Abstract
Kupffer cells, the liver tissue resident macrophages, are critical in the detection and clearance of cancer cells. However, the molecular mechanisms underlying their detection and phagocytosis of cancer cells are still unclear. Using in vivo genome-wide CRISPR-Cas9 knockout screening, we found that the cell-surface transmembrane protein ERMAP expressed on various cancer cells signaled to activate phagocytosis in Kupffer cells and to control of liver metastasis. ERMAP interacted with β-galactoside binding lectin galectin-9 expressed on the surface of Kupffer cells in a manner dependent on glycosylation. Galectin-9 formed a bridging complex with ERMAP and the transmembrane receptor dectin-2, expressed on Kupffer cells, to induce the detection and phagocytosis of cancer cells by Kupffer cells. Patients with low expression of ERMAP on tumors had more liver metastases. Thus, our study identified the ERMAP–galectin–9-dectin-2 axis as an ‘eat me’ signal for Kupffer cells.
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Data availability
CRISPR screen sequencing datasets have been deposited in the Gene Expression Omnibus database under accession code GSE232126. Source data are provided with this paper.
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Acknowledgements
We thank X. Liu from the Core Facility of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine and J. Ding from Clinical Cancer Institute, Center for Translational Medicine, Naval Medical University for technical assistance. This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81830085 to S.S.; 92059111 and 81972738 to J.Y.), the Science and Technology Innovation Action Plan of Shanghai (22140902100 to J.Y.; 23ZR1477300 to J.L.), the Shanghai Key Laboratory of Cell Engineering (14DZ2272300 to J.Y.), and the Shanghai Municipal Health Commission Clinical Research Program (20214Y0014 to J.L.).
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J.Y., S.S. J.L. and G.C. designed the experiments. J.L., J.Y., X.L., R.G., Y.Y., Y.L., W.L., M.H. and X.H. performed the experiments. J.Y., J.L., X.L., R.G., Y.Y., J.W. and G.C. analyzed the data. J.Y. and J.L. wrote the manuscript. J.Y. and S.S. supervised the project. All authors discussed the results and commented on the manuscript.
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Nature Immunology thanks André Veillette, Bing Su and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Ioana Visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the Nature Immunology team.
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Extended data
Extended Data Fig. 1 ERMAP is a suppressor of liver metastasis.
a, Representative liver images of nude mice at four weeks post-intrasplenic injection of Hepa1-6 cells transduced with the GeCKO library. b, Flow cytometry showing the expression of ERMAP in wild-type (WT) and ERMAPKO Hepa1-6 cells. c, Flow cytometry showing the expression of ERMAP in ERMAPEV and ERMAPOE Hepa1-6 cells. d, Representative HE-stained images of liver tissues isolated from C57BL/6 mice at day 19 post-intrasplenic injection of 5 × 106 wild-type or ERMAPKO Hepa1-6 cells and percentages of tumor-replaced areas in liver tissues. Scale bars, 2 mm. e, Representative HE-stained images of liver tissues isolated from C57BL/6 mice at day 49 post-intrasplenic injection of 5 × 106 ERMAPEV or ERMAPOE Hepa1-6 cells and percentages of tumor-replaced areas in liver tissues. Scale bars, 1 mm. f, Flow cytometry showing the expression of ERMAP in ERMAPshCtrl and ERMAPKD SNU-398 cells. g, Flow cytometry showing the expression of ERMAP in ERMAPEV and ERMAPOE SNU-398 cells. h, Flow cytometry showing the expression of ERMAP in ERMAPshCtrl and ERMAPKD B16F10 cells. i, Representative HE-stained images of liver tissues isolated from nude mice at day 10 post-intrasplenic injection of 2×106 ERMAPshCtrl or ERMAPKD B16F10 cells and percentages of tumor-replaced areas in liver tissues. Scale bars, 1 mm. j, Flow cytometry showing the expression of ERMAP in ERMAPEV and ERMAPOE B16F10 cells. k, Representative HE-stained images of liver tissues isolated from nude mice at day 14 post-intrasplenic injection of 2×106 ERMAPEV or ERMAPOE B16F10 cells and percentages of tumor-replaced areas in liver tissues. Scale bars, 1 mm. l, Representative HE-stained images of liver tissues isolated from C57BL/6 mice at day 14 post-intrasplenic injection of 2 × 106 ERMAPshCtrl or ERMAPKD B16F10 cells and percentages of tumor-replaced areas in liver tissues. Scale bars, 1 mm. m, Representative HE-stained images of liver tissues isolated from C57BL/6 mice at day 14 post-intrasplenic injection of 2 × 106 ERMAPEV or ERMAPOE B16F10 cells and percentages of tumor-replaced areas in liver tissues. Scale bars, 1 mm. Results are shown as mean ± s.e.m. or representative images of n = 3 independent experiments (b, c, f-h, j) or n = 6 (d, e, i, k-m) mice in each group and were analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparisons test (d, i, l) or two-sided Mann–Whitney test (e, k, m).
Extended Data Fig. 2 ERMAP had no effect on lung metastasis, but repressed subcutaneous tumor growth.
a, Representative HE-stained images of lung tissues isolated from nude mice at day 28 post-tail vein injection of 5 × 106 wild-type (WT) or ERMAPKO Hepa1-6 cells and numbers of lung metastases per mouse. Scale bars, 1 mm. b, Representative HE-stained images of lung tissues isolated from nude mice at day 28 post-tail vein injection of 5 × 106 ERMAPOE Hepa1-6 cells and numbers of lung metastases per mouse. Scale bars, 1 mm. c, Representative HE-stained images of lung tissues isolated from nude mice at day 14 post-tail vein injection of 2 × 106 ERMAPKD B16F10 cells and percentages of tumor-replaced areas in lung tissues. Scale bars, 1 mm. d, Representative HE-stained images of lung tissues isolated from nude mice at day 14 post-tail vein injection of 2 × 106 ERMAPOE B16F10 cells and percentages of tumor-replaced areas in lung tissues. Scale bars, 1 mm. e, Subcutaneous tumor volume at days 5, 8, 11, 14, and tumor weight and tumor image at day 14 post-subcutaneous injection of 5 × 106 wild-type or ERMAPKO1 Hepa1-6 cells into nude mice. f, Subcutaneous tumor volume at days 5, 8, 11, 14, and tumor weight and tumor image at day 14 post-subcutaneous injection of 5 × 106 wild-type or ERMAPKO2 Hepa1-6 cells into nude mice. g, Subcutaneous tumor volume at days 5, 8, 11, 14, 21, and tumor weight and tumor image at day 21 post-subcutaneous injection of 5 × 106 ERMAPOE Hepa1-6 cells into nude mice. h, Subcutaneous tumor volume at days 3, 7, 14, 21, tumor weight and tumor image at day 21 post-subcutaneous injection of 5 × 106 ERMAPOE SNU-398 cells into nude mice. i, Subcutaneous tumor weight and tumor image at day 13 post-subcutaneous injection of 5 × 106 ERMAPOE B16F10 cells into nude mice. Results are shown as mean ± s.e.m. of n = 6 (a-d, h) mice, n = 8 (e, g, i) mice, or n = 7 (f) mice in each group and were analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparisons test (a, c) or two-sided Mann–Whitney test (b, d-i).
Extended Data Fig. 3 ERMAP promotes in vitro phagocytosis of cancer cells by Kupffer cells.
a,b, Live-cell microscopy showing in vitro phagocytosis of pHrodo Red+ GFP+ ERMAPKO (a) or ERMAPOE (b) Hepa1-6 cells by Kupffer cells at 0, 6, 12, 24 hours post co-culture. Scale bars, 100 µm. c,d, Flow cytometry (c) or fluorescence microscopy (d) showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPKD SNU-398 cells after co-culture for 2 h. e,f, Flow cytometry (e) or fluorescence microscopy (f) showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPKD B16F10 cells after co-culture for 2 h. g,h, Flow cytometry (g) or fluorescence microscopy (h) showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE SNU-398 cells after co-culture for 2 h. i,j, Flow cytometry (i) or fluorescence microscopy (j) showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE B16F10 cells after co-culture for 2 h. Results are shown as mean ± s.e.m. of n = 3 independent experiments and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test (a, c-f) or two-sided Student’s t-test (b, g-j).
Extended Data Fig. 4 ERMAP-induced phagocytosis is not dependent on CD47, PD-L1 and CD24, and is tumor cell specific.
a-c, Flow cytometry showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPKO Hepa1-6 cells (a), ERMAPOE Hepa1-6 cells (b), or ERMAPOE B16f10 cells (c) after co-culture for 2 h in the presence of CD47 or PD-L1 blocking Ab or IgG control. d, Flow cytometry showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE SNU-398 cells after co-culture for 2 h in the presence of CD24 blocking Ab or IgG control. e, Flow cytometry showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE hepatocytes, T cells, B cells, NK cells, MC38, and CT26 cells after co-culture for 2 h. Results are shown as mean ± s.e.m. of n = 3 independent experiments and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test (a) or two-sided Student’s t-test (b-e).
Extended Data Fig. 5 ERMAP promotes in vivo phagocytosis of cancer cells by Kupffer cells.
a, Flow cytometry representative plots and percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPKD SNU-398 cells 12 h post-intrasplenic inoculation. b, Confocal microscopy showing percentages of CFSE-labeled ERMAPKD SNU-398 cells phagocytosed by F4/80+ Kupffer cells 12 h post-intrasplenic inoculation. c, Flow cytometry representative plots and percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE SNU-398 cells 12 h post-intrasplenic inoculation. d, Confocal microscopy showing percentages of CFSE-labeled ERMAPOE SNU-398 cells phagocytosed by F4/80+ Kupffer cells 12 h post-intrasplenic inoculation. e, Flow cytometry representative plots and percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPKD B16F10 cells 12 h post-intrasplenic inoculation. f, Confocal microscopy showing percentages of CFSE-labeled ERMAPKD B16F10 cells phagocytosed by F4/80+ Kupffer cells 12 h post-intrasplenic inoculation. g, Flow cytometry representative plots and percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE B16F10 cells 12 h post-intrasplenic inoculation. h, Confocal microscopy showing percentages of CFSE-labeled ERMAPOE B16F10 cells phagocytosed by F4/80+ Kupffer cells 12 h post-intrasplenic inoculation. Results are shown as mean ± s.e.m. of n = 5 mice in each group and were analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparisons test (a, b, e, f) or two-sided Mann–Whitney test (c, d, g, h).
Extended Data Fig. 6 Kupffer cells phagocytosis of cancer cells controls liver metastasis.
a,b, Luciferase signal intensities at days 0, 1, 3 after intrasplenic injection of ERMAPOE Hepa1-6 (a) or SNU-398 (b) cells in nude mice that received intraperitoneal injection with clodronate liposomes on day -1. c,d, Flow cytometry showing the apoptosis of non-phagocytosed Hepa1-6 cells using Annexin V (c) or cleaved caspase-3 (d) staining after co-culture of CFSE-labeled wild-type (WT) or ERMAPKO Hepa1-6 cells with Kupffer cells for 12 h. e,f, Flow cytometry showing the apoptosis of non-phagocytosed Hepa1-6 cells using Annexin V (e) or cleaved caspase-3 (f) staining after co-culture of CFSE-labeled ERMAPOE Hepa1-6 cells with Kupffer cells for 12 h. g-i, Confocal microscopy showing percentages of cleaved caspase-3 (g), Ki67 (h), or PCNA (i) positive non-phagocytosed CFSE-labeled ERMAPKO or ERMAPOE Hepa1-6 cells 12 h post-intrasplenic inoculation. Scale bars, 5 µm. j,k Representative HE-stained images of liver tissues isolated from C57BL/6 mice that received intraperitoneal injection with clodronate liposomes on day -1 before the intrasplenic injection with ERMAPOE Hepa1-6 (j) or B16F10 (k) cells and analyzed at day 28 (j) or 10 (k) post-intrasplenic injection and percentages of tumor-replaced areas in liver tissues. Scale bars, 1 mm. Results are shown as mean ± s.e.m. of n = 3 independent experiments (c-f), n = 6 (a, b, j, k) or n = 5 (g-i) mice in each group and were analyzed by two-sided Mann–Whitney test (a, b, comparisons between ERMAPEV and ERMAPOE of g-i, j, k), one-way ANOVA followed by Dunnett’s multiple comparisons test (c, d), two-sided Student’s t-test (e, f), or Kruskal–Wallis test followed by Dunn’s multiple comparisons test (comparisons between ERMAPKO1, ERMAPKO2 and WT of g-i).
Extended Data Fig. 7 ERMAP–Gal-9 interaction promotes the in vitro phagocytosis of cancer cells by Kupffer cells.
a, Flow cytometry showing the expression of ERMAP in ERMAPOE or ERMAPN135AOE Hepa1-6 cells. b, Flow cytometry showing the expression of ERMAP in ERMAPOE or ERMAPN132AOE SNU-398 cells. c,d, Flow cytometry (c) or fluorescence microscopy (d) showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE or ERMAPN132AOE SNU-398 cells after co-culture for 2 h in the presence or absence of Gal-9 neutralizing antibody. e,f, Flow cytometry (e) or fluorescence microscopy (f) showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPKD SNU-398 cells after co-culture for 2 h in the presence or absence of Gal-9 neutralizing antibody. g, Flow cytometry showing the expression of ERMAP in ERMAPOE or ERMAPN135AOE B16F10 cells. h,i, Flow cytometry (h) or fluorescence microscopy (i) showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE or ERMAPN135AOE B16F10 cells after co-culture for 2 h in the presence or absence of Gal-9 neutralizing antibody. Results are shown as mean ± s.e.m. or representative images of n = 3 independent experiments in each group and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test.
Extended Data Fig. 8 ERMAP–Gal-9 interaction promotes the in vivo phagocytosis of cancer cells by Kupffer cells and represses liver metastasis.
a, Flow cytometry representative plots and percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE or ERMAPN132AOE SNU-398 cells 12 h post-intrasplenic inoculation. b, Confocal microscopy showing percentages of CFSE-labeled ERMAPOE or ERMAPN132AOE SNU-398 cells phagocytosed by F4/80+ Kupffer cells 12 h post-intrasplenic inoculation. c, Flow cytometry representative plots and percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE or ERMAPN135AOE B16F10 cells 12 h post-intrasplenic inoculation. d, Confocal microscopy showing percentages of CFSE-labeled ERMAPOE or ERMAPN135AOE B16F10 cells phagocytosed by F4/80+ Kupffer cells 12 h post-intrasplenic inoculation. e, Representative HE-stained images of liver tissues isolated from C57BL/6 mice at day 49 post-intrasplenic injection with ERMAPOE or ERMAPN135AOE Hepa1-6 cells and percentages of tumor-replaced areas in liver tissues. Scale bars, 1 mm. f, Representative HE-stained images of liver tissues isolated from nude mice at day 14 post-intrasplenic injection with ERMAPOE or ERMAPN135AOE B16F10 cells and number of hepatic metastases per mouse. Scale bars, 1 mm. Results are shown as mean ± s.e.m. or representative images of n = 5 (a-d) or n = 6 (e, f) mice in each group and were analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparisons test.
Extended Data Fig. 9 ERMAP–Gal-9–dectin-2 ligation promotes the phagocytosis of cancer cells by Kupffer cells and represses liver metastasis.
a,b, Flow cytometry (a) or fluorescence microscopy (b) showing percentages of wild-type (Clec6a+/+) or Clec6a-/- Kupffer cells phagocytizing CFSE-labeled ERMAPOE SNU-398 cells after co-culture for 2 h. c,d, Flow cytometry (c) or fluorescence microscopy (d) showing percentages of wild-type or Clec6a-/- Kupffer cells phagocytizing CFSE-labeled ERMAPKD SNU-398 cells after co-culture for 2 h. e,f, Flow cytometry (e) or fluorescence microscopy (f) showing percentages of wild-type or Clec6a-/- Kupffer cells phagocytizing CFSE-labeled ERMAPOE B16F10 cells after co-culture for 2 h. g, Flow cytometry representative plots and percentages of wild-type or Clec6a-/- F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE B16F10 cells 12 h post-intrasplenic inoculation. h, Confocal microscopy showing percentages of CFSE-labeled ERMAPOE B16F10 cells phagocytosed by wild-type or Clec6a-/- F4/80+ Kupffer cells12h post-intrasplenic inoculation. i, Representative HE-stained images of liver tissues isolated from wild-type or Clec6a-/- mice at day 14 post-intrasplenic injection with ERMAPOE B16F10 cells and percentages of tumor-replaced areas in liver tissues. Scale bars, 1 mm. j, Flow cytometry showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPOE B16F10 cells after co-culture for 2 h in the presence of the indicated inhibitors, control [dimethyl sulfoxide (DMSO)], Syk kinase inhibitor (piceatannol), mTOR inhibitor (Torin1), Src family kinase inhibitor (PP2), PI3K inhibitor (LY294002), NF-κB inhibitor (JSH-23), P38 inhibitor (SB203580), BTK inhibitor (LFM-A13), or MEK inhibitor (PD98059). k, Flow cytometry showing percentages of F4/80+ Kupffer cells phagocytizing CFSE-labeled ERMAPEV or ERMAPOE B16F10 cells in the presence of Syk kinase inhibitor (piceatannol). Results are shown as mean ± s.e.m. of n = 3 independent experiments (a-f, j, k), n = 5 (g, h) or n = 6 (i) mice in each group and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test (a-f), two-sided Mann–Whitney test (g-i) or two-sided Student’s t-test (j, k).
Extended Data Fig. 10 ERMAP–Gal-9–dectin-2 ligation doesn’t mediate the phagocytosis of cancer cells by BMDMs.
a, b, Flow cytometry (a) or immunofluorescence using FITC-conjugated antibody against human Fc (b) showing the binding of ERMAP-Fc to wild-type (Clec6a+/+) or Clec6a-/- BMDMs. Scale bars, 100 µm. c,d, Flow cytometry (c) or fluorescence microscopy (d) showing percentages of wild-type or Clec6a-/- BMDMs phagocytizing CFSE-labeled ERMAPOE Hepa1-6 cells after co-culture for 2 h. e,f, Flow cytometry (e) or fluorescence microscopy (f) showing percentages of wild-type or Clec6a-/- BMDMs phagocytizing CFSE-labeled ERMAPOE SNU-398 cells after co-culture for 2 h. Results are shown as mean ± s.e.m. or representative images of n = 3 independent experiments and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test.
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Supplementary Information
Supplementary Figures 1-6, Supplementary Tables 3 and 4, and Supplementary Video legends.
Supplementary Video
Kupffer cells preferentially phagocytizes wild-type Hepa1-6 cells compared to ERMAPKO Hepa1-6 cells. Kupffer cells were labeled with Dil dye (red), wild-type Hepa1-6 cells were labeled with CFSE (green), and ERMAPKO Hepa1-6 cells were labeled with Violet (blue). The phagocytoses of Hepa1-6 cells by Kupffer cells were detected by time-lapse imaging using the Nikon CSU-W1 spinning disc microscope. Scale bars, 10 µm.
Supplementary Table
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Li, J., Liu, Xg., Ge, Rl. et al. The ligation between ERMAP, galectin-9 and dectin-2 promotes Kupffer cell phagocytosis and antitumor immunity. Nat Immunol 24, 1813–1824 (2023). https://doi.org/10.1038/s41590-023-01634-7
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DOI: https://doi.org/10.1038/s41590-023-01634-7
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