H1975 cells

Manufactured by American Type Culture Collection

Sourced in United States

H1975 cells are a non-small cell lung cancer (NSCLC) cell line derived from a human lung adenocarcinoma. These cells express the EGFR L858R and T790M mutations, which are associated with resistance to certain EGFR tyrosine kinase inhibitors.

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14 protocols using h1975 cells

Cell Culture Protocol: Maintenance of Common Cancer Cell Lines

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HEK293FT cells, 95D cells, H1299 cells, H1975 cells, BEAS-2B and A549 cells were purchased from ATCC. The cells were cultured in high glucose-containing DMEM (ThermoFisher, #12491-015) supplemented with 10% fetal bovine serum (ThermoFisher, #10099133), 100 units/ml penicillin, and 100 µg/ml streptomycin. All cells were cultured at 37°C under a humidified atmosphere containing 5% CO_2.

Cui A., Xue Y., Wang X., Huang Y., Han X., Li X., Niu D., Niu S., Zhao Y., Yang X, & Yu W. (2020). Knockdown of CRAD suppresses the growth and promotes the apoptosis of human lung cancer cells via Claudin 4. Bioscience Reports, 40(10), BSR20201140.

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Establishment of EGFR-Mutant Lung Cancer Models

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Poly(ethylene glycol) (PEG)-OH with molecular weight (MW) of 2 kDa was purchased from J&K (Beijing, China). D,L-lactide and Tin 2-ethylhexanoate were purchased from Energy Chemical (Shanghai, China). 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DGPE) and glacial acetic acid were purchased from Aladdin (Shanghai, China). ELTN and FDTN were purchased from Selleck Chemicals (Houston, TX, United States).
Non-small cell lung cancer cell lines, including H1650 (bearing a deletion in exon 19 of the EGFR gene, i.e., DE746-A750, EGFR 19delE746-A750) and H1975 cells (EGFR L858R/T790M, EGFR Exon21L858R+T790M+Exon20T790M) (ATCC, Rockville, MD, United States) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin (Gibco BRL, Life Technologies, NY, United States).
Male athymic nude mice (4–6 weeks) were purchased from Shanghai Slaccas Experimental Animal Co., Ltd, and were housed in an SPF room. All animal study protocols were reviewed and approved by the Institutional Animal Care and Use Committee, Soochow University.

Chen D., Zhang F., Wang J., He H., Duan S., Zhu R., Chen C., Yin L, & Chen Y. (2018). Biodegradable Nanoparticles Mediated Co-delivery of Erlotinib (ELTN) and Fedratinib (FDTN) Toward the Treatment of ELTN-Resistant Non-small Cell Lung Cancer (NSCLC) via Suppression of the JAK2/STAT3 Signaling Pathway. Frontiers in Pharmacology, 9, 1214.

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Cell Culture Protocols for Multiple Cell Lines

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HEK-293 cells (ATCC), HeLa Cells (ATCC), A-375 cells (ATCC), HCT-116 cells (ATCC), NCI-H358 cells (ATCC), NCI-H647 cells (ATCC), Mia PaCa-2 Cells (ATCC), and SW-1990 cells (ATCC) were cultured in DMEM (Gibco) + 10% FBS (Seradigm), with incubation in a humidified, 37°C/5% CO₂ incubator. H1975 cells (ATCC) were cultured in RPMI 1640 (GIBCO) + 10% FBS, with incubation in a humidified, 37°C/5% CO₂ incubator.

Vasta J.D., Peacock D.M., Zheng Q., Walker J.A., Zhang Z., Zimprich C.A., Thomas M.R., Beck M.T., Binkowski B.F., Corona C.R., Robers M.B, & Shokat K.M. (2022). KRAS is vulnerable to reversible switch-II pocket engagement in cells. Nature Chemical Biology, 18(6), 596-604.

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Non-Small Cell Lung Cancer Cell Culture

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All NSCLC cell were kept at 37 °C in a humidified atmosphere containing 5% CO₂. The highly aggressive A549-3R lung adenocarcinoma [40 (link)] cells were cultured in DMEM (Sigma-Aldrich, USA) supplemented with 10% fetal calf serum (FCS, Sigma Aldrich). H1975 cells, obtained from American Type Culture Collection were cultured in RPMI1640 supplemented with 10% FCS. H1975 cells are erlotinib-resistant because of a T790M mutation of the EGFR. Lastly, we obtained A549-3R cells that are partially resistant to erlotinib (A549res) as described in [17 (link)].

Todesca L.M., Gerke M., Bulk E.E., Bachmann M., Rudersdorf A., Antonuzzo L., Pillozzi S., Düfer M., Szabo I, & Schwab A. (2024). Targeting KCa3.1 channels to overcome erlotinib resistance in non-small cell lung cancer cells. Cell Death Discovery, 10, 2.

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Establishment of Osimertinib-Resistant NSCLC Clones

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H1975 cells were purchased from American Type Culture Collection We independently established two OR clones (OR1 and OR2) by step-wise increasing doses of osimertinib up to 2 μmol/L over a period of four months. These cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS) and incubated in a humidified atmosphere of 5% CO₂ at 37℃. All cell lines were passaged for ≤ 6 months and were not further tested or authenticated by the authors. Osimertinib resistance was stably maintained during cell culture for six months in the absence of drug. EGFR C797S and KRAS mutation was analyzed using Sanger sequencing (Fasmac DNA Sequence Service, Fasmac Co. Ltd.). To analyze the mutations, exon 20 of the EGFR gene was amplified using the forward primer (5′-CATTCATGCGTCTTCACCTG-3′) and the reverse primer (5′-CATATCCCCATGGCAAACTC-3′), and exon 1 of the KRAS gene was amplified using the forward primer (5′-CGTCGATGGAGGAGTTTGTA-3′) and the revere primer (5′-GGACCCTGACATACTCCCAA-3′).

Murakami Y., Kusakabe D., Watari K., Kawahara A., Azuma K., Akiba J., Taniguchi M., Kuwano M, & Ono M. (2022). AXL/CDCP1/SRC axis confers acquired resistance to osimertinib in lung cancer. Scientific Reports, 12, 8983.

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PEGylated Transferrin-Targeted Nanocarriers

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Soya lecithin (SL, injectable) was provided by Lipoid GmbH (Ludwigshafen, Germany). Compritol^® 888 ATO (ATO) was a gift from Gattefossé (Saint-Priest, Lyon, France). DSPE-PEG₂₀₀₀-COOH was produced by Seebio Biotech (Shanghai) Co., Ltd. (Shanghai, China). Transferrin, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-Hydroxysuccinimide (NHS), and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were bought from Sigma Aldrich (St. Louis, MO). The CIS resistant NSCLC cell line (A549/CIS cells), another NSCLC cell line (H1975 cells), and human normal lung epithelial cells (BEAS-2B cells) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI Medium 1640 supplemented with 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin at 37°C in an atmosphere of 5% CO₂.

Mao K., Zhang W., Yu L., Yu Y., Liu H, & Zhang X. (2021). Transferrin-Decorated Protein-Lipid Hybrid Nanoparticle Efficiently Delivers Cisplatin and Docetaxel for Targeted Lung Cancer Treatment. Drug Design, Development and Therapy, 15, 3475-3486.

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NSCLC Cell Lines with EGFR Mutations

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Four human NSCLC cell lines were used: PC-9 [EGFR exon 19 deletion (delE746-A750)]; PC-9ER [EGFR exon 19 deletion (delE746-A750) + T790M]; BID007 [EGFR exon 20 insertion (A763_Y764insFQEA)]; and H1975 [EGFR L858R + T790M]. PC9 and BID007 cells were a kind gift from Dr. Susumu Kobayashi (Beth Israel Deaconess Medical Center, Boston, MA, USA). H1975 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). PC-9ER cells became resistant to erlotinib after chronic exposure to this molecule and acquisition of the EGFR T790M mutation. Cell authentication for H1975 was performed in June 2015.

Masuzawa K., Yasuda H., Hamamoto J., Nukaga S., Hirano T., Kawada I., Naoki K., Soejima K, & Betsuyaku T. (2017). Characterization of the efficacies of osimertinib and nazartinib against cells expressing clinically relevant epidermal growth factor receptor mutations. Oncotarget, 8(62), 105479-105491.

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Establishment of Osimertinib-Resistant Cell Lines

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PC-9 cells and gefitinib-resistant PC-9GR cells were generously provided by Prof. J. Xu and Dr. M. Liu (Guangzhou Medical University, China). H1975 cells were from the American Type Culture Collection (ATCC). To establish osimertinib-resistant cell lines, the parental cells were treated with osimertinib at the concentration of IC 50 for 2 weeks, with higher drug levels for another 3 weeks. The latter dosage was sufficient to kill all parental cells. When resistant clones were visible, the cells were diluted to a single cell per well, and continuous culture was performed in presence of osimertinib. All cells were cultured in RPMI-1640 (Hyclone) with Earle's salts, supplemented with 10% FBS (Gibco), 2 mmol/L L-glutamine (Gibco), 100U/ml penicillin (HyClone), and 100µg/mL streptomycin (Hyclone) at 37°C, with 5% CO 2 and 90% humidity.

Li L., Wang Y., Jiao L., Lin C., Lu C., Zhang K., Hu C., Ye J., Zhang D., Wu H., Feng M, & He Y. (2019). Protective autophagy decreases osimertinib cytotoxicity through regulation of stem cell-like properties in lung cancer. Cancer letters, 452.

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Characterization of EGFR C797S Mutant

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A549 cells, H1975 cells, and HFBs were purchased from the American Type Culture Collection (Manassas, VA, USA). H1975-MS35 cells carrying EGFR C797S were generated by CRISPR/Cas9 knock-in, as described previously [7 (link)]. The sequencing chromatograms of C797S mutation in H1975-MS35 are shown in Supplementary Figure S1. Short tandem repeat profiling was used to verify the identity of all cell lines.

Huang K.Y., Wang T.H., Chen C.C., Leu Y.L., Li H.J., Jhong C.L, & Chen C.Y. (2021). Growth Suppression in Lung Cancer Cells Harboring EGFR-C797S Mutation by Quercetin. Biomolecules, 11(9), 1271.

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Characterization of Lung Cancer Cell Lines

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The wild-type EGFR (201T) cell line was previously derived from a patient with lung adenocarcinoma (19 (link)). H1975 cells with the EGFR intrinsic point mutation L858R and the acquired EGFRi resistance mutation T790M were purchased from American Type Culture Collection (Manassas, VA). 201T and H1975 were cultured in BME and RPMI respectively containing 10% heat-inactivated fetal bovine serum and 1x penicillin/streptomycin (Thermo fisher, Waltham, MA) and 1x GlutaMax (Life Technologies, Carlsbad, CA). A549 and H3255 cells were also purchased from ATCC and cultured in BME and RPMI, respectively. Human bronchial epithelial cells were purchased from ATCC, and primary normal lung fibroblasts were derived from human lung tissue (20 (link)). Cell lines were authenticated by short tandem repeat DNA profiling and used within 6 months of testing. Frozen cell stocks were passaged a maximum of 15 times and cells were mycoplasma-free.

Njatcha C., Farooqui M., Kornberg A., Johnson D.E., Grandis J.R, & Siegfried J.M. (2018). STAT3 Cyclic Decoy Demonstrates Robust Antitumor Effects in Non-Small Cell Lung Cancer. Molecular cancer therapeutics, 17(9), 1917-1926.

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