Cls3397

Similar to the coculture system reported by Xu et al. [20 (link)], we stimulated PBMCs with anti-CD3e (2.5 μg/mL, #300432, BioLegend, San Diego, CA, USA), anti-CD28 (1.25 μg/mL) (#302923, BioLegend) and ovarian cancer cell lysate. Briefly, PBMCs were plated at a density of 2 × 10⁶ per well in six-well plates and stimulated with anti-CD3e, anti-CD28, as well as ovarian cancer cell lysate for 3 days. Then CD8⁺ T cells were purified using RosetteSep human CD8⁺ T cell Enrichment Cocktail. For the direct co-culture system, activated CD8⁺ T cells were co-cultured with ovarian cancer cells in 12-well plates at a ratio of 5:1 [21 (link),22 (link)] in the presence of different doses of glutaminase inhibitor 968, with or without anti-PD-L1 antibody. IgG was used as a control. After 24 h, the CD8⁺ T cells were removed with phosphate-buffered saline (PBS), and the attached cancer cells were collected and subjected to flow cytometry. For the indirect co-culture system, transwell cell culture chambers (0.4 μm, CLS3397, Corning, Tewksbury, MA, USA) were used to separate CD8⁺ T cells and cancer cells. CD8⁺ T cells were seeded into the upper chamber, and cancer cells were seeded into the lower chamber at a ratio of 5:1. After exposure to different concentrations of 968 for 24 h, the cancer cells were collected for flow cytometry analysis.

FRT cell lines were cultured in F12 Coon’s modification media (Sigma, F6636) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PenStrep). 16hBEge-W1282X cell lines were obtained from the Cystic Fibrosis Foundation (CFF) and cultured according to their instructions in minimum essential medium (MEM) media (32 (link), 72 (link)). Single-cell clones of the original CFF16hBEge-W1282X cell line were created to select for high-resistance clonal cell lines. One clonal cell line, CFF16hBEge-W1282X-SCC:3F2, was selected for analysis. Primary hBE cells isolated from CF patients homozygous for CFTR-W1282X (patient code HBEU10014) and compound heterozygous for CFTR-W1282X and CFTR-F508del (patient code HBEND12112) were also obtained from the CFF. For functional analysis, cells were differentiated by plating on Costar 24-well high-throughput screening filter plates (0.4 μM pore size, Polyester, Corning, catalog No. CLS3397). FRT and 16hBE cells were grown in a liquid/liquid interface (180 μL apical/700 μL basolateral) in a 37 °C incubator with 90% humidity and 5% CO₂ for 1 wk. Primary hBE cells were differentiated in an air/liquid interface for 5 wk. Media was replaced three times a week.

Human choroidal vascular endothelial cells (HCVECS; #36052-03, Celprogen, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 4.5 g/l glucose supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. The human macrophages were derived from human peripheral blood mononuclear cells and cultured, as the previous description^{22 (link)}. The cells were kept at 37 ^°C in a humidified atmosphere containing 5% CO₂. HCVECs were cultured in the lower well and macrophages were cultured in the upper well of the transwell plate (#CLS3397, Corning, USA) and verified by STR profiling.
The cells were culture in CO₂ incubator (#BBD6220, Thermo Fisher Scientific; 1%O₂, 94%N₂, and 5%CO₂) for 24 h (hypoxia group), HMPL-013 (0.05 μmol/l for 24 h), CCL2-neutralizing antibody (#AB-479-NA, R&D Systems, USA; 5 μg/ml for the last 30 min), recombinant human CCL2 protein (#279-MC, R&D Systems; 100 ng/ml for 24 h), geraniin (macrophage polarization modulator; #PHL80994, Merck; 30 μM for the last 2 h), or LPS (macrophage M1-type polarization agonist; Escherichia coli LPS, serotype 0127:B8; #L3129; Merck, USA; 2 μg/ml for 24 h).