Human tumor cells were stained with IL13Rα2 (BioLegend) and human monocyte/macrophages were stained with CD14, CD80, CD86 (BD Biosciences) HLA-Dr (eBioscience). All samples were run on MACSQuant (Miltenyi Biotec).
Mouse tumor cells expanded in vitro were stained with an unconjugated goat anti-mouse IL13Rα2 (R&D Systems) followed by secondary donkey anti-goat NL637 (R&D Systems). Live murine CAR T cells were stained with CD8 (BioLegend), CD3, CD4, CD62L (eBioscience) or CD45RA (BD Biosciences). CD19 (BD Biosciences) was used as a surrogate to detect the CAR.
Brains from euthanized mice were removed at the indicated time-points, and a rodent brain matrix was used to cut along the coronal and saggital planes to obtain a 4x4 mm section, centered around the injection site. These sections were minced manually, then passed through a 40 μm filter. Myelin was removed using Myelin Removal Beads II and LS magnetic columns (Miltenyi Biotec) according to the manufacturer’s instructions, then cells were counted. Cell were stained and analyzed using MACSQuant (Miltenyi Biotec). For flow sorting, cells were stained and sorted using BD AriaSORP (BD Biosciences). For gene expression analysis of TME, the remaining cells were lysed for RNA. Each experiment was repeated independently at least twice. List of all antibodies (mouse and human) are provided inSupplemental Table S1.
Mouse tumor cells expanded in vitro were stained with an unconjugated goat anti-mouse IL13Rα2 (R&D Systems) followed by secondary donkey anti-goat NL637 (R&D Systems). Live murine CAR T cells were stained with CD8 (BioLegend), CD3, CD4, CD62L (eBioscience) or CD45RA (BD Biosciences). CD19 (BD Biosciences) was used as a surrogate to detect the CAR.
Brains from euthanized mice were removed at the indicated time-points, and a rodent brain matrix was used to cut along the coronal and saggital planes to obtain a 4x4 mm section, centered around the injection site. These sections were minced manually, then passed through a 40 μm filter. Myelin was removed using Myelin Removal Beads II and LS magnetic columns (Miltenyi Biotec) according to the manufacturer’s instructions, then cells were counted. Cell were stained and analyzed using MACSQuant (Miltenyi Biotec). For flow sorting, cells were stained and sorted using BD AriaSORP (BD Biosciences). For gene expression analysis of TME, the remaining cells were lysed for RNA. Each experiment was repeated independently at least twice. List of all antibodies (mouse and human) are provided in