Stattic

All GBM tumor samples and subsequent cell line derivations were performed with written consents from patients through Quiñones laboratory, observing Institutional Review Board guidelines as previously described. GBM stem cells with tumorsphere forming capabilities, GBM 276 and GBM 612, were cultured in DMEM/F12 (1:1), 1% HyClone Antibiotic Antimycotic Solution (Thermo Scientific), Gibco B-27 serum free Supplement (Thermo Scientific 17504044), 20 ng/ml EGF, and 20 ng/ml FGF. For ligand treatment experiments, Gibco B27 serum-free supplement, minus insulin (Thermo Scientific A1895601) was used. For adherent culture, dishes were coated with Laminin (Sigma-Aldrich L2020). Coating was done in DMEM/F12 basal media for at least 2 h or overnight, and for every 1 cm² of surface area, 1 μl of Laminin was added. Drugs used in this study are as follows: Cycloheximide (Cell signaling #2112), Bafilomycin A1 (InvivoGen CAS # 88899-55-2), MG-132 (InvivoGen CAS # 133407-82-6), Cryptotanshinone (Selleckchem Catalog No. S2285), Stattic (Selleckchem Catalog No. S7024), and S3I-201 (Selleckchem Catalog No. S1155).

Isolation of CD8⁺ T cells from HD samples was performed with CD8 magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturer’s instructions. Freshly isolated CD8⁺ T cells, anti-CXCR3 (10 ng/mL, Abcam), or Stattic (10 nM; S7024; Selleck Chem, Houston, TX)-treated CD8⁺ T cells as well as peripheral blood mononuclear cells (PBMCs) from HDs were seeded on the upper chambers of a 5.0-μm pore Transwell. Supernatants of tumor tissue, anti-CXCL10 (100 ng/mL, Abcam), or recombinant human (rh) CXCL10 (Peprotech, Rocky Hill, NJ) were added to the lower chambers. CD8⁺ T cells in the upper chambers were pretreated with rhIL-17A (20 ng/mL), Stattic (10 nM), and/or the supernatants of enriched Th17 cells (5 × 10⁶) with or without anti-IL-17A (10 ng/mL) for 48 h in vitro. Migrated cells in the lower chambers were then counted and analyzed by flow cytometry after 12 h.

Primary bone marrow-derived macrophages (BMDMs) were isolated and cultured as previously reported (Bai et al. 2021 (link)). Mature BMDMs were exposed to 20 ng/ml IFN-γ plus 50 ng/ml LPS in the presence or absence of leptin (2 µg/ml, Peprotech, New Jersey, USA) for 24 h before the assessment of M1 macrophage polarization. Mature BMDMs were exposed to 20 ng/ml IL-4 (Peprotech, New Jersey, USA) in the presence or absence of leptin (2 µg/ml) for 24 h before the assessment of M2 macrophage polarization. In some experiments, the leptin receptor antagonist Allo-Aca (1 mM) was included to verify the effects of the leptin receptor. In some experiments, synthetic inhibitors of STAT3 (Stattic, 100 nM, Selleck, Texas, USA), JNK (Sp600, 10 μM, Selleck, Texas, USA), or AKT (MK-2206, 10 μM, Selleck, Texas, USA) were used to verify the effects on M1 macrophages.