SLIT2/ROBO signaling in tumor-associated microglia/macrophages drives glioblastoma immunosuppression and vascular dysmorphia Luiz H. Geraldo, … , Anne Eichmann, Thomas Mathivet J Clin Invest. 2021. https://doi.org/10.1172/JCI141083. Research In-Press Preview Oncology Vascular biology Graphical abstract Find the latest version: https://jci.me/141083/pdf 1 SLIT2/ROBO signaling in tumor-associated microglia/macrophages drives 2 glioblastoma immunosuppression and vascular dysmorphia 3 Luiz Henrique Geraldo1,2, Yunling Xu1, Laurent Jacob1, Laurence Pibouin Fragner1, 4 Rohit Rao3, Nawal Maissa1, Maïté Verreault4, Nolwenn Lemaire4, Camille Knosp1, 5 Corinne Lesaffre1, Thomas Daubon5,6, Joost DeJaegher7,8, Lien Solie7,8, Justine 6 Rudewicz5,6, Thomas Viel1, Bertrand Tavitian1, Steven De Vleeschouwer7,8, Marc 7 Sanson4,9, Andreas Bikfalvi5,6, Ahmed Idbaih4, Q. Richard Lu3, Flavia Regina Souza 8 Lima2, Jean-Leon Thomas4.10, Anne Eichmann1,11,12,*,# and Thomas Mathivet1,*,#. 9 10 1 Université de Paris, PARCC, INSERM, F-75015 Paris, France. 11 2 Biomedical Sciences Institute, Federal University of Rio de Janeiro, Brazil. 12 3 Brain Tumor Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 13 4 Sorbonne Université, Inserm U1127, CNRS UMR 7225, Institut du Cerveau, ICM, AP- 14 HP, Hôpitaux Universitaires La Pitié Salpêtrière - Charles Foix, Service de Neurologie 15 2-Mazarin, F-75013, Paris, France. 16 5 Inserm U1029, 6 Université de Bordeaux, F-33170 Pessac, France. 17 7 Department of Neurosciences, 8 Department of Neurosurgery, UZ Leuven, Leuven, 18 Belgium. 19 9 Onconeurotek Tumor Bank, Institut du Cerveau et de la Moelle épinère - ICM, F-75013 20 Paris, France 21 10 Department of Neurology, 11 Cardiovascular Research Center, Department of Internal 22 Medicine, 12Department of Cellular and Molecular Physiology, Yale University School 23 of Medicine, USA 24 *These authors contributed equally 25 1 26 #Correspondence: 27 Anne Eichmann, PhD 28 Yale Cardiovascular Research Center (YCVRC) 29 300 George Street, 7th Floor 30 06511 - New Haven, CT 31 Phone: +12032188459 32 E-mail: [email protected] 33 34 Thomas Mathivet, PhD 35 Paris Cardiovascular Research Center (PARCC) - European Georges Pompidou Hospital 36 56 Rue Leblanc 37 75015 - Paris, France 38 Phone: +33 1 53 98 80 18 39 E-mail: [email protected] 40 41 Conflict of interest: The authors have declared that no conflict of interest exists. 2 42 Abstract 43 44 SLIT2 is a secreted polypeptide that guides migration of cells expressing ROBO1&2 45 receptors. Herein, we investigated SLIT2/ROBO signaling effects in gliomas. In 46 patients with glioblastoma (GBM), SLIT2 expression increased with malignant 47 progression and correlated with poor survival and immunosuppression. 48 Knockdown of SLIT2 in mouse glioma cells and patient derived GBM xenografts 49 reduced tumor growth and rendered tumors sensitive to immunotherapy. Tumor 50 cell SLIT2 knockdown inhibited macrophage invasion and promoted a cytotoxic 51 gene expression profile, which improved tumor vessel function and enhanced 52 efficacy of chemotherapy and immunotherapy. Mechanistically, SLIT2 promoted 53 microglia/macrophage chemotaxis and tumor-supportive polarization via 54 ROBO1&2-mediated PI3Kg activation. Macrophage Robo1&2 deletion and 55 systemic SLIT2 trap delivery mimicked SLIT2 knockdown effects on tumor growth 56 and the tumor microenvironment (TME), revealing SLIT2 signaling through 57 macrophage ROBOs as a potentially novel regulator of the GBM microenvironment 58 and immunotherapeutic target for brain tumors. 59 60 61 62 63 64 3 65 Introduction 66 Malignant gliomas are the most common primary brain tumors (1, 2). Among 67 those, Glioblastoma (GBM, WHO grade IV glioma) is the most frequent and aggressive 68 tumor that accounts for more than 50% of gliomas, with poor patient prognosis (3). GBMs 69 are molecularly heterogeneous and invasive, angiogenic and proliferative tumors that are 70 largely resistant to current therapies (4). 71 Tumor-associated Microglia and Macrophages (TAMs) are the most abundant 72 cells in the GBM microenvironment, composing up to 25% of the tumor mass (5–7). 73 TAMs are key drivers of GBM immunosuppression and pathological angiogenesis (7). 74 TAMs inhibit T cell responses in the GBM microenvironment by favoring regulatory T 75 cells and suppressing anti-tumor T cell responses (8–11), thereby limiting the efficacy of 76 currently available T cell-oriented immunotherapies in GBM (4, 12–14). TAM-derived 77 signaling also contributes to vascular dysmorphia, and drives blood vessel dilation and 78 leakiness in the GBM microenvironment (15, 16). Non-uniform oxygen delivery via 79 dysmorphic and leaky tumor vessels leads to hypoxia, which upregulates angiogenic 80 factors that induce more dysfunctional vessels, thereby preventing the delivery of 81 cytotoxic agents to kill tumor cells (4, 17). The mechanisms by which TAMs promote 82 vessel dysmorphia and immune evasion are as yet incompletely understood, and the 83 means to prevent them are not available (7, 18, 19). 84 SLITs are evolutionary conserved secreted polypeptides that bind to 85 transmembrane Roundabout (ROBO) receptors (20, 21). In mammals, three SLIT ligands 86 (SLIT1-3) signal via two ROBO receptors, ROBO1 and 2 (22). SLIT ligands bind via the 87 second leucine-rich repeat region (D2) to the Ig1 domain of ROBO1&2 (23), while 88 mammalian ROBO3 and ROBO4 lack the SLIT binding residues and do not bind SLITs 89 (24, 25). SLIT binding triggers recruitment of adaptor proteins to the ROBO cytoplasmic 4 90 domain that modulate the cytoskeleton, in turn regulating cell migration, adhesion and 91 proliferation (22, 26, 27). 92 SLIT/ROBO signaling was discovered in the developing nervous system as a 93 guidance cue for axonal growth cones that regulates pathfinding of commissural axons 94 and motor coordination between the left and right side of the body (20, 21). It is now 95 known that SLIT/ROBO signaling controls several additional biological processes, 96 including angiogenesis and immune cell migration. 97 In endothelial cells, SLIT2 activation of ROBO1&2 signaling promotes retinal 98 and bone angiogenesis by driving tip cell migration and polarization (28–31). In the 99 immune system, SLITs have been described as chemo-attractive for neutrophils (32) and 100 chemorepellent for lymphocytes and dendritic cells (33–36). In macrophages, 101 SLIT/ROBO signaling prevented macropinocytosis and cytotoxic polarization (37). 102 In tumor contexts, SLIT2 exerts a pro-angiogenic role (38–40), and has been 103 reported to enhance tumor cell aggressiveness and migration (41–45), metastatic spread 104 (40, 46) and therapy resistance (47), particularly in colorectal cancer, pancreatic cancer 105 and osteosarcoma. Nevertheless, other studies reported a tumor suppressive role for 106 SLIT2/ROBO signaling in in lung and breast cancers (48–50). In the context of GBM, 107 some studies suggested that SLIT2 signaling could inhibit tumor growth (51–53), while 108 in others SLIT/ROBO signaling correlated with more aggressive GBM behavior (54, 55). 109 Given the various and context dependent effects of SLIT/ROBO signaling in cancer, it 110 remained unclear if this pathway could be used therapeutically to prevent cancer growth. 111 We showed here that high SLIT2 expression in GBM patients and in mouse 112 models induced TAM accumulation and vascular dysmorphia, and that SLIT2 knockdown 113 in glioma cells and systemic SLIT2 inhibition with a ligand trap normalized the TME by 114 preventing TAM tumor-supportive polarization and angiogenic gene expression. As a 5 115 result, anti-tumor immune responses and tumor perfusion were enhanced, and efficacy of 116 temozolomide (TMZ)-based chemotherapy and T cell-based immunotherapy were 117 increased. Inducible genetic deletion of Robo1&2 in macrophages was sufficient to 118 normalize the TME and enhanced response to immunotherapy, revealing a potentially 119 novel macrophage-based immunotherapy approach for GBM. 120 6 121 Results 122 SLIT2 expression correlated with poor glioma patient prognosis. 123 The Cancer Genome Atlas (TCGA) Agilent-4502A microarray data analysis 124 using median expression as cutoff showed that high SLIT2 expression was significantly 125 associated with decreased GBM patient survival (Figure 1A, O.S., 12.9 months for high 126 expression and 15.1 months for low expression). Analysis of other TCGA datasets 127 confirmed an association between high SLIT2 expression and decreased GBM patient 128 survival, even though this association did not reach statistical significance within all the 129 datasets (Supplemental Figure 1A-B). Expression of the other SLIT family members and 130 ROBO receptors was not associated with worse prognosis (Supplemental Figure 1C-F). 131 Analysis of a primary glioma patient cohort (129 patients, 84 Low Grade Gliomas 132 and 45 GBMs) also demonstrated correlation between high SLIT2 expression and worse 133 prognosis in both low-grade gliomas (LGGs) and GBMs (Figure 1B, O.S., for LGG: 79.2 134 months for high expression and 135.2 months for low expression; and Supplemental 135 Figure 1G, O.S., for GBM: 15 months for high expression and 16.5 months for low 136 expression). Analysis from TCGA LGG datasets showed a trend towards an association 137 between higher SLIT2 expression and reduced survival, but these results were not 138 statistically significant (Supplemental Figure 1H, O.S., 75 months for high expression 139 and 94.5 months for low expression,