Gist t1

The human GIST cell line GIST-T1 (code number: GIST01, Cosmo Bio Co.) was purchased in 2014. In addition to negative test results for mycoplasma contamination, cell line authentication and characterization were performed by Cosmo Bio Co. The International Council for Laboratory Animal Science (ICLAS) Monitoring Center, Central Institute for Experimental Animals, tested the cell line for the presence of viruses, with negative results. All experiments using cells in this study were performed for less than 20 passages. GIST-T1 cells were cultured in high glucose (4500 mg/L) Dulbecco’s modified Eagle’s medium (DMEM, Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Biosera, Nuaillé, France) and 1% penicillin-streptomycin-amphotericin B suspension (Wako Pure Chemical Industries) under 5% CO₂ and at 37°C.

The GIST 882 and GIST 48 were kindly provided by Dr. Jonathan Fletcher at Brigham and Women’s Hospital, Boston (MA, USA). The GIST T1 was purchased from Cosmo Bio Co. Ltd. (Tokyo, Japan). The GIST 882 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 15% fetal bovine serum. The GIST T1 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. The GIST 48 cells were grown in F-10 media supplemented with 15% fetal bovine serum, 2.5 µg/mL of MITO plus serum extender (Corning, New York, NY, USA), and 5 µg/mL of bovine pituitary extract (Thermo Fisher Scientific, Waltham, MA, USA). All the cell lines were maintained in a humidified 37 °C incubator with 5% CO₂. The two imatinib-sensitive cell lines, GIST 882 and GIST T1, were used to generate imatinib-resistant derivative cell lines, GIST 882R and GIST T1R, by continually exposing them to 1 µM of imatinib for at least 8 months. The GIST 48 cell line is an established imatinib-resistant cell line [13 (link)]. The cell lines were verified by short tandem repeat profiling performed by the National Genomics Infrastructure in Uppsala (SciLifeLab, Uppsala University, Sweden). The resulting genotypes are detailed in Table S2.

GIST-T1 (Cosmobio, Tokyo, Japan, # PMC-GIST01C-COS) cells were cultured in DMEM (Welgene, # LM001-05) and HMC1.2 (Merck, Darmstadt, Germany, # SCC062) cells were cultured in IMDM (Welgene, # LM004-01). Parental Ba/F3 (DSMZ, Braunschweig, Germany, # ACC300) cells were cultured in RPMI (Welgene, # LM011-51) with 10 ng IL-3/mL (Enzo, # ALX-201-821). The culture media was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution. The cells were maintained in a humidified atmosphere containing 5% CO₂ at 37 °C.

GIST-882 and GIST-T1 are defined as primary mutated and imatinib-sensitive cellular models. They harbor exon 9 (K642E) homozygous mutations and exon 13 heterozygous (V560-Y579) in the KIT gene, respectively. GIST-882 and GIST-T1 were grown in RPMI-1640 supplemented with 15% FBS. GIST-48 is instead reported as an imatinib- and sunitinib-resistant cell line harboring a primary homozygous mutation on KIT exon 11 (V560D) and an additional secondary heterozygous mutation in exon 17 (D820A). GIST-48b was established in vitro, starting from GIST-48 after HSP-90 inhibitor (17-AAG) drug pressure selection, resulting in a subline characterized by nearly undetectable KIT transcript and protein. GIST-48 and GIST-48b were grown in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 15% FBS. All the indicated cell lines were routinely tested to avoid mycoplasma contamination with MycoBlue Mycoplasma Detector (Vazyme, Nanjing, China). GIST-T1 was purchased from CosmoBio (Tokyo, Japan), while GIST-48 and GIST-882 cells were kindly provided by Fletcher JA, MD (Harvard Medical School).

The human GIST cell line, GIST-T1, provided by COSMO BIO (Tokyo, Japan), was established and has been characterized in detail by Taguchi et al. [45 (link)]. GIST-T1 has an activating mutation in KIT exon 11 and is sensitive to imatinib. The cells were originally purchased in 2014, and all experiments in this study were carried out using cells within 20 passages. Cells were authenticated through morphological examination and expression of c-kit. Imatinib-resistant GIST, GIST-IR, was established by culturing cells with increasing concentrations of imatinib (5-80 nM) for 6 months [36 (link), 37 (link)]. GIST-T1 and GIST-IR were cultured in high (4500 mg/L) glucose Dulbecco’s Modified Eagle’s Medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% FBS under 5% CO₂ at 37°C. Microbial contamination test of both cell lines was conducted by an outsourced organization, ICLAS Monitoring Center (Kanagawa, Japan).

Two imatinib-sensitive GIST cell lines were used, i.e., GIST 882 and GIST T1. GIST 882 was kindly provided by Dr. Jonathan Fletcher (Brigham and Women’s Hospital, Boston, MA, USA). This cell line was cultured in RPMI 1640 medium containing 15% fetal bovine serum, and 2 mM L-glutamine at 37 °C with 5% CO₂. GIST T1 was purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan) and cultured in DMEM supplemented with 10% fetal bovine serum. The authenticity of these cell lines was previously confirmed by short tandem repeat (STR) profiling [11 (link)]. GIST cell lines were treated with 1 μM imatinib for the indicated time periods. This imatinib concentration was according to previous publication [11 (link)].