Additional file 1 of Soluble CD146, a biomarker and a target for preventing resistance to anti-angiogenic therapy in glioblastoma
Creators: name: Joshkon, Ahmad
name: Tabouret, Emeline
name: Traboulsi, Wael
name: Bachelier, Richard
name: Simoncini, Stéphanie
name: Roffino, Sandrine
name: Jiguet-Jiglaire, Carine
name: Badran, Bassam
name: Guillet, Benjamin
name: Foucault-Bertaud, Alexandrine
name: Leroyer, Aurelie S.
name: Dignat-George, Françoise
name: Chinot, Olivier
name: Fayyad-Kazan, Hussein
name: Bardin, Nathalie
name: Blot-Chabaud, Marcel
Publisher: name: figshare
Year: 2022
Subjects: subject: Cell Biology
subject: Neuroscience
subject: Pharmacology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: FOS: Biological sciences
subject: Cancer
subject: Hematology
subject: 60506 Virology

description: Additional file 1. Supplementary methods:Peptides and antibodies. Plasmids, siRNA and cell transfection. Western-blot and Immunoprecipitation assays. ELISA experiments. Immunofluorescence experiments. Flow cytometry experiments. Reverse Transcription-quantitative PCR (RT-qPCR). Cell migration assays. Cell Proliferation assays. Transwell invasion assays. Crispr/Cas9 deletion of genes. Experiments on animals and imaging. Immunohistochemistry. Patient cohort. Statistical analysis. Supplementary figures and tables: Supplementary Table 1: Absence of significant impact of age, KPS and steroid dose on progression-free survival or overall survival. Supplementary Table 2: references and application condition of the different antibodies used in the study. Supplementary Figure 1: Expression of CD146 on various types of cancers and effect on overall survival in patients with GBM. Supplementary Figure 2: Glioblastoma cell lines characterization. Supplementary Figure 3: Concentration of VEGF and sCD146 in the culture media of U87, U373, and U118 glioblastoma cell lines. Supplementary Figure 4: Avastin induces proliferation, sCD146 secretion, and EMT/CSC markers in U373 cells. Supplementary Figure 5: Avastin has no effect on CD146-negative U118 glioblastoma cells. Supplementary Figure 6: sCD146 induces U373 cell proliferation, migration and invasion in-vitro and promotes CSC and EMT markers. Supplementary Figure 7: Effect of sCD146 and VEGF on CD146-negative glioblastoma cells. Supplementary Figure 8: sCD146 binds integrin αvβ3 on U373 cells. Supplementary Figure 9: Soluble CD146 induces EMT in U118 cells transfected with integrin αvβ3. Supplementary Figure 10: Knocking-down integrin αvβ3 inhibits sCD146-induced EMT in U87 cells. Supplementary Figure 11: sCD146 mediated its effects on U87 cells through a signalosome containing CD146, αvβ3, and VEGFR2. Supplementary Figure 12: sCD146 mediates its effects on U373 cells through a signalosome containing CD146, αvβ3, and VEGFR2. Supplementary Figure 13: Validating gene knock out in U87 and U373 cells. Supplementary Figure 14: Integrin αvβ3 associates with membrane CD146 and VEGFR2 on HUVECs and binds sCD146. Supplementary Figure 15: CD146/VEGFR2/integrin αvβ3 signalosome mediates sCD146 and VEGF effects in U87 cells and activates common signaling pathways. Supplementary Figure 16: CD146/VEGFR2/integrin αvβ3 signalosome mediates sCD146 and VEGF effects in U373 cells and activates common signaling pathways. Supplementary Figure 17: CD146, integrin αvβ3, and VEGFR2 are essential for mediating sCD146 and VEGF effects on U87 cells. Supplementary Figure 18: CD146, integrin αvβ3, and VEGFR2 are essential for mediating sCD146 and VEGF effects on U373 cells. Supplementary Figure 19: Humanized anti-sCD146 antibody mucizumab significantly decreases U373 cell proliferation, migration and invasion and hampers CSC and EMT in-vitro. Supplementary Figure 20: Humanized anti-sCD146 mucizumab significantly decreases human sCD146 and human VEGF in two different mouse models of glioblastoma. Supplementary Figure 21: Illustrative summary describing the mechanism of resistance to bevacizumab in CD146-positive glioblastoma cells.