Cell lines and their culture conditions

NCI-A549 pulmonary adenocarcinoma cells harboring KRASG12S and NCI-H2228 pulmonary adenocarcinoma cells harboring variant 3a and 3b (v3a/b) EML4-ALK fusion were obtained from the American Type Culture Collection (Manassas, VA, USA). NCI-H3122 pulmonary adenocarcinoma cells harboring variant 1 (v1) EML4-ALK fusion were sourced from the National Institute of Health (NIH) (Rockville, MD, USA). These cells were cultured as monolayers in the Roswell Park Memorial Institute (RPMI) 1640 medium for NCI-H2228 and NCI-H3122, or in Dulbecco’s modified Eagle medium (DMEM) for NCI-A549. The media were supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, and 100 units/mL penicillin. All cells were maintained at 37 °C in an atmosphere of 5% CO2, authenticated using short tandem repeat analysis, and regularly tested for Mycoplasma contamination using the TaKaRa PCR Mycoplasma Detection Set (Takara Bio, Inc., Otsu, Japan).

Patient-derived tumoroid culture

Patient-derived lung adenocarcinoma (LUAD) tumoroids (PDT-LUAD#119) were developed using tumoroid culture systems, as described previously [9]. The research protocol was approved by the Ethics Committee of the Kawasaki Medical School (reference number 3171-5). All participating patients signed an informed consent form that was authorized by the relevant authority.

Next-generation and Sanger sequencing

Next-generation sequencing (whole-exome sequencing and RNA sequencing) was conducted as previously described [9]. Sanger sequencing was conducted by Eurofins Genomics K. K. (Tokyo, Japan) using the following primers: for TP53 Exon 4, 5′-CAAGCAATGGATGATTTGATGCTGTC-3′ and 5′-TAGGTTTTCTGGGAAGGGACAGAAGATG-3′; and for TP53 Exon 10, 5′-ACTAAATGCATGTTGCTTTTGTACCGTCA-3′ and 5′-CAGGATGAGAATGGAATCCTATGGCTTT-3′.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total cDNA of lung cancer cells or lung tumoroids was synthesized using reverse transcription (RT) with the PrimeScript™ RT reagent Kit (Takara Bio, Shiga, Japan). PCR was used to amplify the fusion point of EML4-ALK v1 and v3 mRNA using the primers 5′-GAAAATTCAGATGATAGCCGTAATAAATTGTCGAA-3′ and 5′-GTCTTGCCAGCAAAGCAGTAGTTGGGGTTGTAGT-3′. The primer pair used for the amplification of ACTB mRNA was 5′-AGAGAGGCATCCTCACCCTGAAGT-3′ and 5′-GATAGCACAGCCTGGATAGCAACG-3′.

Fluorescence in situ hybridization (FISH)

Alpha-satellite DNA for all chromosomes was produced as previously described [9] and labeled using Nick Translation Mix (Sigma-Aldrich, St. Louis, MO, USA) with rhodamine (orange). EML4 and ALK probes were obtained from CytoTest (Rockville, MD, USA). A standard protocol was utilized to conduct FISH [10], and the samples were examined under a fluorescence microscope (ECLIPSE Ni, DS-Qi2; Nikon, Tokyo, Japan).

Immunoblotting and immunohistochemistry

Immunoblot analysis and immunohistochemistry were carried out according to established protocols [11]. The primary anti-NKX2-1 antibody (8G7G3/1) was purchased from DAKO (Carpinteria, CA, USA) for immunohistochemical staining. The p40 antibody (Cat. ABS552/ MABS519 11F12.1) used for immunohistochemical staining was obtained from Merck Millipore (Burlington, MA, USA). The primary anti-ALK antibody (D5F3) used for immunohistochemical staining and immunoblotting was purchased from Cell Signaling Technology (Danvers, MA, USA).

Xenograft inoculation of lung tumoroids

Cells from PDT-LUAD#119 lung tumoroids (4.0 × 106 cells) were dissociated with TrypLE™ Express Enzyme (Thermo Fisher Scientific), and then combined with 50 μL of basement membrane extract type 2 (BME type 2) and subsequently injected subcutaneously into 5-week-old NOD/Shi-scid/IL-2Rγnull (NOG) mice (Charles River Laboratories Japan, Atsugi, Japan). The mice were euthanized when the subcutaneous tumor diameter reached 15 mm. The duration from xenograft initiation to euthanasia was approximately 120 days. All experimental procedures were approved by the Animal Research Committee of Kawasaki Medical School (Reference Number: 23-047), and animal care and handling were conducted in accordance with committee regulations.

Luminescence cell viability assay

Tumoroids were enumerated and suspended in BME type 2; 4-μL droplets were seeded in clear-bottom, white-walled flat-bottom 96-well culture plates (PerkinElmer, Waltham, MA, USA), and then medium was added. Twenty-four hours post-seeding, ALK TKI inhibitors were added to the medium. Viability assessment was conducted 72 h post-treatment using the Celltiter-GloR 2.0 Cell Viability Assay (Promega, Madison, WI, USA), following the manufacturer's instructions. Luminescence readings were obtained using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Rockford, IL, USA).