Investigating 2-DG Effects on Lung Metastasis and Intratumoral IL-1β

Briefly, C57BL/6 male mice (5–8 weeks old) were intravenously injected with 5 × 10⁵ B16F10 cells to establish a lung metastasis model [29 (link)]. On the next day, the mice were intraperitoneally injected with PBS (vehicle control) or 2-DG (50 mg/kg) in PBS for 5 days. The mice were sacrificed by cervical dislocation on day 14 after tumor cell injection. The lungs and cardiac blood were obtained from these mice for ELISA and western blotting assays. To evaluate effects of 2-DG on tumor progression and intratumoral IL-1β levels, C57BL/6 mice (5–8 weeks old) were subcutaneously injected with 5 × 10⁵ B16F10 cells. PBS or 2-DG (50 mg/kg) in PBS was subcutaneously injected into the tumoral regions on days 7–14, and the tumor tissues were extracted on day 14. Following tumor volume measurements using the Ellipsoid formula (a × b × c × π × 4/3), B16F10-derived tumor tissues were frozen in FSC 22 (Surgipath, Richmond, IL, USA). Next, 5-μm cryosections were cut using a Leica CM1100 cryostat (Leica Microsystems GmbH, Wetzlar, Germany) with microtome blades (Leica 818, Leica Microsystems GmbH, Wetzlar, Germany). Then, the tumor tissues were mounted on poly-L-lysine-coated glass slides (Muto Pure Chemicals, Tokyo, Japan). Tumor tissues were stained with anti-IL-1β, IB-4, and DAPI and analyzed by confocal microscopy.