2.1. In Vitro Generation of Murine Bone Marrow-Derived Macrophages (BMDM)

Whole bone marrow was harvested from 10- to 12-week-old female mice by flushing RPMI medium through femurs and tibias using a 27-gauge needle. Animal use for bone marrow collection has been authorized by the Italian Ministry of Health, Animal and Veterinary Office (authorization No. 597/2019-PR). Following red blood cell lysis, cells were cultured overnight in 10% RPMI in 10-cm plates. Non-adherent cells were collected and seeded in Petri dishes in medium containing 20 ng/mL macrophage colony-stimulating factor (M-CSF) (PeproTech, supplied by Tebu-Bio S.r.l., Magenta MI, Italy). After 3 days of culture, cell media were replenished with fresh medium and M-CSF. BMDM were harvested on day 4, and used for all in vitro assays. Polarization was obtained by culturing BMDM with medium containing IL4 (20 ng/mL; PeproTech) and M-CSF (20 ng/mL; PeproTech), or 100 ng/mL LPS (PeproTech) for 24 h. Hereinafter, IL4+ M-CSF-stimulated macrophages will be referred to as M2-like, and LPS-stimulated macrophages as M1-like cells. When specifically indicated, gemcitabine (1 mg/mL), or anti-IL17A antibody (20 μg/mL; BE0173, BioXCell, supplied by D.B.A. Italia S.r.l., Segrate MI, Italy) or its isotype control (BE0083, BioXCell), or recombinant IL17A (PeproTech) were added parallelly to the polarizing stimuli for 24 h. If not differently specified, all metabolic and molecular biology assays were performed at 24 h after treatment.