Cell lines and culture conditions

NCI-H358, NCI-A549, and NCI-H2122 pulmonary adenocarcinoma cells harboring a KRAS mutation and NCI-H3255 pulmonary adenocarcinoma cells harboring the EGFRL858R mutation were obtained from the American Type Culture Collection (Manassas, VA) and were grown as monolayers in RPMI 1640 (NCI-H358, NCI-H2122, and NCI-H3255) or DMEM (NCI-A549) supplemented with 10% heat-inactivated fetal bovine serum and 100 μg/ml of streptomycin and 100 units/ml of penicillin. All cells were grown under 5% CO2 at 37 °C, were authenticated by using short tandem repeats (STRs), and were routinely tested for mycoplasma using a TaKaRa PCR Mycoplasma Detection Set (Takara Bio, Inc., Otsu, Japan).

Patient-derived tumoroid culture

Patient-derived lung adenocarcinoma (LUAD) tumoroids, PDT-LUAD#19, PDT-LUAD#99, and PDT-LUAD#119, were generated using tumoroid culture systems, as previously described [10]. The research protocol received approval from the Ethics Committee of the Kawasaki Medical School, with the assigned reference number 3171‐5. STR profile analysis was performed in PDT-LUAD#119 tumoroids to investigate genomic stability through future passages with authentication. The patient who participated in the present study signed an informed consent form approved by the responsible authority.

Next-generation sequencing, Sanger sequencing, quantitative real-time polymerase chain reaction (q-PCR), and  fluorescence in situ hybridization

Next-generation sequencing, including whole exome sequencing and RNA-seq, along with fluorescence in situ hybridization (FISH), was carried out following previously established procedures [10]. MUC5AC mRNA expression was confirmed using a StepOnePlus Real-Time PCR system, with a specific probe for MUC5AC (assay reference: Hs00873651_mH).

Sanger sequencing was performed by Eurofins Genomics K. K. (Tokyo, Japan) using the primers for TP53 Exon 4: 5′-CAAGCAATGGATGATTTGATGCTGTC-3′ and 5′- TAGGTTTTCTGGGAAGGGACAGAAGATG-3′, and for TP53 Exon 7: 5′- GACAGAGCGAGATTCCATCTCAAAAA-3′ and 5′- ATGAGAGGTGGATGGGTAGTAGTATGGAA-3′.

Immunoblot analysis, immunohistochemistry, periodic acid–Schiff staining, and Alcian blue staining

Immunoblot analysis and immunohistochemistry were conducted following previously established protocols [10]. The primary anti-MUC5AC antibody (45M1) was obtained from Thermo Fisher Scientific (Rockford, IL, USA), anti-NKX2-1 antibody (8G7G31) was purchased from DAKO (Carpinteria, CA, USA), and anti-HNF4A antibody (H-1) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MAC5AC from tumoroids was detected by plating cells at 2.5 × 105 cells per well in a 24-well cell culture plate with 20 μl of basement membrane extract type 2 (BME type 2, R&D Systems, Minneapolis, MN, USA). Following this, the culture medium was replaced with 200 μl of Advanced DMEM/F12 (Thermo Fisher Scientific) without additives, and tumoroids were cultured for 24 h. For the detection of secreted MAC5AC from cell lines, cells were plated at 5 × 105 cells per well in a 6-well cell culture plate. The next day, the medium was replaced with 500 μl of Advanced DMEM/F12, and cells were cultured for 24 h. Subsequently, the supernatant was collected, and after measuring protein concentrations, 10 μg of protein was subjected to immunoblot analysis.

Periodic acid–Schiff (PAS) staining was performed by incubating the slides in 1% periodic acid solution for 15 min and rinsing with running water for 2–3 min. Subsequently, the slides were immersed in Schiff reagent for 20 min, rinsed with a sulfurous acid solution for 1 min and repeated three times, then 2 min and repeated three times, and then rinsed with running water for 10 min. The slides were then counterstained with hematoxylin, dehydrated, and cover-slipped with a mounting medium. Alcian blue staining was performed with a 3 min incubation in hydrochloric acid solution (0.1 N), followed by a 20 min incubation in Alcian blue solution at pH 1.0 (Muto Pure Chemical. Tokyo, Japan). Slides were incubated with a hydrochloric acid solution (0.1 N) for 1 min, repeated this process three times, and then rinsed with running water for 1 min. This was followed by a 2 min immersion in hematoxylin, dehydration, and coverslipping with mounting media.

Xenograft inoculation of lung tumoroids

Cells from lung tumoroids PDT-LUAD#99 (5 × 106 cells) were dissociated using TrypLE™ Express Enzyme (Thermo Fisher Scientific), mixed with 50 μl of basement membrane extract type 2 (BME type 2, R&D Systems, Minneapolis, MN, USA) and subcutaneously injected into 5-week-old NOD/Shi-scid/IL-2Rγnull (NOG) mice (Charles River Laboratories Japan, Atsugi, Japan). The mice were sacrificed when the diameter of the subcutaneous tumor reached 15 mm. The period from initiation of the xenografts to the point of euthanasia was around eighty days. All studies were approved by the animal research committee of Kawasaki Medical School (Reference Number: 23‐047). The care and use of animals were conducted in accordance with the committee regulations.