bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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 #Corresponding authors ([email protected];

25 [email protected])

1 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

26 Abstract

27

28 SLIT2 is a secreted polypeptide that guides migration of cells expressing ROBO1&2

29 receptors. Herein, we investigated SLIT2/ROBO signaling effects in gliomas. In

30 patients with glioblastoma (GBM), SLIT2 expression increased with malignant

31 progression and correlated with poor survival and immunosuppression.

32 Knockdown of SLIT2 in mouse glioma cells and patient derived GBM xenografts

33 reduced tumor growth and synergized with immunotherapy to prolong survival.

34 Tumor cell SLIT2 knockdown inhibited macrophage invasion and promoted a

35 cytotoxic expression profile, which improved tumor vessel function and

36 enhanced efficacy of chemotherapy and immunotherapy. Mechanistically, SLIT2

37 promoted microglia/macrophage chemotaxis and tumor-supportive polarization via

38 ROBO1&2-mediated PI3Kg activation. Macrophage Robo1&2 deletion and

39 systemic SLIT2 trap delivery mimicked SLIT2 knockdown effects on tumor growth

40 and the tumor microenvironment (TME), revealing SLIT2 signaling through

41 macrophage ROBOs as a novel regulator of the GBM microenvironment and a

42 potential immunotherapeutic target for brain tumors.

43

44

45

46

47

48

2 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

49 Introduction

50 Malignant gliomas are the most common primary brain tumors (1, 2). Among

51 those, Glioblastoma (GBM, WHO grade IV glioma) is the most frequent and aggressive

52 tumor that accounts for more than 50% of gliomas, with poor patient prognosis (3). GBMs

53 are molecularly heterogeneous and invasive, angiogenic and proliferative tumors that are

54 largely resistant to current therapies (4).

55 Tumor-associated Microglia and Macrophages (TAMs) are the most abundant

56 cells in the GBM microenvironment, composing up to 25% of the tumor mass (5–7).

57 TAMs are key drivers of GBM immunosuppression and pathological angiogenesis (7).

58 TAMs inhibit T cell responses in the GBM microenvironment by favoring regulatory T

59 cells and suppressing anti-tumor T cell responses (8–11), thereby limiting the efficacy of

60 currently available T cell-oriented immunotherapies in GBM (4, 12–14). TAM-derived

61 signaling also contributes to vascular dysmorphia, and drives blood vessel dilation and

62 leakiness in the GBM microenvironment (15, 16). Non-uniform oxygen delivery via

63 dysmorphic and leaky tumor vessels leads to hypoxia, which upregulates angiogenic

64 factors that induce more dysfunctional vessels, thereby preventing the delivery of

65 cytotoxic agents to kill tumor cells (4, 17). The mechanisms by which TAMs promote

66 vessel dysmorphia and immune evasion are as yet incompletely understood, and the

67 means to prevent them are not available (7, 18, 19).

68 SLITs are evolutionary conserved secreted polypeptides that bind to

69 transmembrane Roundabout (ROBO) receptors (20, 21). In mammals, three ligands

70 (SLIT1-3) signal via two ROBO receptors, ROBO1 and 2 (22). SLIT ligands bind via the

71 second leucine-rich repeat region (D2) to the Ig1 domain of ROBO1&2 (23), while

72 mammalian ROBO3 and ROBO4 lack the SLIT binding residues and do not bind SLITs

73 (24, 25). SLIT binding triggers recruitment of adaptor to the ROBO cytoplasmic

3 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

74 domain that modulate the cytoskeleton, in turn regulating cell migration, adhesion and

75 proliferation (22, 26, 27).

76 SLIT-ROBO signaling was discovered in the developing nervous system as a

77 guidance cue for axonal growth cones that regulates pathfinding of commissural axons

78 and motor coordination between the left and right side of the body (20, 21). It is now

79 known that SLIT-ROBO signaling controls several additional biological processes,

80 including angiogenesis and immune cell migration.

81 In endothelial cells, SLIT2 activation of ROBO1&2 signaling promotes retinal

82 and bone angiogenesis by driving tip cell migration and polarization (28–31). In the

83 immune system, SLITs have been described as chemo-attractive for neutrophils (32) and

84 chemorepellent for lymphocytes and dendritic cells (33–36). In macrophages, SLIT-

85 ROBO signaling prevented macropinocytosis and cytotoxic polarization (37).

86 In tumor contexts, SLIT2 exerts a pro-angiogenic role (38–40), and has been

87 reported to enhance tumor cell aggressiveness and migration (41–45), metastatic spread

88 (40, 46) and therapy resistance (47), particularly in colorectal cancer, pancreatic cancer

89 and osteosarcoma. Nevertheless, other studies reported a tumor suppressive role for

90 SLIT2-ROBO signaling in in lung and breast cancers (48–50). In the context of GBM,

91 some studies suggested that SLIT2 signaling could inhibit tumor growth (51–53), while

92 in others SLIT-ROBO signaling correlated with more aggressive GBM behavior (54, 55).

93 Given the various and context dependent effects of SLIT-ROBO signaling in cancer, it

94 remained unclear if this pathway could be used therapeutically to prevent cancer growth.

95 We showed here that high SLIT2 expression in GBM patients and in mouse

96 models induced TAM accumulation and vascular dysmorphia, and that SLIT2 knockdown

97 in glioma cells and systemic SLIT2 inhibition with a ligand trap normalized the TME by

98 preventing TAM tumor-supportive polarization and angiogenic . As a

4 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

99 result, anti-tumor immune responses and tumor perfusion were enhanced, and efficacy of

100 temozolomide(TMZ)-based chemotherapy and checkpoint inhibitor-based

101 immunotherapy were increased. Inducible genetic deletion of Robo1&2 in macrophages

102 was sufficient to normalize the TME and enhanced response to immunotherapy, revealing

103 a novel macrophage-based immunotherapy approach for GBM.

104

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105 Results

106 SLIT2 expression correlated with poor glioma patient prognosis.

107 The Cancer Genome Atlas (TCGA) RNA sequencing data analysis showed that

108 high SLIT2 expression was significantly associated with decreased GBM patient survival

109 (Figure 1A, O.S., 9.86 months for high expression, 14.69 months for low expression, and

110 16.79 months for medium expression, log‐rank test), whereas higher expression of the

111 other SLIT family members and ROBO receptors was not (Supplemental Figure 1A-D).

112 Analysis of TCGA Agilent-4502A microarray confirmed that high SLIT2 expression

113 correlated with poor survival (Supplemental Figure 1E, O.S., 12.9 months for high

114 expression and 15.1 months for low expression). Analysis of a primary glioma patient

115 cohort (129 patients, 84 Low Grade Gliomas and 45 GBMs) also demonstrated

116 correlation between high SLIT2 expression and worse prognosis in both low-grade

117 gliomas (LGGs) and GBMs (Figure 1B, O.S., for LGG: 64.73 months for high expression

118 and 209.10 months for low expression; and Supplemental Figure 1F, O.S., for GBM:

119 14.75 months for high expression and 16.25 months for low expression).

120 Further analysis of RNA sequencing data demonstrated higher SLIT2 expression

121 in the most aggressive and angiogenic mesenchymal GBM subtype (Verhaak et al., 2010)

122 and lower expression in classical GBMs (Supplemental Figure 1G). High SLIT2

123 expression was also associated with poor survival in patients with mesenchymal GBM in

124 this cohort (Supplemental Figure 1H, O.S., 10.4 months for high expression and 17.9

125 months for low expression, log‐rank test). Finally, qPCR analysis of patient samples also

126 revealed higher expression levels of SLIT2 in WHO grade IV GBM compared to WHO

127 grade I, II and III glioma patients (Figure 1C), while expression of other SLITs and

128 ROBOs was not changed between glioma grades (Supplemental Figure 1I-L). Expression

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129 levels of SLIT1 and SLIT3 were significantly lower compared to SLIT2 in GBM patients

130 (Supplemental Figure 1M).

131 Isocitrate dehydrogenase 1 and 2 (IDH-1/2) mutations are known prognostic

132 factors in malignant gliomas. Patients with grade III gliomas and no IDH mutations (IDH-

133 WT) have comparable prognosis to those of GBM patients, while patients with IDH

134 mutations have better survival prognosis (56–58). We compared glioma patients

135 classified by IDH-status, and observed increased SLIT2 expression in patients with IDH-

136 WT tumors in either grade III and IV gliomas (Figure 1D) or in all gliomas (Supplemental

137 Figure 1N).

138 To determine the source of SLIT2 in the GBM microenvironment, we analyzed

139 single cell RNA sequencing (scRNAseq) data from human GBM patients (Figure 1E-F).

140 SLIT2 expression was highest in tumor cells and oligodendrocytes (Figure 1G), while

141 ROBO1 and ROBO2 expression were highest in tumor cells but also detected in other cell

142 types in the TME, particularly in TAMs (Figure 1H-I).

143 We next generated a mouse model of GBM by intra-cerebral inoculation of

144 syngenic CT-2A mouse glioma tumor cells expressing green fluorescent (GFP)

145 into adult C57BL/6 mice (16, 59). Expression of Slit ligands and their Robo receptors was

146 tested 21 days after tumor cell inoculation by qPCR on FACS-sorted tumor cells (GFP+),

147 endothelial cells (ECs, CD31+), TAMs (CD45+CD11b+CD3-), and Tumor-associated T

148 Lymphocytes (TALs, CD45+CD11b-CD3+). The major source of Slit2 were the tumor

149 cells themselves (Figure 1J). By contrast, Robo1 and Robo2 receptors were mainly

150 expressed by ECs and recruited TAMs and TALs (Figure 1K-L). Slit1 and Slit3

151 expression levels in mouse tumor cells were much lower when compared to Slit2

152 (Supplemental Figure 1O). These data suggested that interactions between tumor cell-

153 derived SLIT2 and stromal cells expressing ROBOs could affect GBM growth.

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154 Slit2 silencing slowed GBM growth and increased TMZ sensitivity.

155 To determine if tumor cell-derived Slit2 affected GBM growth, we infected CT-

156 2A and GL261 glioma cells with lentivirus encoding GFP-tagged control scrambled

157 shRNA (shCTRL) or Slit2 targeting shRNA (shSlit2) alone or combined with an shRNA-

158 resistant human SLIT2 construct (shSlit2 + hSLIT2). Slit2 knockdown significantly

159 decreased Slit2 protein and mRNA expression, while shSlit2 + hSLIT2 cells expressed

160 more Slit2 than controls (Figure 2A-B and Supplemental Figure 2A-F). Expression of

161 other Slits or Robo1 and 2 was not altered (data not shown). In vitro growth rates of

162 shCTRL and shSlit2 CT2A and GL261 knockdown cells were similar (Supplemental

163 Figure 2G-H). Slit2 did not induce tumor cell chemotaxis in a transwell chamber assay

164 (Supplemental Figure 2I-J). Nevertheless, migration of shSlit2 cells towards a serum

165 gradient in the lower chamber was reduced (Supplemental Figure 2K-L).

166 Individual 250-μm diameter tumor cell spheroids were implanted through cranial

167 windows into Tomato-fluorescent reporter mice (ROSAmT/mG) and followed

168 longitudinally. Compared to shCTRL, Slit2 knockdown tumors exhibited reduced

169 volumes after 21 days (Figure 2C). FDG-PET imaging showed that tumor metabolic

170 volume and FDG total uptake were similar between shSlit2 and shCTRL at 14 days, but

171 reduced in shSlit2 tumors at 21 days (Figure 2D-F), demonstrating that Slit2 knockdown

172 delayed tumor growth in vivo.

173 We investigated if Slit2 knockdown affected survival in combination with low-

174 dose chemotherapy with the DNA alkylating agent TMZ, a classical treatment for GBM

175 (Figure 2G). Compared to shCTRL, Slit2 knockdown increased overall survival of tumor-

176 bearing mice, while Slit2 overexpression tended to decrease survival (Figure 2H, O.S.,

177 22.5 days for shCTRL, 30 days for shSlit2 and 20 days shSlit2 + hSLIT2). TMZ treatment

178 further increased overall survival of shSlit2 glioma-bearing mice (Figure 2H, O.S., 28

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179 days TMZ for shCTRL+ TMZ, 39 days for shSlit2 + TMZ and 27.5 days for shSlit2 +

180 hSLIT2 + TMZ). shSlit2 did not affect TMZ sensitivity of tumor cells in vitro

181 (Supplemental Figure 2M), but significantly increased TMZ-induced pH2AX+ double

182 strand DNA breaks in tumors in vivo (Supplemental Figure 2N-O), suggesting that

183 changes in the TME might contribute to enhanced TMZ sensitivity in vivo.

184

185 SLIT2 silencing slowed GBM growth and invasiveness in a Patient-derived

186 Xenograft model (PDX).

187 To determine whether SLIT2 had similar effects on human GBM tumors, we used

188 N15-0460 patient-derived GBM cells that were established from a biopsy and grown as

189 tumor spheres. We infected these cells with lentivirus encoding a luciferase reporter and

190 GFP-tagged control scrambled shRNA (shCTRL) or SLIT2 targeting shRNA (shSLIT2).

191 SLIT2 knockdown significantly decreased SLIT2 protein and mRNA expression, without

192 altering expression of other SLITs or ROBO1 and 2 (Supplemental Figure 3A-G). In vitro

193 growth rates of shCTRL and shSLIT2 cells and sensitivity to TMZ were similar

194 (Supplemental Figure 3H-I). SLIT2 did not induce tumor cell chemotaxis in a transwell

195 chamber assay, but migration of shSLIT2 cells towards a serum gradient in the lower

196 chamber was reduced (Supplemental Figure 3J-K). Next, we analyzed sphere formation,

197 and observed that shCTRL and shSLIT2 cells formed similar numbers of spheres after

198 48hs in culture, but the size of shSLIT2 spheres was reduced when compared to shCTRL

199 (Supplemental Figure 3L-M). Analysis of tumor sphere invasion in fibrin gels showed

200 that shSLIT2 decreased spheroid invasion after 24 and 48 hours in culture when compared

201 to shCTRL (Supplemental Figure 3N-O).

202 To determine the effect of shSLIT2 on human GBM growth, we implanted

203 shCTRL and shSLIT2 N15-0460 cells in Hsd:Athymic Nude-Foxn1nu mice and followed

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204 tumor growth by bioluminescence analysis every 2 weeks after tumor implantation. 170

205 days after tumor implantation, 80% of the mice injected with shCTRL cells developed

206 tumors, while only 20% of shSLIT2-injected mice had tumors (Supplemental Figure 4A).

207 Analysis of the bioluminescence curves of shCTRL and shSLIT2 tumors demonstrated

208 that more mice developed tumors in the shCTRL group, and that the shCTRL tumors

209 were bigger than shSLIT2 tumors (Supplemental Figure 4B-C). Histological analysis of

210 GFP+ tumor cells on vibratome sections 170 days after tumor implantation showed that

211 shCTRL cells either developed tumor masses or spread throughout the entire brain, while

212 shSLIT2 cells remained restrained to the injection site or migrated through the corpus

213 callosum, but did not form tumor masses (Supplemental Figure 4D-E). SLIT2 shRNA

214 also reduced the expression of SOX2 and PML involved in GBM tumor cell malignancy

215 (55, 60, 61) (Supplemental Figure 4F-H).

216

217 Slit2 knockdown improved tumor vessel function.

218 To determine if tumor-secreted SLIT2 affected the GBM microenvironment, we

219 used 2-photon in vivo imaging of red fluorescent ROSAmT/mG mice. We observed that

220 blood vessels in shCTRL CT2A and GL261 tumors became abnormally enlarged and lost

221 branching points between day 14 and day 21, while shSlit2 tumor vessels dilated less and

222 remained more ramified (Figure 3A-C, Supplemental Figure 5A-D). Conversely, SLIT2

223 overexpressing tumor vessels dilated and lost branchpoints earlier, at day 18 after

224 injection (Figure 3D-F, Supplemental Figure 5E), just prior to their death at 20 days post

225 tumor implantation.

226 Functionally, in vivo imaging after intravenous Alexa Fluor 647-labeled dextran

227 injection revealed significantly improved perfusion in shSlit2 CT2A tumor vessels when

228 compared to shCTRL tumors (Figure 3G-H). Quantification of Evan’s blue extravasation

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229 showed reduced vascular leakage in shSlit2 tumors compared to shCTRL (Figure 3I).

230 Along with improved vascular function in shSlit2 knockdown tumors, glucose transporter

231 1 immunostaining (Glut1)-positive hypoxic areas within the tumor mass were reduced,

232 and Glut1 coverage of blood vessels was increased in shSlit2 knockdown tumors

233 compared to shCTRL, indicating partially improved blood-brain barrier function (Figure

234 3J-L). QPCR analysis of sorted tumor endothelial cells (CD45-CD31+) showed

235 downregulation of immunosuppressive IL-6, PD-L1 and PD-L2 in Slit2 shRNA

236 transfected tumors compared to CTRL tumors (Figure 3M).

237

238 Slit2 silencing reduced myeloid immunosuppression

239 In vivo imaging also revealed that immune cell infiltration was increased in SLIT2

240 overexpressing tumors, and decreased in Slit2 silenced tumors when compared to CTRL

241 tumors (Supplemental Figure 6A-C). Immunofluorescence analysis of tumor sections

242 showed a decrease in the numbers of F4/80+ myeloid cells in day 21 shSlit2 tumors

243 compared to day 21 shCTRL or day 18 SLIT2-overexpressing tumors (Figure 4A-B,

244 Supplemental Figure 6D-E). Activated MHC-II+ antigen-presenting cells (APCs) were

245 increased in shSlit2 tumors, and MRC1(CD206)+ tumor-supportive infiltrating immune

246 cells were decreased (Figure 4A-B, Supplemental Figure 6D-E). FACS sorted

247 CD45+CD11b+F4/80+Ly6G- TAMs accounted for about 12% of the total cells in shCTRL

248 tumors, but only 6% in the Slit2 knockdown tumors (Figure 4C, Supplemental Figure 6F).

249 Half of the TAMs in shSlit2 CT2A tumors had a cytotoxic activation profile and

250 expressed MHC-II and CD11c, while <20% of TAMs in the shCTRL condition expressed

251 MHCII and CD11c and >80% expressed the tumor supportive marker MRC1 (Figure

252 4D). shSlit2 tumors also showed increased infiltration of Dendritic Cells (CD45+CD11b-

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253 CD11c+MHC-II+F4/80-) and neutrophils (CD45+CD11b+Ly6G+) that were much less

254 abundant when compared to TAMs (Supplemental Figure 6G-H).

255 Molecularly, when compared to FACS-sorted shCTRL, TAMs from shSlit2

256 tumors exhibited decreased expression of tumor supportive Mrc1, Vegfa, Tgfβ1,

257 Mmp9, Cd209a, Ccl19, Arg1 and Il10, increased expression of cytotoxic genes Il-12, Il-

258 1b, Ccr7, Cxcl10 and Tnfα, and reduced expression of Pd-l1 and Pd-l2 inhibitors of T cell

259 activation (Figure 4E). ELISA analysis showed increased IFNγ and confirmed reduced

260 IL-10 and VEGFa protein levels in TAMs sorted from shSlit2 tumors when compared to

261 controls (Figure 4F-H). In line with reduced VEGFa expression, in vivo binding of soluble

262 VEGFR1 (sFlt1) showed that only about 40% of stromal cells in shSlit2 tumors bound

263 sFlt1, while >80% of CTRL and SLIT2 overexpressing cells bound Flt1 (Figure 4I).

264

265 SLIT2 inhibition increased T cell infiltration and improved checkpoint inhibitor

266 treatment.

267 In contrast to the decreased number of TAMs in shSlit2 tumors, the total number

268 of TALs was increased 3-fold (Figure 5A, Supplemental Figure 7A-D), with an increase

269 in both CD4+ and CD8+ T lymphocytes within the tumor mass when compared to controls

270 (Figure 5B-C and Supplemental Figure 7E-I). Furthermore, the CD4+ TALs in shSlit2

271 tumors showed increased expression of Th1 response related genes (Ifnγ, Cxcl11 and Il-

272 2) and of IL-17a, but decreased expression of Th2 response related genes (Il-10 and

273 Cxcl10) and PD-1 and CTLA4 (Figure 5D). CD8+ TALs in shSlit2 tumors also showed

274 increased expression of activation markers (IFNγ and GZMB), and reduced expression of

275 genes related to CD8 T cell exhaustion (Tim3 and Lag3) (62) (Figure 5E). In tumor

276 sections, we observed more infiltrating GZMB+ CD8+ activated anti-tumor T cells in

277 shSlit2 compared to shCTRL tumors (Figure 5F-G). ELISA analysis of these sorted CD8+

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278 TALs also showed increased IFNγ (Figure 5H) and reduced IL-10 and VEGFa protein

279 levels (Figure 5I-J) in cells sorted from shSlit2 tumors.

280 Given this shift towards a less immunosuppressive GBM microenvironment, we

281 hypothesized that shSlit2 tumors would be more sensitive to treatment with immune

282 checkpoint inhibitors using anti-PD-1 and anti-4-1BB antibodies (11, 59). We treated

283 mice with 200ug of each antibody at D7, D9, D11 and D13 after tumor implantation.

284 Combining immune checkpoint inhibitor therapy with Slit2 silencing led to powerful anti-

285 tumor responses, with 100% of the mice alive at 90 days after implantation (Figure 5K,

286 O.S.= 25 days for shCTRL, 33 days for shCTRL + Anti-PD-1 + Anti-4-1BB, 33 days for

287 shSlit2 and Undetermined for shSlit2 + Anti-PD-1 + Anti-4-1BB).

288 The changes in the immune cell microenvironment that we observed in the murine

289 GBM models are also likely to occur in GBM patients, as shown by positive correlation

290 between SLIT2 and MRC1 and VEGFA mRNA expression in patient samples from our

291 GBM patient cohort and TCGA database cohorts (Supplemental Figure 8A-C). SLIT2

292 expression also correlated with genes related to tumor-supportive macrophages (CCL19,

293 CD209, MMP9 and PD-L2), inhibition of anti-tumor T cell responses (PD-1, CTLA4,

294 CCL17, CXCL11, LAG3 and TIM3) and IL-6 for example (Supplemental Figure 8D-O).

295

296 SLIT2 promoted microglia and macrophage migration and polarization via

297 ROBO1&2.

298 To determine how SLIT2 affected myeloid cells, we tested microglia and

299 macrophage migration in Transwell chambers. Slit2 in the bottom chamber induced

300 chemotaxis of isolated mouse microglial cells, bone-marrow-derived macrophages

301 (BMDM) and peritoneal macrophages (PM) in a dose-dependent manner, with a

302 maximum response at 6nM (Figure 6A-C). Adding Slit2 to both top and bottom chambers

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303 inhibited macrophage migration, indicating a chemotactic response (Supplemental Figure

304 9A-B).

305 To determine if SLIT2 signaled through ROBO receptors to promote macrophage

306 migration, we silenced Robo1&2 in cultured RAW264.7 macrophages using siRNAs,

307 which inhibited Slit2-induced macrophage migration (Figure 6D-E, Supplemental Figure

308 9C-D). Migration could be rescued by adenoviral-induced expression of a siRNA-

309 resistant full-length rat Robo1 construct (Robo1FL) but not by a construct lacking the

310 cytoplasmic signaling domain (Robo1DCD, Figure 6D-E).

311 To identify SLIT2 downstream signaling pathways in macrophages, we treated

312 BMDM and microglial cells with 6nM Slit2, which led to PLCγ, Erk1/2 and Akt

313 phosphorylation (Figure 6F, Supplemental Figure 9E-G, J). SLIT2 also induced

314 phosphorylation of Stat6 and CEBPβ1 that polarize tumor-infiltrating macrophages

315 towards a tumor supportive phenotype (63) (Figure 6F, Supplemental Figure 9H-J),

316 suggesting that SLIT2 induced tumor-supportive gene expression changes.

317 Conditioned medium of Slit2-treated microglia and macrophages increased levels

318 of IL-10 and VEGFa compared to cells not treated with Slit2 (Figure 6G-H, Supplemental

319 Figure 9K-L). The expression of genes characteristic of a tumor supportive macrophage

320 phenotype, including Mrc1, Vegfa, Mmp9, Tgfβ1, Ccl19, Cd209a, Il-10 and Arg1, were

321 all increased by Slit2 treatment, while cytotoxic response-related genes Il-1β, Cxcl10,

322 Ccr7 and Tnfα were unaffected by Slit2, but increased by LPS (Figure 6I). Slit2-induced

323 gene expression changes were ROBO1&2-dependent, as shown by siRNA silencing of

324 Robo1/2, which abrogated Slit2 induced changes in protein phosphorylation and gene

325 expression (Supplemental Figure 10A-G).

326

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327 Slit2-Robo induced tumor-supportive macrophage/microglia polarization via

328 PI3Kγ

329 Previous studies have shown that Stat6 and CEBPβ1 activation in TAMs occur

330 downstream of PI3Kγ (63), leading us to ask if Slit2-Robo1&2 signaled upstream of

331 PI3Kγ to induce macrophage tumor-supportive polarization. First, we observed Robo1

332 and PI3Kγ co-immunoprecipitation in BMDMs, which was enhanced after Slit2 treatment

333 for 15 minutes (Figure 7A). Second, Slit2-induced BMDM migration was abrogated by

334 pre-treatment with a specific PI3Kγ inhibitor IPI-549 (1µM) (Figure 7B). Third, Slit2-

335 induced phospho-Stat6 nuclear translocation in cultured BMDMs was prevented by pre-

336 treatment with IPI-549 (Figure 7C-D). Slit2-induced Akt and Stat6 phosphorylation

337 (Supplemental Figure 11A), as well as IL-10 and VEGFa secretion in ELISA from

338 BMDM conditioned medium were also reduced by PI3Kγ inhibition (Figure 7E-F).

339 Finally, the Slit2-induced expression of genes characteristic of a tumor supportive

340 macrophage phenotype (Mrc1, Vegfa, Mmp9, Tgfβ1, Ccl19, Cd209a, Il-10 and Arg1) was

341 disrupted by IPI-549 pretreatment, while LPS-induced cytotoxic response-related genes

342 were unaffected in both BMDMs and microglial cells by PI3Kγ inhibition (Figure 7G,

343 Supplemental Figure 11B).

344

345 Robo deficiency in TAMs inhibited glioma growth and vascular dysmorphia.

346 To determine if SLIT2 signaling effects in macrophages were sufficient to drive

347 the stromal response, we developed mice with genetic Robo receptor deletions in

348 macrophages. To do so, we intercrossed Robo1-/-Robo2fl/fl mice (28) with CSF-1RCreERT2

349 mice (64) on a ROSAmT/mG background, generating Robo1-/-Robo2fl/flCSF-

350 1RCreERT2ROSAmT/mG mice (hereafter named iRoboMacKO mice). Littermate Robo1+/-

351 Robo2+/fl CSF-1RCreERT2ROSAmT/mG or Robo1-/-Robo2fl/flROSAmT/mG mice were used as

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352 controls. Mice were implanted with CT-2A-BFP glioma cells and followed longitudinally

353 during tumor growth. Tamoxifen injections to induce gene deletion were done every 3

354 days starting 7 days after tumor implantation, and induced robust gene deletion, assessed

355 by qPCR of GFP+ macrophages extracted from the bone marrow (Supplemental Figure

356 12A-B).

357 MRI imaging and histological analysis 21 days after tumor implantation

358 converged to show reduced tumor size in iRoboMacKO tumors when compared to

359 controls (Figure 8 A-C). T1-weighted imaging after Gadolinium injection showed more

360 homogeneous contrast signal in iRoboMacKO tumors, while control GBMs displayed

361 predominantly peripheral and heterogenous contrast distribution, suggesting improved

362 perfusion in iRoboMacKO tumors (Figure 8A). In vivo two-photon imaging revealed that

363 blood vessels in iRoboMacKO tumors dilated less and remained more ramified when

364 compared to controls (Figure 8D-F). Glut1+ hypoxic zones within the tumor mass were

365 reduced in iRoboMacKO tumors, confirming improved perfusion when compared to

366 controls (Figure 8G-H). Most of the Glut1 staining in iRoboMacKO tumors colocalized

367 with Tomato+ blood vessels, attesting to the qualitative improvement of iRoboMacKO

368 tumor vessels (Figure 8G).

369 Compared to controls, iRoboMacKO displayed reduced overall numbers of intra-

370 tumor Iba1+ myeloid cells, with a significant increase of cytotoxic MHCII+ cells and a

371 reduction in tumor-supportive MRC1+ cells (Figure 8I, Supplemental Figure 12D).

372 Soluble Flt1 binding was reduced in iRoboMacKO tumors (Figure 8J, Supplemental

373 Figure 12D), and Robo1/2-deleted macrophages extracted from the bone marrow of

374 tumor-bearing mice showed decreased Vegfa expression (Supplemental Figure 12C).

375 T cell infiltration was increased in iRoboMacKO tumors (Figure 8K,

376 Supplemental Figure 12D), suggesting that SLIT-ROBO signaling inhibition in

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377 macrophages was sufficient to shift the GBM microenvironment towards a cytotoxic, T

378 cell enriched phenotype. This effect could be due to increased circulation of antigen-

379 presenting cells (APCs) to the tumor draining lymph-nodes, where they can activate anti-

380 tumor T cell responses (59). Analysis of glioma-draining deep cervical and mandibular

381 lymph nodes (DCLN and MLN, respectively) for the presence of BFP tumor antigen in

382 immune cells revealed an important increase in CD11b+BFP+ cells in both deep cervical

383 (DCLN) and mandibular lymph nodes (MLN) of iRoboMacKO tumors when compared

384 to controls (Figure 8L-M, Supplemental Figure 12E).

385 Lymphocyte sequestration in the bone marrow contributes to the T cell-depleted

386 TME and failure of currently available immunotherapy (11). iRoboMacKO mice had

387 significantly increased lymphocyte counts in peripheral blood 21 days after tumor

388 implantation (Figure 8N). Given that total white blood cell (WBC) count was not changed

389 (Supplemental Figure 12F), tumor-bearing iRoboMacKO mice shifted to a predominance

390 of lymphocytes over neutrophils in the blood stream (Figure 8O), revealing a reduction

391 in the systemic immunosuppression after macrophage-specific Robo1&2KO.

392 Given the profound changes observed in the TME observed, we next tested if

393 macrophage-specific Robo1&2 deletion was sufficient to prolong survival and sensitivity

394 to checkpoint inhibitor therapy. Indeed, macrophage-specific Robo1/2 knockout

395 increased tumor-bearing mice survival (Figure 8P, O.S., 21.5 days for Robo1-/-Robo2fl/fl,

396 29 days for iRoboMacKO), and survival benefit was further increased by immune

397 checkpoint inhibitors, with 70% of the iRoboMacKO mice alive after 100 days (Figure

398 8P, O.S., 24 days for Robo1-/-Robo2fl/fl + Anti-PD-1+Anti-4-1BB, Undefined for

399 iRoboMacKO + Anti-PD-1+Anti-4-1BB).

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400 In contrast to macrophage Robo depletion, T cell depletion using anti-CD3 145-

401 2C11 antibodies (65) did not induce significant changes of blood vessels or TAMs in the

402 GBM microenvironment (Supplemental Figure 13).

403

404 Systemic SLIT2 inhibition alleviated GBM immunosuppression.

405 We reasoned that systemic administration of a SLIT2 ligand trap protein

406 (Robo1Fc) might be efficient in a therapeutic setting. Mice with established shCTRL

407 CT2A tumors were intravenously injected 5 times with 2.5 mg/kg of Robo1Fc every

408 second day starting from day 7 after tumor implantation and analyzed at day 23 (Figure

409 9A). Control mice received injections of human control IgG1 Fc fragment. Robo1Fc

410 treatment reduced Slit2 serum levels, as attested by Slit2 ELISA on days 14 and 21 after

411 tumor implantation (Figure 9B). Mice treated with Robo1Fc exhibited a pronounced

412 tumor growth reduction compared to control Fc-treated tumors (Figure 9C-D). MRI

413 analysis 21 days after tumor implantation showed that tumor size was reduced and that

414 tumor perfusion was improved, as seen by the more homogeneous gadolinium uptake in

415 Robo1Fc-treated tumors compared to controls (Supplemental Figure 14A-B). In vivo

416 imaging demonstrated that Robo1Fc treatment reduced vascular dysmorphia (Figure 9E-

417 G) and reduced Glut1+ hypoxic zones within the tumor mass (Figure 9H, Supplemental

418 Figure 14C). Robo1Fc treatment changed immune cell infiltration and reduced overall

419 numbers of intra-tumoral F4/80+ cells, with a significant increase of cytotoxic MHCII+

420 cells and a reduction of tumor-supportive MRC1+ cells compared to controls (Figure 9I,

421 Supplemental Figure 14D). Soluble Flt1 binding was reduced in Robo1Fc treated tumors

422 (Figure 9J, Supplemental Figure 14D), while T cell infiltration was increased compared

423 to controls (Figure 9K, Supplemental Figure 14D).

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424 Analysis of glioma-draining DCLN and MLN showed an increased presence of

425 GFP tumor antigen in APCs (CD45+CD11b+Ly6G-) of Robo1Fc-treated mice when

426 compared to CTRLFc treated ones (Supplemental Figure 14E-G), as we observed in

427 iRoboMacKO mice. Finally, Robo1Fc-treated mice also had significantly increased total

428 WBC and lymphocyte counts in peripheral blood 21 days after tumor implantation, and

429 we observed a shift to a predominance of Lymphocytes over Neutrophils in the blood

430 stream of Robo1Fc treated mice, while other white blood cell counts were unchanged

431 (Supplemental Figure 14H-J).

432 Five injections of Robo1Fc protein during early stages of tumor progression were

433 sufficient to significantly extend survival of tumor-bearing mice and 25% of the treated

434 mice survived 150 days after implantation (Figure 9L, O.S., 24 days for CTRLFc and 41

435 days for Robo1Fc). Combining Robo1Fc with TMZ further increased this survival

436 benefit, with 45% of the mice surviving 150 days after implantation (Figure 9L, O.S., 28

437 days for CTRLFc + TMZ and 119 days for Robo1Fc + TMZ). Combining Robo1Fc with

438 Anti-PD-1 and Anti-4-1BB antibodies further improved anti-tumor responses, with 80%

439 of the mice surviving 90 days after tumor implantation (Figure 9M, O.S., 40.5 days for

440 CTRLFc + Anti-PD-1 + Anti-4-1BB and Undefined for Robo1Fc + Anti-PD-1 + Anti-4-

441 1BB). Mice that survived the immunotherapy were rechallenged by a novel tumor

442 injection in the contralateral hemisphere. Mice that survived after treatment with

443 Robo1Fc and checkpoint inhibitors had the best long-term survival after tumor

444 rechallenge, with more than 80% of mice alive 90 days after tumor re-injection (Figure

445 9N, O.S., 22 days for naïve mice, 53.5 days for Anti-PD-1 + Anti-4-1BB survivors, 63

446 days for Robo1Fc survivors and Undefined days for Robo1Fc + Anti-PD-1 + Anti-4-1BB

447 survivors)

448

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449 Discussion

450 Collectively, our data showed that GBM-derived SLIT2 signaled through

451 ROBO1/2 in TAMs, which resulted in an impairment of anti-tumor immunity and the

452 induction of vascular dysmorphia in the TME. SLIT2-ROBO1&2 signaling is therefore

453 a novel immune evasion mechanism in the TME, and inhibiting this pathway in TAMs

454 could sensitize GBM to immune checkpoint inhibitors, and add to the therapeutic arsenal

455 against GBM.

456 The main findings of our study can be summarized as follows: we showed that

457 SLIT2 expression levels correlated with tumor aggressiveness, poor prognosis and

458 immunosuppression in a variety of glioma patient cohorts. In particular, low-grade glioma

459 patients with low SLIT2 expression levels had a significantly prolonged survival when

460 compared to those with higher SLIT2 expression, suggesting that SLIT2 may be a useful

461 prognostic marker for glioma patients.

462 Our data suggest that GBM tumor cells are a major source of SLIT2. SLIT2

463 expression in human GBM tumors was highest in the tumor cell compartment, and Slit2

464 knockdown in two murine GBM cells lines and in a human PDX model decreased tumor

465 growth, while SLIT2 overexpression in CT2A cells enhanced murine GBM tumor

466 growth. SLIT2 from other cell compartments could also affect GBM growth, but since

467 genetic SLIT2 inhibition in tumor cells and systemic SLIT2 inhibition had similar effects

468 in our mouse models, it is likely that tumor cell SLIT2 plays an important role in GBM.

469 In further support of this idea, silencing tumor cell derived SLIT2 in human PDX GBM

470 tumors reduced tumor development in vivo.

471 We observed that SLIT2 acted on different cell types within GBM. First, both

472 human and mouse GBM tumor cells expressed ROBO1&2 receptors. SLIT2 knockdown

473 did not affect tumor cell proliferation or survival, and SLIT2 did not attract tumor cells

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474 in transwell assays in vitro. However, SLIT2 knockdown decreased tumor cell migration

475 towards a serum gradient in transwell chambers and reduced spheroid invasion in fibrin

476 gels, and patient-derived shSLIT2 GBM cells implanted in Nude mice decreased

477 invasiveness compared to shCTRL. These results are consistent with SLIT2-ROBO

478 signaling driving pro-invasive GBM tumor cell behavior in both mice and patient-derived

479 models. Our data contrast early studies with commercial human GBM cell lines where

480 SLIT2-ROBO1 signaling inhibited migration (51, 52), but they support and extend s

481 studies using murine GBM models (55) and patient-derived tumor spheres and xenograft

482 models (54), which showed that SLIT2-ROBO1 signaling in tumor cells promotes tumor

483 invasiveness.

484 In addition to the tumor cells themselves, SLIT2 exerted major effects in the TME,

485 and remarkably these changes appeared centered around ROBO1&2 signaling in

486 macrophages/microglial cells. We found that in the tumor context, ROBO1&2 signaling

487 inhibition in macrophages was sufficient to recapitulate all major aspects of tumor cell

488 SLIT2 manipulation, or systemic SLIT2 inhibition with a ligand trap, and shifted the

489 entire TME towards a normalized and cytotoxic phenotype. We identified three TME cell

490 types that responded to tumor cell SLIT2, namely TAMs, endothelial cells of blood

491 vessels and T cells. Genetic inhibition of Robo signaling in macrophages reduced

492 macrophage recruitment to the TME, prevented phenotypic conversion into tumor-

493 supportive macrophages, normalized tumor vasculature and induced T-cell based anti-

494 tumor responses. It remains possible that cell-autonomous Robo signaling in endothelial

495 cells, which induces angiogenesis (28, 31), or T-cell Robo signaling contribute to the

496 observed effects in GBM, but clearly macrophage Robo signaling appeared dominant.

497 Mechanistically, SLIT2-mediated TAM migration and polarization were

498 ROBO1&2 dependent and mediated by PI3Kγ signaling. PI3Kγ signaling inhibition has

21 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

499 been previously shown to prevent TAM polarization and tumor progression in different

500 cancer models (63), and this mechanism could be conserved in GBM TAMs. PI3Kγ is

501 traditionally activated by G-protein coupled receptors (GPCRs) or Receptor tyrosine

502 kinases (RTKs), therefore it remains to be established how ROBO activates PI3Kγ

503 mechanistically, via NCK-SOS activation of RAS or other small GTPases that can

504 activate PI3Kγ (66–68). Another possibility is that PI3Kγ activation downstream of

505 ROBO receptors depends on the co-activation of other RTKs or GPCRs and their

506 endocytosis.

507 TAMs are the most abundant cells in the GBM microenvironment, and are known

508 to contribute to immunosuppression in the TME (7–9, 69) and dysmorphic angiogenesis

509 (16, 70–72). Hence, TAMs are key players in the development of resistance to anticancer

510 therapies (17, 73–76). Several attempts have been made to target TAM signaling for

511 GBM treatment, including manipulation of VEGFa and angiopoietins, Neuropilin1 (77,

512 78), CD-47 or CSF-1R (79–81). Combined VEGF/Angiopoietin inhibition led to vascular

513 normalization and cytotoxic TAM polarization, but did not change T cell infiltration or

514 activation profile (82, 83). CD47 inhibition prolonged GBM-bearing mice survival due

515 to increased phagocytosis capacity and cytotoxic TAM polarization, but did not affect

516 other components of the GBM microenvironment (84). CSF-1R inhibition did not change

517 TAM production of pro-angiogenic molecules such as VEGFa and therefore did not lead

518 to vascular normalization in GBM (16, 79, 81). Hence, these strategies changed the TAM

519 component of the GBM microenvironment, but they did not induce the profound changes

520 in angiogenesis and T cell response achieved by SLIT2 inhibition. Systemic SLIT2

521 inhibition via intravenous injection of a SLIT2 ligand trap could be optimized and

522 translated into clinical practice to combat GBM in human patients, especially those with

523 high levels of SLIT2 expression.

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524 Methods

525

526 Bioinformatic analysis

527 For ‘The Cancer Genome Atlas’ (TCGA) dataset, RNAseqV2 normalized data (level 3,

528 log2(x+1) transformed RSEM normalized count, version 2017-10-13) of 151 primary

529 glioblastoma multiforme patients (TCGA Glioblastoma (GBM)) and associated

530 molecular GBM subtypes and clinical data were downloaded from the cBioPortal website

531 datapages (https://www.cbioportal.org/study/summary?id=gbm_tcga). The cohort was

532 split into 3 groups of patients defined by the level of their expression. Overall survival (in

533 months) was used to estimate survival distributions using the Kaplan–Meier method and

534 the distributions were compared using the log‐rank test.

535

536 Patient Samples

537 Frozen tumors samples were obtained from 25 patients after informed consent and

538 approval by UZ Leuven ethical committee for the Brain-Tumor-Imm-2014 study; and

539 tumor RNA was obtained from 104 patients of the Pitié-Salpêtrière tumor bank

540 Onconeurotek.

541 RNA was purified from liquid nitrogen frozen tissue samples using RNeasy-kit (Qiagen).

542 0.5μg of RNA were reverse transcribed using SuperScript IV Reverse Transcriptase and

543 Random Primers (Invitrogen) for qPCR reactions.

544

545 QPCR reactions

546 Real-time quantitative PCR (qPCR) reactions were performed in duplicate using the

547 MyIQ real-time PCR system (Bio-Rad), with iQ SYBR Green Supermix (Bio-Rad) and

548 QuantiTect qPCR primers (Qiagen). Each reaction contained 10 ng of cDNA and 250 nM

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549 forward and reverse primers. Fold changes were calculated using the comparative CT

550 method.

551

552 Single-cell RNA sequencing analysis

553 We downloaded the following published datasets for single cell RNA-seq analysis from

554 GEO: GSE138794, GSE131928, and GSE84465 (85–87). Gene expression matrices were

555 combined and were visualized using the Seurat v3 (88) package in R. Based on the

556 ElbowPlot function, we chose around 43 principal components for UMAP driven

557 visualizations. Markers for each cluster were defined from a combination of literature

558 knowledge and the FindMarkers function in Seurat. For removal of batch effects between

559 different datasets, we used the harmony package (89).

560

561 Cell lines

562 RAW264.7 mouse macrophages, CT-2A and GL261 glioma cells were cultured in

563 DMEM Gluta-MAX (Gibco) supplemented with 10% FBS (Gibco), 1%

564 penicillin/streptomycin (Gibco) until a maximum of 10 passages. Glioma spheroids were

565 obtained by seeding the glioma cells for 48 h on non-adherent culture dishes.

566

567 Animal procedures and glioma implantation

568 All in vivo experiments were conducted in accordance to the European Community for

569 experimental animal use guidelines (L358-86/609EEC) with protocols approved by the

570 Ethical Committee of INSERM (n°MESRI23570 and #17503 2018111214011311 v5).

571 Animals were housed with free access to food and water in a 12h light/dark cycle. For

572 survival experiments, mice were euthanized if they exhibited signs of neurological

573 morbidity or if they lost > 20% of their body weight. C57bl6J and ROSAmT/mG mice were

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574 used for survival and live imaging experiments, respectively. For generation of

575 macrophage-specific Robo1/2 KO, Robo1-/-Robo2fl/fl mice (28) were bred with CSF1-R-

576 CreERT2, ROSAmT/mG mice (64). Gene deletion was induced by injections of 80mg/Kg

577 of tamoxifen every 2 days starting 7 days after tumor implantation and was verified on

578 GFP+ bone marrow monocytes/macrophages.

579

580 Murine Glioma model

581 Craniotomy and glioblastoma spheroid implantation were done as previously described

582 (16). Briefly, a 5-mm circle was drilled between sutures of the skull on ketamine/xylazine

583 anesthetized mice. A 250-μm diameter CT-2A or GL261 spheroid was injected in the

584 cortex and sealed with a glass coverslip. For survival experiments involving PD-1 and 4-

585 1BB inhibition, tumor cells were inoculated as cell suspension in the mice striatum

586 instead as cortical spheroids as previously described (59). Following intramuscular

587 administration of analgesic (buprenorphine 1 mg/kg), mice were placed in a heated cage

588 until full recovery.

589 For Temozolomide (Sigma) treatment, mice were injected intraperitoneally with 40mg/kg

590 in 0,2 mL of PBS at days 7, 11, 15 and 19 after tumor implantation. For anti-PD1 (clone

591 RMP1-14, BioXCell) and anti-4-1BB (clone LOB12.3, BioXCell) treatment, glioma-

592 bearing mice were injected intraperitoneally with 0.2 mg of antibodies on days 7, 9, 11

593 and 13 after tumor implantation.

594 For anti-CD3 145-2C11 monoclonal antibody (BioXCell) treatment, 7-day glioma-

595 bearing mice were injected intravenously with 0.2 mg of antibodies every 3 days and

596 analyzed at 23 days of tumor growth.

597 For Robo1Fc (R&D Systems) treatment, 1-week growth glioma-bearing mice were

598 injected intravenously with 2.5 mg/kg of Robo1Fc or human control IgG1 Fc fragment at

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599 days 7, 9, 11, 13 and 15 after tumor implantation. For this experiment, 6 different series

600 of mice were implanted and treated: 2 for tumor volume measurement and histological

601 analysis and 4 for survival analysis.

602 At the defined time points, blood samples were obtained by retro-orbital bleeding with

603 EDTA-coated capillaries and complete blood cell counts were obtained with a HemaVet

604 (Drew Scientific). 21 or 23 days after tumor implantation, anesthetized mice were

605 transcardially perfused with 2% PFA solution. The mouse brain was harvested and fixed

606 overnight in 4% PFA at 4°C. For immunohistochemistry, brains were washed with PBS

607 and sectioned with a vibratome (200um-400μm sections). Tumor volume was measured

608 on serial 400μm sections of the whole tumor under a stereo-microscope using Leica

609 software according to Cavalieri’s principle.

610

611 Slit2 shRNA knockdown and overexpression

612 CT-2A and GL261 glioma cell lines were infected with Slit2 mouse shRNA lentiviral

613 particles ( ID 20563, Origene TL511128V) in accordance with the manufacturer’s

614 instructions. After infection, cells were polyclonally selected by Puromycin and GFP+

615 cells were sorted by FACS. Slit2 knockdown was verified by qPCR and Western Blot

616 analysis, and cells were implanted after a maximum of 5 passages. For Slit2 re-

617 expression, shSlit2 CT-2A cells were infected with SLIT2 (NM_004787) Human Tagged

618 ORF Clone Lentiviral Particle (Origene) in accordance with manufacturer’s instructions.

619 Cells were implanted after a maximum of 3 passages.

620

621 FDG PET-CT Imaging

622 Mice were fasted overnight with free access to water. Mice were anesthetized with

623 isoflurane, weighed and glycemia was measured in blood drawn from the caudal ventral

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624 artery using an Accu-Chek® Aviva Nano A (Accu-Chek, France). A 26G needle catheter

625 (Fischer Scientific, France) connected to a 5cm polyethylene tubing (Tygon Microbore

626 Tubing, 0.010" x 0.030"OD; Fisher Scientific, France) was inserted in the caudal vein for

627 radiotracer injection. 9.2±1.5 MBq of 2'-deoxy-2'-[18F]fluoro-D-glucose (FDG;

628 Advanced Applied Applications, France) in 0.2mL saline was injected via the catheter.

629 Mice were left on a warming pad for 30 min and then installed into the PET-CT dedicated

630 bed. Respiration and body temperature were registered. Body temperature was

631 maintained at 34±2 °C and anesthesia was controlled on the breathing rate throughout the

632 entire PET-CT examination. CT was acquired in a PET-CT scanner (nanoScan PET-CT;

633 Mediso Medical Imaging Systems, Hungary) using the following acquisition parameters:

634 semi-circular mode, 50kV tension, 720 projections full scan, 300ms per projection,

635 binning 1:4. CT projections were reconstructed by filtered retro-projection (filter: Cosine;

636 Cutoff: 100%) using the software Nucline 3.00.010.0000 (Mediso Medical Imaging

637 Systems, Hungary). 55 min post tracer injection, PET data were collected for 10 min in

638 list mode and binned using a 5ns time window, with a 400-600keV energy window and

639 a 1:5 coincidence mode. Data were reconstructed using the Tera-Tomo reconstruction

640 engine (3D-OSEM based manufactured customized algorithm) with expectation

641 maximization iterations, scatter and attenuation correction. Volumes-of-interest (VOI)

642 were delineated on the tumor and the contralateral brain on PET/CT fusion slices using

643 the PMOD software package (PMOD Technologies Ltd, Zürich, Switzerland). Total FDG

644 uptake was estimated as the product from the volume by the mean uptake of the

645 segmented region.

646

647

648

27 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

649 Live imaging

650 For multiphoton excitation of endogenous fluorophores in experimental gliomas, we used

651 a Leica SP8 DIVE in vivo imaging system equipped with 4tune spectral external hybrid

652 detectors and an InSightX3 laser (SpectraPhysics). The microscope was equipped with in

653 house designed mouse holding platform for intravital imaging (stereotactic frame,

654 Narishige; gas anesthesia and body temperature monitoring/control, Minerve).

655 Acquisition of ROSAmTmG reporter mice was performed at 1040-nm fixed wavelength.

656 GFP signal from genetically modified tumor cells was acquired at 925-nm wavelength.

657 Alexa Fluor 647 coupled Dextran was acquired at 1200-nm wavelength.

658

659 Flow-cytometric staining of tumor-infiltrating immune cells

660 Day 21 CT-2A shCTRL and shSlit2 tumors were harvested, dissociated and incubated

661 with anti-CD45 Alexa Fluor 594 (R&D Systems) or BUV805 (BD), anti-CD11b BV450

662 (BD), anti-Ly6G PerCP/Cy5.5 (BD), anti-Ly6C APC/Cy7 (BD), anti-F4/80 PE (BD),

663 anti-CD11c (APC), anti-MHCII PE/Cy7 (Biolegend), anti-MRC1 BV711 (Biolegend),

664 anti-CD3 PE/Cy5 (Biolegend), anti-CD19 PE/Texas Red (BD), anti-CD4 PE

665 (Biolegend), and anti-CD8 PerCP/Cy5.5 (Biolegend). As a control, cells were stained

666 with the appropriate isotype control. Data acquisition was performed on the BD

667 LSRFortessa X20 and analysis was performed with FlowJo_V10.

668

669 Slit2 ELISA

670 Slit2 concentrations in mice serum were determined by the sandwich ELISA method with

671 the DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems) according to the

672 manufacturer’s instructions, using serum samples obtained either from healthy mice or

673 from tumor-bearing mice. Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone

28 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

674 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and

675 sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a

676 concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were

677 used for revelation.

678

679 MRI

680 Magnetic resonance imaging (MRI) was performed 21 days after tumor implantation in

681 mice under Isofluorane anesthesia (2 to 2,5% mixed in ambient air) in a 4.7-T magnetic

682 resonance scanner (Bruker BioSpec 47/40USR). Brain images were obtained using a

683 Fast-Spin-Echo (FSE) T2 weighted (TE/TR: 15/2000 ms; matrix: 128×128; slice

684 thickness: 1 mm; with no gap; 12 averages) and a Spin-Echo (SE) T1 weighted (TE/TR:

685 15/250 ms; matrix: 128x128; slice thickness: 1 mm; with no gap; 12 averages) sequences

686 in axial and coronal planes. T1 weighted images were acquired before and T2 weighted

687 images after intraperitoneal injection of gadoteric acid (200uL, 0.01mmol/mL, 0.05

688 M/Kg).

689

690 siRNA transfection

691 Robo1, Robo2 and control siRNAs were purchased from Origene. We transfected RAW

692 264.7 macrophages with 10nM final siRNA concentration using siTran1.0 transfection

693 reagent (Origene), according to the manufacturer's instructions. Cells were used for

694 experiments 72 h after transfection. For qPCR experiments, RNAs were purified using

695 RNeasy-kit (Qiagen). 500 or 750 ng of RNA were reverse transcribed using SuperScript

696 II Reverse Transcriptase and Random Primers (Invitrogen). Quantitative PCR were

697 assayed as described for patient samples.

698 For adenoviral Robo1 rescue, we used previously described methods (28, 31).

29 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

699

700 Transwell Migration Assay

701 For chemotactic migration assays with 8.0μm Polycarbonate Membrane Transwell inserts

702 (Corning Inc), 20.000 primary cells were plated in 125 μL of serum-free DMEM medium

703 on the top chambers. When stated, 1.000ng/mL of rmSlit2 was also added to the top

704 chambers. Then, bottom chambers were filled with 500 μL of serum-free DMEM with

o 705 chemoattractants (R&D Systems). Cells were cultured overnight at 37 C and 5% CO2,

706 then incubated for 30 minutes with Calcein AM (Invitrogen) to stain live cells. Then the

707 wells were washed and 10 pictures per well were acquired at 10x magnification using a

708 Leica DMIRB inverted epifluorescence microscope. Migrated cells per field were

709 counted using ImageJ software.

710 For Transwell migration assay in direction to tumor cells, 30.000 tumor cells were plated

711 in the bottom chamber and starved in 500uL of serum-free media for 8 hours before

712 plating cells on the top chamber.

713

714 Western blot analysis

715 After siRNA transfection and/or treatments, cells were lysed in RIPA lysis buffer

716 including phosphatase and protease inhibitors (Invitrogen). Equal amounts of proteins

717 were separated on 4–15% Criterion precast gel (Bio-rad) and transferred on nitrocellulose

718 membrane with Transblot Turbo (Bio-rad). Then membranes were blocked in 5% non-

719 fat milk in TBS-T for 30 minutes at room temperature and incubated with primary

720 antibodies against Robo1 (R&D Systems, 1:500), Robo2 (R&D Systems, 1:500), Actin

721 (Sigma, 1:4000), anti-phospo p44/42 MAP kinase (phospho-ERK, Cell Signaling,

722 1:1000), anti-p44/42 MAP kinase (total ERK, Cell Signaling, 1:1000), anti-pAkt Ser473

723 (Cell Signaling, 1:1000), anti-Akt (Cell Signaling, 1:1000), anti-pPLCγ Ser 1248 (Cell

30 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

724 Signaling, 1:1000), anti-PLCγ (4511T, Cell Signaling, 1:1000) overnight at 4oC under

725 agitation. After washing with TBS-T membranes were incubated with proper HRP-

726 conjugated secondary antibodies for 3 hours at room temperature under agitation.

727 Western blots were developed with chemiluminescence HRP substrate (Bio-rad) on a

728 Luminescent image analyser, ChemiDoc XRS+ (Bio-rad).

729

730 Statistical analysis and quantification

731 For continuous variables, data are presented as mean ± s.e.m. Between-group

732 comparisons used the Mann–Whitney U-test or t-test depending on the sample size for

733 continuous variables. In cases where more than two groups were compared, one-way

734 ANOVA test was performed, followed by Turkey’s multiple comparison test, and results

735 were considered significantly different if P < 0.05. For comparisons involving grouped

736 data, two-way ANOVA test was performed, followed by Turkey’s multiple comparison

737 test, and results were considered significantly different if P < 0.05.

738 For survival experiment, log-rank (Mantel–Cox) tests and multiple comparison tests were

739 performed. A two-tailed value of P < 0.05 was considered statistically significant. All the

740 analyses were performed using Prism 6.0 software (GraphPad).

741 For mice in vivo imaging quantification, four to nine fields per animal were pictured in

742 the tumor center and blood vessel caliber, branching and vessel perfusion were analyzed

743 using Fiji software.

744 For mice ex vivo imaging quantification, five fields per individual were pictured in the

745 tumor center and number of macrophages, overlapping stainings, hypoxic area and tumor

746 double-strand DNA damages were quantified using Fiji software.

31 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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40 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

972 Harmony. Nat. Methods 2019;16(12). doi:10.1038/s41592-019-0619-0

973

41 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

974 ACKNOWLEDGEMENTS

975 This study was supported by the Fondation ARC pour la Recherche sur le Cancer–Institut

976 National du Cancer (ARC-INCa, A.E., JLT and A.B.), ERC (grant agreement No. 834161

977 to AE) and NHLBI (1R01HLI125811 to AE.). L.H.G. was founded by fellowships of the

978 ‘Coordenação de Aperfeiçoamento de Pessoal de Nível Superior’ (CAPES) and the

979 European Society of Cardiology (ESC Basic Research Fellowship). We thank the Paris

980 Cardiovascular Research Center (PARCC) Flow and Image Cytometry facility (Drs.

981 Camille Brunaud and Camille Knosp), FDG PET and MRI imaging platform (France Life

982 Imaging network (grant ANR-11-INBS-0006) and ‘Infrastructures Biologie Santé’

983 (IBISA), and multiphoton imaging platform (Leducq Foundation RETP: Visualisation

984 des pathologies vasculaires). We thank Dr. Holger Gerhardt for providing GL261, CT-

985 2A and RAW264.7 cells and CSF1-R-CreERT2 mice and Dr. Alain Chedotal for

986 providing Robo1-/-Robo2fl/fl mice used in this study.

987

988 AUTHOR CONTRIBUTIONS

989 LHG, AE, JLT and TM conceived and designed the project. LHG, YX, LJ, LPF, RR,

990 NM, MV, CK, CL, TD, JR, TV and TM performed experiments, collected and analyzed

991 data. JD, LS, SDV and MS collected patient samples and provided clinical data for the

992 study. BT, AB, AI, RL, FRSL contributed to study design and reviewed and revised the

993 manuscript. FRSL, AE and TM supervised the project; LHG, TM and AE wrote the

994 manuscript. All authors revised the manuscript.

995

42 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

996 FIGURES AND LEGEND

Figure 1

A B C 25 Low-GradeGBM Gliomas Low-GradeLow-Grade Gliomas Gliomas ** 1.00 1.00 1.0 Low 20 1.0 1.0 Low SLIT2: Medium SLIT2: 0.75 High 0.75 High 15 ** p=0.0577 p<0.01 0.50.50 0.50 p<0.01 0.5 (Fold change) 10 0.5 0.25 0.25 * SLIT2 5 Survival probability Survival probability 0.00.00 Survival probability 0.00 50 100 150 0.00 0 0 50 100 150 200 250 LGG (I-III) GBM (IV)

0 20 40 60 Survival probability 0 50 100 150 200 250 Time (Months) TimeTime ((Months)Months) 0.0 Time (Months) 0 50 100 150 200 250 Time (Months) D E F Tumor Cells T Cells Proliferating Cells Pericytes Macrophages Astrocytes Vascular Cells Oligodendrocytes 16 ** 5 5 12 2 2 0 0 8 (Fold change) UMAP UMAP 4 -5 -5 SLIT2

0 -10 -10 IDH-WT IDH-mutated -10 -5 0 5 -10 -5 0 5 UMAP 1 UMAP 1 G H ROBO1 I SLIT2 ROBO2 6 4 4 Level Level 3 3 Level 4

2 2 2 1 1 Expression Expression Expression Expression Expression Expression 0 0 0

T Cells T Cells T Cells Pericytes Pericytes Pericytes Astrocytes Astrocytes Astrocytes Tumor Cells Tumor Cells Tumor Cells Macrophages Vascular Cells MacrophagesVascular Cells MacrophagesVascular Cells Proliferating Cells Oligodendrocytes Proliferating Cells Oligodendrocytes Proliferating Cells Oligodendrocytes

J K L p<0.01** p<0.001*** p<0.01** p<0.05*

) p<0.01 ) ** 20 p<0.01 1.5 p<0.05 2.0 ) ** * p<0.01** p<0.05* 15 1.5 change 1.0 change change 10 1.0 (Fold (Fold (Fold (Fold 0.5 (Fold change) (Fold (Fold 5 0.5 Slit2 Robo2 Robo1 Slit2

0 0.0 change) (Fold ROBO2 0.0

ECs TALs ECs TAMs TALs ECs TAMs TALs TAMsTAMs TALs Tumor Tumor TAMs TALs Tumor Cells Cells Cells Tumor Cells Tumor Cells 997 ECs TAMs TALs

Tumor Cells 43 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.12.438457; this version posted April 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

998 Figure 1. Slit2 expression correlates with glioma aggressiveness and poor patient

999 prognosis.

1000 A. In silico analysis of TCGA glioblastoma RNAseq patient database (n = 51 high, 50

1001 medium and 50 low Slit2 expressing patients; O.S., 9.86 months for high expression,

1002 14.69 months for low expression, and 16.79 months for medium expression, log‐rank

1003 test). B. Survival analysis of Low-Grade Gliomas (LGG, Grades I to III) patients grouped

1004 by their levels of SLIT2 expression (n = 41 high and 41 low SLIT2 expressing patients;

1005 O.S., 64.73 months for high expression and 209.13 months for low expression, log‐rank

1006 test). C. SLIT2 qPCR expression in glioma patient samples from (B) (GBM, n = 45; LGG,

1007 n = 84; Student’s t test). D. SLIT2 qPCR expression in Grades III and IV glioma patient

1008 samples classified by their IDH-1/2 status (IDH-WT, n = 51; IDH-mutated, n = 34;

1009 Mann-Whitney U test). E-F. UMAP plots of single cell RNA sequencing (scRNAseq) of

1010 32 GBM patients showing different samples (E) and clustering of the different cell types

1011 in the GBM microenvironment (F). G-I. Expression plots of SLIT2 (G), ROBO1 (H) and

1012 ROBO2 (I) in scRNAseq data from (E). J-L. qPCR analysis of Slit2 (J), Robo1 (K) and

1013 Robo2 (L) expression in endothelial cells (ECs), tumor associated macrophages (TAMs),

1014 tumor associated T lymphocytes (TALs) and tumor cells FACS-sorted from late-stage

1015 CT-2A mice glioblastomas (n = 3 independent tumors, day 21 after implantation, One-

1016 Way ANOVA). All data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P

1017 < 0.001.

1018

1019

1020

1021

1022

44 Figure 2 A B C p<0.001***

) p<0.001

3 *** 2.5 p<0.01** p<0.001*** 260 shSlit2 shSlit2 + shCTRL hSLIT2 2.0 195 1.5 Slit2 130 1.0

Slit2/Actin 65 0.5 Actin (Fold change)

0.0 (mm Volume Tumor 0

shSlit2 shSlit2 shCTRL shCTRL

shSlit2 + hSLIT2

D E p<0.001*** p<0.05 FDG-PET - Day 14 FDG-PET - Day 21 * 300 p<0.001*** p<0.001*** ) 3 shCTRL 200 shSlit2

100 Volume (mm Volume shCTRL Tumor Metabolic Metabolic Tumor 0 14 days100 21 days F p<0.001*** 0.4 p<0.001*** p<0.001***

0.3 shCTRL 50 shSlit2 0.2 shSlit2

0.1 Percent survival ***

Total FDG uptake FDG Total ** n.s. ** 0.0 [({SUVbw}g/ml)*(ccm)] 0 *** 14 days 0 21 days 20 40 60 G H Time (Days) 100 shCTRL shCTRL + TMZ shSlit2 shSlit2 + TMZ 50 shSlit2 + hSLIT2 shSlit2 + hSLIT2 + TMZ

Percent survival *** ** n.s. ** 0 *** 0 20 40 60 Time (Days) shCTRL shCTRL + TMZ shSlit2 shSlit2 + TMZ shSlit2 + hSLIT2 shSlit2 + hSLIT2 + TMZ

1023

45 1024 Figure 2. Slit2 promotes Glioblastoma growth and resistance to TMZ.

1025 A-B. Western blot analysis (A) and quantification (B) of Slit2 expression in shCTRL,

1026 shSlit2 and shSlit2+hSLIT2 CT-2A cells (n = 5, One-Way ANOVA). C. Tumor volume

1027 quantification at 21 days (n = 10 for shCTRL and n = 8 for shSlit2, Student’s t- test). D.

1028 FDG-PET imaging over CT-2A shCTRL and shSlit2 glioma growth (n = 5 shCTRL and

1029 n = 4 shSlit2). E-F. Quantification of tumor metabolic volume (E) and total tumor

1030 glucose uptake (F) from (D) (n = 5 for shCTRL and n = 4 for shSlit2, One-Way

1031 ANOVA). G. Survival trial design: 8-week-old mice were engrafted with CT-2A

1032 shCTRL, shSlit2 or shSlit2+hSLIT2 spheroids and randomly assigned to vehicle or TMZ

1033 treatment (40 mg/kg on days 7, 11, 15 and 19 after tumor implantation). H. Survival

1034 curves of the mice in (G) (n = 10 mice per group, O.S.= 22.5 days for shCTRL, 28 days

1035 for shCTRL + TMZ, 30 days for shSlit2, 39.5 days for shSlit2 + TMZ, 20 days for

1036 shSlit2+hSLIT2 and 27 days for shSlit2+hSLIT2 + TMZ; Multiple comparisons log-rank

1037 test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

1038

1039

1040

1041

1042

1043

1044

1045

1046

1047

46 m) 80 µ p<0.001 150 p<0.001 Figure 3 60

40 shCTRL100 shCTRL A shCTRL shSlit2 B C shSlit2 shSlit2 20 m) 80 50 150 p<0.001*** µ p<0.001 0 *** Vessel diameter ( diameter Vessel 14 days 21 days60 0 100 shCTRL Branchpoints per field 21 40 14 days 21 daysshCTRL shSlit2 shSlit250 Day Day 20 0 0

150µm ( diameter Vessel 14 days 21 days Branchpoints per field 14 days 21 days

D E F shSlit2 shSlit2 + hSLIT2 shSlit2 shSlit2 shSlit2+ shSlit2+

m) 40 hSLIT2 p<0.001 150 hSLIT2 µ *** p<0.001 30 *** 100 shSlit2 18 20 shSlit2 shSlit2 shSlit2+50

Day Day + hSlit2 10 hSlit2

150µm 0 0 Vessel diameter ( diameter Vessel 10 days 18 days Branchpoints per field 10 days 18 days

G Tomato H I Evans Blue Dextran 647 20 p<0.001*** 1.5 p<0.01** * 15 * 1.0 * * * 10 * *

per field 0.5 * 5

** (fold change) * 0 0.0 shCTRL Unperfused vessels Evans blue extravasation * shSlit2 shSlit2 * shCTRL shCTRL

J 80 p<0.01** K 80 p<0.01** 60 60 * * 40 40 *

shSlit2 * 20 20 %Glut1 blood %Glut1+ Area

0 vessel coverage 0 200µm 1mm *

shSlit2 shSlit2 shCTRL shCTRL L Tomato Glut1 M

2

1 shCTRL

to shCTRL 0 p<0.05* p<0.01** p<0.05* Fold change relative -1

IL-6

shSlit2 PD-L1 PD-L2

150µm

1048

47 1049 Figure 3. Slit2 promotes blood vessel dysmorphia in GBM.

1050 A. In vivo two-photon images of ROSAmTmG mice bearing day 21 CT-2A shCTRL or

1051 shSlit2 tumors. B-C. Quantification of vessel diameter (B) and branchpoints (C) (n = 8

1052 mice per group, One-way ANOVA). D. In vivo two-photon images of ROSAmTmG mice

1053 bearing day 18 CT-2A shSlit2 or shSlit2+hSLIT2 tumors. E-F. Quantification of vessel

1054 diameter (E) and branchpoints (F) (n = 7 mice per group, One-way ANOVA). G-I. Left

1055 panels: Two-photon in vivo imaging following intravenous injection of Alexa Fluor 647

1056 conjugated Dextran highlighting unperfused blood vessel segments in the tumor core

1057 (asterisks) of day 21 CT-2A shCTRL and shSlit2 tumors. Right panels: representative

1058 pictures of whole brains of day 21 shCTRL or shSlit2 CT-2A tumors following Evans

1059 blue injection. H. Quantification of unperfused blood vessel segments in the tumor mass

1060 presented in (G) (n = 5 mice per group, Mann-Whitney U test). I. Quantification of Evans

1061 Blue extravasation in (G) (n = 5 mice per group, Mann-Whitney U test). J-L.

1062 quantifications of Glut1+ hypoxic areas in the tumor (J) and Glut1 blood vessel coverage

1063 (K) from immunohistochemistry on sections (L) (n = 5 mice per group, Mann-Whitney

1064 U test). M. qPCR analyses from FACS-sorted endothelial cells (n = 3 tumors/group,

1065 Mann-Whitney U test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, ***

1066 P < 0.001

1067

1068

1069

1070

1071

1072

1073

48 Figure 4

A B *** *** *** *** shCTRL shSlit2 shSlit2 + hSLIT2 250 *** *** F4/80+ *** *** *** 200 MHCII+ 150 *** MRC1+

F4/80 100 50 Cells per field 0 Tomato

shSlit2 shCTRL

MHCII shSlit2 + hSLIT2 C D Tumor-supportive Tumor-associated TAMs (MRC1+) -

Tomato Microglia/Macrophages Cytotoxic TAMs (TAMs) (MHCII+CD11c+)

Ly6G 20 100 + p< ***0.001 *** 15 Tumor-supportive MRC1 + F4/80 TAMs (MRC1 ) + 10 50 *** Cytotoxic TAMs (MHCII+CD11c+) (% total) % of TAMs Tomato 125µm 5 CD11b + 0 0 CD45

shSlit2 shCTRL shSlit2 shCTRL

E F G

8 Tumor supportive Cytotoxic 3 40 * p<0.001*** p<0.001*** 6 * 30 2 4 *** 20 /mg protein

* γ 2 * 1 to shCTRL 10 0 ** *** *** *** *** *** ** *** *** *** ng IFN Fold change relative 0 ng IL-10/mg protein 0 1 β α β Arg1 IL-10 IL-12IL-1 CCR7 TNF MRC1VEGFaTGF MMP9 CCL19 PD-L1PD-L2 CD209a CXCL10

shSlit2 TAMs shSlit2 TAMs shCTRL TAMs shCTRL TAMs H I shCTRL shSlit2 shSlit2 + hSLIT2 100 p<0.001*** 20 p<0.001*** p<0.001*** p<0.001*** 80

15 cells

+ 60 10 40

5 % sFlt1 20 sFlt1binding 0 125µm 0 ng VEGFa/mg protein

shSlit2 shCTRL

shSlit2 TAMs shCTRL TAMs shSlit2 + hSLIT2

1074

49 1075 Figure 4. Slit2 promotes TAM recruitment and polarization in mouse gliomas.

1076 A. Immunohistochemistry on sections of late-stage CT-2A shCTRL, shSlit2 or

1077 shSlit2+hSLIT2 tumors for F4/80, MHC-II and MRC1+ cells (green). B. Quantifications

1078 of (A) (n = 7 mice per group, 5 fields per tumor, Two-Way ANOVA). C-D. FACS

1079 analysis of 21 days CT-2A shCTRL and shSlit2 for quantification of TAMs (n = 10

1080 tumors/group; Student’s t-test and Two-way ANOVA). E. qPCR analysis from FACS-

1081 sorted TAMs (n = 6 tumors/group, Mann-Whitney U test). F-H. ELISA from protein

1082 samples extracted from FACS-sorted TAMs from shCTRL and shSlit2 tumors to quantify

1083 IFNγ (F), IL-10 (G) and VEGFa (H) (n = 5 tumors/group, Mann-Whitney U test). I.

1084 Representative images and quantification of soluble-Flt1 binding to sections of day 21

1085 CT-2A shCTRL, shSlit2 and day 18 shSlit2+hSLIT2 tumors (n = 7 mice per group, 5

1086 fields per tumor, One-Way ANOVA). * P < 0.05, ** P < 0.01, *** P < 0.001

1087

1088

1089

1090

1091

1092

1093

1094

1095

1096

1097

1098

1099

50 Figure 5 A Tumor-associated B C T Lymphocytes (TALs) p<0.01 + p<0.01** 2.0 ** 6 p<0.05* 3

CD3 1.5 - 2 4 1.0 CD11b Lymphocytes 0.5 Lymphocytes + (% total) (% total) + + (% total) 2 1 0.0 CD45 T CD8 0 T CD4 -0.5 0

shCTRL shSlit2 shCTRL shSlit2 shCTRL shSlit2 D E Th1 Th2 Th17 25 10 Activated Exhausted *** 20 *** 15 5 *** 10 *** ** to shCTRL to shCTRL 5 *** Fold change relative Fold change relative 0 0 ** *** * *** *** *** *** γ α γ IL-2 IFN TNF IL-10 PD-1 IFN Tim3 Lag3 CCL17IL-17a CTLA4 GZMB CXCL11 CXCL10

F G H 10 p<0.05 15 shCTRL shSlit2 * p<0.001*** 8 T cells

+ 10 6 100 *** /mg protein CD8 γ

+ 4 per field

GZMB 5 2 ng IFN GZMB CD8 CD8 50µm 0 50 0

shSlit2Percent survival shSlit2 shCTRL ***shCTRL*** 0 I J K 0 30 60 90 4 2.0 100 Time*** (days) p<0.001*** p<0.001*** 3 1.5 shCTRL shCTRL + Anti-PD-1 + Anti-41BB 2 1.0 50 shSlit2 shSlit2 + Anti-PD-1 + Anti-41BB 1 0.5 Percent survival

ng IL-10/mg protein *** *** ng VEGFa/mg protein 0 0.0 0 0 30 60 90 Time (days) shSlit2 shSlit2 shCTRL shCTRL shCTRL shCTRL + Anti-PD-1 + Anti-41BB shSlit2 shSlit2 + Anti-PD-1 + Anti-41BB

1100

51 1101 Figure 5. Slit2 inhibits T lymphocyte responses in the glioma microenvironment.

1102 A-C. T lymphocyte FACS analysis of day 21 CT-2A shCTRL and shSlit2 tumors for total

1103 CD3+ TALs (A), CD4+ TALs (B) and CD8+ TALs (C) (n = 8 tumors/group; Student’s t-

1104 test). D. qPCR analyses from FACS-sorted CD4+ T lymphocytes (n = 10 tumors/group,

1105 Mann-Whitney U test). E. qPCR analyses from FACS-sorted CD8+ T lymphocytes (n =

1106 6 tumors/group, Mann-Whitney U test). F. Representative images of CD8 and GZMB

1107 staining on sections of day 21 CT-2A shCTRL and shSlit2 tumors. G. Quantification of

1108 (F) (n = 4 mice per group, 5 fields per tumor, Mann-Whitney U test). H-J. ELISA from

1109 protein samples extracted from FACS-sorted CD8+ TALs from shCTRL and shSlit2

1110 tumors to quantify IFNγ (H), IL-10 (I) and VEGFa (J) (n = 5 tumors/group, Mann-

1111 Whitney U test). K. 8-week-old mice were engrafted with CT-2A shCTRL or shSlit2 and

1112 randomly assigned to vehicle or Anti-PD-1 + Anti-4-1BB treatment (200ug each on days

1113 7, 9, 11 and 13 after tumor implantation) (n = 10/11 mice per group, O.S.= 25 days for

1114 shCTRL, 33 days for shCTRL + Anti-PD-1 + Anti-4-1BB, 33 days for shSlit2 and

1115 Undetermined for shSlit2 + Anti-PD-1 + Anti-4-1BB; Multiple comparisons log-rank

1116 test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

1117

1118

1119

1120

1121

1122

1123

1124

52 Figure 6 Bone-MarrowBone-Marrow Derived Derived Microglia Peritoneal Macrophages (PM) A B MacrophagesMacrophages (BMDM) (BMDM) C 30 5050 30 25 p<0.001*** p<0.001*** *** 4040 20 20 * ** 30 p<0.01* 20 p<0.05 15 30 p<0.05 * * 20 p<0.05 10 20 10 Cells per field Cells per field Cells per field 10 10 5 Cells per field 10 Cells per field 0 0 0 0 0 CTRL CTRL CTRL CTRL Slit 6nM Slit 6nM Slit 6nM Slit2 3nM Slit2 3nM Slit2 3nM Slit 6nMSlit2 12nM CTRL Slit2 12nM Slit2 12nM Slit2 0,6nMSlit2 1,2nMSlit2 3nM Slit2 0,6nMSlit2 1,2nM Slit 6nM Slit2 0,6nMSlit2 1,2nM Slit2 0,6nMSlit2 1,2nM Slit2 12nM Slit2 3nM Slit2 0,6nMSlit2 1,2nM Slit2 12nM siScr siRobo1/2 D F Slit2 6nM (time) G p<0.001 CTRL Slit2 Slit2 *** 0’ 5’ 10’ 15’ 30’ 60’ p<0.001*** p<0.001 Calcein *** pPLCγ 80 p<0.001***

CTRL 60 PLCγ p<0.001*** 40 pErk1/2

[IL-10] (ng/mL) 20 Erk1/2 Robo1FL 0 CTRL LPS IL-10 Slit2 pAkt CD Δ

Akt *** 50µm Robo1 H *** p<0.001 8 * *** E p<0.001 ***p<0.001 pCEBPβ 50 50 *** *** p<0.001 *** 6 40 p<0.00140 *** CTRLCTRL *** * 30 30 Slit2Slit2 CEBPβ 4 20 20 2 [VEGFa] (ng/mL) Cells per field

Cells per field 10 10 pStat6 0 0 0 CTRL LPS IL-10 Slit2 Stat6

CTRL AV siScr CTRL AV siScr CD AV siRobo1+2 CTRL AV siRobo1+2 Δ CD AV siRobo1+2 CTRL AV siRobo1+2Robo1 FLΔ AV siRobo1+2 Robo1 Robo1 FL AV siRobo1+2 I IlRobo1-1β Cxcl10 Mrc1 Mmp9 Ccl19 Il-10 4000 *** 80 8 5 5 4 * * 4 60 *** 6 4 * 3 *** 3 3 3000 40 4 2 2 2 Fold change Fold change Fold change Fold change Fold change 2 Fold change 20 2 1 1 1 0 0 0 0 0 0

LPS Slit2 LPS Slit2 Slit2 Slit2 Slit2 Slit2 CTRL CTRL CTRL CTRL CTRL CTRL Ccr7 Tnfα Vegfa Tgfβ1 Cd209a Arg1 60 *** 30 5 5 4 800 *** *** ** *** 4 4 3 600 ** 40 20 3 3 2 400 2 2 20 10 Fold change Fold change Fold change Fold change Fold change Fold change 1 1 1 200 0 0 0 0 0 0

LPS Slit2 LPS Slit2 Slit2 Slit2 Slit2 Slit2 CTRL CTRL CTRL CTRL CTRL CTRL Cytotoxic Tumor supportive

1125

53 1126 Figure 6. Slit 2 drives microglia and macrophage migration and tumor supportive

1127 polarization.

1128 A-C. Transwell assay of microglial cells (A), bone marrow derived macrophages

1129 (BMDM) (B) and peritoneal macrophages (PM) (C) in response to Slit2 or carrier

1130 (CTRL) in the bottom chamber (n = 4, One-way ANOVA). D-E. Transwell assay of

1131 RAW macrophages treated or not with Robo1/2 siRNA and infected with adenovirus

1132 encoding CTRL (GFP construct), Robo1FL or Robo1DCD constructs and stained with

1133 Calcein. E. Quantification of (D) (n = 3, Two-way ANOVA). F. Western blot analysis

1134 of Slit2 downstream signaling in cultured BMDM (n = 6). G-H. ELISA from conditioned

1135 medium from LPS, Il-10 or Slit2-treated BMDMs to quantify IL-10 (G) and VEGFa (H)

1136 (n = 3 independent cultures, Mann-Whitney U test). I. qPCR analysis of BMDM cultures

1137 following Slit2 or LPS treatment (n = 4, One-Way ANOVA or Mann-Whitney U test).

1138 Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

1139

1140

1141

1142

1143

1144

1145

1146

1147

1148

1149

1150

54 Figure 7 A C CTRL PI3Kγ inhibitor CTRL Slit2 CTRL Slit2 pStat6 DAPI

Robo1 PI3Kγ IP CTRL Robo1

PI3Kγ Input

Actin Slit2

20µm

p<0.001*** B p<0.001 D *** p<0.001*** p<0.001*** 120 p<0.001 p<0.001 40 *** *** CTRL 90 Slit2 30 CTRL Slit2 60 20

Cells per field 30 10

Nuclear pStat6 (A.U.) CTRL 0 0 CTRL PI3KCTRLγ inhibitor CTRLPI3KPI3Kγ inhibitorγ inhibitor PI3Kγ inhibitor E F p<0.001*** 8 p<0.001*** p<0.001*** 100 p<0.001*** p<0.001*** p<0.001*** p<0.001*** 6 80 CTRL 60 CTRL 4 PI3Kγ inhibitor PI3Kγ inhibitor p<0.001 40 *** p<0.001*** ns 2 ns ns [IL-10] (ng/mL) [VEGFa] (ng/mL) 20 ns ns ns 0 0 CTRL LPS Slit2 CTRL LPS Slit2 G Il-1β Cxcl10 Mrc1 Mmp9 Ccl19 Il-10 CTRL 1000 PI3Kγ inhibitor 7000 20 400 100 *** 6 *** *** *** 750 5000 15 80 300 4 1000 500 60 3000 10 40 750 250 200 2 5 Fold change Fold change Fold change Fold change Fold change Fold change 20 5 50µm 10 20 500 0 0 0 0 0 0 250 CTRLCTRLLPS Slit2 CTRL LPS Slit2 CTRL Slit2 CTRL Slit2 CTRL Slit2 CTRL Slit2 PI3Kγ inhibitor Fold change 20 Ccr7 Tnfα Vegfa Tgfβ1 Cd209a Arg1 10 900 350 25 8 60 *** 800 *** *** 0 *** CTRL LPS Slit2 275 20 6 700 40 15 600 600 200 4 10 500 125 20

Fold change 2 Fold change Fold change Fold change Fold change 5 Fold change 2 5 5 0 0 0 0 0 0 CTRL LPS Slit2 CTRL LPS Slit2 CTRL Slit2 CTRL Slit2 CTRL Slit2 CTRL Slit2

Cytotoxic Tumor supportive

1151

55 1152 Figure 7. Slit2 driven microglia/macrophage polarization via PI3Kγ.

1153 A. PI3Kγ immunoprecipitation in BMDMs treated or not with Slit2 for 15 minutes and

1154 WB for Robo1 (n = 3 independent experiments). B. Transwell assay of BMDMs in

1155 response to Slit2 or carrier (CTRL) in the bottom chamber after pretreatment with vehicle

1156 control (DMSO) or PI3Kγ inhibitor IPI-549 (1uM). C-D. Phospho-Stat6

1157 immunofluorescent staining of BMDMs treated or not with Slit2 and PI3Kγ inhibitor and

1158 quantification of nuclear pStat6 intensity (n = 4 independent cultures, 2-way ANOVA).

1159 E-F. ELISA from conditioned medium from LPS or Slit2-treated BMDMs with vehicle

1160 control (DMSO) or PI3Kγ inhibitor, to quantify IL-10 (E) and VEGFa (F) (n = 3

1161 independent cultures, 2-way ANOVA). G. qPCR analysis of BMDM cultures following

1162 Slit2 or LPS treatment with vehicle control or PI3Kγ inhibitor (n = 4 independent

1163 cultures, 2-way ANOVA). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01,

1164 *** P < 0.001

1165

1166

1167

1168

1169

1170

1171

1172

1173

1174

1175

1176

56 Figure 8 A T2 Pre- T1 Post- B C D gadolinium gadolinium Day 14 Day 21 p<0.01 ) ) ** 3 180 p<0.05* 3 260

135 195 CTRL CTRL 90 130

45 65 Tumor volume (mm volume Tumor Tumor volume (mm volume Tumor 0 0

CTRL CTRL

iRoboMacKO iRoboMacKO iRoboMacKO iRoboMacKO 150µm

E G H J 60 p<0.01** 80 p<0.001 CTRL *** 400 Tomato GFP Glut1 60 Area) iRoboMacKO cells

+ 40 + 300 40 p<0.001 p<0.01** p<0.001 20 400 20 p<0.001 p<0.001 Hypoxic Area

200 % sFlt1 (%Glut1 0 0 100 300 CTRL CTRL CTRL Branchpoints per field 0 200 14 days 21 days iRoboMacKO 100 iRoboMacKO

F I Macrophages per field 500 *** m) 50 p<0.01** 0 µ *** CTRL iRoboMacKO 40 400 *** *** Iba1 30 p<0.01** 300 MHC-II 20 200 MRC1 iRoboMacKO 10 150µm Cells per field 100

Vessel diameter ( diameter Vessel 0 0 14 days 21 days CTRL iRoboMacKO Ib K L M N O a1 80 MHC-II ) ) MRC1 L) + + 70 40 p<0.001*** 30 20 p<0.05* µ 4 p<0.01** p<0.05* 60 * * 30 15 3 50 CTRL 20 40 iRoboMacKO 20 10 2

% of WBC 3 macrophages macrophages 10 + 10 + 5 1 2 1 BFP BFP CD3+ cells per field in MLN (% CD11b Lymphocytes (K/ Lymphocytes 0 in DCLN (% CD11b 0 0 0 0

CTRL CTRL CTRL CTRL Basophils Neutrophils MonocytesEosinophils Lymphocytes iRoboMacKO iRoboMacKO iRoboMacKO iRoboMacKO P Robo1-/-Robo2flox/flox Robo1-/-Robo2flox/flox + Anti-PD-1 + Anti-41BB Anti-PD-1 + Anti-4-1BB 100 iRoboMacKO (200ug) iRoboMacKO + Anti-PD-1 + Anti-41BB *** D0 D7 D9 D11 D13 50

*** Tumor Percent survival ns Implantation TAM 80mg/Kg every 3 days from D7 0 0 15 30 45 60 75 90 Time (days)

Robo1-/-Robo2flox/flox Robo1-/-Robo2flox/flox + Anti-PD-1 + Anti-41BB iRoboMacKO iRoboMacKO + Anti-PD-1 + Anti-41BB 1177

57 1178 Figure 8. Macrophage-specific Robo1/2 KO normalizes the TME.

1179 A. T2-weighted pre-gadolinium and T1-weighted post-gadolinium MRI images of CTRL

1180 and iRoboMacKO mice 21 days after tumor spheroid implantation. B-C. Quantification

1181 of tumor size 21 days after tumor spheroid implantation on MRI images (B, n = 4 tumors

1182 per group, Mann-Whitney U test) and serial vibratome sections (C, n = 7 CTRL and 6

1183 iRoboMacKO tumors, Mann-Whitney U test). D-F. In vivo two-photon images of tumor

1184 bearing mice in (D) and quantification of vessel diameter (E) and branchpoints (F) (n =

1185 6 mice per group, One-way ANOVA). G-H. Glut1 (blue) immunohistochemistry on day

1186 21 tumor bearing CTRL and iRoboMacKO mice (G), and quantification of hypoxic areas

1187 in the tumor (H) (n = 6 CTRL and 5 iRoboMacKO tumors, Mann-Whitney U test). I-K.

1188 Quantification of immunohistochemistry on sections of day 21 CT-2A CTRL and

1189 iRoboMacKO tumors for F4/80, MHC-II and MRC1+ cells (I), VEGFa-expressing

1190 (sFLT1+) GFP+ cells (J), and total TALs (CD3+) (K) (n = 6 CTRL and 5 iRoboMacKO

1191 tumors, 2-way ANOVA or Mann-Whitney U test). L-M. FACS analysis of deep cervical

1192 and mandibular lymph nodes (DCLN and MLN, respectively) from day 21 CTRL and

1193 iRoboMacKO tumor-bearing mice (n = 5 CTRL and 4 iRoboMacKO mice; Mann-

1194 Whitney U test). N-O. Lymphocyte counts (N) and differential WBC counts (O) from

1195 peripheral blood of day 21 CTRL and iRoboMacKO tumor-bearing mice (n = 5

1196 mice/group; Mann-Whitney U test). P. 8-week-old mice were engrafted with CT-2A BFP

1197 and gene deletion was achieved by 80mg/kg Tamoxifen intraperitoneal injection every 3

1198 days starting 7 days after tumor implantation. Robo1-/-Robo2flox/flox and iRoboMacKO

1199 mice were randomly assigned to vehicle or Anti-PD-1 + Anti-4-1BB treatment (200ug

1200 per dose on days 7, 9, 11 and 13 after tumor implantation) (n = 10/11 mice per group,

1201 O.S.= 21.5 days for Robo1-/-Robo2flox/flox, 24 days for Robo1-/-Robo2flox/flox + Anti-PD-1

1202 + Anti-4-1BB, 29 days for iRoboMacKO and Undetermined for iRoboMacKO + Anti-

58 1203 PD-1 + Anti-4-1BB; Multiple comparisons log-rank test). Data are presented as mean ±

1204 s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

1205

1206

1207

1208

1209

1210

1211

1212

1213

1214

1215

1216

1217

1218

1219

1220

1221

1222

1223

1224

59 Figure 9 A C D E Tumor CTRLFc Robo1Fc p<0.001 ) 450 *** 3

300 14 CTRLFc B p<0.001

*** Day 250 Robo1Fc CTRLFc 150 200

150 (mm volume Tumor 0 3

100 2 Slit2 (ng/mL)

CTRLFc Day 50 Robo1Fc Robo1Fc 3mm 150µm 0 p<0.001 p<0.001 14 21 120 120 Time (Days) 90 90 F G CTRLFc H CTRLFc p<0.001*** 50 120 Robo1Fc 100 Robo1Fc

m) CTRLFc p<0.00160 60 p<0.001

µ *** *** 40 Robo1Fc 90 80 30 30 CTRLFc Area) 30 Robo1Fc+ 60 60 Branchpoints per field 20 p<0.001 Branchpoints per field 0 0 40 *** 14 days 23 days 14 days 23 days

30 Hypoxic Area

10 (% Glut1 20 Vessel diameter ( diameter Vessel 0 Branchpoints per field 0 0 14 days 23 days 14 days 23 days

I J 50 K CTRLFc Robo1Fc50

m) CTRLFc p<0.001 m) CTRLFc p<0.001 250 p<0.001µ µ 100 *** 40 Robo1Fc 20 40 Robo1Fc *** F4/80 200 p<0.001 MHC-II 80 30 ***30 *** 15 150 MRC1 cells + 60 20 p<0.001 20 p<0.001 *** *** 10 100 *** 40 10 10 Vessel diameter ( diameter Vessel ( diameter Vessel % sFlt1 Cells per field 5 50 20 0 0

14 days 23 days CD3+ cells per field 14 days 23 days 0 0 0 CTRLFc Robo1Fc L Robo1Fc M Robo1Fc CTRLFc CTRLFc Robo1Fc + TMZ Robo1Fc Robo1Fc + Anti-PD-1Robo1 Fc + Anti-41BB 100 CTRLFc 100 CTRLFc CTRLFc + TMZ CTRLFc + Anti-PD-1 + Anti-41BB ***

50 *** 50

Percent survival *** *** Percent survival ** ** 0 0 0 30 60 90 120 150 0 30 60 90 Time (days) Time (days) N 100 CTRLFc *** Robo1Fc + Anti-PD-1 + Anti-41BB Survivors CTRLFc + TMZ Robo1Fc SurvivorsCTRLFc Robo1Fc Anti-PD-1 + Anti-41BB Survivors CTRLFc + Anti-PD-1 + Anti-41BB Robo1Fc + TMZ Treatment Naive 50 Robo1Fc ** Robo1Fc + Anti-PD-1 + Anti-41BB Percent survival * 0 0 30 60 90 Time (days)

Treatment Naive 1225 Anti-PD-1 + Anti-41BB Survivors Robo1Fc Survivors Robo1Fc + Anti-PD-1 + Anti-41BB Survivors 60 1226 Figure 9. Robo1Fc treatment limits glioma growth by shifting the

1227 microenvironment.

1228 A. Experimental design: 8-week-old mice were engrafted with CT-2A spheroids and

1229 randomly assigned to CTRLFc or Robo1Fc treatment (2.5 mg/kg) every other day

1230 between day 7 and day 15 after tumor implantation. B. ELISA dosage of serum Slit2 in

1231 CTRLFc- and Robo1Fc-treated mice at days 14 and 21 (n = 4 mice per group, Two-Way

1232 ANOVA). C. Representative CTRLFc- and Robo1Fc-treated tumors at 23 days. D.

1233 quantification of (C) (n = 6, Student’s T test). E. In vivo two-photon images of day 23

1234 CTRLFc and Robo1Fc treated CT-2A tumors. F-G. Quantification of vessel diameter (F)

1235 and branchpoints (G) from (E) (n = 6 mice per group, One-way ANOVA). H.

1236 Quantification of hypoxic areas (Glut1+) on stained tumor sections of day 23 tumor-

1237 bearing mice treated with CTRLFc or Robo1Fc (n = 6 mice per group, Mann-Whitney U

1238 test). I-K. Quantification of F4/80, MHC-II and MRC1 (I), soluble-Flt1 binding (J) and

1239 CD3 (K) immunostaining of day 23 tumor-bearing mice treated with CTRLFc or

1240 Robo1Fc (n = 6 mice per group, Two-way ANOVA or Student’s t-test). L. 8-week-old

1241 wild-type mice were injected with CT-2A cells and assigned randomly to one of the

1242 following treatments: CTRLFc + vehicle (n = 20), CTRLFc + TMZ (40mg/kg on days 7,

1243 11, 15 and 19 after tumor implantation) (n = 15), Robo1Fc + vehicle (n = 24) or Robo1Fc

1244 + TMZ (n = 22, Multiple comparisons Mantel–Cox log-rank; O.S., CTRLFc: 24 days;

1245 CTRLFc + TMZ: 28 days; Robo1Fc: 41 days; Robo1Fc + TMZ: 119 days). M. 8-week-

1246 old wild-type mice were injected with CT-2A cells and assigned randomly to one of the

1247 following treatments: CTRLFc + vehicle, CTRLFc + Anti-PD-1 + Anti-4-1BB treatment

1248 (200ug of each on days 7, 9, 11 and 13 after tumor implantation), Robo1Fc + vehicle or

1249 Robo1Fc + Anti-PD-1 + Anti-4-1BB treatment (n = 10/11 mice per group; O.S.,

1250 CTRLFc: 25.5 days; CTRLFc + Anti-PD-1 + Anti-4-1BB treatment: 40 days; Robo1Fc:

61 1251 39 days; Robo1Fc + Anti-PD-1 + Anti-4-1BB treatment: Undetermined; Multiple

1252 comparisons log-rank test). N. 90 days after tumor implantation, surviving mice from

1253 Figure 9M (n = 2 Anti-PD-1 + Anti-4-1BB survivors, 3 Robo1Fc survivors and 8

1254 Robo1Fc + Anti-PD-1 + Anti-4-1BB survivors) or 8-weeks-old tumor naïve mice (n =

1255 10 mice) were re-challenged by implantation of CT-2A cells in the contralateral

1256 hemisphere from the first injection (O.S., Naïve mice: 21 days; CTRLFc + Anti-PD-1 +

1257 Anti-4-1BB survivors: 53.5 days; Robo1Fc survivors: 63 days; Robo1Fc + Anti-PD-1 +

1258 Anti-4-1BB treatment: Undetermined; Multiple comparisons log-rank test). Data are

1259 presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

1260

1261

1262

1263

1264

1265

1266

1267

1268

1269

1270

1271

1272

1273

1274

1275

62 1276 Supplemental Methods

1277

1278 Bioinformatic analysis

1279 For ‘The Cancer Genome Atlas’ (TCGA) dataset Agilent-4502A microarray, data of 488

1280 glioblastoma patients and associated clinical data were downloaded from GlioVis data

1281 portal (https://gliovis.bioinfo.cnio.es) (1). Cohort was split into 2 groups of patients

1282 defined by the level of Slit2 expression. Overall survival (in months) was used to estimate

1283 survival distributions using the Kaplan–Meier method and the distributions were

1284 compared using the log‐rank test.

1285

1286 Patient Samples

1287 For the patient samples analyzed in Figure 1C/D and Supplemental Figure 1 I-N, central

1288 review histopathology of the patients classified the samples as follows:

1289 45 patients were diagnosed with glioblastoma multiforme (GBM) grade IV, 18 patients

1290 with primary anaplastic oligodendroglioma grade III, 6 patients with primary anaplastic

1291 astrocytoma grade III, 1 patient with primary anaplastic oligoastrocytoma grade III, 16

1292 patients with grade III mixed anaplasic gliomas, 26 patients with primary

1293 oligodendroglioma grade II, 1 patient with recurrent oligodendroglioma grade II, 4

1294 patients with grade II astrocytomas, 9 patients with grade II mixed gliomas, 1 patient with

1295 primary xanthoastrocytoma grade II and 1 patient with primary subependymoma grade I.

1296 Associated IDH-1/2 mutation status and relevant clinical data from all the 129 patients

1297 were used in this study.

1298

1299

1300

63 1301 Patient-derived GBM xenograft model (PDX)

1302 N15-0460 patient-derived cell line (PDCL) was established by Gliotex team from GBM

1303 tissue sample that was provided by the neuropathology laboratory of Pitie-Salpetriere

1304 University Hospital, and obtained as part of routine resections from patients under their

1305 informed consent (ethical approval number AC-2013-1962). The parental tumor was

1306 IDH-WT and MGMT methylated. Cells are cultivated in DMEM/F12 supplemented with

1307 B27, EGF (20 ng/ml), FGF (20 ng/ml), penicillin/streptomycin 1% and plasmocin 0.2%,

1308 and dissociated with Accutase. Cells were transduced with luciferase/mkate2 lentiviral

1309 particles (in-house produced) at MOI of 3 then shRNA-GFP lentiviral particles (SLIT2,

1310 Locus ID 9353, Origene TL309262V) at MOI of 3. After infection, cells were

1311 polyclonally selected by Puromycin and mKate+ and GFP+ cells were sorted by FACS

1312 (BioRad S3e Cell Sorter).

1313 For intracranial xenografts, 1.4 x 105 cells were injected in 2 µL of HBSS in Hsd:Athymic

1314 Nude-Foxn1nu mice (Envigo) by stereotaxic injection at Bregma AP : +0.1 ; ML : -0.15

1315 ; DV : -0.25 under isoflurane anesthesia (protocol #17503 2018111214011311 v5).

1316 Tumor growth was monitored every 15 days by bioluminescence imaging following

1317 100µL luciferin subcutaneous injection at 30mg/mL, and image acquisition with IVIS®

1318 Spectrum in vivo imaging system (Perkin Elmer). The development of tumors (Tumor

1319 take) was evaluated by determining the day when bioluminescence signal doubled

1320 compared to the first bioluminescence measured 8 days post-graft.

1321

1322 In vitro spheroid formation and invasion assays

1323 For spheroid formation, 1,000 N15-0460 shCTRL or shSLIT2 cells were plated in non-

1324 adherent 96 well plates for 48hs and then imaged by fluorescence using a standard FITC

1325 filter to detect endogenous GFP. For invasion assays, spheroids were then resuspended in

64 1326 fibrinogen solution (2.5 mg/ml fibrinogen (Sigma) in DMEM/F12 supplemented with

1327 B27, EGF (20 ng/ml), FGF (20 ng/ml) and 50 mg/ml aprotinin (Sigma)) and clotted with

1328 1 U thrombin (Sigma-Aldrich) for 20 min at 37 °C. Cultures were topped with medium

1329 and incubated at 37 °C, 5% CO2. After 1 and 2 days, cultures were imaged by

1330 fluorescence using a standard FITC filter.

1331

1332 Extraction of tumor-associated macrophages, lymphocytes and endothelial cells and

1333 qPCR analysis

1334 Ketamine/Xylazine anaesthetized tumor-bearing mice were transcardially perfused with

1335 30 ml of ice-cold PBS. Tumors were harvested and incubated with DMEM containing

1336 2.5 mg/ml collagenase D, and 5 U/ml DNase I for 20 min at 37°C. The digested tissue

1337 was passed through a 40μm nylon cell strainer (Falcon) and red blood cells were lysed

1338 (Red Blood Cells Lysis buffer, Merck).

1339 After blocking with mouse FcR Blocking Reagent (MACS Miltenyi Biotec) cells were

1340 stained with the following monoclonal antibodies: anti-CD45 BUV 805 (BD), anti-

1341 CD11b BV450 (BD), anti-CD31 PE/CF594 (BD), and anti-CD3 BUV395 (BD), anti-

1342 CD4 PE (BD) and anti-CD8 PerCP/Cy5.5 (BD) antibodies. TAMs (CD45+CD11b+CD3-

1343 ), TALs (CD45+CD11b-CD3+, either CD4+ or CD8+), endothelial cells (CD45-CD31+)

1344 and tumor cells (CD45-GFP+) were sorted on a BD FACS Aria II. The cells were then

1345 shock-frozen in liquid nitrogen and stored at -80°C until further use.

1346 Total RNA was isolated using the NucleoSpin RNA XS kit from Macherey-Nagel. For

1347 protein extraction, frozen cells were resuspended in RIPA Buffer with protease and

1348 phosphatase inhibitors and sonicated 3x for 15 seconds each time. Protein concentration

1349 was determined by the BCA method and ELISAs were performed according to the

65 1350 manufacturer’s instructions (Mouse VEGF, IL-10 and IFNγ DuoSet ELISA, R&D

1351 Systems).

1352

1353 Cell Growth Determination

1354 Cell viability was determined using the Cell Growth Determination Kit, MTT based

1355 (Sigma) according to manufacturer’s instructions. Briefly, 20.000 cells were plated in 24

1356 well plates and grown in normal supplemented medium over 3 days, for determination of

1357 their growth curve. After each 24-hour period, cells were incubated with 10% MTT

1358 solution for 3 hours, then MTT formazan crystals were dissolved and absorbance was

1359 spectrophotometrically measure at 570 nm. Background absorbance measured at 690 nm

1360 was subtracted to the first value.

1361 For TMZ sensitivity test, cells were treated for 24 hours with increasing concentrations

1362 of TMZ in serum-free medium. The same procedure was performed on untreated cells,

1363 and values were normalized and expressed in comparison to untreated cells.

1364

1365 Vessel perfusion and permeability assay

1366 Glioma-bearing mice from 3 weeks growth were anesthetized and injected intravenously

1367 with 100 μL of Alexa Fluor 647 labeled 2,000,000 MW dextran (Life Technologies).

1368 Blood vessel perfusion was visualized in vivo using the live imaging settings.

1369 For Miles assay, glioma-bearing mice were anesthetized and injected intravenously with

1370 100 μL 1% Evan’s blue solution (Sigma). Thirty minutes after injection, mice were

1371 sacrificed and transcardially perfused with 2% PFA solution. Dissected tumors were

1372 weighed and incubated in formamide solution at 56°C overnight to extract the dye. The

1373 absorbance of the solution was measured with a spectrophotometer at 620 nm. Five mice

66 1374 per group were analysed. Data are expressed as fold change compared to shCTRL glioma

1375 growth with tumor weight normalization.

1376

1377 Immunofluorescence staining

1378 Vibratome sections of tumors injected in ROSAmTmG reporter mice were blocked and

1379 permeabilized in TNBT buffer (0.1 M Tris pH 7.4; NaCl 150 mM; 0.5% blocking reagent

1380 from Perkin Elmer, 0.5% Triton X-100) overnight at 4oC. Tissues were then incubated

1381 with primary antibodies anti-F4/80 (Life Technologies, 1:100), anti-MRC1 (R&D

1382 Systems, 1:100), anti-CD3 (R&D Systems, 1:100), anti-MHCII (Thermo Scientific,

1383 1:100), anti-Glut1 (Millipore, 1:200), anti-Iba1 (Wako, 1:200), anti-Ki67 (Abcam,

1384 1:200), anti-pH2AX (Cell Signaling, 1:100) diluted in TNBT overnight at 4oC, washed

1385 in TNT buffer (0.1 M Tris pH 7.4; NaCl 150 mM; 0.5% Triton X-100) at least 7 times

1386 and incubated with appropriate Alexa Fluor 647 conjugated antibody (Life Technologies,

1387 1:400) diluted in TNBT overnight at 4oC. Samples were then washed at least 7 times in

1388 TNT and mounted on slices in fluorescent mounting medium (Dako). Images were

1389 acquired using a Leica SP8 inverted confocal microscope.

1390

1391 Soluble Flt-1 binding assay

1392 For detection of VEGF expression, vibratome sections were blocked and permeabilized

1393 in TNBT overnight at 4oC. Tissues were then incubated with 1μg/ml recombinant mouse

1394 soluble Flt-1 FC chimera (R&D Systems) diluted in TNBT for 2.5 h at room temperature.

1395 Samples were rinsed three times in TNT and subjected to 4% PFA fixation for 3 min.

1396 Samples were washed at least 7 times in TNT and incubated in Alexa Fluor 647 coupled

1397 anti-human IgG secondary antibodies (Life Technologies, 1:200) diluted in TNBT

1398 overnight at 4°C. Tissues were washed at least 7 times and mounted on slides in

67 1399 fluorescent mounting medium (Dako). Images were acquired using a Leica SP8 inverted

1400 confocal microscope.

1401

1402 Flow-cytometric analysis of tumor-antigen in lymph node immune cells

1403 Deep cervical and mandibular lymph nodes (DCLN and MLN) were dissected from tumor

1404 bearing mice 21 days after injection of CT-2A BFP or CT-2A GFP tumor spheroids. The

1405 2 DCLNs and 6 MLNs of each mice were pooled for analysis. LNs were digested for 30

1406 minutes in 1mg/mL Collagenase I diluted in DMEM at 37oC and after RBC lysis, single

1407 cell suspensions were prepared by filtering dissociated tissue on 40uM nylon cell

1408 strainers. Single cell suspensions were incubated with anti-CD45 APC or BUV805 (BD),

1409 anti-CD11b BV650 or BV450 (BD) antibodies. As a control, cells were stained with the

1410 appropriate isotype control. Data acquisition was performed on the BD LSRFortessa X20

1411 and analysis was performed with FlowJo_V10.

1412

1413 Primary cell cultures

1414 Bone-marrow derived macrophages (BMDMs) were isolated from C57BL/6 mice by

1415 flushing the femur and tibia with PBS. The bone marrow cells were resuspended in

1416 DMEM GlutaMax (Gibco) containing 1% Pen/Strep (Gibco), 20% FBS (Gibco) and

1417 100 ng/mL M-CSF (R&D Systems). Cells were incubated for 2 days at 37 °C and 5%

1418 CO2 in non-treated bacterial dishes for adhesion of bone-marrow resident macrophages,

1419 and then changed for treated plastic dishes and culture for 6 days with medium change

1420 every 2 days. Before experiments, cells were starved in serum- and CSF-free medium

1421 overnight. For PI3Kγ inhibition experiments, cells were pre-treated with 1uM IPI-549 for

1422 30 minutes as previously described (2) and then treatments were performed as described

1423 for all other experiments.

68 1424 Microglial cells were obtained as described previously (3, 4). Peritoneal macrophages

1425 (PMs) were isolated from peritoneal lavage as previously described (5).

1426

1427 Immunoprecipitation

1428 After Slit2 treatments for 15 minutes, BMDMs were lysed using NP40 lysis buffer

1429 (Boston bioproducts, BP-119X) supplemented with protease and phosphatase inhibitor

1430 cocktails (Roche, 11836170001 and 4906845001). Protein concentrations were

1431 quantified by BCA assay (Thermo Scientific, 23225) according to the manufacturer’s

1432 instructions. 300ug of protein were diluted in 1ml of NP40 buffer containing protease and

1433 phosphatase inhibitors for each condition. In the meantime, protein A/G magnetic beads

1434 (Thermo fischer, 88802) were washed 5x 10min with NP40 buffer. Protein lysates were

1435 incubated for 2 hours at 4°C under gentle rotation with 10ug of PI3Kγ antibodies (Cell

1436 Signaling Tecnologies). Then, 50ul of A/G magnetic beads were added to each protein

1437 lysate for 2 hours at 4°C under gentle rotation. Beads were then isolated using magnetic

1438 separator (Invitrogen) and washed 5 x with NP40 buffer. After the last wash, supernatants

1439 were removed and beads were resuspended in 40ul of Laemmli buffer (Bio-Rad,

1440 1610747), boiled at 95°C for 5min and loaded onto 4-15% gradient gels. Western blotting

1441 was performed as described above.

1442

1443 GFP+ macrophage isolation

1444 We collected mouse femoral bone-marrows (BMs) before the sacrifice of tumor-bearing

1445 mice as previously described for BMDM cultures. In the meantime, rabbit anti-GFP

1446 antibodies (Invitrogen) were incubated with sheep anti-rabbit IgG magnetic dynabeads

1447 (Invitrogen) in a solution of sterile PBS 0.1%BSA (120ul of beads, 24ul of antibodies in

1448 12ml PBS 0.1%BSA). Solutions were place under gentle rotation at room temperature for

69 1449 2hours to allow proper coupling of antibodies and beads. Coupled beads were next

1450 isolated using a magnetic separator and incubated in the resuspended BMs for 30min.

1451 After 5 washes with PBS 0.1%BSA, beads were separated using magnetic separator and

1452 RNA was extracted as previously described using RNeasy-kit (Qiagen). RNA samples

1453 were and reverse transcribed using SuperScript IV RT (Invitrogen) for gene-deletion

1454 verification by qPCR.

1455

1456 Supplemental References

1457 1. Bowman RL, Wang Q, Carro A, Verhaak RGW, Squatrito M. GlioVis data portal for

1458 visualization and analysis of brain tumor expression datasets. Neuro. Oncol. 2017;19(1).

1459 doi:10.1093/neuonc/now247

1460 2. Kaneda MM et al. PI3Kγ 3 is a molecular switch that controls immune suppression.

1461 Nature 2016;539(7629). doi:10.1038/nature19834

1462 3. Lima FRS et al. Regulation of microglial development: A novel role for thyroid

1463 hormone. J. Neurosci. 2001;21(6). doi:10.1523/jneurosci.21-06-02028.2001

1464 4. do Amaral RF et al. Microglial lysophosphatidic acid promotes glioblastoma

1465 proliferation and migration via LPA1 receptor. J. Neurochem. [published online ahead

1466 of print: 2020]; doi:10.1111/jnc.15097

1467 5. Pontes B et al. Membrane Elastic Properties and Cell Function. PLoS One 2013;8(7).

1468 doi:10.1371/journal.pone.0067708

1469

1470

1471

1472

1473

70 1474 Supplemental Figures

1475

1476

1477

71 GBM Low-Grade Gliomas Supplemental1.01.00 Figure 1 1.0 Low A 0.75 GBM B GBM C GBM Low-GradeROBO1: GliomasMedium Low-Grade Gliomas Low-Grade Gliomas 1.01.00 1.00 1.01.00 p=0.0577 LowHighp<0.01 1.0 Low 1.00.50.50 Low 1.0 Low 1.0 0.5 SLIT1:SLIT1: Medium SLIT3: Medium Low 0.75 Medium 0.75 SLIT3: Medium 0.75 Low High High ROBO1:ROBO1: Medium 0.25 High High Medium p=0.0577 p<0.01 p=0.0577 p<0.01 p=0.0577 HighHighp<0.01 0.50.50 0.50.50 0.50.50 Survival probability

0.5Survival probability 0.5 0.00.00 0.5 0.0 0 50 100 150 0.25 0 0 50 20100 15040 20060 250 80 0.25 0.25 TimeTimeTime (Months) (Months)(Months) Survival probability Survival probability Survival probability Survival probability Survival probability 0.00.00 Survival probability 0.00.00 0.00.00 0.0 0 50 100 150 0.0 0 50 100 150 0.0 0 50 100 150 0 0 50 20100 15040 200 60 250 80 0 0 50 20100 15040 20060 250 80 0 0 50 20100 15040 20060 250 80 TimeTimeTime (Months) (Months)(Months) TimeTimeTime (Months) (Months)(Months) TimeTimeTime (Months) (Months)(Months)

D E GBM F GBM Low-Grade Gliomas TCGA Agilent-4502A GBM 1.01.00 1.0 1.0 1.0 0.75 Low GBM SLIT2: Low Low-GradeSLIT2: GliomasLow ROBO2:ROBO2: Medium High 1.01.00 High p=0.0577 Highp<0.01 0.50.50 High 0.5 p<0.001 1.0 0.5 ** 0.5 P = 0.0577 0.75 Low ROBO2: 0.25 Medium p=0.0577 Highp<0.01 0.50.50 Survival probability Survival probability Survival probability 0.5 0.00.00 Survival probability 0.0 0 50 100 150 0.0 0.0 0 0 50 20100 15040 20060 250 80 0 20 40 60 80 100 120 0.25 0 30 60 90 TimeTimeTime (Months) (Months)(Months) Time (Months) Survival probability Time (Months) Survival probability 0.00.00 0.0 0 50 100 150 0 0 50 20100 15040 20060 250 80 Mesenchymal GBM TimeTimeTime (Months) (Months)(Months) G H RNAseq I 1.0 Mesenchymal GBM 10 * RNAseq 1.0 10 5 p<0.01** SLIT2: Low 0.5 High p<0.01 5

0 0.5 (Fold change) expression (log2) Survival probability -5 0.0 0 SLIT2 ROBO1 Survival probability 0 20 40 60 80 0.0 0 Time20 (Months)40 60 80 Grade 4 Classical Proneural Time (Months) Grades 1-3 J Mesenchymal K L 30 6 10 8 4 20 6 4 (Fold change) (Fold change) 10 (Fold change) 2 2 SLIT3 0 SLIT1 0 0 ROBO2

Grade 4 Grade 4 Grade 4 Grades 1-3 Grades 1-3 Grades 1-3 M N 16 ** O

12 45 * * 6 * ** 36 8 Slit1 SLIT1 4 27 (Fold change) 18 2 4 Expression SLIT2 Expression 9 relative to relative to 0 0 0 IDH-WT IDH-mutated

SLIT1 SLIT2 SLIT3 Slit1 Slit2 Slit3

1478

72 1479 Supplemental Figure 1. Impact of Slit2 on GBM patient survival.

1480 A-D. In silico analysis of TCGA glioblastoma RNAseq patient database demonstrating

1481 that expression of SLIT1 (A), SLIT3 (B), ROBO1 (C) or ROBO2 (D) do not affect patient

1482 survival. E. In silico analysis of TCGA GBM patient microarray Agilent-4502A database

1483 showing expression of SLIT2 is significantly associated with decreased patient survival

1484 (n = 244 high and 244 low SLIT2 expressing patients; O.S., 12.9 months for high

1485 expression and 15.1 months for low expression, log‐rank test). F. Survival analysis of

1486 GBM patients from (Figure 1C) grouped by their levels of SLIT2 expression (n = 22 high

1487 and 22 low Slit2 expressing patients; O.S., 14.75 months for high expression and 16.25

1488 months for low expression, log‐rank test). G. In silico analysis of TCGA glioblastoma

1489 RNAseq patient database demonstrating SLIT2 expression in different GBM molecular

1490 subtypes (n = 59 classical, 51 mesenchymal and 46 proneural tumors; One-Way

1491 ANOVA). H. In silico analysis of TCGA glioblastoma RNAseq patient database

1492 demonstrating that SLIT2 expression is significantly associated with decreased patient

1493 survival in mesenchymal GBM patients (n = 26 high and 25 low SLIT2 expressing

1494 patients; O.S., 10.4 months for high expression and 17.9 months for low expression, log‐

1495 rank test). I-L. qPCR expression of ROBO1 (I), ROBO2 (J), SLIT1 (K) and SLIT3 (L)

1496 in glioma patient samples (GBM, n = 45; LGG, n = 84; Student’s t test). M. qPCR

1497 comparison of SLIT1, SLIT2 and SLIT3 expression in GBM patient samples (Grade IV, n

1498 = 14 patients; One-Way ANOVA). N. SLIT2 qPCR expression in all glioma patient

1499 samples from (Figure 1C) classified by their IDH-1/2 status (IDH-WT, n = 67; IDH-

1500 mutated, n = 59; Student’s t test). O. qPCR comparison of Slit1, Slit2 and Slit3 expression

1501 in CT-2A tumors (n = 3 independent tumors, One-Way ANOVA). Data are presented as

1502 mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

1503

73 Supplemental Figure 2

A B mSlit2 C hSLIT2 D p<0.01 1.5 1.6 ** p<0.001*** 3 p<0.001*** 15

1.0 2 10 0.8

0.5 Ct SLIT2 (Fold change)

(Fold change) 1 5 (Fold change) ΔΔ Slit2 Slit2 0.0 Slit2 0 0 N/A 0.0

shSlit2 shSlit2 shSlit2 shScr shCTRL shSlit2

shSlit2 + hSLIT2 shSlit2 + hSLIT2

E F 120 G CT-2A 120 H GL261 p<0.01** shCTRL shSlit2 1.5 0.5 shCTRL shCTRL 100 shSlit2 100 0.8 shSlit2 0.4 Slit2 1.0 80 80 CT-2A shCTRL0.6 GL261 shCTRL 0.3 CT-2A shSlit2 GL261 shSlit2 60 60 0.4 0.2

0.5 CTRL) (% Viability Viability (% CTRL) (% Viability Slit2/Actin 40 40 0.2 Actin (Fold change) 0.1 Optical Density (AU) 0.0 CTRL 0.0 CTRL Optical Density (AU) 0.0 0 1 2 3 0 1 2 3 TMZ 100uMTMZ 600uM TMZ 100uMTMZ 600uM TMZ 1.000uMTime (days) TMZTime 1.000uM (days) shSlit2 shCTRL I CT-2A J GL261 K CT-2A L GL261 ns 30 30 40 40 ns *** *** 30 30 20 20 20 20 10 10 Cells per field Cells per field Cells per field Cells per field 10 10

0 0 0 0 CTRL Slit2 CTRL Slit2

shSlit2 shSlit2 shCTRL shCTRL M CT-2A N 120 shCTRL shSlit2 shCTRL 100 shSlit2 Tomato pH2AX 80

60

Viability (% CTRL) (% Viability 40

CTRL Vehicle

TMZ 100uMTMZ 600uM TMZ 1.000uM O p<0.001*** 150 p<0.001*** p<0.001*** 100 cells/field + 50 p<0.05* p<0.05* TMZ pH2AX 0

shCTRL shSlit2 150µm

shSlit2 + TMZ shCTRL + TMZ

1504

74 1505 Supplemental Figure 2. Slit2 silencing does not change tumor cell proliferation or

1506 sensitivity to TMZ in vitro, but increases TMZ-induced tumor cell death in vivo.

1507 A. Slit2 qPCR expression in CT-2A shSlit2 and shCTRL (n = 10 shCTRL and n = 7

1508 shSlit, Student’s t-test). B-C. qPCR analysis of murine (B, n = 8) and human (C, n = 4)

1509 Slit2 expression in cells infected with a human SLIT2 construct (Mann-Whitney U test).

1510 D-F. qPCR analysis (D), western blot analysis (E) and protein quantification (F) of

1511 shRNA Slit2 silencing in GL261 cells (n = 4, Mann-Whitney U test). G-H. Kinetics of

1512 shCTRL and shSlit2 treated CT-2A (G) and GL261 (H) glioma cell growth over 72 hours

1513 in complete medium (n = 3, multiple comparison linear regression). I-J. Transwell assay

1514 quantification of CT-2A (I) and GL261 (J) cells migration towards a Slit2 gradient (n =

1515 4, Mann-Whitney U test). K-L. Transwell assay quantification of CT-2A (K) and GL261

1516 (L) shCTRL or shSlit2 cells invasion towards a serum gradient (n = 4, Mann-Whitney U

1517 test). M. In vitro shCTRL and shSlit2 treated CT-2A glioma cell response to TMZ

1518 treatment (n = 4, One-way ANOVA). N. Phospho-H2AX (pH2AX) immunostainings

1519 (green) on 23 days tumor sections of CT-2A shCTRL and shSlit2 mice treated or not with

1520 TMZ in order to evaluate double-stranded DNA breaks (pH2AX+, green) in response to

1521 TMZ treatment. O. Quantification of (N) (n = 4 mice per group, 5 fields per tumor, One-

1522 way ANOVA). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P <

1523 0.001.

1524

1525

1526

1527

1528

1529

75 Supplemental Figure 3 A B C 1.2 ** 1.6 * shCTRL shSlit2

1.2 Slit2 0.8 0.8

Actin 0.4 (Fold change) 0.4 SLIT2

Slit2/Actin (Fold change) 0.0 0.0 shCTRLshSLIT2 shCTRLshSLIT2 D E F G 1.6 ns 2.0 ns 1.6 ns 2.5 ns 1.6 2.0 1.2 1.2 1.2 1.5 0.8 0.8 (Fold change) (Fold change)

(Fold change) 1.0

(Fold change) 0.8 0.4 0.4 0.4 0.5 SLIT1 SLIT3 ROBO1 ROBO2 0.0 0.0 0.0 0.0 shCTRLshSLIT2 shCTRLshSLIT2 shCTRLshSLIT2 shCTRLshSLIT2

H I J K

1.0 150 300 300 shCTRL ns shCTRL ns ns *** 0.8 shSLIT2 shSlit2 100 200 200 0.6

0.4 50 100 100 Cells per field Cells per field

0.2 CTRL) (% Viability Optical Density (AU) 0.0 0 0 0 0 1 2 3 CTRL SLIT2 shCTRLshSLIT2

Time (days) CTRL

TMZ 100uMTMZ 600uM TMZ 1.000uM L M N O 24hs 48hs 50 ns 1500 ) 0.018 *** 400 2 ** * 40 0.015 300 0.012 1000 30 0.009 200 20 (% of area) 0.006 (% of area) 500 100

10 Spheroid invasion Spheroid invasion Spheroids per well 0.003 Spheroids size (mm 0 0.000 0 0 shCTRLshSLIT2 shCTRLshSLIT2 shCTRLshSLIT2 shCTRLshSLIT2

1530

76 1531 Supplemental Figure 3. Slit2 silencing reduces invasion of Patient-derived GBM

1532 cells.

1533 A-C. Western blot analysis (A), protein quantification (B) and qPCR analysis (C) of

1534 shRNA SLIT2 silencing in N15-0460 GBM patient-derived cells (n = 6, Mann-Whitney

1535 U test). D-G. qPCR expression of SLIT1 (D), SLIT3 (E), ROBO1 (F) and ROBO2 (G) in

1536 N15-0460 cells after shRNA SLIT2 silencing (n = 4, Mann-Whitney U test). H. shCTRL

1537 and shSLIT2 treated N15-0460 growth over 72 hours in complete medium (n = 3,

1538 multiple comparison linear regression). I. shCTRL and shSLIT2 treated N15-0460 cells

1539 response to TMZ treatment (n = 4, Two-way ANOVA). J. Transwell assay quantification

1540 of N15-0460 cell migration towards a SLIT2 gradient (n = 4, Mann-Whitney U test). K.

1541 Transwell assay quantification of N15-0460 shCTRL or shSLIT2 cell migration towards

1542 a serum gradient (n = 4, Mann-Whitney U test). L-M. Spheroid formation assay

1543 quantification of shCTRL and shSLIT2 N15-0460 cells. Number (L) and size (M) of

1544 spheroids formed after 48 hours in culture were quantified (n = 6 cultures per group,

1545 Mann-Whitney U test). N-O. Quantification of spheroid invasion assay in fibrin gels of

1546 shCTRL and shSLIT2 N15-0460 cells after 24 (N) and 48 hours (O). Data are presented

1547 as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

1548

1549

1550

1551

1552

1553

1554

1555

77 Supplemental Figure 4 A B C shCTRL (n=15) ) shCTRL (n=15) 3 * ) 4 10

shSLIT2 (n=15) 10 100 shSLIT2 (n=15) ** *** 3 80 2 2 60 1 40 1 Normalized 0 20 Last normalized 0 Tumor negative (%) negative Tumor bioluminescence (log 0 -1 bioluminescence (log -1 0 50 100 150 0 50 100 150 shCTRLshSLIT2 Time post graft (days) Days post graft

D E GFP (Tumor cells)

36 *** 30 24 shCTRL 18

per brain 12 tumors cell slices + 6

GFP 0 shCTRLshSLIT2 shSLIT2

F G H shCTRL shSLIT2

1.5 1.2 ** PML * 1.2 0.9 0.9 SOX2 0.6 0.6 0.3 0.3 PML/Actin (Fold change) Actin 0.0 SOX2/Actin (Fold change) 0.0 shCTRLshSLIT2 shCTRLshSLIT2

1556

1557 Supplemental Figure 4. SLIT2 silencing slows tumor growth in a GBM Patient-

1558 derived Xenograft (PDX) model.

1559 A. Tumor development curve after injection of shCTRL and shSLIT2 N15-0460 GBM

1560 patient-derived cells in nude mice (n = 15 mice per group, log-rank test). B.

78 1561 bioluminescence signal over time after tumor injection (n = 15 mice per group, One-way

1562 ANOVA). C. bioluminescence signal at the end-point of experiment for each of the

1563 injected mice (n = 15 mice per group, Mann-Whitney U test). D. Tile-scan images of

1564 vibratome sections from implanted mice demonstrating GFP+ tumor cell spread. E.

1565 Quantification of GFP+ tumor cell spread (n = 10 shCTRL and 11 shSLIT2 mice, Mann-

1566 Whitney U test). F-H. Western blot analysis (F) and protein quantification of PML (G)

1567 and SOX2 (H) expression in shCTRL and shSLIT2 N15-0460 GBM cells (n = 6, Mann-

1568 Whitney U test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P <

1569 0.001.

1570

1571

1572

1573

1574

1575

1576

1577

1578

1579

1580

1581

1582

1583

1584

1585

79 Supplemental Figure 5 A B CT-2A GL261

shCTRL shSlit2 shCTRL shSlit2 Day 14 Day Day 21 Day

100µm 100µm

C D E CT-2A p<0.001*** 40 80 shSlit2 shSlit2 + hSLIT2

m) p<0.001

µ *** 30 60

20 40 1 10 20 Day 1 Day Vessel diameter ( diameter Vessel 0 Branchpoints per field 0 100µm

shSlit2 shSlit2 shCTRL shCTRL 1586

1587 Supplemental Figure 5. Slit2 drives vessel dysmorphia and vascular dysfunction in

1588 CT-2A and GL261 glioma models.

mTmG 1589 A. In vivo two-100photonµm imaging3mm of ROSA mice bearing day 14 CT-2A shCTRL or

1590 shSlit2 tumors. B. In vivo two-photon imaging of ROSAmTmG mice bearing day 21 GL261

1591 shCTRL or shSlit2 tumors. C-D. Quantification of blood vessel diameter (C) and

1592 branchpoints (D) of the GL261 tumors shown in (B) (n = 7 mice per group, Student’s t-

1593 test). E. In vivo two-photon imaging of ROSAmTmG mice bearing day 11 CT-2A shSlit2

1594 or shSlit2+hSLIT2 tumors. Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01,

1595 *** P < 0.001 125µm 1596

1597

80 Supplemental Figure 6 A B C CT-2A p<0.001***

shCTRL shSlit2 shSlit2 + hSLIT2 field 250 200 p<0.001*** field 200 150 per per 150 per 100

in vivo 100 in vivo cells 50 cells 50 0 0 Immune cells per field Immune cells per field Tomato Immune shSlit2 Immune shCTRL shSlit2 shCTRL shSlit2 shSlit2 150µm shSlit2+ shSlit2 +hSLIT2 hSlit2

D E GL261 p<0.001*** p<0.001*** shCTRL shSlit2 200 p<0.001***

150 p<0.001*** +

F4/80 F4/80 100 p<0.01** MHC-II+ MRC1+ 50 Cells per field Tomato 125µm 0 125µm II -

shSlit2

MHC shCTRL F Tomato MRC1

Tomato 100µm

G H - Dendritic cells (DCs) Neutrophils

F4/80 p<0.05* + 4 p< 0.01 8 ** +

3 6 Ly6G MHC-II + +

2 4 CD11c CD11b (% total) (% total) - + 1 2 CD45 CD11b + 0 0 CD45

shCTRL shSlit2 shCTRL shSlit2

1598

81 1599 Supplemental Figure 6. Slit2 silencing favors macrophage cytotoxic polarization in

1600 CT-2A and GL261 glioma models.

1601 A-C. In vivo imaging (A) and quantification (B-C) of host-derived tumor infiltrating

1602 immune cells (red) in late-stage CT-2A tumors (n = 7 shCTRL, n = 8 shSlit2 and

1603 shSlit2+hSLIT2 mice, Student’s t-test). D. Immunohistochemistry on sections of day 21

1604 GL261 shCTRL or shSlit2 tumors with antibodies recognizing F4/80, MHC-II and MRC1

1605 (green). E. Quantifications of (D) (n = 7 mice per group, 5 fields per tumor, Two-Way

1606 ANOVA). F. Flow cytometry-gating strategy example for macrophage counting shown

1607 in Figure 4C-E. G-H. FACS quantification of Dendritic Cells (G, DCs,

1608 CD45+CD11b+CD11c+MHC-II+F4/80-) and Neutrophils (H, CD45+CD11b+Ly6G+).

1609 Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

1610

1611

1612

1613

1614

1615

1616

1617

1618

1619

1620

1621

1622

1623

82 Supplemental Figure 7 A B CT-2A 30 *** *** shCTRL shSlit2 shSlit2 + hSLIT2 20

cells per field 10 + CD3 CD3 0 Tomato shSlit2 shCTRL 100µm

shSlit2 + hSLIT2 C D

GL261 20 p<0.001*** shCTRL shSlit2 15

10 cells per field per cells +

CD3 5 CD3 0 Tomato

shSlit2 100µm shCTRL E F

p < 0.001 60 *** ) + 40 Q3 + + + + (%CD45 CD3 CD4 CD3 CD8 + (CD45+CD11b-) 20 CD3

0

shCTRL shSlit2

G H I

40 p<0.05* 100 5 ) ) + + 80 4

30 + 60 3 /CD4

20 + (% CD3 (% CD3 + + 40 2

10 CD8 CD8 CD4 20 1

0 0 0

shCTRL shSlit2 shCTRL shSlit2 shCTRL shSlit2

1624

83 1625 Supplemental Figure 7. Slit2 drives T cell depletion in CT-2A and GL261 models.

1626 A. Anti-CD3 staining (green) on sections of late stage CT-2A shCTRL, shSlit2 or

1627 shSlit2+hSLIT2 tumors. B. Quantification of (A) (n = 7 mice per group, 5 fields per

1628 tumor, One-Way ANOVA). C. Anti-CD3 staining (green) on sections of late stage GL261

1629 shCTRL and shSlit2 tumors. D. Quantification of (C) (n = 7 mice per group, 5 fields per

1630 tumor, Student’s t-test). E-H. Extension of flow-cytometry analysis from Figure 5. When

1631 considering only the immune cell compartment of the tumor microenvironment (CD45+

1632 cells), there is a 10-fold increase in the proportion of TALs (from 4,4% to 43,5%) in

1633 shSlit2 tumors (F). Analysis of the percentage of CD4+ T helper cells (G) and CD8+

1634 cytotoxic T cells (H) among the TALs (n = 8 mice per group, Mann-Whitney). I. Ratio

1635 between CD8+ and CD4+ TALS (n = 8 mice per group, Mann-Whitney). Data are

1636 presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

1637

1638

1639

1640

1641

1642

1643

1644

1645

1646

1647

1648

1649

84 Supplemental Figure 8 A B C 1000 12 30 800 R=0,265 p<0.0001 9 20 R=0,575 600 p<0.0001 R=0,25 150 p<0.01 10 100 6 50

0 0 MRC1 (Fold change) MRC1 (Fold change) VEGFA (Fold change) 3 0 10 20 0 10 20 30 3 6 9 12 SLIT2 (Fold change) SLIT2 (Fold change) SLIT2 (Fold change) D E F 9 7 6 R=0,245 R=0,276 p<0.0001 R=-0,193 p<0.0001 p<0.001 7 5

5 CD209 (Fold change) CCL19 (Fold change) 3 3 IL-12A (Fold change) 3 4 6 8 10 12 3 6 9 12 3 6 9 12 SLIT2 (Fold change) SLIT2 (Fold change) SLIT2 (Fold change)

G H I

15 5 12 R=0,127 R=0,1 R=0,241 p<0.01 p=0.06 p<0.0001 12 10

8 9 4

6 6 4 MMP9 (Fold change) IL-6 (Fold change) 3 PDCD1LG2 (Fold change) 3 3 6 9 12 3 6 9 12 4 6 8 10 12 SLIT2 (Fold change) SLIT2 (Fold change) SLIT2 (Fold change) J K L

5 7 5 R=0,123 R=0,224 R=0,1 p<0.01 p<0.0001 p<0.05

4 5 4 CCL17 (Fold change) PDCD1 (Fold change) 3 CTLA4 (Fold change) 3 3 6 9 12 3 6 9 12 3 6 9 12 SLIT2 (Fold change) SLIT2 (Fold change) SLIT2 (Fold change)

M N O

8 4 R=-0,105 R=0,178 R=0,249 p<0.05 4 p<0.001 6 p<0.0001 2 4 2

2 0 0 0 CXCL11 (Fold change) HAVCR2 (Fold change) -2 -2 Lag3 (Fold change) -2 -4 -2 0 2 4 6 -4 -2 0 2 4 6 -4 -2 0 2 4 6 SLIT2 (Fold change) SLIT2 (Fold change) SLIT2 (Fold change)

1650

85 1651 Supplemental Figure 8. Slit2 expression correlated with immunosuppression in

1652 GBM patients.

1653 A-B. Correlation analysis of MRC1 (A) or VEGFA (B) and SLIT2 expression in GBM

1654 patients (n = 129 patients, Spearman’s correlation test). C-O. Correlation analysis of

1655 SLIT2 expression with the indicated genes in GBM patients from TCGA cohort (n = 489

1656 patients, Spearman’s correlation test). * P < 0.05, ** P < 0.01, *** P < 0.001.

1657

1658

1659

1660

1661

1662

1663

1664

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86 Supplemental Figure 9 A B 60 p<0.001 p<0.001 CTRL in both Slit2 in bottom Slit2 in both *** *** chambers chamber chambers 40

Calcein 20 Cells per field 0

50µm 50µm 50µm CTRL

Slit2 bottom

Slit2 both chambers C D E F 3 2.5 2.5 1.5 *** p<0.001 γ * * ** ** 2.0 *** * 2.0

PLC 2 1.0 / 1.5 1.5 γ 1.0 1.0 0.5 1

pPLC 0.5 (Fold change) 0.5 (Fold change) N/A pErk1/2 / Erk1/2 0.0 0.0 0 0.0 ROBO2 (Fold change) ROBO1 (Fold change) 0' 5' 0' 5' 10' 15' 30' 60' 10' 15' 30' 60' siScr siScr

siRobo1+2 siRobo1+2 G H I 1 2.5 6 ** β 5 ** * *** 2.0 * * 4 4 ** 1.5 3 * 1/CEBP 1.0 β 2

pAkt/Akt 2

pStat6/Stat6 1 (Fold change)

0.5 (Fold change) (Fold change)

0.0 0 pCEBP 0 0' 5' 0' 5' 0' 5' 10' 15' 30' 60' 10' 15' 30' 60' 10' 15' 30' 60' J K L Microglia Slit2 6nM 0’ 5’ 15’ 30’ *** *** *** 8 pAkt 100 *** *** *** 80 Akt 6 *** 60 pErk1/2 4 40 Erk1/2 ** [IL-10] (ng/mL) 2 20 [VEGFa] (ng/mL) pStat6 0 0 CTRL LPS IL-10 Slit2 CTRL LPS IL-10 Slit2 Stat6

pCEBPβ

CEBPβ

Clathrin

1676

87 1677 Supplemental Figure 9. Slit2 induces chemotactic migration and signaling in

1678 primary macrophages and microglial cells.

1679 A-B. Representative images (A) of calcein-stained (green) Transwell assays with Slit2

1680 treatment in the bottom chamber or in both chambers and quantification (B). Slit2-

1681 induced migration is chemotactic, as treatment with Slit2 in both chambers disrupts the

1682 gradient and abrogates migration (n = 4, One-way ANOVA). C-D. qPCR analysis of

1683 Robo1 (C) and Robo2 (D) expression after siRNA treatment of cultured RAW264.7

1684 macrophages for 72hs (n = 4, Mann Whitney). E-I. Quantifications of the Western Blots

1685 shown in Figure 6F. (n = 6, One-Way ANOVA). J. Western blot analysis of Slit2

1686 downstream signaling in cultured microglial cells (n = 3). K-L. ELISA from conditioned

1687 medium from LPS or Slit2-treated microglial cells quantifying the secretion of IL-10 (K)

1688 and VEGFa (L) (n = 3 independent cultures, Mann-Whitney U test). Data are presented

1689 as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

1690

1691

1692

1693

1694

1695

1696

1697

1698

1699

1700

1701

88 Supplemental Figure 10 A siScr siRobo1+2 B C Slit2 2.0 1.5 *** 6nM 0’ 5’ 10’ 15’ 30’ 60’ 0’ 5’ 10’ 15’ 30’ 60’ 1.5 *** Robo1 1.0 1.0 0.5 Robo2 0.5 Robo2/Actin Robo1/Actin (Fold change) pPLCg (Fold change) 0.0 0.0

PLCg siScr siScr

pAkt siRobo1+2 siRobo1+2 D 2.0 Akt siScr 1.5 * ** siRobo1+2 * pErk1/2 1.0

Erk1/2 0.5

pPLCg/PLCg (AU) 0.0 Actin 0' 5' 10' 15' 30' 60' 2.0 2.0 E 2.0 siScr F 1.5 siScr 1.5 * ** *** siRobo1+2 1.5 * ** siRobo1+2 1.5 *** * * 1.0 *** 1.0 * 1.0 * 1.0 * * * 0.5 0.5 0.5 pPLCg/PLCg (AU) pPLCg/PLCg (AU) pAkt/Akt (AU) 0.0 0.0

0.0 0' 5' pErk1/2 / Erk1/2 (AU) 0.0 0' 5' 10' 15' 30' 60' 10' 15' 30' 60' 0' 5' 0' 5' 10' 15' 30' 60' 10' 15' 30' 60' G 10 *** 8 siScr 6 siRobo1+2 4 * ** *

Fold change 2 * relative to siCTRL 0

Arg1 MRC1 CCL19 VEGFa CD209a 1702 Supplemental Figure 10. Slit2 induces macrophage migration and downstream

1703 signaling via Robo1/2.

1704 A. Western blot analysis of PLCγ, Akt and Erk1/2 phosphorylation induced by Slit2 in

1705 control and Robo1/2 knockdown RAW264.7 macrophages (n = 5). B-C. Quantification

1706 of Robo1 (A) and Robo2 (B) protein expression after Robo1 and Robo2 knockdown. D-

1707 F. Quantification of (A) (n = 5, Two-way ANOVA). G. qPCR analysis of genes related

1708 to the tumor supportive phenotype (Mrc1, Vegfa, Arg1, Cd209a and Ccl19) in RAW264.7

1709 macrophages after Robo1 and Robo2 knockdown and Slit2 treatment (n = 4, Mann

1710 Whitney U test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P <

1711 0.001.

89 Supplemental Figure 11 A

PI3K! inhibitor Slit2 CTRL 6nM 0’ 5’ 15’ 30’ 0’ 5’ 15’ 30’ pAkt

Akt

pStat6

Stat6

Clathrin

B Il-1β Cxcl10 Mrc1 Mmp9 Ccl19 Il-10 CTRL 4000 500 16 40 *** 60 20 PI3Kγ inhibitor *** *** *** 350 12 30 45 15 1000 2000 8 20 30 10 200 750 4 15 5 Fold change 10 Fold change Fold change Fold change Fold change Fold change 10 10 5 5 500 0 0 0 0 0 0 CTRL LPS Slit2 CTRL LPS Slit2 CTRL Slit2 CTRL Slit2 CTRL Slit2 CTRL Slit2 250 Ccr7 Tnfα Vegfa Tgfβ1 Cd209a Arg1 Fold change 20 275 175 32 *** 6 *** 10 *** 80 10 *** 0 24 8 60 125 4 CTRL LPS Slit2 6 16 40 150 4 75 2 8 20 Fold change Fold change Fold change Fold change Fold change 5 Fold change 5 2 0 0 0 0 0 0 CTRL LPS Slit2 CTRL LPS Slit2 CTRL Slit2 CTRL Slit2 CTRL Slit2 CTRL Slit2 Cytotoxic Tumor supportive

1712

1713 Supplemental Figure 11. PI3Kγ inhibiton disrupts Slit2-induced macrophage and

1714 microglia polarization.

1715 A. WB analysis of Akt and Stat6 phosphorylation in BMDMs induced by Slit2 after

1716 PI3Kγ inhibitor IPI-549 pretreatment (n = 3 independent cultures). B. qPCR analysis of

1717 microglial cultures following Slit2 or LPS treatment after pre-treatment with PI3Kγ

1718 inhibitor (n = 4 independent cultures, 2-way ANOVA). Data are presented as mean ±

1719 s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

1720

1721

90 Supplemental Figure 12 A B C 1.5 p<0.01 2.0 2.0 p<0.01** ** p<0.01** 1.5 1.5 1.0 1.0 1.0 (Fold change) (Fold change) 0.5 (Fold change) 0.5 0.5 Vegfa Robo2 Robo1 0.0 0.0 0.0

CTRL CTRL CTRL

iRoboMacKO iRoboMacKO iRoboMacKO

D Tomato GFP Tomato GFP Iba1 Tomato GFP MRC1 Tomato GFP sFlt1 Tomato GFP CD3 MHC-II CTRL iRoboMacKO 200µm

E A A - - BV510 – FSC CTRL SSC Count CFP

FSC-A FSC-H CD11b BV650 CD11b BV650 A A - - BV510 – FSC SSC Count CFP iRoboMacKO

FSC-A FSC-H CD11b BV650 CD11b BV650 F 8

L) 6 µ

4

WBC (K/ 2

0

CTRL

iRoboMacKO

1722

91 1723 Supplemental Figure 12. Analysis of iRoboMacKO

1724 A-C. qPCR of Robo1 (A), Robo2 (B) and Vegfa (C) in GFP+ macrophages extracted

1725 from the bone-marrow of CTRL and iRoboMacKO tumor-bearing mice 21 days after

1726 tumor implantation. D. Immunohistochemistry images related to quantifications shown

1727 in Figure 8I-K. E. Flow cytometry-gating strategy example for graphs shown in Figure

1728 8M-N. F. Total white blood cells (WBC) counts from peripheral blood of late-stage

1729 CTRL and iRoboMacKO tumor-bearing mice (n = 5 mice/group; Mann-Whitney U test).

1730 Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

1731

1732

1733

1734

1735

1736

1737

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1739

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1742

1743

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92 Supplemental Figure 13 A B C shSlit2 + CTRL Ab shSlit2 + CD3 Ab 20 *** 15 cells + 10 per field

CD3 5

CD3 0

100µm

CD3 Antibody CTRL Antibody

D E F shSlit2 + CTRL Ab shSlit2 + CD3 Ab 30 CTRL Ab CD3 Ab 20 m) µ 300 ( 10 Pp=0.06= 0.06 4 1 225 Vessel diameter Vessel 0 150 Day 14 days 23 days 75 G 0 CTRL Ab Tumor Volume(mm3) Tumor 150 CD3 Ab 100 3 50 CD3 Antibody per field CTRL Antibody 2 Day Branchpoints 0 100µm 150µm

14 days 23 days

H I Tomato F4/80 Tomato MHC-II Tomato MRC1 Ab

120 80 F4/80 ** ** MHC-II 40 MRC1 shSlit2+ CTRL Cells per field 0 Ab

CD3 Antibody CTRL Antibody

100µm shSlit2+ CD3

1748

93 1749 Supplemental Figure 13. In vivo T cell-depletion does not affect the TME.

1750 A. Experimental design for T cell depletion by intravenous injection with 145-2C11 anti-

1751 CD3 antibodies (mice were treated with 100ug of 145-2C11 antibodies every 3 days

1752 starting 7 days after tumor implantation). B. CD3 immunostainings performed on sections

1753 of late stage CT-2A tumors. C. Quantification from (B) (n = 5 mice per group, 5 fields

1754 per staining, Student’s t test). D. Tumor volume quantification at 23 days following anti-

1755 CD3 mAb treatment (n = 5 mice per group, Mann-Whitney U test). E. In vivo two-photon

1756 imaging of ROSAmTmG mice bearing early (14 days) and late stage (23 days) CT-2A

1757 shSlit2 tumors with or without anti-CD3 mAb treatment. F-G. Quantification of Blood

1758 vessel diameter (F) and branchpoints (G) from € (n = 5 mice per group, One-way

1759 ANOVA). H. F4/80, MHC-II and MRC1 Immunohistochemistry on sections of late stage

1760 (23 days) CT-2A shSlit2 tumors treated with control mAb or anti-CD3 mAb. I.

1761 Quantification from (H, n = 5 mice per group, 5 fields per tumor, Two-way ANOVA).

1762 Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

1763

1764

1765

1766

1767

1768

1769

1770

1771

1772

1773

94 Supplemental Figure 14 A B C CTRLFc Robo1Fc Tomato Glut1 )

3 200 **

150

100 Fc CTRL 150µm 50

Tumor volume (mm volume Tumor 0 D21 post implantation post D21 CTRLFc Robo1Fc Robo1Fc

D Tomato F4/80 Tomato MHC-II Tomato MRC1 Tomato sFlt1 Tomato CD3 CTRL Fc CTRL 150µm Robo1Fc - E F H 10 I 6 * DCLN * L) µ

Ly6G 10

+ 8

* L) 8 µ 4 6 6 CD11b

+ 4 4 2 WBC (K/ (% total) 2 2 CD45 + Lymphocytes (K/ Lymphocytes 0 0 0 GFP

CTRLFc Robo1Fc CTRLFcRobo1Fc CTRLFcRobo1Fc

G - J MLN 80 ***

Ly6G 25 *** CTRLFc + * 60 20 Robo1Fc 40 15 CD11b + 10 20 % of WBC (% total) 4 5 CD45 2 + 0 0 GFP

CTRLFc Robo1Fc Basophils Neutrophils MonocytesEosinophils Lymphocytes

1774

95 1775 Supplemental Figure 14. Robo1Fc treatment slowed GBM growth by inducing

1776 systemic long-term anti-tumor immune responses.

1777 A. T2-weighted post-gadolinium MRI images of CTRLFc and Robo1Fc treated mice 21

1778 days after tumor implantation. B. Quantification of tumor size from (A) (n = 7 CTRLFc

1779 and 9 Robo1Fc tumors). C. Immuno-staining for Glut1 (quantified in Figure 9H). D.

1780 Immunohistochemistry images related to quantifications shown in Figure 9I-K. E. Flow

1781 cytometry-gating strategy example for graphs shown in (F-G). F-G. FACS analysis of

1782 deep cervical and mandibular lymph nodes (DCLN and MLN, respectively) from late-

1783 stage CTRLFc- and Robo1Fc-treated mice (n = 4 mice/group; Mann-Whitney U test).

1784 H-J. Total white blood (WBC, H), lymphocyte (I) and differential WBC (J) counts from

1785 peripheral blood of late-stage CTRLFc- and Robo1Fc-treated tumor-bearing mice (n = 4

1786 mice/group; Mann-Whitney U test and Two-way ANOVA). Data are presented as mean

1787 ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

1788

96 Primer Cat No HS_ROBO3_1_SG QT00055951 Hs_ROBO2_2_SG QT01007664 Hs_ROBO4_1_SG QT00237741 Hs_SLIT2_1_SG QT00007784 Hs_SLIT3_1_SG QT00018795 Hs_SLIT1_1_SG QT00071113 Hs_ROBO1_2_SG QT01668982 Hs_ACTB_1_SG QT00095431 Mm_ACTB_1_SG QT00095242 Mm_GAPDH_3_SG QT01658692 Mm_CCR7_1_SG QT00240975 Mm_MRC1_1_SG QT00103012 Mm_VEGFA_1_SG QT00160769 Mm_CCL19_2_SG QT02532173 Mm_TNF_1_SG QT00104006 Mm_MMP9_1_SG QT00108815 Mm_TGFB1_1_SG QT00145250 Mm_IL1B_2_SG QT01048355 Mm_PDCD1IG1_1_SG QT00148617 Mm_PDCD1IG2_1_SG QT00136640 Mm_CXCL10_1_SG QT00093436 Mm_IL12B_1_SG QT00153643 Mm_CD209A_1_SG QT00116312 Mm_ARG1_1_SG QT00134288 Mm_IL10_1_SG QT00106169 Mm_IL12M_1_SG QT00101108 Mm_IL2_1_SG QT00112315 Mm_CXCL11_1_SG QT00265041 Mm_IL17A_1_SG QT00103278 Mm_IFNg_1_SG QT01038821 Mm_CCL17_1_SG QT00131572 Mm_PDCD1_1_SG QT00111111 Mm_ROBO1_1_SG QT00146853 Mm_SLIT1_1_SG QT01044925 Mm_SLIT2_1_SG QT00163828 Mm_SLIT3_1_SG QT00283416 Mm_ROBO3_1_SG QT00136605 1789 Mm_ROBO2_1_SG QT00143255

1790 Supplemental Data Table. 1. List of qPCR Primers used in this study.

1791 1792

97