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Tumors evade immune cytotoxicity by altering the surface topology of NK cells

Nature Immunology volume 24, pages 802–813 (2023)Cite this article

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Abstract

The highly variable response rates to immunotherapies underscore our limited knowledge about how tumors can manipulate immune cells. Here the membrane topology of natural killer (NK) cells from patients with liver cancer showed that intratumoral NK cells have fewer membrane protrusions compared with liver NK cells outside tumors and with peripheral NK cells. Dysregulation of these protrusions prevented intratumoral NK cells from recognizing tumor cells, from forming lytic immunological synapses and from killing tumor cells. The membranes of intratumoral NK cells have altered sphingomyelin (SM) content and dysregulated serine metabolism in tumors contributed to the decrease in SM levels of intratumoral NK cells. Inhibition of SM biosynthesis in peripheral NK cells phenocopied the disrupted membrane topology and cytotoxicity of the intratumoral NK cells. Targeting sphingomyelinase confers powerful antitumor efficacy, both as a monotherapy and as a combination therapy with checkpoint blockade.

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Fig. 1: Intratumoral NK cells lack abundant membrane protrusions.
Fig. 2: Intratumoral NK cells are impaired in recognizing tumor cells.
Fig. 3: Decreased SM level in intratumoral NK cell membranes.
Fig. 4: Inhibition of SM biosynthesis disrupts membrane topology of NK cells.
Fig. 5: Dysregulated serine metabolism contributes to SM decrease in NK cells.
Fig. 6: Inhibition of SM catabolism promotes the antitumor function of NK cells.

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Data availability

The clinical characteristics of patients are shown in Supplementary Tables 1–3 and 5. All data are present in the article and Supplementary information files or are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the Natural Science Foundation of China (grant reference nos. 82122055, 82241216, 81872318 and 22025405), the National Key Research and Development Program of China (grant no. 2019YFA0405603), the CAMS Innovation Fund for Medical Sciences (CIFMS; grant no. 2019-I2M-5-073) and the Strategic Priority Research Program of the Chinese Academy of Sciences (grant nos. XDB04050200, XDPB1002 and XDB39020700).

Author information

Author notes

  1. These authors contributed equally: Xiaohu Zheng, Zhuanghao Hou.

Authors and Affiliations

  1. Hefei National Research Center for Physical Sciences at the Microscale, CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Xiaohu Zheng, Xuben Wang, Yiqing Shen, Yonggang Zhou, Binqing Fu, Rui Sun, Zhigang Tian & Haiming Wei

  2. Institute of Immunology, Biomedical Sciences and Health Laboratory of Anhui Province, University of Science and Technology of China, Hefei, China

    Xiaohu Zheng, Xuben Wang, Yiqing Shen, Yonggang Zhou, Binqing Fu, Rui Sun, Zhigang Tian & Haiming Wei

  3. The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China

    Xiaohu Zheng, Zhuanghao Hou, Guangming Huang & Haiming Wei

  4. School of Chemistry and Materials Science and National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, China

    Zhuanghao Hou & Guangming Huang

  5. Department of General Surgery, First Affiliated Hospital of Anhui Medical University, Hefei, China

    Yeben Qian, Yongwei Zhang & Quanwei Cui

  6. Core Facility Center for Life Sciences, University of Science and Technology of China, Hefei, China

    Zhenbang Liu

  7. Research Unit Of NK Cells, Chinese Academy Of Medical Sciences, Hefei, China

    Zhigang Tian & Haiming Wei

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Contributions

H.W., G.H. and Z.T. conceived and conducted the project. H.W., X.Z. and G.H. supervised the project. X.Z. and Z.H. wrote the paper. X.Z. and Z.H. performed the experiments and data analysis. X.Z. contributed to mouse models and cell culture. Y.Q., Y.S., Q.C. and Y.W.Z. collected tissue samples and information from patients. R.S., Y.G.Z, Z.L., X.W. and B.F. contributed to imaging analysis and interpreted the data.

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Correspondence to Xiaohu Zheng, Zhigang Tian, Guangming Huang or Haiming Wei.

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Nature Immunology thanks Thomas Braun, David Finlay, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. N. Bernard was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Imaging of membrane protrusions of NK cells.

a, Gating strategy of NK cells for flow cytometry. b, Representative density plots show the purity of NK cells. c–d, Related to Fig. 1a–g, The mean diameter of membrane protrusions (c, TEM; d, SEM) in each sample is shown. Each dot represents one sample. n = 10 in each group. e–g, CLSM shows the membrane protrusions of purified NK cells. e, Representative 2D CLSM images (up) and 3D resultant ‘cartoon’ images (down). Scale bar, 1 μm. f–g, The mean number of membrane protrusions and mean length of membrane protrusions (2D CLSM) in each sample are shown. Each dot represents one sample. n = 8 in each group. Data are the mean ± s.d. Data were analysed by two-way ANOVA.

Source data

Extended Data Fig. 2 SEM showing the membrane protrusions of intratumoral NK cells from multiple cancers.

a–c, SEM showing the membrane protrusions of the NK cells purified from resected tumors and outside the tumor of lung-cancer, colon-cancer, and ovarian-cancer patients. The mean number of membrane protrusions and the mean length of membrane protrusions in each sample are shown. Each dot represents one sample. n = 5 in each group. Data are the mean ± s.d. Scale bar, 1 μm. Data were analysed by two-tailed, unpaired Student’s t-tests.

Source data

Extended Data Fig. 3 Analyses of binding and immunological synapses of human liver-cancer cells and NK cells.

a, SEM showing the immunological synapses of human liver-cancer cells and NK cells. Primary NK cells purified from resected tumors of liver-cancer patients, of liver NK cells from outside the tumor of these liver-cancer patients, and of NK cells isolated from peripheral blood of normal donors and were co-cultured with liver-cancer cells (HuH7 cell line) for 1, 2 and 4 h. The large cells on the left represent HuH7 cells. The small cells on the right represent NK cells. Scale bar, 0.5 μm. Data are representative of 9 samples. b, (related to Fig. 2c), TEM showing the interface of human liver-cancer cells and NK cells at low magnification. NK cells freshly purified from liver cancer patients (tumor and liver tissue outside tumor) and normal donor peripheral blood and were co-cultured with HepG2 cells for 1, 2 or 4 h. The large cells are HepG2 cells; the small cells are NK cells. The red arrows guides the junction of human liver-cancer cells and NK cells. Scale bar, 1 μm. Data are representative of 5 samples in each group. c, Super resolution microscopy (Leica, STED) show the polymerized actin filaments (microclusters) of purified NK cells. Representative 3D images were shown. Scale bar, 1 μm. d, Fluorescence photobleaching recovery (FPR) was used to assess membrane fluidity (DiI membrane stain) based on CLSM imaging. Data are representative of 2 samples in each group. e, CLSM showing the binding of human liver-cancer cells and NK cells. NK cells freshly purified from liver cancer patients (tumor and liver tissue outside tumor) and normal donor peripheral blood and were co-cultured with HepG2 (liver cancer cell line) cells. The large cells on the left are HepG2 cells; the small cells on the right are NK cells. Green, F-actin staining. Scale bar, 0.5 μm. f, K562 cells were used as target cells in cytotoxicity assays with NK cells isolated from liver cancer patients (tumor and liver tissue outside tumor) and normal donor peripheral blood. % target cell death presents the percent of dead target cells among total target cells (7AAD + CFSE + ) assessed by flow cytometry. n = 3 samples per group. g–h, CLSM showing GZMB and CD107a stain in NK cells. NK cells freshly purified from liver cancer patients (tumor and liver tissue outside tumor) and normal donor peripheral blood and were co-cultured with HepG2 (liver cancer cell line) cells. Scale bar, 0.5 μm. Data are the mean ± s.d. Data were analyzed by two-way ANOVA.

Source data

Extended Data Fig. 4 MS/MS assays sphingomyelins of NK cells.

a–f, Representative MS/MS spectra for membrane sphingomyelins (a, SM(d34:1); b, SM(d38:0); c, SM(d36:1); d, SM(d36:0); e, SM(d42:2); f, SM(d43:2)) obtained from NK-cell lysates. Fragment ions correspond to relative neutral loss from the headgroup of SMs are marked in red. g, The p values for Fig. 3F. Data are the mean ± s.d. Data were analysed by two-way ANOVA.

Extended Data Fig. 5 Detection of the components of sphingolipin in NK cells by sIC-MS and bulk LC-MS.

a, sIC-MS and bulk LC-MS in detecting standard of sphingomyelin (SM(d34:1) and SM(d36:1)). The range of concentration from 0.2 to 50 mg/L. With sIC-MS, for the standard curve of SM(d34:1), the slope was 2.021 × 10−2, R2 = 0.9925 and the standard curve of SM(d36:1)), the slope was 2.012 × 10−2, R2 = 0.9977. With bulk LC-MS, for the standard curve of SM(d34:1), the slope was 1.977 × 10−2, R2 = 0.9981. And the standard curve of SM(d36:1)), the slope was 1.979 × 10−2, R2 = 0.9923. b, LC-MS was used to analyse the SM content of purified peripheral NK cells from healthy donors. Freshly purified NK cells were treated with D609 (25, 100, 400 μM) or PBS (control) for 24 h. n = 5 samples per group. Data are the mean ± s.d. Data were analysed by two-way ANOVA.

Source data

Extended Data Fig. 6 The phenotype of NK cells after inhibition of sphingomyelin synthesis.

a, CLSM showing the binding of human liver-cancer cells and NK cells. NK cells freshly purified from normal donor peripheral blood and were co-cultured with HepG2 (liver cancer cell line) cells. Peripheral NK cells were treated with or without the small molecule sphingomyelin synthase inhibitor D609 (25, 100, 400 μM) or PBS (control) for 24 h. The large cells on the left are HepG2 cells; the small cells on the right are NK cells. Green, F-actin staining. Scale bar, 0.5 μm. b–c, Flow cytometry showing apoptosis (annexin-V+) in NK cells after treatment with D609 (25, 100, 400 μM) or PBS (control) for 24 h or knockdown of sphingomyelin synthase 1 (si-SGMS1). n = 5 samples per group. c (left), Western blotting analysis of SGMS1 expression in primary NK cells isolated from normal donor peripheral blood. The siRNA-SGMS1 and siRNA-mock were transfected into peripheral NK cells for 24 h until analysis. d, Flow cytometry assaying proliferation (Ki67+) in NK cells after treatment with D609 (25, 100, 400 μM) or PBS (control) for 24 h or knockdown of sphingomyelin synthase 1 (si-SGMS1). n = 5 samples per group. e–f, Flow cytometry assay of the percentage of IFN-γ, perforin, and granzyme-B (GZMB) producing cells among IL-12+IL-18 or PBS (Control) stimulated NK cells from normal donor peripheral blood. The MFI of IFN-γ in IL-12+IL-18-stimulated IFN-γ+ NK cells, perforin in IL-12+IL-18-stimulated perforin+ NK cells, and GZMB in IL-12+IL-18-stimulated GZMB+ NK cells are presented as the mean ± s.d.. Peripheral NK cells pre-treated with D609 (25, 100, 400 μM) or PBS (control) for 24 h before IL-12 and IL-18 stimulation. n = 5 samples per group. N.D., not detected. g, FACS assay of CD107a levels in peripheral NK cells. Peripheral NK cells were treated with D609 or PBS for 24 h. Then, NK cells were co-cultured (Co-cultured group) with or without (Control, single-cultured group) HepG cells before FACS assay. n = 5 donor samples per group. h, i (related to Fig. 5m and n) HepG2 cells and (J) K562 cells were used as target cells in cytotoxicity assays with indicated NK cells. % cytolysis is shown (n = 5 per group). h, j, Purified NK cells were co-cultured with HepG2 cells or K562 cells for cytotoxicity assays after treatment with the small molecule sphingomyelin synthase inhibitor D609 (25, 100, 400 μM) or PBS (control) for 24 h. i, Purified NK cells were co-cultured with HepG2 cells for cytotoxicity assays after knockdown of sphingomyelin synthase 1 (si-SGMS1) or control (si-mock) for 24 h. j, n = 3 per group. k, The ADCC (antibody-dependent cell-mediated cytotoxicity) activity assays of NK cells. Target cells, HepG2 cells (An EpCAM+ liver cancer cell line); Ab, humanized antibodies against EpCAM; peripheral NK cells pre-treated with D609 (25, 100, 400 μM) or PBS (control) for 24 h. n = 3 samples. l, SEM images showing the membrane protrusions of purified NK cells. Representative SEM imaging (left) and the mean number of membrane protrusions (right) in each sample are shown. NK cells were treated with IL-2, IL-2 + IL-15, or IL-2 + IL-15 + IL-12. Each dot represents one sample. n = 6 in each group. Scale bar, 1 μm. m, Fresh NK cells isolated from liver cancer patients (tumor and liver tissue outside tumor) and normal donor peripheral blood (n = 8 in each group). Peripheral NK cells were treated with or without the small molecule sphingomyelin synthase inhibitor D609 (25, 100, 400 μM) or PBS (control) for 24 h (n = 5 in each group). Using a Transwell™ apparatus, we tested the migration of the NK cells from the upper compartment to the lower compartment. Counting the numbers of NK cell migrating to the lower layer. n, Flow cytometry assay of CD29 and CD18 levels in intratumoral NK cells, liver NK cells and peripheral NK cells. n = 5 samples per group. Data are the mean ± s.d. Data were analysed by two-way ANOVA.

Source data

Extended Data Fig. 7 The potential relationships between cholesterol and sphingomyelin levels in NK cells.

a, CLSM showing the cholesterol signals for purified intratumoral NK cells, liver NK cells and peripheral NK cells by filipin III staining. Scale bar, 1 μm. b–c, CLSM of filipin III stained purified peripheral NK cells staining (cyclodexrin treated or untreated (vehicle, PBS); D609 treated or untreated (vehicle, PBS) to assess cholesterol levels. sIC-MS was used to analyse the membrane SM content of purified peripheral NK cells (cyclodexrin treated or PBS). Scale bar, 1 μm. n = 9 in each group. d, SEM image showing the membrane protrusions of purified peripheral NK cells. Peripheral NK cells were treated with cyclodextrin. Each dot represents one sample. n = 5 samples per group. Scale bar, 1 μm. Data are the mean ± s.d. Data were analyzed by two-tailed unpaired Student’s t-test. e, Purified peripheral NK cells were co-cultured with HepG2 cells for cytotoxicity assays after treatment of cyclodextrin. n = 4 samples per group. f, Single-cell MS was employed to analyze the cholesterol in intratumoral NK cells, peripheral NK cells, and liver NK cells. n = 5 samples per group. Data are the mean ± s.d. Data were analyzed by two-tailed unpaired Student’s t-test.

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Extended Data Fig. 8 The phenotype of intratumoral NK cells after blockade of SM catabolism.

a, SEM image showing the membrane protrusions of purified intratumoral NK cells from liver cancer patients. Intratumoral NK cells were subjected to knockdown of an acidic SMase (si-ASM), of neutral SMases (si-NSMASE1/2/3), or control (si-mock). Each dot represents one patient sample. n = 5 samples. Scale bar, 1 μm. b–e, SEM image showing the membrane protrusions of purified peripheral and liver NK cells. Representative SEM imaging is presented. The mean number of membrane protrusions and the mean length of membrane protrusions in each sample are shown. Each dot represents one sample. n = 5 samples. Scale bar, 1 μm. f, Flow cytometry assaying proliferation (Ki67+) and apoptosis (annexin-V+) of intratumoral NK cells. g, Flow cytometry assaying the expression of IFN-γ, granzyme-B (GZMB), CD107a and perforin in intratumoral NK cells. n = 5 per group. f–h, Intratumoral NK cells were treated with GW4869 (1 μM), LCL521 (1 μM), or DMSO (control) for 24 h. h–i, (Related to Fig. 6g and i) human liver-cancer cells (HepG2) were employed as targets in cytotoxicity assays with indicated intratumoral cells by real-time Cell Index measurements (xCELLigence). % cytolysis is shown (n = 6 per group). Intratumoral NK cells cells were co-cultured with liver-cancer cells for cytotoxicity assays after treatment with GW4869 (1 μM) + anti-Tim3-blocking antibody (10 μg/mL), LCL521 (1 μM)+anti-Tim3-blocking antibody (10 μg/mL), GW4869 (1 μM), LCL521 (1 μM), anti-Tim3-blocking antibody (10 μg/mL), or DMSO (control) for 24 h. Data are the mean ± s.d. Data were analysed by two-way ANOVA.

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Extended Data Fig. 9 Analyses of the membrane protrusions of mouse liver cancer NK cells.

a–f, A genetically engineered mouse model of liver cancer developed with weakened PTEN signaling and with an activated K-RAS signal. a, Images of the liver from liver cancer model and normal mice. b, Representative density plots showing the purity of sorted mouse NK cells. c, SEM showing the membrane protrusions of the mouse NK cells from normal liver, spleen, and liver cancer. Representative SEM imaging (left). The mean number of membrane protrusions and the mean length of membrane protrusions (SEM) in each sample are shown (middle and right). n = 5 per group. d, SEM showing the membrane protrusions of the mouse NK cells from liver cancer. Freshly purified NK cells were treated with sphingomyelinase inhibitors (GW4869; 1 μM) or DMSO (control) for 24 h. n = 5 per group. e, Mouse liver-cancer cells (K-RASG12DPTEN−/− primary liver caner cell and Hep1-6 cell line) were employed as targets in cytotoxicity assays with the indicated NK cells, assessed with real-time Cell Index measurements (xCELLigence). % cytolysis is shown. After treatment with GW4869 (1 μM), or DMSO (control) for 24 h, liver cancer NK cells were co-cultured with target cells for cytotoxicity assays. n = 4 per group. f, Two mouse liver cancer models were established by subcutaneous injection of liver cancer cells (K-RASG12DPTEN−/− primary liver caner cell and Hep1-6 cell line). Freshly purified normal spleen NK cells were treated with D609 or PBS (control) for 24 h, followed by treated with GW4869 (1 μM), or DMSO (control) for 24 h. n = 6 per group. g, Single-cell MS was employed to analyse membrane sphingomyelins (SMs) of purified mouse NK cells from normal liver, spleen, and liver cancer. Normalized intensities of membrane SMs are shown. n = 5 in each group. h, SEM showing the membrane protrusions of the mouse NK cells from normal spleen. Freshly purified NK cells were treated with D609 (25, 100, 400 μM) or PBS (control) for 24 h. Each dot represents one mouse. n = 5 in each group. Data are the mean ± s.d. Data were analysed by two-way ANOVA. Scale bar, 1 μm.

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Dynamical imaging for coincubation with intratumoral NK cells and HepG2 cells.

Dynamical imaging for coincubation with liver NK cells and HepG2 cells.

Dynamical imaging for coincubation with peripheral NK cells and HepG2 cells.

Dynamical imaging for coincubation with NK (PBS) cells and HepG2 cells.

Dynamical imaging for coincubation with NK (25 μM D609) cells and HepG2 cells.

Dynamical imaging for coincubation with NK (100 μM D609) cells and HepG2 cells.

Dynamical imaging for coincubation with NK (400 μM D609) cells and HepG2 cells.

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Zheng, X., Hou, Z., Qian, Y. et al. Tumors evade immune cytotoxicity by altering the surface topology of NK cells. Nat Immunol 24, 802–813 (2023). https://doi.org/10.1038/s41590-023-01462-9

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