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Reciprocal interactions between innate immune cells and astrocytes facilitate neuroinflammation and brain metastasis via lipocalin-2

Nature Cancer volume 4, pages 401–418 (2023)Cite this article

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Abstract

Brain metastasis still encompass very grim prognosis and therefore understanding the underlying mechanisms is an urgent need toward developing better therapeutic strategies. We uncover the intricate interactions between recruited innate immune cells and resident astrocytes in the brain metastatic niche that facilitate metastasis of melanoma and breast cancer. We show that granulocyte-derived lipocalin-2 (LCN2) induces inflammatory activation of astrocytes, leading to myeloid cell recruitment to the brain. LCN2 is central to inducing neuroinflammation as its genetic targeting or bone-marrow transplantation from LCN2−/− mice was sufficient to attenuate neuroinflammation and inhibit brain metastasis. Moreover, high LCN2 levels in patient blood and brain metastases in multiple cancer types were strongly associated with disease progression and poor survival. Our findings uncover a previously unknown mechanism, establishing a central role for the reciprocal interactions between granulocytes and astrocytes in promoting brain metastasis and implicate LCN2 as a prognostic marker and potential therapeutic target.

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Fig. 1: Systemic LCN2 signaling is upregulated in plasma and CSF in brain metastases and correlates with metastatic burden.
Fig. 2: LCN2 is systemically secreted by the microenvironment of primary tumors, upregulated in brain-tropic tumors and corelates with patient survival.
Fig. 3: LCN2 signaling is functionally important for brain metastases formation and originates mainly from granulocytes and endothelial cells.
Fig. 4: Inflammatory activation of astrocytes is partially mediated by specific LCN2 receptor signaling.
Fig. 5: LCN2 signaling facilitates recruitment of immune suppressive myeloid cells into brain metastases.
Fig. 6: LCN2 signaling from bone-marrow-derived cells plays a key functional role in facilitating brain metastasis.
Fig. 7: Microenvironment-derived LCN2 signaling is operative in human patients and correlates with decreased survival in patients with brain metastases from melanoma, breast and lung primary origin.
Fig. 8: Graphical summary.

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Data availability

Source data for Figs. 1–7 and Extended Data Figs. 1–7 have been provided as Source Data files.

All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

The authors thank Y. Zilberstein from the Faculty Cellular and Molecular Imaging Center for her help with intracardiac injections and mouse imaging. We thank the Rabin Medical Center Biobank and A. Levy-Barda for their support of this research. We also thank E. Vollmer, E. Ostermeier and K. Kronenberg from Regensburg University Hospital and L. Siam, M. Schaffrinski and D. Egert from Göttingen University Hospital, Germany, for their contribution in collecting and processing human samples. The study was supported by grants to N.E. from the Melanoma Research Alliance (award ID 826222), a Breakthrough Award from the US Department of Defense (BCRP award ID W81XWH2110394), Israel Science Foundation Personalized Medicine Program (IPMP no. 3495/19) and a research grant from the Tel Aviv University Cancer Biology Research Center. N.E. and T.P. were supported by a grant from the German Research Foundation (PU 355/4-1). T.P. was supported by SFB/TRR 305/1 (B03).

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Author notes

  1. These authors contributed equally: Omer Adler, Yael Zait.

Authors and Affiliations

  1. Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Omer Adler, Yael Zait, Noam Cohen, Hila Doron, Lea Monteran, Yeela Scharff, Tamar Shami, Dhanashree Mundhe & Neta Erez

  2. Department of Internal Medicine III, Hematology and Medical Oncology, University Hospital Regensburg, Regensburg, Germany

    Raquel Blazquez & Tobias Pukrop

  3. Department of Surgery, University Hospital Regensburg, Regensburg, Germany

    Gunther Glehr & James A. Hutchinson

  4. Department of Neurosurgery, Rabin Medical Center and Sackler Faculty of Medicine Tel Aviv University, Tel Aviv, Israel

    Andrew A. Kanner

  5. Department of Pathology, Rabin Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Suzana Horn

  6. Blood Services & Apheresis Institute, Rabin Medical Center and Tel Aviv University, Tel Aviv, Israel

    Vered Yahalom

  7. Department of Dermatology, University Hospital Regensburg, Regensburg, Germany

    Sebastian Haferkamp

  8. Department of Hematology/Medical Oncology, University Medical Center Göttingen, Göttingen, Germany

    Annalen Bleckmann

  9. Medical Clinic A, Haematology, Haemostasiology, Oncology and Pulmonology, University Hospital Münster, Münster, Germany

    Annalen Bleckmann

  10. West German Cancer Center, University Hospital Münster, Münster, Germany

    Annalen Bleckmann

  11. The Shmunis School of Biomedicine and Cancer Research, the George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

    Limor Nahary & Itai Benhar

  12. Neuro-Oncology Unit, Davidoff Cancer Center at Rabin Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Shlomit Yust Katz

  13. Division of Personalized Tumor Therapy, Fraunhofer Institute for Toxicology and Experimental Medicine, Regensburg, Germany

    Tobias Pukrop

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  1. Omer Adler
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Contributions

O.A., Y.Z., H.D. and N.E. conceived of and designed this study. O.A., Y.Z., N.C., T.P. and N.E. developed the methods. O.A., Y.Z., R.B., L.M. and N.C. performed the experiments. G.G., Y.S., T.S., D.M. and L.M. performed formal analysis. A.A.K., S.H., V.Y., S.H., J.A.H., A.B., S.Y.K., L.N, I.B. and T.P. provided resources. O.A., Y.Z., N.C. and N.E. wrote the original draft. O.A., Y.Z., N.C. and N.E. revised and edited the manuscript. N.C. and N.E. administered this project. N.E. supervised the study. All authors discussed the results and provided feedback on the manuscript.

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Correspondence to Neta Erez.

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Nature Cancer thanks Frank Winkler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Proteomic analysis of secreted proteins in blood of mice with melanoma or breast cancer brain metastasis.

Heatmap showing fold change values of secreted proteins measured by Proteome profiler Mouse XL Cytokine Array. Mean grey value was quantified by ImageJ. Heatmap represents Log2 (BrM/Normal) values.

Extended Data Fig. 2 LCN2 expression is induced by tumor cell-secreted factors, and is not necessary for primary tumor growth.

a. Gating strategy for FACS isolation of different cell populations from primary tumors 2.5 weeks following BT-RMS or BT-EO771 orthotopic injection. b–d. qPCR analysis of Lcn2 from BMDM, dermal fibroblasts and C166 endothelial cells treated in vitro with BT-RMS CM (BT_RMS CM) versus serum free medium (SFM). 3 biological repeats, error bars represent mean ± SD (Student’s t-test, two-sided). e. qPCR analysis of Lcn2 in primary dermal fibroblasts treated with secreted factors from RMS and sBT-RMS, error bars represent mean ± SD (SFM n = 3, RMS CM n = 3, SBT CM n = 3 biological replicates) (one-way ANOVA). f. Primary tumor growth curve of WT and Lcn2−/− mice orthotopically injected with BT-RMS melanoma cells, measured manually by caliper, (WT n = 10, Lcn2−/− n = 8 mice) (Repeated measure ANOVA). g. Primary tumor weight at time of resection of WT and Lcn2−/− mice orthotopically injected with BT-EO771 cells, (WT n = 10, Lcn2−/− n = 10 mice), dots represent individual mice, error bars represent s.e.m. (Student’s t-test, two-sided).

Extended Data Fig. 3 Brain metastatic burden and survival in mice injected with breast cancer cells.

a. Experimental scheme analyzed in (b,c). b. Survival curve analysis of WT and Lcn2-/- mice injected intracardially with BT-EO771 cells, (WT n = 10, Lcn2-/- n = 9 mice) (Kaplan–Meier curve, log-rank test). c. Quantification of brain metastatic burden for mice in (a), quantified as % CD45- mCherry+ tumor cells/live cells, (ctrl n = 9, WT n = 9, Lcn2-/- n = 7 mice) (one-way ANOVA). d. Gating strategy for isolation by FACS of different cells populations from WT and Lcn2-/- BrM mice injected intracardially with BT-RMS or BT-EO771 cells 18 days after injection.

Extended Data Fig. 4 Astrocytes activate pro-inflammatory signaling in an LCN2-dependent manner, and SLC22A17 is required for astrocyte response to LCN2.

a. Expression of SLC22A17 in bulk RNA-seq of different cell populations isolated from samples of human gliomas (CD45- n = 23, MG n = 21, MDM n = 17, Neutrophils n = 16, T cells n = 22 patients) (one-way ANOVA), (Brain TIME dataset). b. LCN2 is sufficient to induce pro-inflammatory signaling in primary astrocytes. qPCR analysis of inflammatory gene signature in primary astrocytes incubated with 10ug/ml rLCN2. Error bars represent SD, dots represent two biological repeats with technical replicates (two-way ANOVA). c. qPCR analysis of inflammatory gene signature in primary astrocytes transfected with siRNA targeting Slc22a17 or with control siRNA (siSlc22a17 or siScramble). Astrocytes were then treated with 10ug/ml rLCN2 or SFM for 24 h. Error bars represent SD, 4 biological repeats in duplicates. d. LCN2 measured in CM of BT-RMS cells or granulocytes activated by BT-RMS CM, error bars represent SD, dots represent 3 biological repeats (two-way ANOVA). e, f. Activation of astrocytes was quantified by number of GFAP + cells/ field. Representative images are shown from n = 6,8,8 mice per group, error bars represent mean ± SD. 6 fields X 1 sections per mouse were analyzed (one-way ANOVA). g, h. Expression level of inflammatory gene signature measured by qPCR in RNA of FACS sorted astrocytes in vivo from WT or Lcn2-/- mice with BrM following BT-RMS or BT-EO771 injection. Dots represent individual mice, line indicates median, plot shows mean to max (melanoma: Ctrl n = 4, WT n = 7,10, Lcn2-/- n = 5,9 mice), (breast: Ctrl n = 6, WT n = 5, Lcn2-/- n = 5,7 mice) (two-way ANOVA). i. Expression level of inflammatory gene signature measured by qPCR of FACS sorted astrocytes in vivo from WT or Lcn2-/- normal mice, (WT n = 3, Lcn2-/- n = 2 mice). j, k. Expression level of immunosuppressive gene signature measured by qPCR of sorted MG/MDM in vivo from WT or Lcn2-/- mice, 3 weeks following BT-RMS or BT-EO771 intracardiac injection, line indicates median, plot shows mean to max (Melanoma WT n = 7, Lcn2-/- n = 5, Breast WT n = 5, Lcn2-/- n = 6 mice) (two-way ANOVA).

Extended Data Fig. 5 LCN2 induces recruitment of immune suppressive granulocytes to brain in the in the context of BrM.

a. Quantification of migrated Ly6G+ granulocytes or Ly6C+ Ly6G− monocytes toward normal astrocytes or astrocytes treated 24 h with 10ug/ml rLCN2. Dots represent individual wells, 4 biological repeats in duplicates, error bars represent mean ± SD (Student’s t-test, two-sided). b. Immune profiling of bone marrow cell populations by flow cytometry of 8-week-old C57BL/6 WT or Lcn2−/− mice, (WT n = 5, Lcn2−/− n = 5 mice). c. Immune profiling of CD11b+ myeloid cells by flow cytometry of normal WT or Lcn2−/− mice (male WT n = 5, Lcn2−/− n = 3, female WT n = 6, Lcn2−/− n = 3 mice). d,e. Expression level of immunosuppressive gene signature measured by qPCR of FACS sorted granulocytes in vivo from WT or Lcn2−/− mice, 3 weeks following BT-RMS intracardiac injection, line indicates median, plot shows mean to max (Melanoma WT n = 11, Lcn2−/− n = 9, Breast WT n = 5, Lcn2−/− n = 6 mice) (two-way ANOVA).

Extended Data Fig. 6 Bone marrow-derived granulocyte induce increasing systemic LCN2 levels in plasma and inflammatory activation of astrocytes.

a. qPCR analysis of Lcn2 expression in different cell populations sorted from bone marrow of normal C57BL/6 males: CD45-, CD45+CD11b+Ly6CinterLy6G+ granulocytes, CD45+CD11b+Ly6C+Ly6G- monocytes, CD45+CD11b-CD3+ B220- T cells, CD45+CD11b-CD3-B220+ B cells. Dots represent individual mice (n = 4 mice per group), line indicates median, whisker shows mean to max (one-way ANOVA). b. ‘LCN2 BM contribution’ calculated by expression of LCN2 in different cell populations from a, multiplied by their abundance in BM from Extended Data Fig. 5b. Results are presented as percent of total. c. LCN2 ELISA in blood, one and two weeks following BMT. Dots represent individual mice, error bars represent s.e.m., (WT n = 9, Lcn2-/- n = 9 mice) (one-way ANOVA). d. LCN2 levels in blood of mice at endpoint measured by ELISA, (WT n = 7, Lcn2-/- n = 7 mice), dots represent individual mice, error bars represent s.e.m. (Student’s t-test, two-sided). e. Expression level of inflammatory gene signature measured by qPCR in RNA of FACS sorted astrocytes in vivo from WT mice that underwent BMT as described in (Fig. 4a). Dots represent individual mice, line indicates median, plot shows mean to max (Ctrl n = 4, WT n = 6, Lcn2-/- n = 6 mice) (two-way ANOVA).

Extended Data Fig. 7 LCN2 is highly expressed in granulocytes in human BrM and can help direct patient’s care in combination with clinically used prognostic factors.

a. Immunofluorescence staining in frozen sections of resected human brain metastases from melanoma, breast and lung primary origin. Co-localization of LCN2 with CD66b (granulocytes). Representative images of separate staining are shown from n = 2 patients samples stained per cancer type. b. Five-year survival curve analysis of patients with low versus high LCN2 levels in patients with lung cancer BrM. The cutoff between high and low levels was defined as the median LCN2 level (Kaplan–Meier curve, log-rank test). c. Five-year survival curve analysis of patients with KPS score over or under 70, in patients with lung cancer BrM. d. 5-year survival curve analysis of patients with KPS score < 70, stratified to low versus high LCN2 levels in patients from Fig. 7h.

Extended Data Table. 1 Data of patients with brain metastasis from melanoma, breast and lung cancer
Full size table
Extended Data Table. 2 Data of patients with untreated stage IV melanoma
Full size table
Extended Data Table. 3 Data of patients with brain metastasis from melanoma and lung cancer
Full size table

Supplementary information

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Adler, O., Zait, Y., Cohen, N. et al. Reciprocal interactions between innate immune cells and astrocytes facilitate neuroinflammation and brain metastasis via lipocalin-2. Nat Cancer 4, 401–418 (2023). https://doi.org/10.1038/s43018-023-00519-w

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