F12 medium

Samples obtained from primary tumor resection (NB#1 and NB#2) were dissociated. Briefly, tissues were washed with Ca²⁺-, Mg²⁺-free PBS (Life Technologies), crushed with a scalpel in F12 medium (ThermoFisher Scientific) and then dissociated with 156 Units/ml type IV Collagenase (Sigma–Aldrich), 25 mM HEPES (Life technologies), 3% fetal calf serum (FCS, Sigma) and 20 Units/ml Dnase I (Sigma) for 30 min at 37 C. A 5 min more incubation with 4.9 mg/ml trypsin (Sigma) at 37 °C was performed. Non-dissociated tissue was removed by filtration through a 40 mm nylon Cell Strainer (BD Falcon).
Bone marrow sample (NB#3) was harvested under general anesthesia at diagnosis, taken from posterior and anterior iliac crests and collected on EDTA tube. Mononuclear marrow cells were separated by density gradient centrifugation (Pancoll, PAN-Biotech). The percentage of malignant cells in the samples was evaluated by a two-colour fluorochrome staining as described in^{54 (link)}. The mononuclear marrow cells showing tumoral invasion were frozen in RPMI containing 20% PBS and 10% DMSO. Cell suspensions obtained from dissociated tumors or bone marrow aspirates were labeled with an 8 µM CFSE solution (Life Technologies).

Primary normal human bronchial epithelial (NHBE) cells from healthy adults and high-risk adults (deidentified) were obtained from Dr. Kristina Bailey at the University of Nebraska Medical Center (UNMC) (Omaha, NE) under an approved material transfer agreement (MTA) between the University of North Dakota (UND) and UNMC (Omaha, NE). The protocol for obtaining cells was reviewed by the UNMC IRB and was determined to not constitute human subject research (#318-09-NH). In this study, we used cells from three donors: nonsmoker healthy adults (donors #1 and #2) and adult with chronic obstructive pulmonary disease (COPD) (donor #3). The protocols for subculturing primary NHBE cells were published previously (Osan et al., 2020 , 2021 (link), 2022 (link); Talukdar et al., 2022 ). Human lung epithelial A549 cells was maintained in F-12 medium (Thermo Fisher Scientific) as described previously (Mehedi et al., 2016 (link)). SARS-CoV-2 (USA/WA-CDC-WA1/2020 isolate, GenBank accession no. MN985325; kindly provided by CDC), SARS-CoV (Urbani strain, GenBank accession no. AY278741; kindly provided by Rocky Mountain Laboratories (RML), NIAID, NIH), and MERS-CoV (GenBank accession no. NC_019843.3; kindly provided by the Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands) were used for in vitro infections described below.

RLE-6TN alveolar epithelial cells (ATCC, CRL-2300, USA) were cultured in F12 medium (Thermo Fischer Scientific, # 11765054, USA) supplemented with 2 mM L-glutamine (Pan Biotech, P04–80100, Germany), 0.01 mg/ml bovine pituitary extract (Thermo Fischer Scientific #13028014, USA), 0.005 mg/ml insulin (Sigma-Aldrich, # I0516, Germany), 2.5 ng/ml insulin-like growth factor (Sigma-Aldrich, # I3769, Germany), 0.00125 mg/ml transferrin (Sigma-Aldrich, # T1147, Germany), and 2.5 ng/ml epidermal growth factor (Sigma-Aldrich, # E4127, Germany), 10% fetal bovine serum (heat-inactivated, PAN Biotech, P30–1506, Germany), 100 U/mL penicillin and 100 μg/mL streptomycin (PAN Biotech, P06–07100, Germany). Cells were detached using 2.5 ml Accutase solution (Sigma Aldrich, A6964-500ML, Germany) and sub cultivated with a ratio of 1:5 twice a week.