Nci h1299

Manufactured by American Type Culture Collection

Sourced in United States, United Kingdom, China, Germany

The NCI-H1299 is a cell line derived from a non-small cell lung carcinoma. It is a widely used model for cancer research.

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306 protocols using nci h1299

Lung Adenocarcinoma Cell Lines: Cultivation and Characterization

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Lung adenocarcinoma cell lines, including NCI-H1650, NCI-H1299, LTEP-a 2, NCI-H1975, CaLu-3, A549, PG49, NCI-H358, NCI-H1299 and HEY-293T, were obtained from ATCC. The cell lines were maintained in Roswell Park Memorial Institute (RPMI) -1640 containing 10% fetal bovine serum (FBS; Invitrogen, USA).
The lung carcinoma tissues and clinical data obtained from our institute were approved by the Institutional Review Board of China (approval ID 81470137). We also subjected the cell lysates from the clinical specimens to Western blot analysis.

Zhang L., Wang Y., Zhang B., Zhang H., Zhou M., Wei M., Dong Q., Xu Y., Wang Z., Gao L., Qu Y., Shi B., Zhu J., Yin Y., Chen Y., Sun L., Zhang W., Xu S., Ying G, & Wang C. (2017). Claudin-3 expression increases the malignant potential of lung adenocarcinoma cells: role of epidermal growth factor receptor activation. Oncotarget, 8(14), 23033-23047.

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Evaluation of Antigen Presentation by CD11b+ Cells

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Antigen-presentation capacity of CD11b⁺ cells was tested by co-culturing with autologous naïve CD3⁺ T-cells and a human lung cancer cell line, NCI-H1299 (ATCC^® CRL-5803). Verified mycoplasma – negative NCI-H1299 cells were purchased from ATCC (Lot number # 58483200) and passaged according to ATCC recommendation less than 6 passages. CD14⁺ myeloid cells were differentiated for 5 days under the listed conditions, collected, counted and co-cultured at a 1:1 ratio with autologous naïve CD3⁺ T-cells and adherent H1299. After 48 hr, supernatants containing non-adherent immune cells were discarded, and tumor cells trypsinized for annexin-V flow cytometry.

Gonzalez-Junca A., Driscoll K.E., Pellicciotta I., Du S., Lo C.H., Roy R., Parry R., Tenvooren I., Marquez D.M., Spitzer M.H, & Barcellos-Hoff M.H. (2018). Autocrine TGFβ is a Survival Factor for Monocytes and Drives Immunosuppressive Lineage Commitment. Cancer immunology research, 7(2), 306-320.

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Lung Cancer Cell Line Cultivation

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Lung cancer cell lines NCI-H460, A549 were purchased from cell resource center of Chinese academy of medical sciences, and NCI-H345, NCI-H1299, NCI-H146, NCI-H1341 cells were purchased from ATCC. Mouse Lewis Lung Cancer cell LLT was obtained from cell resource center of Chinese academy of medical sciences. NCI-H345, NCI-H1299, NCI-H146, NCI-H1341 cells were purchased from ATCC. Cells were maintained in RPMI-1640 (Gibco, USA) or DMEM (Gibco, USA) medium supplement with 10% FBS (Gibco, USA). All cells were cultured at 37°C under a humidified atmosphere of 5% CO2.

Meng Y., Wang W., Chen M., Chen K., Xia X., Zhou S, & Yang H. (2021). GBP1 Facilitates Indoleamine 2,3-Dioxygenase Extracellular Secretion to Promote the Malignant Progression of Lung Cancer. Frontiers in Immunology, 11, 622467.

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Generation of PD-L1 Cell Line Panel

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Five cell lines with various PD-L1 expression levels (NCI-H446, PC-3, NCI-H1299, A549, and NCI-H460) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). PC-3 and A549 cells were cultured in F-12K (ATCC 30-2004), whereas NCI-H1299, H460, and H446 cells were cultured in RPMI 1640 (ATCC 30-2001) with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Nacalai Tesque, Kyoto, Japan). Cells were maintained in a humidified incubator with 5% CO₂ at 37°C.
A formalin-fixed, paraffin-embedded (FFPE) cell block was constructed for the five cell lines. The specimens were centrifuged for 10 min at 2,000 rpm. The resulting pellet was fixed in 10% buffered formalin for 6 h and embedded in paraffin.

Fujisawa T., Tsuta K., Yanagimoto H., Yagi M., Suzuki K., Nishikawa K., Takahashi M., Okada H., Nakano Y, & Iwai H. (2019). Quantitative immunohistochemical assay with novel digital immunostaining for comparisons of PD-L1 antibodies. Molecular and Clinical Oncology, 10(3), 391-396.

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Cell Line Diversity for Research

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The following human cell lines were used in this study: SW13 (adrenocortical carcinoma) [SW13(vim-) was used as a subtype of SW13 that is deficient in Brm and BRG1]16 (link); HuTu80 (duodenum carcinoma; the previous nomenclature, AZ521, was corrected according to the instructions of the American Type Culture Collection); NCI-H522, A549, and NCI-H1299 (non-small-cell lung carcinoma); C33A and HeLaS3 (cervical carcinoma); KB (recently shown to be a derivative of HeLaS3); Panc-1 (pancreatic carcinoma); and DLD-1, HT29, and HCT116 (colon carcinoma). All cultures were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. A549, NCI-H522, NCI-H1299, C33A, A549, KB, Panc-1, DLD-1, HT29, and HCT116 cell lines were purchased from the American Type Culture Collection. AZ521 (HuTu80) and HeLaS3 cell lines were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Japan.

Kobayashi K., Sakurai K., Hiramatsu H., Inada K.I., Shiogama K., Nakamura S., Suemasa F., Kobayashi K., Imoto S., Haraguchi T., Ito H., Ishizaka A., Tsutsumi Y, & Iba H. (2015). The miR-199a/Brm/EGR1 axis is a determinant of anchorage-independent growth in epithelial tumor cell lines. Scientific Reports, 5, 8428.

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NSCLC Surgical Resection and Cell Culture

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Six patients diagnosed with advanced NSCLC underwent surgery to remove tumor tissues, which were connected with para-carcinoma tissues. Tumor tissues and para-carcinoma tissues were identified and separated by an experienced oncologist who performed the surgeries. The surgeries were conducted in Taizhou Hospital of Zhejiang Province Hospital. The collection of tissues was approved by the ethics committee of Taizhou Hospital of Zhejiang Province Hospital. The NSCLC cell lines, including A549, NCI-H1299, and H460 cells, and the normal human lung epithelial cell line, BEAS-2B, were obtained from ATCC (Maryland, USA) and cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum at 5% CO₂ and 37°C. Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (Manassas, VA, USA) and cultured in endothelial cell medium at 5% CO₂ and 37°C.

Zhang B., Hu Q., Zhang J., Jin Z., Ruan Y., Xia L, & Wang C. (2022). Silencing of A-kinase anchor protein 4 inhibits the metastasis and growth of non-small cell lung cancer. Bioengineered, 13(3), 6895-6907.

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Transfection of TROAP and RASGRF1 in Cell Lines

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BEAS-2B, A549, and NCI-H1299 cell lines were purchased from ATCC. GLC-82 and LTEP-A2 cell lines were purchased from the Tumor Cell Bank of Chinese Academy of Medical Science (Shanghai, China). All the cell lines were cultured in the indicated media as instructed. Cells were transfected by small interfering RNA (siRNA) specifically targeted TROAP using Lipofectamine RNAiMAX, or RASGRF1 plasmid using Lipofectamine 3000 transfection reagent (Invitrogen, USA). The sequences of TROAP siRNA were provided in Table S2.

Li B., Huang Z., Yu W., Liu S., Zhang J., Wang Q., Wu L., Kou F, & Yang L. (2021). Molecular subtypes based on CNVs related gene signatures identify candidate prognostic biomarkers in lung adenocarcinoma. Neoplasia (New York, N.Y.), 23(7), 704-717.

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Culturing of Lung and Breast Cancer Cell Lines

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Lung cancer cell lines NCI-H1299 (non-small cell lung carcinoma, CRL-5803), NCI-H23 (adenocarcinoma, CRL-5800), A549 (lung carcinoma, CCL-185), and MCF7 (breast adenocarcinoma, HTB-22) were purchased from ATCC (Manassas, VA, USA). Cells were cultured at 37 °C under standard conditions (5% CO₂, 95% humidity, 21% O₂ concentration) in RPMI (NCI-H1299, NCI-H23; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), DMEM-F12 (MCF7; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) or DMEM HG (A549; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) medium supplemented with 10% fetal bovine serum (EuRx, Gdańsk, Poland) and antibiotics (gentamycin or penicillin-streptomycin). All experiments were performed in the absence of antibiotics. Cells were regularly checked for mycoplasm contamination.

Sojka D.R., Hasterok S., Vydra N., Toma-Jonik A., Wieczorek A., Gogler-Pigłowska A, & Scieglinska D. (2021). Inhibition of the Heat Shock Protein A (HSPA) Family Potentiates the Anticancer Effects of Manumycin A. Cells, 10(6), 1418.

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Metabolic Reprogramming in Lung Cancer

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The normal human bronchial epithelioid cell (HBE) and the non-small-cell lung cancer cell lines including NCI-H1650, NCI-H1299, A549, NCI-H460, HCC4006, NCI-H1975, and NCI-H358 were obtained from ATCC and cultured with recommended culture medium. The primary antibodies for western blotting including anti-TRAF6 (#8028), hexokinase-1 (#2024), hexokinase-2 (#2867), GLUT1 (#12939), PKM2 (#4053), LDHA (#35 82), VDAC-1 (#4661), phosphor-Akt (#4060), Akt (#8596), phosphor-S6 (#4858), and ubiquitin (#58395) as well as the secondary anti-rabbit IgG HRP (#7074) were products of Cell Signaling Technology Inc. (Danvers, MA). β-Actin (A5316) was obtained from Sigma-Aldrich. In immunohistochemistry staining, the primary antibodies against hexokinase-2 (ab227198) and Ki67 (ab15580) were products of Abcam. TRAF6 shRNA#1 (TRCN0000007350) and shRNA#2 (TRCN0000007351) were purchased from the Sigma Mission shRNA library. The constitutively active Akt (CA-Akt) plasmid (Cat. #10841) and pLKO.1 GFP shRNA (Cat. #30323) were purchased from Addgene (Cambridge, MA, USA). Recombinant human insulin-like growth factor 1 (IGF-1) was a product of R&D (Cat. 291-G1-200). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA).

Feng L., Feng S., Nie Z., Deng Y., Xuan Y., Chen X., Lu Y., Liang L, & Chen Y. (2021). TRAF6 Promoted Tumor Glycolysis in Non-Small-Cell Lung Cancer by Activating the Akt-HIFα Pathway. BioMed Research International, 2021, 3431245.

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Cell Line Authentication and Validation

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The NCI‐H23, NCI‐H441, NCI‐H1299, and NCI‐H2009 lung adenocarcinoma cell lines were purchased from ATCC (Manassas, VA, USA), whereas PC‐9 was obtained from RIKEN Cell Bank (Tsukuba, Japan). ACC‐LC‐319 and ACC‐LC‐94 lung adenocarcinoma cell lines were established by our group. An immortalized lung epithelial cell line, BEAS‐2B, was a generous gift from Curtis C. Harris (National Cancer Institute, Bethesda, MD, USA). The conditions used to culture these cell lines have been previously reported.18 Verification of all cell lines was carried out by short tandem repeat profiling at the Japanese Collection of Research Bioresources, National Institute of Biomedical Innovation of Japan (Osaka, Japan) in February 2015. All cell lines were confirmed to be absent of mycoplasma contamination (MycoAlert; Lonza, Tokyo, Japan).

Griesing S., Kajino T., Tai M.C., Liu Z., Nakatochi M., Shimada Y., Suzuki M, & Takahashi T. (2017). Thyroid transcription factor‐1‐regulated microRNA‐532‐5p targets KRAS and MKL2 oncogenes and induces apoptosis in lung adenocarcinoma cells. Cancer Science, 108(7), 1394-1404.

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