The GIST-T1 cell line, obtained from Cosmo Bio (Japan), was established from a metastatic human GIST that harbors a heterozygous deletion of 57 bases in exon 11 of KIT [41 (link)]. The imatinib-resistant GIST-T1/670 cell clone was derived from GIST-T1 cells upon culture in the presence of 5 μM imatinib and acquired a secondary missense T670I mutation in exon 14 of KIT [11 (link),31 (link),32 (link)]. GIST-T1 cell lines that stably express control shRNA (GIST-T1-Scramble) or shRNAs targeting two distinct regions of LIX1 (GIST-T1-ShLIX1#1 and GIST-T1-ShLIX1#2) were previously developed [29 (link)]. Their characterization by RT-qPCR analysis has confirmed LIX1 downregulation in GIST-T1-ShLIX1 cells with a higher efficacy of ShLIX1#2 than ShLIX1#1 [29 (link)]. All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, France) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and routinely tested for the absence of mycoplasma contamination (Venor-GeM OneStep Test, BioValley). GIST cell lines were incubated with imatinib mesylate (STI571, Euromedex, France) and sunitinib (SU11248) malate (SE-S1042, Euromedex, France) at the concentrations indicated in the figure legends.
Gist t1 cell line
Manufactured by Cosmo Bio
Sourced in Japan
The GIST-T1 cell line is a commercially available cell line derived from a gastrointestinal stromal tumor (GIST). It is a well-characterized model used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (2 mentions)
Dmem medium, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Imatinib mesylate, by Euromedex (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Glutamax, by Lonza (1 mentions)
Fb25015, by Clark Bioscience (1 mentions)
15 017 cvr, by Corning (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Gist t1, by American Type Culture Collection (1 mentions)
Ly294002, by Selleck Chemicals (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Verteporfin, by Selleck Chemicals (1 mentions)
Accutase solution, by Merck Group (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
11 protocols using gist t1 cell line
1
GIST-T1 Cell Line Characterization
2
GIST Cell Lines Characterization
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The GIST882 cell line was established from an untreated human GIST with a homozygous missense mutation in KIT exon 13, and was kindly provided by Dr Fletcher from Harvard Medical School. The GIST‐T1 cell line was purchased from Cosmo Bio. The GIST‐T1 cell line is characterized by a heterozygous deletion of 57 bases in KIT exon 11. GIST882 cells were cultured in RPMI‐1640 (ATCC) supplemented with 15% FBS and 1% l ‐glutamine, and GIST‐T1 cells were cultured in DMEM (ATCC) supplemented with 10% FBS.
3
Diverse Cell Lines Cultured and Maintained
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We tested 19 cell lines in this study, which are all adherent cells. The GIST-T1 cell line (gastrointestinal stromal tumor) was from Cosmo Bio Co. Ltd. (Tokyo, Japan). All other cell lines were from American Type Culture Collection (Manassas, VA, USA).
MCF-7, MDA-MB-231, GIST-T1, HeLa (cervix epithelial adenocarcinoma), HCT116 (colon epithelial carcinoma), PC3 (prostate adenocarcinoma), C6 (rat brain glial), HepG2 (hepatocellular carcinoma), RPE1 (retina epithelial), 293T (kidney epithelial), differentiated PC-12 (rat pheochromocytoma), NIH-3T3 (mouse embryo fibroblast), and CHO (Chinese hamster ovary) cells were cultured in DMEM medium without L-glutamine (15-017-CVR, Corning, NY, USA), supplemented with 10% (v/v) FBS (fetal bovine serum) (FB25015, Clark Bioscience, Richmond, VA, USA), 1% GlutaMAX (35050-061, Gibco, Carlsbad, CA, USA), and 1% (v/v) P/S (penicillin/streptomycin) (SV30010, HyClone, Logan, UT, USA). CNE-2Z (nasopharyngeal cancer), EJ1 (bladder cancer), and U251 (brain glioblastoma) cells were cultured in RPMI 1640 without L-glutamine (15-040-CVR, Corning) and supplemented with 10% FBS, 2 mM GlutaMAX, and 1% P/S. Three human noncancer lung cells (HSAEC2-KT, HSAEC30-KT, and HBEC30-KT) were cultured in SAGM (Lonza, CC-3118). All cells were maintained in a cell incubator (BC-J160S, Shanghai Boxun, Shanghai, China) at 37.0°C and 5% CO2.
MCF-7, MDA-MB-231, GIST-T1, HeLa (cervix epithelial adenocarcinoma), HCT116 (colon epithelial carcinoma), PC3 (prostate adenocarcinoma), C6 (rat brain glial), HepG2 (hepatocellular carcinoma), RPE1 (retina epithelial), 293T (kidney epithelial), differentiated PC-12 (rat pheochromocytoma), NIH-3T3 (mouse embryo fibroblast), and CHO (Chinese hamster ovary) cells were cultured in DMEM medium without L-glutamine (15-017-CVR, Corning, NY, USA), supplemented with 10% (v/v) FBS (fetal bovine serum) (FB25015, Clark Bioscience, Richmond, VA, USA), 1% GlutaMAX (35050-061, Gibco, Carlsbad, CA, USA), and 1% (v/v) P/S (penicillin/streptomycin) (SV30010, HyClone, Logan, UT, USA). CNE-2Z (nasopharyngeal cancer), EJ1 (bladder cancer), and U251 (brain glioblastoma) cells were cultured in RPMI 1640 without L-glutamine (15-040-CVR, Corning) and supplemented with 10% FBS, 2 mM GlutaMAX, and 1% P/S. Three human noncancer lung cells (HSAEC2-KT, HSAEC30-KT, and HBEC30-KT) were cultured in SAGM (Lonza, CC-3118). All cells were maintained in a cell incubator (BC-J160S, Shanghai Boxun, Shanghai, China) at 37.0°C and 5% CO2.
4
Evaluation of TKI Therapy in GIST PDX
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PDX models were established by transplanting (sc) 2–3 tumor pieces into the left flank of 6-week-old male athymic nude (nu/nu) mice. When the sc tumors reached a size of 200 mm3 (day 0), three TKI-resistant PDX or xenograft models established using imatinib-sensitive GIST-T1 cell lines were randomly allocated to one of the following treatment groups: Control (vehicle treated, DW or imatinib, citrate-buffered (pH 3.5) solution for sunitinib, and polyethylene glycol 400 (PEG400)/125 mM aqueous methanesulfonic acid (80/20) for regorafenib), imatinib, sunitinib, and regorafenib. These compounds were purchased from SelleckChem (Houston, TX, USA). Imatinib (50 mg/kg, twice daily [bid]), sunitinib (20 mg/kg, daily [qd]), regorafenib (10 mg/kg, qd), and the vehicle were administered orally (po) for 21 days. LY294002 (50 mg/kg per each dose) purchased from SelleckChem (Houston, TX, USA) was administered intraperitoneally (ip) twice a week. Tumors were measured using a caliper twice weekly and calculated as volume (mm3), (length × width2)/2 and the body weights were also monitored. The GIST-T1 cell line for the imatinib-sensitive model was obtained from Cosmo Bio (Tokyo, Japan).
5
GIST-T1 Cell Line Establishment and Manipulation
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The GIST-T1 cell line was obtained from Cosmo Bio (Japan). This cell line was established from a metastatic human GIST that harbors a heterozygous deletion of 57 bases in exon 11 of KIT [67 (link)]. HeLa cells were a kind gift of V. Baldin and A. Debant (CRBM, Montpellier France). Human gastric SMCs, provided by Innoprot Innovative (Spain), were grown at low density to maintain a synthetic undifferentiated phenotype, or at high density to induce their differentiation. All cells were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. GIST-T1 stable cell lines (GIST-T1-Scrambled and GIST-T1-ShLIX1#1 and GIST-T1-ShLIX1#2) were generated as previously reported [23 (link)]. For linoleic acid supplementation, stable GIST-T1 cell lines were incubated with EtOH (vehicle) or with 50 μM linoleic acid for 12 h. For NAC experiments, GIST-T1 cells were incubated with 3 mM NAC for 18 h. All cell lines were routinely tested for the absence of mycoplasma contamination (VenorGeM OneStep Test, BioValley). For mitochondrial CATALASE overexpression, GIST-T1 cells were transfected with the pcDNA3-mitoCat plasmid using lipofectamine (Invitrogen), as previously described [40 (link),41 (link)]. CATALASE-overexpressing GIST-T1 cells were selected using G418 (250 μg/ml) (Invitrogen).
6
Establishment of Imatinib-Resistant GIST Cell Line
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GIST882 and GIST48 cell lines were kindly provided by Dr. Jonathan Fletcher (Harvard Medical School), and the GIST‐T1 cell line was purchased (Cosmo Bio). In an agreement with data from other studies, our GIST48 cell line is not fully imatinib resistant.17 , 18 , 19 Authenticity of the GIST882, GIST48 and GIST‐T1 cell lines was confirmed with DNA sequencing. The GIST cells were cultured in a humidified 5% CO2 atmosphere at 37°C, GIST882 and GIST48 cells in the RPMI 1640 medium (GIBCO) supplemented with 20% fetal bovine serum with 2% penicillin/streptomycin (GIBCO), and GIST‐T1 cells in a DMEM medium (Lonza) supplemented with 10% fetal bovine serum with 2% penicillin/streptomycin (GIBCO).
To establish an imatinib‐resistant version of GIST‐T1 cell line, the GIST‐T1‐IRO cell line, imatinib was first administered to the DMEM medium at a concentration of 20 nM during the logarithmic phase of the GIST‐T1 cell growth. The imatinib‐containing medium was applied onto the cells, followed by culture for 48 h, after which the medium was replaced with drug‐free DMEM medium. The administration of imatinib was continued until the cells started to grow normally at the 20 nM imatinib concentration. Thereafter, 20 to 100 nM higher imatinib concentrations were stepwise administered until the cells eventually grew at imatinib concentration of 2 µM suggesting imatinib resistance (FigureS2 ).
To establish an imatinib‐resistant version of GIST‐T1 cell line, the GIST‐T1‐IRO cell line, imatinib was first administered to the DMEM medium at a concentration of 20 nM during the logarithmic phase of the GIST‐T1 cell growth. The imatinib‐containing medium was applied onto the cells, followed by culture for 48 h, after which the medium was replaced with drug‐free DMEM medium. The administration of imatinib was continued until the cells started to grow normally at the 20 nM imatinib concentration. Thereafter, 20 to 100 nM higher imatinib concentrations were stepwise administered until the cells eventually grew at imatinib concentration of 2 µM suggesting imatinib resistance (Figure
7
GIST-T1 Cell Line Transfection and Verteporfin Treatment
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The GIST‐T1 cell line was from Cosmo Bio. It was established from a metastatic human GIST sample with a heterozygous deletion of 57 bases in exon 11 of KIT.16 Human gastric SMCs, provided by Innoprot Innovative, were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin. GIST‐T1 cells were resuspended in Accutase™ solution (Sigma‐Aldrich) before electroporation of different constructs using the Neon Transfection System (Life Technologies), according to the manufacturer's instructions. GIST‐T1 stable cell lines were generated by selection in 500 ng/mL puromycin. All cell lines were routinely tested for the absence of mycoplasma contamination (VenorGeM OneStep Test, BioValley). Verteporfin (Sellekchem) was added to GIST‐T1 cells at a final concentration of 2 μmol/L.
8
Establishing Imatinib-Resistant GIST Cell Lines
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The GIST882 cell line was kindly provided by Dr. Fletcher from Harvard Medical School. The GIST882 cell line is established from an untreated human GIST with a homozygous missense mutation in KIT exon 13, encoding a K642E mutant KIT protein. The GIST-T1 cell line was purchased from Cosmo Bio Co. LTD (Tokyo, Japan). The GIST-T1 cell line is derived from a metastatic plural tumor from the stomach of a Japanese woman and is characterized by a heterozygous deletion of 57 bases in KIT exon 11. GIST882 cells were cultured in RPMI-1640 (ATCC) supplemented with 15% FBS and 1% L-glutamine, and GIST-T1 cells were cultured in DMEM (ATCC) supplemented with 10% FBS. The identity of the cell lines was confirmed periodically throughout the study using SNP fingerprinting, and the KIT mutations were confirmed with RNA sequencing. Imatinib-resistant sublines of GIST-882 and GIST-T1 cells were obtained by culturing the cells in gradually increasing doses of imatinib. Cells that grew in 0.2, 0.4 and 1μM imatinib were obtained after 1, 2 and 4 months of culture with imatinib, respectively. The stability of the resistant phenotype was determined by culturing continuously in medium with corresponding concentrations of imatinib and assessing relative resistance for up to 5 months.
9
GIST Cell Line Generation and Culture
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GIST‐T1‐T670I cell line was generated using CRISPR/Cas9 system as described previously [31 (link)]. GIST‐T1 cell line was purchased from Cosmo Bio Co., Ltd, Tokyo, Japan. GIST‐882 and GIST‐48B were a gift from J. A. Fletcher (Brigham and Women’s Hospital in Boston, USA). GIST‐5R cell line was a gift from B. Rubin (Lerner Research Institute, USA). GIST‐5R, GIST‐T1‐T670I and GIST‐T1 cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM; Corning, Manassas, VA, USA) with 10% FBS (ExCell Bio, Guangzhou, China). GIST‐882 and GIST‐48B cell lines were cultured in IMDM (Gibco by Life Technologies, Carlsbad, CA, USA) with 10% FBS (GIBCO). BaF3 isogenic cell lines were cultured in RPMI1640 with 10% FBS. Primary cells were cultured in DMEM/F12 with 5% FBS (GIBCO), Glutamax‐I (GIBCO), primocin (InvivoGen, San Diego, CA, USA), 5 μg·mL−1 insulin (GIBCO), 25 μg·mL−1 hydrocortisone (Sigma, St. Louis, MO, USA), 125 ng·mL−1 EGF (Sigma) and 10 μm Rho kinase inhibitor y27632 (Haoyuan Chemexpress Inc, Shanghai, China) [30 (link)].
10
Characterization of GIST Cell Lines
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GIST882 and GIST48 cell lines were a kind gift from Dr. Jonathan A. Fletcher (Harvard Medical School, Boston, MA, USA). GIST‐T1 cell line was purchased from Cosmo Bio (Tokyo, Japan). GIST882 is a primary human GIST cell line with a homozygous missense mutation in KIT exon 13, encoding a p.K642E mutant KIT oncoprotein 28 . GIST48 is a GIST cell line that progressed after an initial response to imatinib and harbours a primary KIT exon 11 p.V560D missense mutation and a secondary exon 17 missense mutation p.D820A 29 . The GIST‐T1 cell line was established from a metastatic pleural tumour from a primary gastric GIST. GIST‐T1 is a KIT exon 11 mutant cell line that has a heterozygous in‐frame deletion of 57 bases 30 . The authenticity of the GIST882, GIST48 and GIST‐T1 cell lines was confirmed with DNA sequencing. The cells were cultured in a humidified 5% CO2 atmosphere at 37°C. GIST882 and GIST48 cells were cultured in the RPMI 1640 medium (GIBCO, Carlsbad, CA, USA), supplemented with 20% foetal bovine serum with 2% penicillin/streptomycin (GIBCO), and GIST‐T1 cells were cultured in a DMEM medium (Lonza, Walkersville, MD, USA) supplemented with 10% foetal bovine serum with 2% penicillin/streptomycin (GIBCO).
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