Flll32

Human osteosarcoma cell lines Saos-2 and MG-63 were purchased from the American Type Culture Collection (ATCC, USA). All tumor cells were cultured using RPMI1640 complete medium, containing 10% fetal bovine serum (Gibco, MA, USA) under 37°C and 5% CO₂. For 2D collagen culture, type I collagen (Solarbio, Beijing, China) was diluted to 2.5 mg/mL with sterile water. Then, 25μL 10×phosphate-buffered saline (PBS) and 20μL 1N NaOH solution (Solarbio, Beijing, China) were subsequently added into the mixture. The 295μL of the collagen mixture was seeded into each well of a 24-well plate and mixed thoroughly. After 37°C incubation for 2 hours, the collagen mixtures became solid and cancer cells were seeded on the 2D collagen gel for 2D cells culture. α2β1 Integrin Ligand Peptide and STAT3 inhibitor FLLL32 were purchased from Med Chem Express (NJ, USA). Other reagents are of HPLC standard and purchased from Transgene biological Company (Beijing, China).

P6, AG490, peficitinib, tofacitinib, ruxolitinib, FLLL32, IQDMA, baricitinib, and WP1066 were obtained from MedChemExpress (CA, USA; Table 1). All inhibitors were dissolved in an appropriate volume of DMSO to make a 10 mM stock concentration. These stocks were then diluted 1000-fold in complete RPMI 1640 to prepare 10 μM working solutions of each. Those solutions were then diluted threefold serially in RPMI 1640 to prepare 3.3, 1.1, and 0.37 μM working solutions.