NVs (Fig. 4E).

Fig. 4.

Fig. 4

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L-TG/GW-NVs Re-activate T Cells. (A) Schematic diagram of L-TG/GW-NVs acting on T cells. (B) The attachment of fluorescence-labeled L-TG/GW-NVs (green) to the surface of T cells (red) was observed via LCSM. Scale bar, 2 μm. (C) ELISA detects changes in IL-2, IFN-γ and TNF-α secretion by T cells. (D) FCM quantitative of the expression of TIM-3 and PD-1 in T cells. (E) FCM results of the expression of CD4 and CD8 in T cells. (F) Schematic diagram of L-TG/GW-NVs acting on DCs. (G) The attachment of fluorescence-labeled L-TG/GW-NVs (green) to the surface of DCs (red) was observed via LCSM. Scale bar, 10 μm. (H) ELISA detects changes in IL-12, IL-6 and TNF-α secretion by DCs. (n = 3, ns: no significance, *p < 0.1,**p < 0.01, ***p < 0.001, ****p < 0.0001)

Next, we investigated the inhibitory effect of PGE2 on DCs and verified the reactivating effect of TG/GW-NVs on DCs (Fig. 4F and S10). Upon exposure to PGE2, DCs fall into a dysfunctional state where their antigen-presenting capability remains unaffected, but their cytokine secretion is significantly reduced [40]. As we can see from CCK8 result in Fig. S11, the proliferation of DCs was inhibited by PGE2. The ELISA results also demonstrated that under the influence of PGE2, the secretion of IL-12, IL-6, and TNF-α from DCs decreases (Fig. S12), showing an inverse correlation with the concentration of PGE2. L-TG/GW-NVs could be taken up by DCs, enabling targeted delivery of PGE2 receptor antagonists and activation of DCs. The LCSM results showed that L-TG/GW-NVs could be successfully combined with DCs (Fig. 4G). PGE2 was incubated with DCs for 48 h, and then different materials were added to observe its effect on DCs. CCK8 results showed that L-TG/GW-NVs effectively alleviated the loss of DCs proliferative activity caused by PGE2 (Fig. S13). At the same time, L-TG/GW-NVs restored the secretion capacity of IL-12, IL-6 and TNF-α of DCs (Fig. 4H).

Taken together, the aforementioned experimental results indicate that L-TG/GW-NVs can reverse the immunosuppressive effects of PGE2 on DCs and T cells, as well as enhance the immune activity of them.

Therapeutic efficacy of L-TG/GW-NVs to suppress postoperative metastasisof TNBC in vivo

TNBC is classified as one of the breast cancer subtypes that are currently deficient in effective treatment modalities, and it is highly metastatic, highly invasive and highly recurrent [41]. Breast cancer cells in situ remodel the lung immune microenvironment by secreting tumor exosomes, which makes the lung a metastatic target organ for TNBC [42]. To test whether L-TG/GW-NVs can interfere with premetastatic niche formation in the lung of tumor-bearing mice and thus prevent lung metastasis of breast cancer, we developed a breast cancer lung metastasis model in BALB/c mice using 4T1-GFP cells (Fig. 5A). The excellent results of in vitro experiments encourage us to further study the anti-tumor metastasis effect of L-TG/GW-NVs in vivo. Freeze-dried L-TG/GW-NVs powder with DiD labeling was applied to female BALB/c mice through lung inhalation, and fluorescence images of lung showed persistent red fluorescence in the lungs (Fig. 5B). These results indicated that the lyophilized powder of L-TG/GW-NVs was successfully accumulated in the lungs of mice and exerted anti-tumor effect. The survival curve of mice showed that L-TG/GW-NVs could significantly prolong the survival time of mice (Fig. S14) and ensure the weight gain of mice (Fig. S15). The mice were sacrificed 14 days after tail vein injection of 4T1-GFP cells. The results showed that there were fewer lung nodules in L-TG/GW-NVs group (Fig. 5C and S16). The inhibitory effect of L-TG/GW-NVs on lung metastasis was also confirmed by histological examination. H&E staining results showed that L-TG/GW-NVs mice had fewer metastatic foci represented by cell clusters with deep nuclear staining (Fig. 5D). At the same time, by detecting GFP fluorescence in the lung of mice, it was found that the fluorescence intensity of L-TG/GW-NVs group was the weakest, which proved that the number of cancer cell metastasis in L-TG/GW-NVs group was less (Fig. 5E). The expression of Ki67 in the lung of mice was detected by immunofluorescence method, and it was found that the red fluorescence intensity representing Ki67 in the L-TG/GW-NVs group was weaker than other groups (Fig. 5F). L-TG/GW-NVs also significantly inhibited lung metastasis of TNBC compared with LA-NVs or TG/GW. This inhibitory effect was not only reflected in lung colonization of cancer cells in mice, but also reflected in the survival of mice.

The effects of L-TG/GW-NVs reactivating immune cells in vivo

It has been shown in previous studies that PGE2 has a direct inhibitory effect on DCs and T cells, leading to the formation of an immunosuppressive microenvironment [43, 44]. Here we used L-TG/GW-NVs to abolish the PGE2 effect and reactivate T cells in the exhausted state [45]. Immunofluorescence sections of lung tumor tissues showed that the number of T cells in the lung tumors of L-TG/GW-NVs group was significantly increased, especially CD8+ T cells, which were increased in number and infiltration (Fig. 6A and B). The FCM results of mouse lung homogenates showed that L-TG/GW-NVs successfully activated T cells (Fig. 6C and D) and attenuated the depletion effect of PGE2 on T cells (Fig. S17). It has been shown that PGE2 does not affect the maturation, number and antigen presentation of DCs, but it can impair the ability of DCs to secrete cytokines and thus make DCs into a state of dysfunction, unable to recruit enough CD8+ T cells. In this study, we selected LA-NVs loaded with PGE2 antagonists. On the one hand, the inhibitory effect of PGE2 on DCs was eliminated through the action of antagonists; on the other hand, the external vesicles from DCs, LA-NVs, could activate DCs [46]. The levels of TNF-α, IL-6, IFN-γ, Gzms-b and IL-2 in lung tissue homogenate of L-TG/GW-NVs group were detected by ELISA. The results showed that DCs in lung tissue of L-TG/GW-NVs group recovered the function of secreting cytokines (Fig. 6E). The biological safety of L-TG/GW-NVs was determined by histological examination of major organs and blood biochemical examination. The results showed that no serious tissue damage was found in the heart, liver, spleen and kidney of mice (Fig. 6F and S18).

Fig. 6.

Fig. 6

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L-TG/GW-NVs reactivates immune cells and interferes with PMN formation A. Immunofluorescence analysis images of CD8+ in macroscopic lung metastatic nodules. B. Immunofluorescence analysis images of CD4+ T cells in macroscopic lung metastatic nodules. Scale bar, 50 μm. C. FCM results of CD8+ T cells in lungs from different groups. D. FCM results of CD4+ T cells in lungs from different groups. E. ELISA analysis of TNF-α, IL-6, IFN-γ, Gzms-b and IL-2 in lungs from different groups (n = 3). F. Concentrations of liver (ALT, ALP) and kidney (UREA, UA, Crea) function-related indexes in the serum from mice. (n = 3, ns: no significance, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Conclusions

In this study, a freeze-dried powder of artificial drug-loaded nanovesicles (L-TG/GW-NVs) was developed for inhalation delivery to the lungs. This formulation activates pulmonary T cells and DCs, thereby disrupting the formation of the PMN. Compared with conventional extracellular vesicles, lyophilized L-TG/GW-NVs offer improved storage convenience and enhanced stability.

L-TG/GW-NVs are derived from DCs and present co-stimulatory molecules CD80, CD86, and MHC II on their surface, enabling effective targeting and activation of DCs and T cells. They are additionally loaded with the PGE2 inhibitors TG and GW, which block the immunosuppressive effects of PGE2 on immune cells. Thus, L-TG/GW-NVs exert dual immunostimulatory effects: relieving PGE2-mediated immune suppression and reactivating DCs and T cells to disrupt pulmonary PMN. In vitro, L-TG/GW-NVs restored the function of PGE2-primed DCs and T cells, exhibiting both reactivation and anti-exhaustion effects. In a breast cancer lung metastasis mouse model, inhaled L-TG/GW-NVs achieved targeted delivery to the lungs, reinstated cytokine secretion by DCs, and activated T cells. This significantly increased CD4+ and CD8+ T cell populations, effectively disrupting PMN and preventing lung metastasis.

In conclusion, this study presents a promising strategy for disrupting pulmonary PMN, providing both mechanistic insight and foundational data with important implications for cancer metastasis prevention.

Supplementary Information

Supplementary Material 1 (2.1MB, docx)

Author contributions

Q. L.: conducted experiments and data analysis. Investigation, validation, formal analysis, and writing - original draft. Z. Y.: conducted experiments and software. Y. Z.: Supervision. Y. S.: conducted experiments and software. Q. J.: conducted experiments and software. Q. Z.: conducted experiments and writing original draft. Y. Y.: conducted experiments and software. M. J.: conducted experiments and software. H. Y.: conducted experiments and writing original draft. L. R.: conducted experiments and software. G. J.: conducted experiments and software. X. W.: conducted experiments and software. J. Z.: Funding acquisition and project administration. Z. L.: Methodology and supervision. H. L.: Conceptualization, supervision, formal analysis and revised - original draft.

Funding

This work was supported by the National Key R&D Program of China (2022YFB3808300) and National Natural Science Foundation of China (32322045, 32471460), Science Fund for Creative Research Groups of Nature Science Foundation of Hebei Province (B2025201113), Natural Science Foundation of Hebei Province (B2023201108). Funded by Science Research Project of Hebei Education Department (CYZD202501), Research and Innovation Team of Hebei University (IT2023A01).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

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Contributor Information

Jinchao Zhang, Email: zjc@hbu.edu.cn.

Zhenhua Li, Email: zhenhuali@hbu.edu.cn.

Huifang Liu, Email: liuhuifang@hbu.edu.cn.

References

  • 1.Pan Y, Cheng J, Zhu Y, et al. Immunological nanomaterials to combat cancer metastasis. Chem Soc Rev. 2024;53(12):6399–444. 10.1039/d2cs00968d. [DOI] [PubMed] [Google Scholar]
  • 2.Waks AG, Winer EP. Breast cancer treatment: a review. J Am Med Assoc. 2019;321(3):288–300. 10.1001/jama.2018.19323. [DOI] [PubMed] [Google Scholar]
  • 3.Esposito M, Ganesan S, Kang Y. Emerging strategies for treating metastasis. Nat Cancer. 2021;2(3):258–70. 10.1038/s43018-021-00181-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ortiz A, Stavrou A, Liu S, et al. NUPR1 packaged in extracellular vesicles promotes murine triple-negative breast cancer in a type 1 interferon-independent manner. Extracell Vesicles Circulating Nucleic Acids. 2024;5(1):19–36. 10.20517/evcna.2023.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Zhao L, Ma X, Yu J. Exosomes and organ-specific metastasis. Molecular Therapy-methods &. Clin Dev. 2021;22:133–47. 10.1016/j.omtm.2021.05.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Patras L, Shaashua L, Matei I, et al. Immune determinants of the pre-metastatic niche. Cancer Cell. 2023;41(3):546–72. 10.1016/j.ccell.2023.02.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Lin S, Mo H, Li Y, et al. Clinicopathological characteristics and survival outcomes in patients with synchronous lung metastases upon initial metastatic breast cancer diagnosis in Han population. BMC Cancer. 2021;21(1):1330. 10.1186/s12885-021-09038-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Bayerl F, Meiser P, Donakonda S, et al. Tumor-derived prostaglandin E2 programs cDC1 dysfunction to impair intratumoral orchestration of anti-cancer T cell responses. Immunity. 2023;56(6):1341–e135811. 10.1016/j.immuni.2023.05.011. [DOI] [PubMed] [Google Scholar]
  • 9.Gong Z, Li Q, Shi J, et al. Immunosuppressive reprogramming of neutrophils by lung mesenchymal cells promotes breast cancer metastasis. Sci Immunol. 2023;8(80):eadd5204. 10.1126/sciimmunol.add5204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Santiso A, Heinemann A, Kargl J. Prostaglandin E2 in the tumor microenvironment, a convoluted affair mediated by EP receptors 2 and 4. Pharmacol Rev. 2024;76(3):388–413. 10.1124/pharmrev.123.000901. [DOI] [PubMed] [Google Scholar]
  • 11.Finetti F, Travelli C, Ercoli J, et al. Prostaglandin E2 and cancer: insight into tumor progression and immunity. Biology. 2020;9(12):434. 10.3390/biology9120434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kwok AJ, Allcock A, Ferreira RC, et al. Neutrophils and emergency granulopoiesis drive immune suppression and an extreme response endotype during sepsis. Nat Immunol. 2023;24(5):767–79. 10.1038/s41590-023-01490-5. [DOI] [PubMed] [Google Scholar]
  • 13.Parida S, Parekh A, Dey G, et al. Molecular inhibition of prostaglandin E2 with GW627368X: therapeutic potential and preclinical safety assessment in mouse sarcoma model. Cancer Biol Ther. 2015;16(6):922–32. 10.1080/15384047.2015.1040953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Qiu J, Li Q, Li J, et al. Complementary roles of EP2 and EP4 receptors in malignant glioma. Br J Pharmacol. 2023;180(20):2623–40. 10.1111/bph.16148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zheng M, Zhang W, Chen X, et al. The impact of lipids on the cancer-immunity cycle and strategies for modulating lipid metabolism to improve cancer immunotherapy. Acta Pharm Sin B. 2023;13(4):1488–97. 10.1016/j.apsb.2022.10.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Knudsen NH, Manguso RT. Tumor-derived PGE2 gives NK cells a headache. Immunity. 2020;53(6):1131–2. 10.1016/j.immuni.2020.11.018. [DOI] [PubMed] [Google Scholar]
  • 17.Gong Z, Li Q, Shi J, et al. Lung fibroblasts facilitate pre-metastatic niche formation by remodeling the local immune microenvironment. Immunity. 2022;55(8):1483–e15009. 10.1016/j.immuni.2022.07.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Elewaut A, Estivill G, Bayerl F, et al. Cancer cells impair monocyte-mediated T cell stimulation to evade immunity. Nature. 2025;637(8046):716–25. 10.1038/s41586-024-08257-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Sanchez-Trincado JL, Pelaez-Prestel HF, Lafuente EM, et al. Human oral epithelial cells suppress T cell function via prostaglandin E2 secretion. Front Immunol. 2022;12:740613. 10.3389/fimmu.2021.740613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Liu J, Shen T, Zhang Y, et al. Cell dehydration enables massive production of engineered membrane vesicles with therapeutic functions. J Extracell Vesicles. 2024;13(7):e12483. 10.1002/jev2.12483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Tuluwengjiang G, Rasulova I, Ahmed S, et al. Dendritic cell-derived exosomes (Dex): underlying the role of exosomes derived from diverse DC subtypes in cancer pathogenesis. Pathol Res Pract. 2024;254:155097. 10.1016/j.prp.2024.155097. [DOI] [PubMed] [Google Scholar]
  • 22.Xia J, Chen X, Dong M, et al. Antigen self-presenting dendrosomes swallowing nanovaccines boost antigens and STING agonists codelivery for cancer immunotherapy. Biomaterials. 2025;316:122998. 10.1016/j.biomaterials.2024.122998. [DOI] [PubMed] [Google Scholar]
  • 23.Li X, Wei Y, Zhang Z, et al. Harnessing genetically engineered cell membrane-derived vesicles as biotherapeutics. Extracell Vesicles Circ Nucleic Acids. 2024;5(1):44–63. 10.20517/evcna.2023.58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Huang L, Rong Y, Tang X, et al. Engineered exosomes as an in situ DC-primed vaccine to boost antitumor immunity in breast cancer. Mol Cancer. 2022;21(1):45. 10.1186/s12943-022-01515-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Ansari FJ, Tafti HA, Amanzadeh A, et al. Comparison of the efficiency of ultrafiltration, precipitation, and ultracentrifugation methods for exosome isolation. Biochem Biophys Rep. 2024;38:101668. 10.1016/j.bbrep.2024.101668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Charoenviriyakul C, Takahashi Y, Nishikawa M, et al. Preservation of exosomes at room temperature using lyophilization. Int J Pharm. 2018;553(1–2):1–7. 10.1016/j.ijpharm.2018.10.032. [DOI] [PubMed] [Google Scholar]
  • 27.Gong Z, Li Q, Shi J, et al. Lipid-laden lung mesenchymal cells foster breast cancer metastasis via metabolic reprogramming of tumor cells and natural killer cells. Cell Metabol. 2022;34(12):1960–e19769. 10.1016/j.cmet.2022.11.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Amaradhi R, Banik A, Mohammed S. Potent, selective, water soluble, brain-permeable EP2 receptor antagonist for use in central nervous system disease models. J Med Chem. 2020;63(3):1032–50. 10.1021/acs.jmedchem.9b01218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Liang X, Gupta D, Xie J, et al. Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors. Nat Commun. 2025;16(1):4028. 10.1038/s41467-025-59377-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Gholami Farashah MS, Javadi M, Soleimani Rad J, et al. 17β-estradiol-loaded exosomes for targeted drug delivery in osteoporosis: a comparative study of two loading methods. Adv Pharm Bull. 2023;13(4):736–46. 10.34172/apb.2023.072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Colja S, Jovčevska I, Šamec N, et al. Sonication is a suitable method for loading nanobody into glioblastoma small extracellular vesicles. Heliyon. 2023;9(5):e15674. 10.1016/j.heliyon.2023.e15674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Chen L, Zhang J, Huang Y, et al. Drug delivery systems based on dendritic-cell-derived exosomes. Pharmaceutics. 2025;17(3):326. 10.3390/pharmaceutics17030326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Yu Z, Liu W, He Y, et al. HLA-A2.1-restricted ECM1-derived epitope LA through DC cross-activation priming CD8+ T and NK cells: a novel therapeutic tumour vaccine. J Hematol Oncol. 2021;14(1):158. 10.1186/s13045-021-01176-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lacher SB, Dörr J, de Almeida GP, et al. PGE2 limits effector expansion of tumour-infiltrating stem-like CD8+ T cells. Nature. 2024;629(8011):417–25. 10.1038/s41586-024-07254-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Punyawatthananukool S, Matsuura R, Wongchang T, et al. Prostaglandin E2-EP2/EP4 signaling induces immunosuppression in human cancer by impairing bioenergetics and ribosome biogenesis in immune cells. Nat Commun. 2024;15(1):9464. 10.1038/s41467-024-53706-3v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Zitvogel L, Regnault A, Lozier A, et al. Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med. 1998;4(5):594–600. 10.1038/nm0598-594. [DOI] [PubMed] [Google Scholar]
  • 37.Tkach M, Kowal J, Zucchetti AE. Qualitative differences in T‐cell activation by dendritic cell‐derived extracellular vesicle subtypes. EMBO J. 2017;36(20):3012–28. 10.15252/embj.201696003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Baessler A, Vignali DAA. T cell exhaustion. Annu Rev Immunol. 2024;42(1):179–206. 10.1146/annurev-immunol-090222-110914. [DOI] [PubMed] [Google Scholar]
  • 39.Sen DR, Kaminski J, Barnitz RA, et al. The epigenetic landscape of T cell exhaustion. Science. 2016;354(6316):1165–9. 10.1126/science.aae0491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Brown M, Tran MA, Bhardwaj N. Cancer immunity derailed: PGE2 misconducts cDC1s. Immunity. 2023;56(6):1165–7. 10.1016/j.immuni.2023.05.015. [DOI] [PubMed] [Google Scholar]
  • 41.Liu X, Adorno-Cruz V, Chang YF, et al. EGFR inhibition blocks cancer stem cell clustering and lung metastasis of triple negative breast cancer. Theranostics. 2021;11(13):6632–43. 10.7150/thno.57706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Yu P, Han Y, Meng L, et al. The incorporation of acetylated LAP-TGF-β1 proteins into exosomes promotes TNBC cell dissemination in lung micro-metastasis. Mol Cancer. 2024;23(1):82. 10.1186/s12943-024-01995-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Li L, Chao Z, Peng H, et al. Tumor ABCC4-mediated release of PGE2 induces CD8 + T cell dysfunction and impairs PD-1 blockade in prostate cancer. Int J Biol Sci. 2024;20(11):4424–37. 10.7150/ijbs.99716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Xu X, Lu Y, Cao L, et al. Tumor-intrinsic P2RY6 drives immunosuppression by enhancing PGE2 production. Cell Rep. 2024;43(7):114469. 10.1016/j.celrep.2024.114469. [DOI] [PubMed] [Google Scholar]
  • 45.Lyu F, Wu K, Wu SY, et al. Functional evaluation of dendritic cells and extracellular vesicles as immunotherapy for breast cancer. Oncogene. 2024;43(5):319–27. 10.1038/s41388-023-02893-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Fan M, Liu H, Yan H, et al. A CAR T-inspiring platform based on antibody-engineered exosomes from antigen-feeding dendritic cells for precise solid tumor therapy. Biomaterials. 2022;282:121424. 10.1016/j.biomaterials.2022.121424. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (2.1MB, docx)

Data Availability Statement

No datasets were generated or analysed during the current study.

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