The echinoderm microtubule associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene was identified in patients with non-small cell lung cancer. To the best of our knowledge, there are only three cell lines harboring the EML4-ALK fusion gene, which have contributed to the development of therapeutic strategies. Therefore, we tried to establish a new lung cancer cell line harboring EML4-ALK.
A 61-year-old Japanese female presented with chest discomfort. She was diagnosed with left lung adenocarcinoma with T4N3M1 Stage IV. Although she was treated with chemotherapy, her disease progressed with massive pleural effusion. Because the EML4-ALK rearrangement was found in a biopsied specimen using fluorescence in situ hybridization, she was treated with crizotinib. She did well for 3 months.
Tumor cells were obtained from the malignant pleural effusion before treatment with crizotinib. Cells continued to proliferate substantially for several weeks. The cell line was designated ABC-11. The EML4-ALK fusion protein and genes were identified in ABC-11 cells using fluorescence in situ hybridization and immunohistochemistry, respectively. ABC-11 cells were sensitive to crizotinib and next-generation ALK inhibitors (ceritinib and AP26113), as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Phosphorylated ALK protein and its downstream signaling were suppressed by treatment with crizotinib in western blotting. Furthermore, we could transplant ABC-11 cells subcutaneously into BALB/c nu/nu mice.
We successfully established a new lung adenocarcinoma cell line harboring the EML4-ALK fusion gene. This cell line could contribute to future research of EML4-ALK-positive lung cancer both in vivo and in vitro.
The discovery in 2007 of the echinoderm microtubule associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene in non-small cell lung cancer (NSCLC) (1) highlighted the importance of ALK tyrosine kinase inhibitors (TKIs). A first generation ALK-TKI, crizotinib, which was initially formulated as a c-MET inhibitor, caused dramatic responses in patients with EML4-ALK-positive tumors in early clinical trials (2). The US Food and Drug Administration approved crizotinib (Xalkori®, Pfizer Inc., NY, USA) <4 years after the discovery of the fusion gene. Subsequently, crizotinib has resulted in superior progression-free survival compared with platinum-based chemotherapy (3).
Clinical samples have been employed in most ALK studies, including those assessing fusion gene variants, fusion genes with a positive ratio in fluorescence in situ hybridization (FISH), and mechanisms of resistance to crizotinib (4–7). The results of these studies suggest that ALK-positive lung cancers are heterogeneous. Therefore, ALK-positive lung cancers should be assessed in several different ways. To the best of our knowledge, only three cell lines harboring the EML4-ALK fusion gene have been reported: H2228 (EML4-ALK variant 3a/b E6; A20), H3122 and DFCI032 (EML4-ALK variant 1 E13; A20) (5). Therefore, additional ALK-positive cell lines are needed as tools for basic research to develop novel therapeutic strategies for this disease. In this study, we established a new cell line derived from a patient with NSCLC harboring the EML4-ALK fusion gene. This cell line could be useful for investigating ALK-positive lung cancer.
PATIENTS AND METHODS
A 61-year-old Japanese female never-smoker presented with chest discomfort. She was diagnosed with left lung adenocarcinoma with T4N3M1 Stage IV. Her Eastern Cooperative Oncology Group performance status (PS) was one. Because her tumor cells did not harbor any epidermal growth factor receptor (EGFR) mutations, she was treated with chemotherapy consisting of carboplatin and pemetrexed. During this first-line chemotherapy, an EML4-ALK rearrangement in the tumor was detected using FISH. The tumor progressed after three cycles of chemotherapy. Subsequently, she received docetaxel as a second-line therapy; however, her disease progressed rapidly. Her PS deteriorated to two, and her left pleural effusion was drained to relieve her respiratory condition. She was then treated with 250 mg crizotinib twice per day. During the few days after beginning the treatment, the pleural effusion increased on chest radiography. Her respiratory distress then improved gradually, and the pleural effusion was reduced further at Day 24 (Fig. 1A). However, the tumor regrew with massive pleural effusion 3 months after crizotinib treatment. Crizotinib was discontinued, and she received best supportive care.
Establishing the ALK-positive Lung Cancer Cell Line
Pleural effusion was drained to control the patient's respiratory condition before crizotinib treatment was started. Numerous tumor clusters were observed cytologically in the effusion. Mononuclear cells were isolated from the malignant effusion using the Ficoll-Hypaque method and were washed twice with RPMI 1640 medium (8). The cells were suspended in a Petri dish in ACL-4, which is serum-free medium described by Gazdar et al. (9), and cultured for 2 months. Subsequently, the culture media were changed to RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and the cells were cultured at 37°C in a humidiﬁed atmosphere with 5% CO2. The cells began to grow within a week and proliferated consistently thereafter. The cell line was designated ABC-11.
Cell Lines and ALK Inhibitors
The lung adenocarcinoma cell lines, H2228 (EML4-ALK variant 3a/b E6; A20) and A549 (KRAS G12S) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). PC-9 (EGFR del E746_A750) was purchased from Immuno-Biological Laboratories (Takasaki, Gunma, Japan). Crizotinib and AP26113 were purchased from Selleck Chemical (Houston, TX, USA). Ceritinib was purchased from Chemietek (Indianapolis, IN, USA).
ABC-11, H2228, A549 and PC-9 cells were not contaminated with mycoplasma, confirmed using the luminescent MycoAlert™ Mycoplasma Detection Kit (Lonza, Basel, Switzerland) and a compact luminometer Gene Light GL-200A (Microtec, Chiba, Japan), following the manufacturer's instructions.
Genomic DNAs were extracted from cell line using a QIA-amp DNA Mini Kit (Qiagen, Valencia, CA, USA). The genomic DNAs were enriched using GeneRead DNAseq Targeted Panels V2 (Human Lung Cancer Panel) (Qiagen) and sequenced using Miseq (illumine, San Diego, CA, USA). The data were analyzed using Illumine VariantStudio (illumina).
Drug Sensitivity Assay
Drug sensitivities were determined using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (10). Cells were seeded on 96-well plates at a density of 3000 cells per well and exposed continuously to each drug for 96 h. Following treatment, cells were incubated at 37°C with MTT reagent for 4 h, and the absorbance at 570 nm was measured using a 680 Microplate Reader (Bio-Rad, Hercules, CA, USA). The absorbance values were expressed as a ratio of treated to untreated cells. The concentration required to inhibit the growth of tumor cells by 50% (IC50) was used to evaluate the effect of the drug. Assays were performed in triplicate, and the mean and standard error (SE) were calculated.
Cells were lysed using radioimmunoprecipitation assay buffer (1% Triton X-100, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM glycerol-phosphate, 10 mM NaF and 1 mM Na-orthovanadate) containing protease inhibitor tablets (Roche, Tokyo, Japan). Proteins were separated by electrophoresis on polyacrylamide gels and transferred to nitrocellulose membranes. Subsequently, the membranes were incubated with the appropriate antibodies overnight at 4°C. The bands were then detected using ECL Plus (GE Healthcare, Fairfield, CT, USA) and imaged using the LAS-4000 (Fujiﬁlm, Tokyo, Japan).
Rabbit antisera against phospho-ALK (Tyr1604), STAT3, phospho-STAT3 (Tyr705) (D3A7), Akt, phospho-Akt (pSer473), ERK1/2, phospho-ERK1/2 (pT202/pY204) and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Polyclonal antibodies against ALK were purchased from Invitrogen (Carlsbad, CA, USA).
Xenograft Mouse Model
Female BALB/c nu/nu mice at 7 weeks of age were purchased from Japan Charles River Co. (Yokohama, Japan). Cells (2 × 106) were injected subcutaneously into the backs of the mice.
Characteristics of ABC-11
The EML4-ALK fusion protein and gene were identified in ABC-11 cells by immunohistochemistry (anti-ALK antibody (5A4) was purchased from Abcam (Cambridge, UK)) and FISH, respectively (SRL, Tokyo, Japan) (Fig. 2A). EML4-ALK was observed; specifically, exons 20–29 of ALK were fused to exons 1–6b of EML4, variant 3b, as determined using multiplex reverse transcription-PCR and exon array analyses (Mitsubishi Chemical Medience, Tokyo, Japan) (data not shown) (11). EGFR, KRAS, LKB1, TP53 and p16INK4A mutations, which had been reported in NSCLC so far, were not observed in ABC-11 cells according to next-generation sequencing.
Microscopic images of ABC-11 cells revealed that they exhibited an amoeboid form (Fig. 2B). The IC50 value (mean ± SE) of crizotinib in ABC-11 cells was 0.17 ± 0.018 μm, which was similar to H2228 cells (0.18 ± 0.066 μm) (Fig. 2C). ABC-11 cells were also sensitive to next-generation ALK-TKIs, ceritinib and AP26113 (Table 1). The levels of pALK and its downstream proteins (pSTAT3, pAkt and pERK1/2) were suppressed in the presence of crizotinib (Fig. 2D).
|ABC-11||0.13 ± 0.052||0.073 ± 0.0097|
|H2228||0.84 ± 0.021||0.19 ± 0.016|
|PC-9||2.19 ± 0.21||0.25 ± 0.0039|
|ABC-11||0.13 ± 0.052||0.073 ± 0.0097|
|H2228||0.84 ± 0.021||0.19 ± 0.016|
|PC-9||2.19 ± 0.21||0.25 ± 0.0039|
IC50, 50% inhibitory values of ALK TKIs in ABC-11, H2228 and PC-9 cells.
ABC-11 cells that had been injected into the backs of the mice grew steadily. Figure 3A shows the tumors at 20 days after injection. The tumor of the xenograft (Fig. 3a) was harvested and compared with the primary lesion of the patient (Fig. 3b). Both samples were determined to be adenocarcinomas.
We established a new lung adenocarcinoma cell line harboring the EML4-ALK fusion gene from the pleural effusion of an ALK-positive lung cancer patient. We also successfully established a mouse model bearing ABC-11 cells xenografts. The cell line was sensitive to crizotinib as well as H2228 cells. Recently, next-generation ALK-TKIs, including ceritinib (Novartis), alectinib (Chugai) and AP26113 (Ariad), have been developed (12). The effectiveness of such novel compounds should be assessed using multiple cell lines including ABC-11 cells.
The tumor in the patient initially responded very well to crizotinib; however, it progressed within 3 months of the initial crizotinib treatment (Supplementary data, Figure S1). This suggests that ABC-11 cells might possess the ability to acquire resistance to crizotinib. To date, mechanisms underlying crizotinib resistance, such as ALK amplification, ALK secondary mutations (L1197M, C1156Y, G1202R, S1206Y, 1151Tins or G1269A), and the activation of alternative receptor tyrosine kinases (KIT amplification, EGFR mutation or KRAS mutation) have been reported using preclinical models and clinical samples (4–6, 13–16). Therefore, the ABC-11 cell line will be a useful tool to investigate acquired resistance to crizotinib. We are currently planning to establish an ALK-TKI-resistant ABC-11 cell line.
In conclusion, we established a new cell line derived from a patient with lung adenocarcinoma harboring an ALK fusion gene. This cell line could contribute to future studies of EML4-ALK-positive lung cancer both in vivo and in vitro.
Ministry of Education, Culture, Sports, Science, and Technology, Japan grants 24591182 (N.T.) and 23390221 (K.K.).
Conflict of interest statement
Katsuyuki Kiura received honoraria from Chugai pharmaceutical Co., Ltd, Pfizer Inc., Japan and Novartis Pharma K.K. Nagio Takigawa received honoraria from Pfizer Inc., Japan. Katsuyuki Hotta received honoraria from Chugai pharmaceutical Co., Ltd.
We thank the patient and her family, colleagues in our laboratory for the useful discussions, Dr Koichi Ichimura (Department of Pathology, Okayama University Hospital, Okayama, Japan) for the pathological review, and Mr Takehiro Matsubara and Ms Yayoi Kubota (Department of Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama, Japan) for next-generation sequencing technical support.