SLIT2/ROBO signaling in tumor-associated microglia and macrophages drives glioblastoma immunosuppression and vascular dysmorphia
Luiz H. Geraldo, Yunling Xu, Laurent Jacob, Laurence Pibouin-Fragner, Rohit Rao, Nawal Maissa, Maïté Verreault, Nolwenn Lemaire, Camille Knosp, Corinne Lesaffre, Thomas Daubon, Joost Dejaegher, Lien Solie, Justine Rudewicz, Thomas Viel, Bertrand Tavitian, Steven De Vleeschouwer, Marc Sanson, Andreas Bikfalvi, Ahmed Idbaih, Q. Richard Lu, Flavia R.S. Lima, Jean-Leon Thomas, Anne Eichmann, Thomas Mathivet
Luiz H. Geraldo, Yunling Xu, Laurent Jacob, Laurence Pibouin-Fragner, Rohit Rao, Nawal Maissa, Maïté Verreault, Nolwenn Lemaire, Camille Knosp, Corinne Lesaffre, Thomas Daubon, Joost Dejaegher, Lien Solie, Justine Rudewicz, Thomas Viel, Bertrand Tavitian, Steven De Vleeschouwer, Marc Sanson, Andreas Bikfalvi, Ahmed Idbaih, Q. Richard Lu, Flavia R.S. Lima, Jean-Leon Thomas, Anne Eichmann, Thomas Mathivet
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Research Article Oncology Vascular biology

SLIT2/ROBO signaling in tumor-associated microglia and macrophages drives glioblastoma immunosuppression and vascular dysmorphia

Abstract

SLIT2 is a secreted polypeptide that guides migration of cells expressing Roundabout 1 and 2 (ROBO1 and ROBO2) receptors. Herein, we investigated SLIT2/ROBO signaling effects in gliomas. In patients with glioblastoma (GBM), SLIT2 expression increased with malignant progression and correlated with poor survival and immunosuppression. Knockdown of SLIT2 in mouse glioma cells and patient-derived GBM xenografts reduced tumor growth and rendered tumors sensitive to immunotherapy. Tumor cell SLIT2 knockdown inhibited macrophage invasion and promoted a cytotoxic gene expression profile, which improved tumor vessel function and enhanced efficacy of chemotherapy and immunotherapy. Mechanistically, SLIT2 promoted microglia/macrophage chemotaxis and tumor-supportive polarization via ROBO1- and ROBO2-mediated PI3K-γ activation. Macrophage Robo1 and Robo2 deletion and systemic SLIT2 trap delivery mimicked SLIT2 knockdown effects on tumor growth and the tumor microenvironment (TME), revealing SLIT2 signaling through macrophage ROBOs as a potentially novel regulator of the GBM microenvironment and immunotherapeutic target for brain tumors.

Authors

Luiz H. Geraldo, Yunling Xu, Laurent Jacob, Laurence Pibouin-Fragner, Rohit Rao, Nawal Maissa, Maïté Verreault, Nolwenn Lemaire, Camille Knosp, Corinne Lesaffre, Thomas Daubon, Joost Dejaegher, Lien Solie, Justine Rudewicz, Thomas Viel, Bertrand Tavitian, Steven De Vleeschouwer, Marc Sanson, Andreas Bikfalvi, Ahmed Idbaih, Q. Richard Lu, Flavia R.S. Lima, Jean-Leon Thomas, Anne Eichmann, Thomas Mathivet

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Figure 8

Macrophage-specific Robo1/2 knockout normalizes the TME.

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Macrophage-specific Robo1/2 knockout normalizes the TME. (A) MRI images ...
(A) MRI images of CTRL and iRoboMacKO mice 21 days after tumor implantation. (B and C) Quantification of day 21 tumor size on MRI images (B, n = 4 tumors per group, Mann-Whitney U test) and serial vibratome sections (C, n = 7 CTRL and 6 iRoboMacKO tumors, Mann-Whitney U test). (D–F) In vivo 2-photon images of tumor-bearing mice (D) and quantification of vessel diameter (E) and branchpoints (F) (n = 6 mice per group, 1-way ANOVA). (G and H) Glut1 (blue) immunohistochemistry on day 21 tumor-bearing mice (G), and quantification of tumor hypoxic areas (H) (n = 6 CTRL and 5 iRoboMacKO tumors, Mann-Whitney U test). (I–K) Quantification of F4/80, MHC-II, and MRC1+ cells (I), sFLT1+ GFP+ cells (J), and total TALs (CD3+) (K) (n = 6 CTRL and 5 iRoboMacKO tumors, 2-way ANOVA or Mann-Whitney U test). (L and M) FACS analysis of deep cervical DCLNs and MLNs from day 21 tumor-bearing mice (n = 5 CTRL and 4 iRoboMacKO mice; Mann-Whitney U test). (N and O) Lymphocyte counts (N) and differential WBC counts (O) from peripheral blood of day 21 tumor-bearing mice (n = 5 mice/group; Mann-Whitney U test). (P) Eight-week-old mice engrafted with CT-2A BFP and treated with 80 mg/kg tamoxifen i.p. every 3 days starting 7 days after tumor implantation were randomly assigned to vehicle or anti–PD-1 + anti–4-1BB treatment (0.2 mg/dose on days 7, 9, 11, and 13 after tumor implantation) (n = 10/11 mice per group, OS 21.5 days for Robo1–/–Robo2fl/fl, 24 days for Robo1–/–Robo2fl/fl + anti–PD-1 + anti–4-1BB, 29 days for iRoboMacKO, and undetermined for iRoboMacKO + anti–PD-1 + anti–4-1BB; multiple comparisons log-rank test). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.