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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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];
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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 gene 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 SLIT 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 proteins 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 gene expression. As a
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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 protein (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 genes 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
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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 (Locus 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
1665
1666
1667
1668
1669
1670
1671
1672
1673
1674
1675
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
1738
1739
1740
1741
1742
1743
1744
1745
1746
1747
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