Journal: The Journal of Clinical Investigation

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

doi: 10.1172/JCI141083

Figure Lengend Snippet: (A) Immunohistochemistry on sections of late-stage CT-2A shCTRL, shSlit2, or shSlit2 + hSLIT2 tumors for F4/80, MHC-II, and MRC1+ cells (green). (B) Quantifications of A (n = 7 mice per group, 5 fields per tumor, 2-way ANOVA). (C and D) FACS analysis of day 21 CT-2A shCTRL and shSlit2 tumors for quantification of TAMs (n = 10 tumors/group; Student’s t test and 2-way ANOVA). (E) qPCR analysis from FACS-sorted TAMs (n = 6 tumors/group, Mann-Whitney U test). (F–H) ELISA from protein samples extracted from FACS-sorted TAMs from shCTRL and shSlit2 tumors to quantify IFN-γ (F), IL-10 (G), and VEGFa (H) (n = 5 tumors/group, Mann-Whitney U test). (I) Representative images and quantification of soluble-Flt1 binding to sections of day 21 CT-2A shCTRL, shSlit2, and day 18 shSlit2 + hSLIT2 tumors (n = 7 mice per group, 5 fields per tumor, 1-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: FACS-sorted CD45 + CD11b + F4/80 + Ly6G – TAMs accounted for about 12% of the total cells in shCTRL tumors, but only 6% in the Slit2 knockdown tumors ( and Supplemental Figure 6F ).

Techniques: Immunohistochemistry, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: The Journal of Clinical Investigation

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

doi: 10.1172/JCI141083

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

Article Snippet: FACS-sorted CD45 + CD11b + F4/80 + Ly6G – TAMs accounted for about 12% of the total cells in shCTRL tumors, but only 6% in the Slit2 knockdown tumors ( and Supplemental Figure 6F ).

Techniques: Tumor Implantation, MANN-WHITNEY, In Vivo, Immunohistochemistry

Journal: The Journal of Clinical Investigation

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

doi: 10.1172/JCI141083

Figure Lengend Snippet: (A) Eight-week-old mice engrafted with CT-2A spheroids were treated with CTRLFc or Robo1Fc as indicated, and analyzed at day 23. (B) ELISA of serum Slit2 (n = 4, 2-way ANOVA). (C and D) Tumor size at day 23 (n = 6, Student’s t test). (E–G) In vivo 2-photon images (E) and quantification of vessel diameter (F) and branchpoints (G) (n = 6 mice per group, 1-way ANOVA). (H) Quantification of Glut1+ area (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 immunostaining (K) (n = 6 mice per group, 2-way ANOVA (I) or Student’s t test). (L) Eight-week-old tumor-bearing mice were assigned to CTRLFc + vehicle (n = 20), CTRLFc + TMZ (n = 15), Robo1Fc + vehicle (n = 24), or Robo1Fc + TMZ (n = 22; OS 24 days CTRLFc; 28 days CTRLFc + TMZ; 41 days Robo1Fc; 119 days Robo1Fc + TMZ; multiple comparisons Mantel-Cox log-rank). (M) Eight-week-old tumor-bearing mice were assigned to CTRLFc + vehicle, CTRLFc + anti–PD-1 + anti–4-1BB, Robo1Fc + vehicle, or Robo1Fc + anti–PD-1 + anti–4-1BB (n = 10/11 mice per group; OS 25.5 days CTRLFc; 40 days CTRLFc + anti–PD-1 + anti–4-1BB; 39 days Robo1Fc; and undetermined for Robo1Fc + anti–PD-1 + anti–4-1BB; multiple comparisons log-rank test). (N) Ninety days after tumor implantation, surviving mice from M (n = 2 anti–PD-1 + anti–4-1BB, n = 3 Robo1Fc, and n = 8 Robo1Fc + anti–PD-1 + anti–4-1BB) or 8-week-old tumor-naive mice (n = 10) were rechallenged in the contralateral hemisphere (OS 21 days naive mice; 53.5 days CTRLFc + anti–PD-1 + anti–4-1BB survivors; 63 days Robo1Fc survivors; and undetermined for Robo1Fc + anti–PD-1 + anti–4-1BB survivors; multiple comparisons log-rank test). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: FACS-sorted CD45 + CD11b + F4/80 + Ly6G – TAMs accounted for about 12% of the total cells in shCTRL tumors, but only 6% in the Slit2 knockdown tumors ( and Supplemental Figure 6F ).

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