Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A. In silico analysis of TCGA glioblastoma RNAseq patient database ( n = 51 high, 50 medium and 50 low Slit2 expressing patients; O.S., 9.86 months for high expression, 14.69 months for low expression, and 16.79 months for medium expression, log-rank test). B. Survival analysis of Low-Grade Gliomas (LGG, Grades I to III) patients grouped by their levels of SLIT2 expression ( n = 41 high and 41 low SLIT2 expressing patients; O.S., 64.73 months for high expression and 209.13 months for low expression, log-rank test). C. SLIT2 qPCR expression in glioma patient samples from ( B) (GBM, n = 45; LGG, n = 84; Student’s t test). D. SLIT2 qPCR expression in Grades III and IV glioma patient samples classified by their IDH-1/2 status (IDH-WT, n = 51; IDH-mutated, n = 34; Mann-Whitney U test). E-F. UMAP plots of single cell RNA sequencing (scRNAseq) of 32 GBM patients showing different samples ( E ) and clustering of the different cell types in the GBM microenvironment ( F ). G-I. Expression plots of SLIT2 ( G ), ROBO1 ( H ) and ROBO2 ( I ) in scRNAseq data from ( E) . J-L. qPCR analysis of Slit2 ( J ), Robo1 ( K ) and Robo2 ( L ) expression in endothelial cells (ECs), tumor associated macrophages (TAMs), tumor associated T lymphocytes (TALs) and tumor cells FACS-sorted from late-stage CT-2A mice glioblastomas ( n = 3 independent tumors, day 21 after implantation, One-Way ANOVA). All data are represented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: In Silico, Expressing, MANN-WHITNEY, RNA Sequencing Assay

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A-D. In silico analysis of TCGA glioblastoma RNAseq patient database demonstrating that expression of SLIT1 ( A ), SLIT3 ( B ), ROBO1 ( C ) or ROBO2 ( D ) do not affect patient survival. E. In silico analysis of TCGA GBM patient microarray Agilent-4502A database showing expression of SLIT2 is significantly associated with decreased patient survival ( n = 244 high and 244 low SLIT2 expressing patients; O.S., 12.9 months for high expression and 15.1 months for low expression, log-rank test). F. Survival analysis of GBM patients from grouped by their levels of SLIT2 expression ( n = 22 high and 22 low Slit2 expressing patients; O.S., 14.75 months for high expression and 16.25 months for low expression, log-rank test). G. In silico analysis of TCGA glioblastoma RNAseq patient database demonstrating SLIT2 expression in different GBM molecular subtypes ( n = 59 classical, 51 mesenchymal and 46 proneural tumors; One-Way ANOVA). H. In silico analysis of TCGA glioblastoma RNAseq patient database demonstrating that SLIT2 expression is significantly associated with decreased patient survival in mesenchymal GBM patients ( n = 26 high and 25 low SLIT2 expressing patients; O.S., 10.4 months for high expression and 17.9 months for low expression, log- rank test). I-L. qPCR expression of ROBO1 ( I) , ROBO2 ( J ), SLIT1 ( K ) and SLIT3 ( L ) in glioma patient samples (GBM, n = 45; LGG, n = 84; Student’s t test). M . qPCR comparison of SLIT1 , SLIT2 and SLIT3 expression in GBM patient samples (Grade IV, n = 14 patients; One-Way ANOVA). N. SLIT2 qPCR expression in all glioma patient samples from classified by their IDH-1/2 status (IDH-WT, n = 67; IDH- mutated, n = 59; Student’s t test). O. qPCR comparison of Slit1, Slit2 and Slit3 expression in CT-2A tumors ( n = 3 independent tumors, One-Way ANOVA). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: In Silico, Expressing, Microarray, Comparison

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A-B. Western blot analysis ( A ) and quantification ( B ) of Slit2 expression in shCTRL, shSlit2 and shSlit2+hSLIT2 CT-2A cells ( n = 5, One-Way ANOVA). C. Tumor volume quantification at 21 days ( n = 10 for shCTRL and n = 8 for shSlit2, Student’s t-test). D. FDG-PET imaging over CT-2A shCTRL and shSlit2 glioma growth ( n = 5 shCTRL and n = 4 shSlit2). E-F. Quantification of tumor metabolic volume ( E ) and total tumor glucose uptake ( F ) from (D) ( n = 5 for shCTRL and n = 4 for shSlit2, One-Way ANOVA). G. Survival trial design: 8-week-old mice were engrafted with CT-2A shCTRL, shSlit2 or shSlit2+hSLIT2 spheroids and randomly assigned to vehicle or TMZ treatment (40 mg/kg on days 7, 11, 15 and 19 after tumor implantation). H. Survival curves of the mice in (G) ( n = 10 mice per group, O.S.= 22.5 days for shCTRL, 28 days for shCTRL + TMZ, 30 days for shSlit2, 39.5 days for shSlit2 + TMZ, 20 days for shSlit2+hSLIT2 and 27 days for shSlit2+hSLIT2 + TMZ; Multiple comparisons log-rank test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: Western Blot, Expressing, Imaging, Tumor Implantation

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A. Slit2 qPCR expression in CT-2A shSlit2 and shCTRL ( n = 10 shCTRL and n = 7 shSlit, Student’s t-test). B-C. qPCR analysis of murine ( B , n = 8) and human ( C , n = 4) Slit2 expression in cells infected with a human SLIT2 construct (Mann-Whitney U test). D-F. qPCR analysis (D), western blot analysis (E) and protein quantification (F) of shRNA Slit2 silencing in GL261 cells ( n = 4, Mann-Whitney U test). G-H. Kinetics of shCTRL and shSlit2 treated CT-2A (G) and GL261 (H) glioma cell growth over 72 hours in complete medium ( n = 3, multiple comparison linear regression). I-J. Transwell assay quantification of CT-2A ( I ) and GL261 ( J ) cells migration towards a Slit2 gradient ( n = 4, Mann-Whitney U test). K-L. Transwell assay quantification of CT-2A ( K ) and GL261 ( L ) shCTRL or shSlit2 cells invasion towards a serum gradient ( n = 4, Mann-Whitney U test). M. In vitro shCTRL and shSlit2 treated CT-2A glioma cell response to TMZ treatment ( n = 4, One-way ANOVA). N. Phospho-H2AX (pH2AX) immunostainings (green) on 23 days tumor sections of CT-2A shCTRL and shSlit2 mice treated or not with TMZ in order to evaluate double-stranded DNA breaks (pH2AX + , green) in response to TMZ treatment. O. Quantification of (N) ( n = 4 mice per group, 5 fields per tumor, One-way ANOVA). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: Expressing, Infection, Construct, MANN-WHITNEY, Western Blot, shRNA, Comparison, Transwell Assay, Migration, In Vitro

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A-C. Western blot analysis (A) , protein quantification (B) and qPCR analysis (C) of shRNA SLIT2 silencing in N15-0460 GBM patient-derived cells ( n = 6, Mann-Whitney U test). D-G. qPCR expression of SLIT1 ( D ), SLIT3 ( E ), ROBO1 ( F ) and ROBO2 ( G ) in N15-0460 cells after shRNA SLIT2 silencing ( n = 4, Mann-Whitney U test). H. shCTRL and shSLIT2 treated N15-0460 growth over 72 hours in complete medium ( n = 3, multiple comparison linear regression). I. shCTRL and shSLIT2 treated N15-0460 cells response to TMZ treatment ( n = 4, Two-way ANOVA). J. Transwell assay quantification of N15-0460 cell migration towards a SLIT2 gradient ( n = 4, Mann-Whitney U test). K. Transwell assay quantification of N15-0460 shCTRL or shSLIT2 cell migration towards a serum gradient ( n = 4, Mann-Whitney U test). L-M. Spheroid formation assay quantification of shCTRL and shSLIT2 N15-0460 cells. Number ( L ) and size ( M ) of spheroids formed after 48 hours in culture were quantified ( n = 6 cultures per group, Mann-Whitney U test). N-O. Quantification of spheroid invasion assay in fibrin gels of shCTRL and shSLIT2 N15-0460 cells after 24 ( N ) and 48 hours ( O ). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: Western Blot, shRNA, Derivative Assay, MANN-WHITNEY, Expressing, Comparison, Transwell Assay, Migration, Tube Formation Assay, Invasion Assay

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A-B. Correlation analysis of MRC1 ( A) or VEGFA ( B ) and SLIT2 expression in GBM patients ( n = 129 patients, Spearman’s correlation test). C-O. Correlation analysis of SLIT2 expression with the indicated genes in GBM patients from TCGA cohort ( n = 489 patients, Spearman’s correlation test). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: Expressing

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A-C. Transwell assay of microglial cells ( A ), bone marrow derived macrophages (BMDM) ( B ) and peritoneal macrophages (PM) ( C ) in response to Slit2 or carrier (CTRL) in the bottom chamber ( n = 4, One-way ANOVA). D-E. Transwell assay of RAW macrophages treated or not with Robo1/2 siRNA and infected with adenovirus encoding CTRL (GFP construct), Robo1FL or Robo1ΔCD constructs and stained with Calcein. E. Quantification of (D) ( n = 3, Two-way ANOVA). F . Western blot analysis of Slit2 downstream signaling in cultured BMDM ( n = 6). G-H. ELISA from conditioned medium from LPS, Il-10 or Slit2-treated BMDMs to quantify IL-10 ( G ) and VEGFa ( H ) ( n = 3 independent cultures, Mann-Whitney U test) . I. qPCR analysis of BMDM cultures following Slit2 or LPS treatment ( n = 4, One-Way ANOVA or Mann-Whitney U test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: Transwell Assay, Derivative Assay, Infection, Construct, Staining, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A-B. Representative images (A) of calcein-stained (green) Transwell assays with Slit2 treatment in the bottom chamber or in both chambers and quantification (B) . Slit2- induced migration is chemotactic, as treatment with Slit2 in both chambers disrupts the gradient and abrogates migration ( n = 4, One-way ANOVA). C-D. qPCR analysis of Robo1 ( C ) and Robo2 ( D ) expression after siRNA treatment of cultured RAW264.7 macrophages for 72hs ( n = 4, Mann Whitney). E-I. Quantifications of the Western Blots shown in . ( n = 6, One-Way ANOVA). J . Western blot analysis of Slit2 downstream signaling in cultured microglial cells ( n = 3). K-L. ELISA from conditioned medium from LPS or Slit2-treated microglial cells quantifying the secretion of IL-10 ( K ) and VEGFa ( L ) ( n = 3 independent cultures, Mann-Whitney U test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: Staining, Migration, Expressing, Cell Culture, MANN-WHITNEY, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A. Western blot analysis of PLCγ, Akt and Erk1/2 phosphorylation induced by Slit2 in control and Robo1/2 knockdown RAW264.7 macrophages ( n = 5). B-C . Quantification of Robo1 ( A ) and Robo2 ( B ) protein expression after Robo1 and Robo2 knockdown. D-F . Quantification of (A) ( n = 5, Two-way ANOVA). G. qPCR analysis of genes related to the tumor supportive phenotype ( Mrc1 , Vegfa, Arg1, Cd209a and Ccl19 ) in RAW264.7 macrophages after Robo1 and Robo2 knockdown and Slit2 treatment ( n = 4, Mann Whitney U test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A. PI3Kγ immunoprecipitation in BMDMs treated or not with Slit2 for 15 minutes and WB for Robo1 ( n = 3 independent experiments). B. Transwell assay of BMDMs in response to Slit2 or carrier (CTRL) in the bottom chamber after pretreatment with vehicle control (DMSO) or PI3Kγ inhibitor IPI-549 (1uM). C-D. Phospho-Stat6 immunofluorescent staining of BMDMs treated or not with Slit2 and PI3Kγ inhibitor and quantification of nuclear pStat6 intensity ( n = 4 independent cultures, 2-way ANOVA). E-F. ELISA from conditioned medium from LPS or Slit2-treated BMDMs with vehicle control (DMSO) or PI3Kγ inhibitor, to quantify IL-10 ( E ) and VEGFa ( F ) ( n = 3 independent cultures, 2-way ANOVA) . G . qPCR analysis of BMDM cultures following Slit2 or LPS treatment with vehicle control or PI3Kγ inhibitor ( n = 4 independent cultures, 2-way ANOVA). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: Immunoprecipitation, Transwell Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A. WB analysis of Akt and Stat6 phosphorylation in BMDMs induced by Slit2 after PI3Kγ inhibitor IPI-549 pretreatment ( n = 3 independent cultures). B. qPCR analysis of microglial cultures following Slit2 or LPS treatment after pre-treatment with PI3Kγ inhibitor ( n = 4 independent cultures, 2-way ANOVA). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2021.04.12.438457

Figure Lengend Snippet: A. Experimental design: 8-week-old mice were engrafted with CT-2A spheroids and randomly assigned to CTRLFc or Robo1Fc treatment (2.5 mg/kg) every other day between day 7 and day 15 after tumor implantation. B. ELISA dosage of serum Slit2 in CTRLFc- and Robo1Fc-treated mice at days 14 and 21 ( n = 4 mice per group, Two-Way ANOVA). C. Representative CTRLFc- and Robo1Fc-treated tumors at 23 days. D . quantification of (C) ( n = 6, Student’s T test). E . In vivo two-photon images of day 23 CTRLFc and Robo1Fc treated CT-2A tumors. F-G. Quantification of vessel diameter ( F ) and branchpoints ( G ) from ( E ) ( n = 6 mice per group, One-way ANOVA). H . Quantification of hypoxic areas (Glut1 + ) on stained tumor sections of day 23 tumor-bearing mice treated with CTRLFc or Robo1Fc ( n = 6 mice per group, Mann-Whitney U test). I-K . Quantification of F4/80, MHC-II and MRC1 ( I ), soluble-Flt1 binding ( J ) and CD3 ( K ) immunostaining of day 23 tumor-bearing mice treated with CTRLFc or Robo1Fc ( n = 6 mice per group, Two-way ANOVA or Student’s t-test). L. 8-week-old wild-type mice were injected with CT-2A cells and assigned randomly to one of the following treatments: CTRLFc + vehicle ( n = 20), CTRLFc + TMZ (40mg/kg on days 7, 11, 15 and 19 after tumor implantation) ( n = 15), Robo1Fc + vehicle ( n = 24) or Robo1Fc + TMZ ( n = 22, Multiple comparisons Mantel–Cox log-rank; O.S., CTRLFc: 24 days; CTRLFc + TMZ: 28 days; Robo1Fc: 41 days; Robo1Fc + TMZ: 119 days). M. 8-week-old wild-type mice were injected with CT-2A cells and assigned randomly to one of the following treatments: CTRLFc + vehicle, CTRLFc + Anti-PD-1 + Anti-4-1BB treatment (200ug of each on days 7, 9, 11 and 13 after tumor implantation), Robo1Fc + vehicle or Robo1Fc + Anti-PD-1 + Anti-4-1BB treatment ( n = 10/11 mice per group; O.S., CTRLFc: 25.5 days; CTRLFc + Anti-PD-1 + Anti-4-1BB treatment: 40 days; Robo1Fc: 39 days; Robo1Fc + Anti-PD-1 + Anti-4-1BB treatment: Undetermined; Multiple comparisons log-rank test). N. 90 days after tumor implantation, surviving mice from Figure 9M ( n = 2 Anti-PD-1 + Anti-4-1BB survivors, 3 Robo1Fc survivors and 8 Robo1Fc + Anti-PD-1 + Anti-4-1BB survivors) or 8-weeks-old tumor naïve mice ( n = 10 mice) were re-challenged by implantation of CT-2A cells in the contralateral hemisphere from the first injection (O.S., Naïve mice: 21 days; CTRLFc + Anti-PD-1 + Anti-4-1BB survivors: 53.5 days; Robo1Fc survivors: 63 days; Robo1Fc + Anti-PD-1 + Anti-4-1BB treatment: Undetermined; Multiple comparisons log-rank test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rat anti-Human/Mice Slit2 monoclonal antibodies (Clone 710305, R&D Systems) was used as capture antibody at a concentration of 1ug/mL and sheep anti-mouse Slit2 polyclonal antibody was used as detection antibody at a concentration of 400ng/mL HRP-linked anti-sheep secondary antibodies (1:1000) were used for revelation.

Techniques: Tumor Implantation, Enzyme-linked Immunosorbent Assay, In Vivo, Staining, MANN-WHITNEY, Binding Assay, Immunostaining, Injection