---
title: "Migrasome-mediated antigen release during metastasis drives CD8+ T cell antitumor immunity"
id: "nature-immunology-1-metastasis-enables-immunogenicity-through-migrasome-mediated-antigen-release"
canonical_url: "https://medichelpline.com/clinical-feed/nature-immunology-1-metastasis-enables-immunogenicity-through-migrasome-mediated-antigen-release"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Nature Immunology"
source_url: "https://www.nature.com/articles/s41590-026-02641-0"
published_at: "2026-09-22T12:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Migrasome-mediated antigen release during metastasis drives CD8+ T cell antitumor immunity
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/nature-immunology-1-metastasis-enables-immunogenicity-through-migrasome-mediated-antigen-release
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Nature Immunology
- **Source URL:** [Original Journal Publication](https://www.nature.com/articles/s41590-026-02641-0)
- **Published At:** 2026-09-22T12:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Metastasis can paradoxically increase tumor immunogenicity through release of **migrasomes**, membranous organelles shed from migrating tumor cells that carry cytosolic contents. - Circulating tumor cells (CTCs) migrating through the vasculature shed abundant migrasomes enriched in tumor-associated antigens, including cancer–testis and mutated antigens, according to the source article. - Migrasomes traffic to secondary lymphoid organs and are preferentially taken up by antigen-presenting cells (APCs); APCs cross-present migrasome-derived antigens to activate **CD8+ T cells**, producing immune responses that limit metastatic outgrowth. - Genetic inhibition of migrasome formation increased metastatic burden in mouse models, indicating migrasomes contribute to antitumor immune surveillance during dissemination. - Conversely, administration of purified cancer-derived migrasomes restored immune-mediated suppression of metastatic growth in experimental systems described by the authors. - The tetraspanin **Tspan4** was investigated in 4T1 mouse mammary carcinoma cells: Tspan4 knockout (_Tspan4_−/−) increased metastasis and reduced survival, while Tspan4 overexpression curtailed metastatic burden and extended survival in syngeneic mice. - Tspan4 manipulation did not affect primary tumor growth in subcutaneous models in either immunocompetent or immunodeficient mice, suggesting its effect is specific to metastatic dissemination rather than primary tumor proliferation. - The study positions migrasomes as a distinct, metastasis-specific platform for endogenous tumor antigen delivery that can either reveal tumor vulnerability to immunity or, context-dependently, carry immune-modulatory cargo. - Source-reported methods and model details include use of the 4T1 triple-negative breast cancer model, luciferase-labelled cells, intravenous injection into BALB/c mice, and in vivo imaging (IVIS) at day 14 post D-luciferin injection; further experimental specifics were reported in the original article sections not fully reproduced here.
## Clinical Analysis & Structured Key Points
Metastasis enables immunogenicity through migrasome-mediated antigen release | Nature Immunology Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement Metastasis enables immunogenicity through migrasome-mediated antigen release Download PDF Download PDF Abstract Antigen release is a critical step in initiating antitumor immune responses, yet its regulation during metastasis is not well understood. Here we show that circulating tumor cells undergoing vascular migration produce migrasomes that serve as a metastasis-specific mechanism of antigen release. These migrasomes are enriched in tumor-associated antigens, including cancer-testis and mutated antigens, and are captured efficiently by antigen-presenting cells in secondary lymphoid organs, where they undergo cross-presentation to elicit CD8 + T cell-mediated immune responses that constrain metastatic progression. Genetic inhibition of migrasome formation enhances metastasis, while administration of purified cancer-derived migrasomes restores immune-mediated suppression of metastatic growth. These findings show paradoxically that metastasis can enhance tumor immunogenicity through migrasome-mediated antigen release, highlighting a link between cancer dissemination and immune activation and establishing migrasomes as a distinct and potent platform for endogenous tumor antigen delivery. Explore related subjects Discover the latest articles and news in related subjects. Antigen-presenting cells CD8-positive T cells Cancer immunotherapy Tumour immunology Main Metastasis is the leading cause of cancer mortality. It marks a pivotal stage of tumor progression where malignant cells disseminate to distant sites and often escape immune elimination 1 . Effective antitumor immunity hinges on the release and presentation of tumor antigens to the immune system. Such antigens are typically liberated when tumor cells die (for example, through immunogenic cell death) 2 , 3 , 4 or shed their contents in extracellular vesicles 5 , 6 , allowing dendritic cells to capture these antigens and prime cytotoxic T lymphocytes against the cancer 6 , 7 , 8 , 9 , 10 , 11 . Tumor-derived extracellular vesicles have emerged as critical mediators of tumor–immune interactions, with a dualistic influence on immunity. On one hand, extracellular vesicles carry immunosuppressive cargo facilitating immune evasion—for example, tumor exosomes displaying programmed cell death ligand 1 (PD-L1) can inhibit cytotoxic T cells 12 . On the other hand, extracellular vesicles can disseminate tumor antigens and proinflammatory signals: exosomes bearing major histocompatibility complex (MHC) class I-restricted tumor antigens can activate T cells and provoke antitumor immune responses 5 , 13 . Thus, context-dependently, tumor-derived extracellular vesicles may serve either as agents of immune escape or as triggers of antitumor immunity 12 , 14 , 15 , 16 , 17 . Migrasomes are a recently identified organelle produced by migrating cells. As cells move, they leave behind long retraction fibers from which migrasomes bud off, carrying diverse cytosolic contents 18 , 19 , 20 . Migrasomes have been implicated in modes of intercellular communication during development and disease 21 . In cancer, migrasomes have been observed accompanying metastasizing tumor cells and ferrying immune checkpoint proteins such as PD-L1, suggesting a role in facilitating immune evasion. However, it is unknown whether metastatic dissemination might also leverage migrasomes to release tumor antigens—a possibility that would represent a previously unrecognized mechanism of antitumor immune activation. Here, we demonstrate that metastasis can paradoxically unmask tumor immunogenicity through migrasome-mediated antigen release. In mouse models, circulating tumor cells (CTCs) migrating through the vasculature shed abundant migrasomes enriched with tumor antigens. These migrasomes traffic to secondary lymphoid organs and are preferentially phagocytosed by antigen-presenting cells (APCs), which then cross-present migrasome-derived antigens to elicit robust, CD8 + T cell-dependent antitumor immune responses that constrain metastatic outgrowth. Our findings uncover a metastasis-dependent mechanism of antigen release that activates antitumor immunity. Thus, rather than serving only as a means of immune escape, the metastatic process can inadvertently engage immune surveillance and reveal tumor vulnerability. Results Tspan4 suppresses breast cancer metastasis The 4T1 mouse mammary carcinoma cell line is highly invasive and can metastasize to multiple organs, including lymph nodes, blood, liver, lung, brain and bone 22 . The 4T1 line is widely regarded as a relevant model for human triple-negative breast cancer 23 , as it lacks estrogen receptor, progesterone receptor and HER2 expression and exhibits aggressive metastasis. Tetraspanins play roles in regulating membrane signaling, adhesion and immune receptor dynamics, potentially impacting cancer progression and therapeutic responses 24 . To investigate the role of Tspan4 in metastasis, we generated and intravenously (i.v.) injected wild-type (WT), Tspan4 -knockout ( Tspan4 −/− ) and Tspan4 -overexpressing ( Tspan4 OE ) 4T1 cells stably expressing luciferase into syngeneic female BALB/c mice. In vivo imaging was performed on day 14 following D -Luciferin injection using the IVIS system. Metastatic burden was significantly increased in mice injected with Tspan4 −/− 4T1 cells and markedly reduced with Tspan4 OE cells compared to WT (Fig. 1a,b ). Tspan4 deficiency shortened, and overexpression extended, survival of tumor-bearing mice (Fig. 1c ). Notably, Tspan4 knockout did not enhance primary tumor growth in subcutaneous models using immunocompetent BALB/c or immunodeficient BALB/c-nude mice (Extended Data Fig. 1a,b ), indicating that Tspan4 selectively suppresses metastatic dissemination without affecting primary tumor growth. Fig. 1: Tspan4 regulates the metastasis of 4T1 cells and the production of migrasomes during this process. Full size image a , Cells (4T1) were injected i.v., and in vivo photo emission of 4T1 cells was detected by d -Luciferin injection and imaged using an IVIS spectrum imaging system on day 14. b , Statistics of the luminescence counts of 4T1 cells. n = 18 mice for each group. c , Survival plots for mice injected with WT, Tspan4 −/− and Tspan4 OE 4T1 cells. Tspan4 OE , n = 13 mice; WT, n = 12 mice; Tspan4 −/− , n = 9 mice. d , H&E-stained lung images of MMTV-PyMT; Tspan4 +/+ and MMTV-PyMT; Tspan4 −/− mice. Scale bar, 2 mm. e , Quantification of the number of metastasis nodules in lung. T4 +/+ , n = 21 mice, T4 −/− , n = 22 mice. f , Quantification of the incidence of lung metastasis. g , Confocal imaging of Tspan4 -GFP expressing 4T1 cells after seeding on fibronectin (10 μg ml −1 ) and laminin (1 μg ml −1 ) for 24 h in vitro. Scale bar, 20 μm. h , Diagram of intravital imaging. i , Tspan4 -GFP stably expressing 4T1 cells were injected into the mice for liver intravital imaging. Vessels were labeled with AF647-conjugated wheat germ agglutinin (AF647-WGA). Scale bar, 10 μm. Arrowheads indicate migrasomes. j , Confocal imaging of WT, Tspan4 −/− and Tspan4 OE 4T1 cells seeded in fibronectin (10 μg ml −1 ) and laminin (1 μg ml −1 )-coated dishes and stained with WGA (1 μg ml −1 ). Scale bar, 10 μm. k , Quantification of the number of migrasomes in WT, Tspan4 −/− and Tspan4 OE 4T1 cells. Tspan4 OE , n = 73 cells; WT, n = 80 cells; Tspan4 −/− , n = 56 cells. l , WT, Tspan4 −/− and Tspan4 OE 4T1 cells were transfected with PH-mCherry and injected into mice for intravital imaging. m , n , Quantification of the number of migrasomes produced by WT, Tspan4 −/− and Tspan4 OE 4T1 cells in vivo. Tspan4 OE , n = 20 cells; WT, n = 24 cells; Tspan4 −/− , n = 26 cells. Statistical data in b , e , k , m and n are presented as the mean ± s.e.m. P values were calculated using a two-tailed, unpaired t -test. Source data To further validate this, we examined Tspan4 in the MMTV-PyMT transgenic model, which recapitulates key features of human breast cancer, including spontaneous lung metastasis 25 , 26 . Tspan4 −/− mice were crossed with MMTV-PyMT mice to generate MMTV-PyMT; Tspan4 +/+ and MMTV-PyMT; Tspan4 −/− littermates. At 16 weeks, we observed a higher incidence of lung metastasis in the Tspan4 -deficient cohort (Fig. 1d–f ). Collectively, these results indicate that Tspan4 functions as a negative regulator of breast cancer metastasis, without altering primary tumor growth. Tspan4 regulates migrasome formation in 4T1 cells We previously showed Tspan4 is essential for migrasome formation in vitro and in chicken and zebrafish embryos 19 , 27 , 28 . To determine whether migrasomes are formed by metastatic 4T1 cells, we examined their presence both in vitro and in vivo. We confirmed that 4T1 cells produce migrasomes in vitro (Fig. 1g ). To investigate migrasome formation during tumor metastasis in vivo, we performed intravital imaging of livers from mice injected with 4T1 cells expressing Tspan4 -GFP (Fig. 1h ). Notably, circulating 4T1 cells generated retraction fibers and migrasomes within blood vessels (Fig. 1i and Supplementary Video 1 ). To assess the role of Tspan4 in migrasome biogenesis, we examined migrasome formation in Tspan4 −/− 4T1 cells in vitro. Tspan4 −/− cells exhibited a significant reduction in migrasome number, whereas Tspan4 OE cells led to enhanced migrasome formation (Fig. 1j,k ). We further examined migrasome formation in vivo by injecting Tspan4 −/− and Tspan4 OE cells and performing intravital imaging. Consistent with in vitro data, migrasome formation was significantly increased in Tspan4 OE cells and markedly decreased in Tspan4 −/− cells within blood vessels (Fig. 1l–n ). Injection of 4T1-derived migrasomes blocks metastasis Next, we investigated whether the accelerated metastasis in Tspan4 −/− 4T1 cells is caused by impaired migrasome formation or by migrasome-independent functions of Tspan4 . We reasoned that if impaired migrasome formation drives the enhanced metastasis, administering WT 4T1 migrasomes should rescue the phenotype. To efficiently obtain sufficient migrasomes, we used Tspan4 OE 4T1 cells, which generate more migrasomes than WT cells. Purified migrasomes (Fig. 2a ) showed the characteristic morphology—a round vesicle connected to a retraction fiber—by transmission electron microscopy (TEM) (Fig. 2b ) 18 . Western blot analysis according to MISEV guidelines 29 confirmed the enrichment of established migrasome markers (INTEGRINS α5 and β1, NDST, PIGK, EOGT and CPQ) and the absence of exosome markers (CD63, TSG101 and SYNTENIN-1) or the ectosome-associated marker KIF23 (Fig. 2c,d and Extended Data Fig. 1c ). Subsequently, we i.v. injected purified migrasomes into BALB/c mice, and 16 h later we injected WT and Tspan4 −/− 4T1 cells. Bioluminescence imaging 14 days later showed that migrasome injection significantly reduced the enhanced metastasis of Tspan4 −/− cells (Fig. 2e,f ). As a control, ultraviolet (UV)-induced dead 4T1 cells did not reduce metastasis of Tspan4 −/− cells (Fig. 2e,f ). We further compared migrasomes head-to-head with small extracellular vesicles (sEVs), tumor lysates and apoptotic bodies; equal protein amounts were injected before 4T1 challenge. Migrasomes significantly suppressed metastasis and prolonged survival, whereas sEVs, lysates and apoptotic bodies failed to inhibit metastatic growth; lysates even appeared to promote metastasis (Extended Data Fig. 1d–f ). Thus, the enhanced metastasis in Tspan4 −/− cells is likely due to reduced migrasome formation, and migrasomes possess distinct immunostimulatory properties superior to other extracellular vesicle populations or tumor-derived materials. Fig. 2: Injection of 4T1-derived migrasomes inhibits metastasis. Full size image a , Purification of migrasomes (mig) from in vitro-cultured 4T1 cells stably expressing Tspan4 -GFP. b , TEM image of purified 4T1 migrasomes. Scale bar, 1 μm. c , Western blot analysis of 4T1 cells and purified 4T1 migrasomes using indicated antibodies. d , Western blot analysis of sEV markers (CD63, TSG101, SYNTENIN-1), ectosome marker (KIF23), migrasome markers (EOGT, PIGK, CPQ), MHC class I and flotillin-1 in migrasome, sEV and ectosome fractions isolated from 4T1 cells. e , Migrasomes (from 4T1 cells), UV-irradiated 4T1 cells (dead cells) or PBS were i.v. injected into mice, and WT or Tspan4 −/− 4T1 cells were i.v. injected, at 16 h after injection of migrasomes. In vivo photo emission of 4T1 cells was detected by d -luciferin injection and imaged with an IVIS spectrum imaging system on day 14. f , Statistics of the luminescence counts of 4T1 cells. PBS + WT, n = 16 mice; PBS + Tspan4 −/− , n = 16 mice; migrasome + Tspan4 −/− , n = 17 mice; Dead-cell + Tspan4 −/− , n = 6 mice. g , Diagram of negative selection of in vivo 4T1-derived migrasomes. h , Confocal imaging of negatively selected 4T1-derived migrasomes. Scale bar, 20 μm. i , Western blot analysis of 4T1 cells, blood cells and IV-migs using the indicated antibodies. j , IV-migs or PBS were i.v. injected into mice, and 4T1 cells were i.v. injected at 16 h after injection of migrasomes. In vivo photo emission of 4T1 cells was detected by d -luciferin injection and photographed with an IVIS spectrum imaging system on day 14. k , Statistics of the luminescence counts of 4T1 cells. n = 10 mice for each group. l , Survival plots for mice from k . m , H&E-stained lung images from mice treated with PBS or 4T1 migrasomes (10 μg each). n , Quantification of the number of metastasis nodules per lung. PBS, n = 18; migrasome, n = 20. Statistical data in f , k and n are presented as the mean ± s.e.m. P values were calculated using a two-tailed, unpaired t -test. s.c., subcutaneous; sup, supernatant; ctrl, control. Source data To further confirm this effect is migrasome dependent rather than Tspan4 specific, we used an orthogonal approach targeting a distinct migrasome biogenesis pathway 30 . Treatment of 4T1 cells with UNC3230, a selective inhibitor of the migrasome-essential kinase PIP5K1C , significantly reduced migrasome formation in vitro (Extended Data Fig. 1g,h ). Moreover, mice injected with UNC3230-pretreated 4T1 cells exhibited markedly enhanced metastasis (Extended Data Fig. 1i,j ), confirming that migrasome deficiency, whether genetic or pharmacological, promotes metastasis. These data further support that the enhanced metastasis in Tspan4 −/− cells results from reduced migrasome formation. To investigate whether in vivo-produced migrasomes (IV-migs) have the same function, we isolated migrasomes from the blood of tumor-bearing mice. After i.v. injecting 4T1 cells, we used negative selection to purify migrasomes 20 (Fig. 2g ). Confocal imaging revealed that the purified fraction are Tspan4-GFP-positive vesicles (Fig. 2h ), and western blot confirmed enrichment of migrasome markers (TSPAN4-GFP, INTEGRIN α5, NDST and CPQ) without HISTONE H3 (Fig. 2i ). Mice were i.v. injected with IV-migs (3 μg per mouse) and challenged 16 h later with 4T1 cells. IV-migs suppressed metastasis (Fig. 2j,k ) and prolonged survival (Fig. 2l ), supporting an essential role for migrasomes in limiting metastasis. To further test the anti-metastatic effect, we used an orthotopic model: 2 × 10 5 4T1 cells were injected into the flank, and migrasomes were administered i.v. on days 8 and 15. Pulmonary metastasis was reduced at day 45 (Fig. 2m,n ), confirming that 4T1-derived migrasomes can prevent breast cancer metastasis in vivo. Migrasomes prime the adaptive immune response Considering the antitumor potential of migrasomes, we hypothesized that migrasomes may inhibit the metastasis of 4T1 cells by activating the adaptive immunity. To test this, we used T cell‑deficient BALB/c‑nude mice. We i.v. injected luciferase-expressing WT, Tspan4 −/− and Tspan4 OE 4T1 cells into syngeneic female BALB/c-nude mice. Twelve days later, bioluminescence imaging revealed that, in contrast to the metastasis observed in WT BALB/c mice, where Tspan4 deficiency significantly enhanced metastasis, knockout of Tspan4 in the immune-deficient nude mice did not enhance metastasis. In fact, metastasis was reduced compared to the WT 4T1 cells. Conversely, Tspan4 -GFP overexpression in 4T1 cells led to a slight enhancement in metastasis (Fig. 3a,b ). Thus, Tspan4 promotes metastasis in nude mice but inhibits it in WT mice (Fig. 1a,b ), indicating Tspan4‑mediated inhibition requires T lymphocytes. To test if migrasomes function in T cell‑deficient conditions, we injected migrasomes into nude mice bearing Tspan4 −/− cells and found no reduction in metastasis (Fig. 3a,b ), confirming the anti‑metastatic effect is T cell dependent. To test CD8 + T cell dependence, we depleted CD8 + T cells (anti‑CD8α) in BALB/c mice before migrasome injection and 4T1 challenge. Migrasomes reduced metastasis in IgG controls but not after CD8 + T depletion (Fig. 3c,d ), confirming a CD8 + T cell‑dependent mechanism. Fig. 3: Migrasomes induce CD8 + T cell priming. Full size image a , PBS or purified migrasomes were i.v. injected into BALB/c-nude mice on day 0. After 16 h, 4T1 cells were i.v. injected, and bioluminescence imaging (IVIS) was performed on day 12. b , Quantification of bioluminescence signals. PBS + Tspan4 OE , n = 11 mice; PBS + WT, n = 11 mice; PBS + Tspan4 −/− , n = 12 mice; Mig + Tspan4 −/− , n = 12 mice. c , Anti-CD8a or IgG (160 μg) was injected intraperitoneally (i.p.) as the time bar indicates. After 24 h, migrasomes or PBS were i.v. injected, and 16 h later WT 4T1 cells were i.v. injected. Bioluminescence imaging was performed on day 14. d , Quantification of bioluminescence. n = 10 mice for each group. e , Schematic diagram of the experimental timeline for bone marrow transplantation and migrasome treatment. Eight‑week‑old C57BL/6J mice were irradiated (10 Gy) and reconstituted with WT or B2m −/− bone marrow. After 8 weeks, all mice received OT‑I CD8 + T cells, followed by migrasomes or vehicle, and LLC‑OVA challenge. Lungs were collected at the endpoint. f , Representative H&E staining of lung tissue sections from each group, revealing metastatic tumor foci. Scale bar, 2.5 mm. g , Quantification of metastatic tumor foci per lung section. n = 12 mice for WT BM + Ctrl, n = 13 mice for WT BM + Mig, n = 11 mice for B2m −/− BM + Ctrl and B2m −/− BM + Mig groups. h , Schematic diagram of the experimental timeline. i , Representative H&E-stained lung sections showing metastatic foci. Scale bar, 2.5 mm. j , Quantification of metastatic foci. n = 6 mice per group. k , In vitro killing assay. Mice were i.v. injected with migrasomes or PBS on days 0 and 7. On day 19, splenic CD8 + T cells were co-cultured with 4T1 cells, stained with annexin V, and imaged. Scale bar, 20 μm. l , Quantification of annexin V-positive cells. n = 15 fields per group. Statistical data are presented as the mean ± s.e.m. P values were calculated using a two-tailed, unpaired t -test. Source data We next investigated how migrasomes activate CD8 + T cells. Proteomic analysis and western blotting revealed that migrasomes are enriched with MHC class I molecules but lack co-stimulatory molecules such as CD80 and CD86 (Extended Data Fig. 2a–c ), suggesting that migrasomes are unlikely to directly prime naive T cells and instead require professional APCs. To identify which APCs capture migrasomes, we incubated purified Tspan4-GFP + migrasomes with mouse splenocytes in vitro. Confocal imaging showed that migrasomes were predominantly engulfed by F4/80 + macrophages and F4/80⁻CD11c + dendriti
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