迁移体介导的抗原释放与抗转移免疫

Fig. 1 | Tspan4 regulates the metastasis of 4T1 cells and the production of migrasomes during this process. 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 Tspan4OE 4T1 cells. Tspan4OE, 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 Tspan4OE 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 Tspan4OE 4T1 cells. Tspan4OE, n = 73 cells; WT, n = 80 cells; Tspan4−/−, n = 56 cells. l, WT, Tspan4−/− and Tspan4OE 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 Tspan4OE 4T1 cells in vivo. Tspan4OE, 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.

Fig. 2 | Injection of 4T1-derived migrasomes inhibits metastasis. 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.

Fig. 3 | Migrasomes induce CD8+ T cell priming. 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 + Tspan4OE, 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.

Fig. 4 | Tumor-associated antigens are enriched in migrasomes. a, Volcano plot showing mass spectrometry-based quantification of TMT-labeled proteins. Upper‑right dots represent a migrasome/cell abundance ratio ≥ 2, P < 0.05; upper‑left dots represent a migrasome/cell abundance ratio < 0.5, P < 0.05. n = 3 independent experiments. P values were calculated using a two-tailed, two-sample unequal variance t-test. b, Heat map plot showing the distribution of migrasome marker proteins in 4T1 cells and migrasomes. c, Heat map plot showing testis-specific proteins and a top 10 list of mutated proteins that are enriched in migrasomes. d, BRDT, DDHD1(D318H) and SDC2(A18S) peptides (100 μg) were mixed with Poly I:C (50 μg) and injected (i.v.) into mice on day 0 and day 12. Seven days later, 4T1 cells 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 33. e, Statistics of the luminescence counts of 4T1 cells. n = 25 mice for each group. f, Genome sequencing of 4T1 cells to identify single-nucleotide mutations. The reference genome sequence of BALB/c was from the UCSC database. Statistical data in e are presented as the mean ± s.e.m. P values were calculated using a two-tailed, unpaired t-test.

Fig. 5 | Tumor-associated antigen depletion aggravates 4T1 metastasis. a, WT, Ddhd1−/− and Sdc2−/− 4T1 cells 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 = 15 mice for each group. c, WT and Brdt−/− 4T1 cells 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. d, Statistics of the luminescence counts of 4T1 cells. n = 15 mice for each group. e, WT, Ddhd1−/−, Sdc2−/− and Brdt−/− 4T1 cells were injected (i.v.) into BALB/c-nude mice and in vivo photo emission of 4T1 cells was detected by d-luciferin injection and imaged using an IVIS spectrum imaging system on day 12. f, Statistics of the luminescence counts of 4T1 cells in BALB/c-nude mice. n = 10 mice for each group. g, Migrasomes were purified from Tspan4-GFP stably expressing WT and Brdt−/− 4T1 cell lines and injected (i.v.) into BALB/c mice. PBS served as a negative control. 4T1 cells were injected (i.v.) on the next day after migrasome injection. In vivo photo emission of 4T1 cells was detected by d-luciferin injection and imaged using an IVIS spectrum imaging system on day 14. h, Statistics of the luminescence counts of 4T1 cells. n = 15 mice for each group. i, Mice were i.v. injected with PBS control or Sdc2-/Ddhd1-deficient migrasomes, followed by i.v. injection with 4T1 cells the next day. In vivo photo emission of 4T1 cells was detected by d-luciferin injection and imaged using an IVIS spectrum imaging system on day 14. j, Statistics of the luminescence counts of 4T1 cells. n = 10 mice for PBS and Sdc2−/− groups, n = 9 mice for Ddhd1−/− group. Statistical data in b, d, f, h and j are presented as the mean ± s.e.m. P values were calculated using a two-tailed, unpaired t-test.

Fig. 6 | Migrasomes have therapeutic vaccine potential for cancer immunotherapy. a, Cells (4T1) were injected (i.v.) at day 0. Migrasomes were purified from Tspan4-GFP expressing 4T1 cells and injected (i.v.) into mice on day 5 and day 7. PBS and UV-irradiated 4T1 cells (Dead-cell) served as controls. 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. PBS, n = 20 mice; migrasome, n = 19 mice; Dead-cell, n = 10 mice. c, 4T1 cells were injected (i.v.) on day 0. Migrasomes were purified from Tspan4-GFP-expressing 4T1 cells and injected (i.v.) into the mice on day 5 and day 7. Anti-PD-1 was also injected (i.p.) on day 5 (2.5 mg per kg body weight). PBS and IgG served as controls. In vivo photo emission of 4T1 cells was detected by d-luciferin injection and imaged using an IVIS spectrum imaging system on day 14. d, Statistics of the luminescence counts of 4T1 cells. n = 12 mice for each group. e, Anatomical lung images of MMTV-PyMT mice treated with PBS or PyMT tumor cell-derived migrasomes (10 μg per mouse, each time) from 10 weeks (day 0) for three times at the indicated time points. Arrowheads indicate the metastasis nodules. f, Representative H&E-stained anatomical and histological lung images from e. Insets: higher-magnification views. g, Quantification of the metastasis nodule number per lung image. PBS, n = 15 mice; migrasome, n = 17 mice. Statistical data in b, d and g are presented as the mean ± s.e.m. P values were calculated using a two-tailed, unpaired t-test.

Fig. 7 | Migrasomes have clinical significance in human cancers. a, Confocal images of OSCC primary tumor cells seeded in collagen-coated dishes and stained with WGA for imaging. Scale bars, 50 μm and 10 μm (enlarged). b, SEM image of purified OSCC-derived migrasomes. Scale bar, 1 μm. c, Heat map plots show migrasome marker proteins enriched in OSCC-derived migrasomes. d, Heat map plots show mutated proteins enriched in OSCC-derived migrasomes. e, Heat map plots show testis-specific proteins enriched in OSCC-derived migrasomes. The values of the mass spectrometry results in e were normalized according to the amount in the cell and then plotted. f, Tspan4 RNA expression in transcripts per million (TPM) in breast invasive carcinoma tumors and normal tissues from the UCSC Xena Toil RNA-sequencing dataset (TCGA + GTEx). In the box plots, the center line denotes the median, the box represents the interquartile range (25th–75th percentiles), whiskers extend to 1.5 times the interquartile range (excluding outliers), and jitter points represent individual samples. Data were analyzed at https://gepia.cancer-pku.cn/. g, The survival curves of Tspan4-low and Tspan4-high groups dichotomously divided by Tspan4 median in breast invasive carcinoma using TCGA dataset. Data were analyzed at https://gepia. cancer-pku.cn/. HR, hazard ratio. h, Diagram illustrating migrasome-mediated antigen release during metastasis to unlock immunogenicity. Illustration in h created in BioRender; Lam, R. https://biorender.com/r8v3lz6 (2026).