Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) Representative flow cytometry graphs of CD11b and GFP in peripheral blood of KDRGFP mice bearing gliomas. Astrocytic tumors were generated by RCAS/TVA-mediated overexpression of PDGF as described in Methods. Low-grade gliomas were observed by week 5 and high grade by weeks 7–8. Controls (CTL) were mice bearing an intact Kdr locus without GFP knockin. (B) Quantification of CD11b+KDRGFP+ cell frequency in peripheral blood of RCAS/TVA tumor mice at low-grade and high-grade stages. ***P < 0.001, Student’s t test. LEU, Leukocytes. (C) Further characterization of murine CD11b+KDRGFP+ cells in peripheral blood by Ly6C and Ly6G staining. (D) Quantification of Ly6G+ and Ly6C+ frequency out of total CD11b+ cells in the CD11b+KDRGFP+ and CD11b+KDRGFP– populations in peripheral blood at high-grade stage. **P < 0.01, for Ly6Chi cells, Student’s t test. (E) CFU assays were performed on Lin–KDRGFP+ hematopoietic cells from BM, and macrophage (M) colonies and macrophage/granulocyte (GM) colonies, but not granulocyte (G) colonies, were observed. Quantification of various colony types formed from lineage-negative KDRGFP– or KDRGFP+ cells. ***P < 0.001, in granulocytes, Student’s t test. (F) Gene expression (quantitative RT-PCR) of various markers, including Kdr in in vitro–cultured BM Lin– cells exposed to GL261-conditioned medium at different time points. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA. Data are shown as mean ± SD.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Flow Cytometry, Generated, Over Expression, Knock-In, Staining, Gene Expression, Quantitative RT-PCR, In Vitro, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: Lethal dose–irradiated C57BL/6 mice were transplanted with Ubc-GFP Rosa26-CreERT2Kdrfl/fl and Rosa26-CreERT2KDRfl/+ BM cells, and GL261 tumors were implanted after BM engraftment. (A) Peripheral white blood cells were analyzed on side scatter (SSC) and GFP by flow cytometry. The GFP+ and GFP– populations were gated for further analysis. CD11b versus GFP (B), Ly6C versus Ly6G (C), and CD3 versus B220 (D) are shown on both GFP+ and GFP– populations. (E) BM cells were also analyzed on side scatter and GFP. (F) Lineage-negative cells were gated for analysis of HSCs and HPCs by c-Kit versus Sca-1 (G). (H) HPCs (Lin–c-Kit+Sca-1–) were further characterized by FcγR versus CD34 within both GFP+ and GFP– populations. The experiment was conducted 5 times.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Irradiation, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) The unsupervised principal component analysis of significantly altered genes in Lin–c-Kit+Sca-1– from naive mice, Lin–c-Kit+Sca-1– KDR-GFP+ cells, and Lin–c-Kit+Sca-1–KDR-GFP– cells from BM of tumor-bearing mice. (B) Lin–c-Kit+Sca-1– cells from naive mice (black), Lin–c-Kit+Sca-1– KDR-GFP+ cells (red), and Lin–c-Kit+Sca-1–KDR-GFP– cells (green) from BM of tumor-bearing mice, based on similarity of gene profiles. PC1, principal component 1. (C) The candidate genes (P < 0.05, >1.5-fold change; Lin–c-Kit+Sca-1– KDR-GFP+ versus Lin–c-Kit+Sca-1–KDR-GFP–) were divided according to the subset with the highest expression and analyzed for categories with significant enrichment (P < 0.05) of categories in GO biologic processes using DAVID tools. Similar categories were grouped accordingly. The presence of association between functions and genes was color highlighted (black [negative] versus green [positive]). (D) Expression of Id2 in various lineages of hematopoietic cells. ***P < 0.001, 1-way ANOVA. (E) Expression of ID2 in HPCs (CD45–CD34+) from patients with low-grade or high-grade gliomas. ***P < 0.001, 1-way ANOVA. n = 21. (F) Expression of ID2 in CD11b+ blood cells from patients with low-grade or high-grade gliomas. ***P < 0.001, 1-way ANOVA. n = 20. Data are shown as mean ± SD.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) Chimeric C57BL/6 mice transplanted with Id2–/– BM cells (Id2+/+ BM cells as control) were implanted with luciferase-labeled GL261 tumors intracranially. Tamoxifen was applied at day 3 after implantation. The tumors were monitored by bioluminescence. Representative images were taken at day 14. The color bar on the right represents photon intensity. The experiments had 2 replicates. The tumor growth curve is based on bioluminescence. n = 10. **P < 0.01, 1-way ANOVA. (B) Flow cytometry analysis of peripheral blood cells on CD11b, Ly6C, and Ly6G in Id2+/+ BMT and Id2–/– BMT groups. Quantification of CD11b+ cell frequency out of total white blood cells and of Ly6Chi cell frequency out of CD11b+ cells. **P < 0.01, 1-way ANOVA, for each group. n = 7. (C) Tubule formations of HCMEC/D3 (GFP) cocultured with lineage-negative Id2+/+, Id2–/–, Id2–/– scrambled sequence (SC) control, or Id2–/– KDR overexpression (OE) HPCs pretreated with TGF-β/GM-CSF. Quantifications of tubule lengths in the indicated group. ***P < 0.0001, 1-way ANOVA. n = 6. (D) Growth factor–reduced Matrigel plugs of lineage-negative Id2+/+, Id2–/–, Id2–/– SC CTL, or Id2–/– KDR overexpression HPCs pretreated with TGF-β/GM-CSF. Blood vessels (red) were perfused with rhodamine-dextran. Lower panels show magnified views to highlight vascular permeability. Quantifications of based blood vessels density (BVD) in each group. **P < 0.01, 1-way ANOVA. n = 6. Data are represented as mean ± SD. Scale bars: 50 μm (D, upper panels); 20 μm (C, D, lower panels).

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Control, Luciferase, Labeling, Flow Cytometry, Sequencing, Over Expression, Permeability