Murine bfgf

GSCs were obtained according to previous studies [18 (link)] by growing GL261 in serum-free DMEM/F12 medium (HyClone, USA) supplemented with 20 ng/mL murine bFGF (PeproTech, USA) and 20 ng/mL EGF (PeproTech, USA) at 37°C with 5% CO₂. The culture medium was refreshed by 50% every 2-3 days. The cells were passaged when they reached 90% confluence. Spherical cells at the third passage (P3) were examined for the expression of CD133 by immunofluorescence staining. After three days, the expression of GFAP in the spheres was detected by an immunofluorescence assay.
We extracted GSC antigens from apoptotic lysates. GSCs of the third passage (P3) were digested with 0.05% trypsin (HyClone, USA) for 5 min and processed overnight with hydrogen peroxide (100 μM),which is known to induce apoptosis. In order to evaluate apoptosis, an apoptosis assay kit manufactured by Beyotime in China was utilized. Then, GSCs were stained with FITC-Annexin V/PI (KeyGen Biotech, China) for 15 min, and apoptotic antigen labelled with FITC-Annexin V was enriched by fluorescence-activated cell sorting (FACS; BD FACSAria III, USA). The purity of these apoptotic GSCs was verified by flow cytometry test. After sorting, the enriched apoptotic GSCs were added to imDCs at a 3 : 1 (stimulator : responder) ratio in RPMI-1640 medium and incubated for 24 h at 37°C with 5% CO₂.

For subcutaneous (s.c.) injection, 10 × 10⁶ BM cells or 1 × 10⁵ tumor cells were re-suspended in a total of 200 μl PBS with 1 μg murine bFGF (Peprotech, Rocky Hill, NJ, USA) mixed with 400 μl of Matrigel (Becton-Dickinson), according to a previously published protocol [18 (link)]. We injected a total of 300 μl (cells plus Matrigel mix) per dorsolateral flank. Tumors were inspected weekly and measured with caliper. Tumor volumes were calculated according to the following formula: Tumor volume (mm³) = L (longest diameter) × l (shortest diameter)² × 0.5 [50 (link)]. Tumor growth was scored when tumor volumes exceeded 10 mm³.
For the Matrigel assay, a total of 5 × 10⁶ of BM cells in 400 μl of Matrigel supplemented with bFGF and/or VEGF were s.c. injected as previously described [18 (link)]. After one week Matrigel plugs were recovered, snap frozen in liquid nitrogen and stored at −80°C.
For in vivo silencing experiments, DTC cells transduced with lentiviral vectors where s.c injected (2.5×10⁵/flank) in 200 μl of Matrigel in NSG mice.

Cells were seeded at clonal density of 1000 cells/mL in ultra-low attachment 6-well plates (ThermoFisher^TM Scientific, Fontenay-sous-Bois, France) under neural crest cell culture conditions: DMEM GlutaMAX (0.5X; Gibco^®), F12 NutMix GlutaMAX (0.5X; Gibco^®), B27 supplement (1X; Gibco^®, Waltham, MA, USA), antibiotics (100 U/mL penicillin and 1000 μg/mL streptomycin; Gibco^®, Waltham, MA, USA), murine EGF (20 ng/mL; peproTech, Rocky Hill, NJ, USA), murine bFGF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA), and insulin (5 μg/mL; Sigma Aldrich Chimie, Saint-Quentin-Fallavier, France).
Cells were dispersed in low-adherent plates and medium was changed twice a week. Spheroids were allowed to grow over the course of 12 days. Spheres were counted and measured every 2–3 days under the microscope.