Computational protocol: Functional invadopodia formed in glioblastoma stem cells are important regulators of tumor angiogenesis

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Protocol publication

[…] GSC cell line (GSC-2) used in this study derived from an adult GBM-patient operated in Poitiers University Hospital was characterized and cultured as previously described [–]. Briefly, the GBM tumor was washed and mechanically dissociated into single cells. Dissociated cells were cultured in Neurobasal medium (NBE) supplemented with 20 ng/mL of basic fibroblast growth factor (bFGF, Invitrogen), 20 ng/mL of epidermal growth factor (EGF, Invitrogen) and the culture supplements N2 (100×, Invitrogen) and B27 (50×, Invitrogen). GSC cells positive for the surface marker CD133 were isolated by magnetic cell sorting. GSCs were cultured as non-adherent spheroids and at the point spheres augmented in size were enzymatically dissociated using accutase (Merck Millipore). GFP expression to GSCs was performed via infection using the lentiviral vector that encodes the green fluorescent protein TRIP/ΔU3-EF1α-GFP as previously described [].To study the potential role of CD44 signaling in invadopodia formation, GSCs were transfected with NT- and CD44- siRNA using Lipofectamine RNAiMAX. Knockdown was performed via two rounds of siRNA transfection at 24-h intervals. NT- and CD44- siRNA transfected GSC-GFP cells were then seeded on matrigel-coated coverslips for 4 h, fixed and stained with phalloidin. Fixed cells were examined using a confocal laser-scanning microscope (IX81; Olympus) equipped with 40× (NA 1.35) UAPO ID/340UV (oil immersion) and 60× (NA 1.4) PLAPO (oil immersion) objectives. The fluorescence images were sampled with FV1000 Viewer software (Olympus). The images were then processed with ImageJ (http://rsb.info.nih.gov/ij/).For the sphere formation assays, GSC dissociated cells were subcultured (160 cells per cm2) in 96-well plates in serum-free neurobasal medium in the presence of LIMKi and vehicle (DMSO) for 15 days. Bright field images of the spheres were taken using an inverted microscope (CKX41; Olympus) equipped with 4× (NA 0.10) PlanC-N, and 10× (NA 0.25) PlanC-N objectives. Images were then sampled with ToupView software (ToupTek Photonics). Morphometric parameters such as spheres surface area and perimeter were quantified using ImageJ.HUVEC cells were purchased from Lonza and cultured in EBM-2 medium (Lonza) supplemented with HUVEC growth factors (Lonza). Only early passages of HUVECs (between 4 and 6) were used. For the tube formation assay, HUVECs were seeded at a density of 8.5 × 104 cells/cm2 in FluroDish™ culture dishes (35 mm, World Precision Instruments Inc) in supplemented EBM-2 media over a gelled basement matrix (Matrigel; 3 mg/mL) and allowed to form capillary-like structures for 16h. In the same manner, NT-, CD44- and OPN- siRNA transfected GSCs were seeded at a density of 8.5 × 104 cells/cm2 in FluroDish™ culture dishes (35 mm, World Precision Instruments Inc) in supplemented NBE media over a gelled basement matrix (Matrigel; 3 mg/mL) and allowed to form capillary-like structures for 16 h. Bright field images of the vascular tubes were taken using an inverted microscope (CKX41; Olympus) equipped with 4× (NA 0.10) PlanC-N, and 10× (NA 0.25) PlanC-N objectives. Images were then sampled with ToupView software (ToupTek Photonics) and processed with ImageJ (http://rsb.info.nih.gov/ij/). [...] For immunofluorescence analysis, GSCs, GSC-spheres and HUVECs seeded on glass coverslips (coated with Matrigel or Poly-L-lysine) or gelled substrates were fixed with 4% paraformaldehyde in PBS, permeabilized or not (0.2% Triton X-100) and blocked in 4% bovine serum albumin (BSA) in PBS. Cells were then incubated for 1 h at room temperature (RT) with primary antibodies and rinsed in PBS. Secondary antibodies were then added for 1 h at RT. Coverslips were permanently mounted in PVA-DABCO. Fixed cells were examined using a confocal laser-scanning microscope (IX81; Olympus) equipped with 10× (NA 0.4) UPLAPO, 20x (NA 0.70) UPLAPO, 40× (NA 1.35) UAPO ID/340UV (oil immersion) and 60× (NA 1.4) PLAPO (oil immersion) objectives. The fluorescence images were sampled with FV1000 Viewer software (Olympus). The images were then processed with ImageJ (http://rsb.info.nih.gov/ij/) and Imaris v7 (Bitplane) software.For live imaging of the tube formation assays, GSC-GFP cells were seeded over a gelled basement matrix in FluroDish™ culture dishes and allowed to form capillary-like structures for 3 h before imaging. Dishes were placed on a heated 37° C stage and imaged with a spinning disk confocal microscope (IX81-ZDC; Olympus) equipped with a 20× (NA 0.75) UPLSAPO objective (). Fluorescence images were sampled with FluoView software (Olympus) using an interval time of 5 min for a total recording of 23,5 h. Time-lapse movies were then processed with ImageJ software.To monitor GSCs transmigration capacities on HUVEC-based monolayers, GFP-GSC spheroids were seeded on top of confluent monolayer and live imaging was performed with a spinning disk confocal microscope (IX81-ZDC; Olympus) equipped with a 20× (NA 0.75) UPLSAPO objective (). Fluorescence images were sampled with FluoView software (Olympus) using an interval time of 5 min for a total recording of 21,5h. Time-lapse movies were then processed with ImageJ software. […]

Pipeline specifications

Software tools ImageJ, Imaris
Databases HUVEC
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Brain Neoplasms, Glioblastoma, Neoplasms