Establishment of a highly metastatic model with a newly isolated lung adenocarcinoma cell line

  • Authors:
    • Yong-Qi Cui
    • Qin Geng
    • Tao Yu
    • Fang-Lin Zhang
    • He-Chun Lin
    • Jing Li
    • Miao-Xin Zhu
    • Lei Liu
    • Ming Yao
    • Ming-Xia Yan
  • View Affiliations

  • Published online on: June 26, 2015     https://doi.org/10.3892/ijo.2015.3065
  • Pages: 927-940
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Abstract

Lung cancer is the leading cause of malignancy-related death worldwide, and metastasis always results in a poor prognosis. However, therapeutic progress is hampered by a deficiency of appropriate pre-clinical metastatic models. To bridge this experimental gap, we developed an in vivo metastatic model via subcutaneous (s.c.) injection. The original cell line (XL-2) adopted in this model was newly isolated from the ascites of a patient with extensive metastases of lung adenocarcinoma, thereby avoiding any alteration of its initial molecular biology features from artificial serial cultivation. After comprehensive phenotypical and histological analysis, it was identified as a lung adenocarcinoma cell line. Additionally, the drug test showed that XL-2 cell line was sensitive to docetaxel, and resistant to doxorubicin, indicating it might serve as a cell line model of drug resistance for identifying mechanisms of tumors resistant to doxorubicin. Through this s.c. model, we further obtained a highly metastatic cell line (designated XL-2sci). The metastatic rate of mice in XL-2 group was 3/10, in contrast to the rate of 9/10 in XL-2sci group. Optical imaging, micro-computed tomography (micro-CT) scanning and Transwell assays were further applied to identify the enhanced metastatic capacity of Xl-2sci cells both in vivo and in vitro. Compared with XL-2 cells, ITRAQ labeled proteomics profiling study showed that some tumor metastasis-associated proteins were upregulated in XL-2sci cells, which also indicated the reliability of our model. Proliferation ability of XL-2 and XL-2sci were also evaluated. Results showed that highly metastatic XL-2sci possessed a decreased proliferation capacity versus XL-2, which demonstrated that its increased metastatic activity was not facilitated by a faster growth rate. In conclusion, we successfully developed an in vivo metastatic model using a newly established lung adenocarcinoma cell line, which will be beneficial to further investigations of lung cancer metastasis and to the development of anti-metastasis drugs.

Introduction

Lung cancer is currently considered the leading cause of malignancy-related death worldwide (1,2) with a 5-year survival rate of ~15% (3). Metastasis is one of the most devastating characteristics of cancer, which leads to almost 90% of all lung cancer deaths (1,2). Although important advances have been made in understanding the pathogenesis and treatment of primary malignancy, little progress has been made in treating metastasis. Exploring the still undiscovered mechanisms of it will improve diagnosis and treatment methods. In this process, pivotal importance is attached to developing metastatic models because they can aid us in illustrating the underlying biological features of metastasis in more detail.

An ideal lung cancer metastatic model should not only mimic all aspects of tumor progression but should also be generally practical, stable and optimal. Subcutaneous (s.c.) injection, which has been widely used in the development of most human tumor models (4,5), was also considered as the most appropriate method for lung cancer in our study, because orthotopic implantation is prone to complications, and its rate of tumorigenicity and metastasis formation in vivo is always unstable (6). Furthermore, unlike breast cancer and melanoma, an orthotopic model for lung cancer is relatively unavailable due to the inaccessibility of the anatomical location. Compared with tail intravenous injection, s.c. injection can adequately mimic the metastases of cancer, because it recapitulates the early biological steps that a primary tumor must undergo to form a distant metastatic site (7,8), such as detachment of tumor cells from the primary site, invasion into the surrounding tissue, lymphatic or blood vessels and intravasation. Therefore, s.c. injection can credibly mimic the selection and evolution of metastasis.

After having been artificially cultivated in vitro for a long time, tumor cells often have altered phenotypes and genotypes, no longer maintaining the initial molecular characteristics and the heterogeneity of patient tumors (9,10). Under such circumstances, s.c. xenograft models may present poor predictive power because of the inconsistencies between laboratory and clinical findings (11,12). To circumvent these issues, new cell lines from patient-derived tumor samples are urgently needed. To fill this need, we established and characterized a new lung cancer cell line (designated XL-2) that was isolated from the ascites fluid of a Chinese patient with systemic lung cancer metastasis. Therefore, this cell line can reliably reproduce some important clinical features, such as metastatic behavior.

Through this in vivo metastatic model, we successfully obtained a highly metastatic subpopulation of XL-2 cell line (termed XL-2sci). ITRAQ label technology together with nano liquid chromatography-mass spectrometry (NanoLC-MS/MS) has proven to be efficient in protein analysis (1315). Hence, the clinic-combined metastatic model and this technological means will increase the likeliness of discovering new metastasis-associated genes (16) and proteins (17) for identifying new antitumor targets.

Materials and methods

Cell lines and cell culture

The human lung cancer A549 cells were maintained in our laboratory and cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum (FBS) (Biowest, South America Origin), 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA), and 100 μg/ml streptomycin (Sigma-Aldrich) at 37°C in a humidified air atmosphere containing 5% CO2.

Patient materials

A 45-year-old Chinese male patient from Ruijin Hospital (Shanghai, China) was diagnosed with adenocarcinoma of the right lung. Despite undergoing a right upper lobe lobectomy, he still suffered from widespread metastases at one year following the operation. The right adrenal gland, both kidneys, bilateral lungs, retroperitoneal lymph nodes besides the liver were all involved in the metastasis progression. The patient's clinical situation continued to deteriorate, and predominant ascites was the concomitant symptom. Written informed consent for the scientific purpose of ascites specimen was obtained from him at the time of surgery, according to an established protocol approved by the Human Ethic Committee of Ruijin Hospital. All procedures adhered to the generally accepted guidelines for utilization of human materials.

Isolation of primary cell line from ascites

Approximately 60 ml of ascites fluid was collected and immediately centrifuged at 1,500 × g for 5 min. The upper supernatant was discarded and the remaining sediment washed with phosphate-buffered saline (PBS, Sangon Biotech, Shanghai, China) at 1,500 × g for 5 min; the deposit was then resuspended in fresh PBS. Subsequently, density gradient centrifugation was performed using Percoll (Biodee Biotechnology, Beijing, China) to isolate the cells as previously described (18,19) with slight modifications. The cell suspension was processed using a 60 and 40% Percoll gradient (1:1:1 volume ratio) at 3,000 × g for 30 min at room temperature. The cream-colored layer at the interface of the two Percoll gradients was collected and washed with PBS. After centrifugation at 1,500 × g for 5 min, the cell pellets were resuspended with DMEM, containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin; then incubated them at 37°C in a humidified atmosphere of 5% CO2. Several days later, the bottom of the cell culture flask began to fill with numerous cells. Once the mixed cells reached confluence of ~80%, differential trypsinization was used to minimize the fibroblasts and mesothelial cells. Finally, a pure cancer cell line named XL-2 by our laboratory was harvested. Aliquots of cells in low passages were regularly stored in liquid nitrogen. Some others were further passaged for future study.

Morphological characteristics

For light microscopy, the morphology of XL-2 cells was observed using phase-contrast microscopy in bright fields as 80% confluent cultures (Olympus, Tokyo, Japan).

Determination of chromosome numbers

For chromosome preparation, XL-2 and XL-2sci cells were seeded in a 75-cm2 culture flask (Corning, Lowell, MA, USA). The cells at 70% confluence were treated with colchicine (0.2 μg/ml) (Biodee Biotechnology) for 2 h before harvesting. After centrifugation, 75 mM KCL hypotonic solution was added to the incubated cell pellets at a concentration of 1×104 cells/ml for 25 min at 37°C, prior to fixation with methanol/glacial acetic acid (3:1) following routine methods (20). The cell suspension was then dropped onto ice-cold slides and stained with Giemsa solution. Approximately 50 mitotic figures per cell were randomly analyzed and recorded.

Immunofluorescence

Immunofluorescence was performed to detect the expression of pan-cytokeratin (2123) to identify the epithelial origin of XL-2. Because surfactant protein C (SP-C) is considered a marker of mature type II alveolar epithelial cells (AEC-II) (2427), immunofluorescence was also used to evaluate its expression level. Cells were cultured on glass coverslips for 18–20 h, fixed with 4% paraformaldehyde for 30 min, and permeabilized in 0.25% Triton X-100 for 15 min. After blocking with immunostaining blocking buffer (Beyotime, Jiangsu, China), cells were subsequently incubated with the primary antibodies: rabbit polyclonal anti-SP-C (1:150, sc-13979; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-pan-cytokeratin (1:100, sc-15367; Santa Cruz Biotechnology) overnight at 4°C. Slides were then incubated with a mixture of FITC-labeled goat anti-rabbit IgG (1:100, Sigma) and Alexa-Fluor-labeled chicken anti-rabbit IgG (1:100, Invitrogen) at 37°C for 1 h. Finally, they were incubated with DAPI (1:200, Invitrogen) for 2 min. Slides were viewed with a Fluoview FV1000 microscope (Olympus).

Western blot analysis

An equal amount of protein samples extracted from XL-2 and A549 (positive control) cells was separated using 15% SDS-PAGE gels. Following electrophoresis, the proteins were transferred onto PVDF filter membranes (Millipore, Billerica, MA, USA). After blocking with 5% (w/v) non-fat dry milk for 1.5 h at room temperature, the membrane was incubated with rabbit polyclonal anti-SP-C (1:400) antibody overnight at 4°C. HRP-conjugated anti-rabbit IgG (1:4,000, Sigma-Aldrich) was used as a secondary antibody for a 2-h incubation. A 10-min PBST wash was conducted three times after each antibody incubation step. Subsequent visualization was performed using SuperSignal West Femto Maximun Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and the loading control was β-actin (1:30,000, Sigma-Aldrich).

Animal experiments

Athymic male BALB/c-nu/nu mice, 6–8 weeks of age at experiments were provided by Shanghai Cancer Institute and maintained under specific pathogen-free (SPF) conditions. Housing and all procedures involving animals were performed in strict compliance with protocols approved by the Shanghai Medical Experimental Animal Care Commission at Shanghai Jiaotong University [approval ID SYXK (S) 2012-0001].

The tumorigenicity assay for XL-2 cell line was conducted as previously described (28). Ten nude mice were used for the s.c. route of inoculation. A total of 2×106 XL-2 cells mixed with 0.2 ml saline were injected into the right upper flank of each mouse, and tumor growth was monitored regularly. When the s.c. tumors developed and reached 2 cm in largest dimension, they were removed, preserved in 10% buffered formalin and embedded in paraffin. Five non-sequential serial sections per animal were sampled for hematoxylin and eosin (H&E) staining to evaluate tumor morphology and for subsequent immunohistochemistry (IHC) examination. Because A549 cell line was chosen as the positive control in IHC analysis, thus, slices derived from the A549 s.c. tumors were prepared in the same way as depicted above.

The model for testing drug-sensitivity of XL-2 was generated as previously reported (29). Briefly, thirty-six mice was randomly divided into two groups. The XL-2 group and A549 positive control group were respectively subcutaneously injected with 2×106 of XL-2 or A549 cells per mouse. When tumor volume reached ~100 mm3, the corresponding group was equally divided into three subgroups (n=6), and this day was designated as day 0. The three subgroups were treated with three different formulations via intravenous injection: PBS (negative control), doxorubicin (15 mg/kg) (twice a week) and docetaxel (4 mg/kg) (once a week). Tumor size were regularly measured twice per week, and tumor volume (mm3) was calculated using the formula: tumor volume = 0.5 × length × width2 (mm3). When body weight of most of the mice in one subgroup declined by 20%, the test of all the three subgroups were stopped, the mice were sacrificed, the tumors excised, and photographed.

To establish the metastatic model, a consecutive in vivo metastasis selection was carried out using XL-2 cell line via s.c. injection as previously described (30,31). Briefly, XL-2 cells were injected subcutaneously into the mice, and primary tumors were excised 4 weeks later to improve the life-span of each mouse, and for checking the appearance of spontaneous lung metastasis. When the mice became moribund, macroscopic metastatic lesions were removed and implanted into the s.c. region of new recipient mice. Then the new flank-grown tumor was dissected to initiate the daughter cells. When a certain number of cells were present, they were re-injected subcutaneously into another recipient for the next round. This procedure was repeated three times. At the fourth and fifth rounds of selection, cells were generated directly from the isolated pulmonary nodules. Nodules were finely minced and then seeded in tissue culture dishes to generate cells. Routinely, primary tumors were no longer needed to be excised during the last two rounds for the shortened metastasis time in this stage.

For comparison of the primary tumor growth and spontaneous metastatic ability of XL-2 and XL-2sci cells, twenty mice were randomly divided equally into two groups. Both cell lines were implanted into the mice as described above. Tumor size was measured, and tumor volume was calculated. Nine weeks post-implantation, all individuals in these two parallel groups were euthanized. Liver, spleen and lung were all harvested at necropsy and processed according to the standard protocol for H&E staining to analyze the presence of metastases.

IHC

Sections from primary s.c. tumors of XL-2 were stained with pan-cytokeratin antibody (1:150) to confirm its epithelial origin. The following antibodies were used to identify the lung adenocarcinoma characteristics: thyroid transcription factor-1 (TTF-1) (1:150, sc-13040; Santa Cruz Biotechnology) and carcinoembryonic antigen (CEA) (1:100, 60053-1-Ig; Santa Cruz Biotechnology). All procedures were performed according to the manufacturer's protocols (Polymer HRP Detection System, Polink-2 plus, ZSGB-BIO, Beijing, China). Results were photographed and observed with a full-automatic slide scanning system (Leica, Solms, Germany).

Cells transduction

The GFP-Luc lentiviral vector was constructed by recombining the plasmid eGFP-2A-CBGr99 that encodes the GFP-Luc fusion gene (donated by Professor G.J. Hammerling) and a pWPXL vector (Addgene). Following the instructions described by Addgene (http://www.addgene.org), the XL-2 and XL-2sci cells were transfected with the recombinant vector expressing the fusion protein of GFP-Luc. Cell sorting (Epics Altra, Beckman Coulter) was further introduced to purify the stable transfectants expressing high levels of EGFP, and then the isolated cells were amplified by conventional culture methods.

Luciferase imaging and GFP imaging

A Berthold LB983 NC320 NightOwl System (Berthold, Bad Wildbad, Germany) was used in the s.c. xenografts model to monitor the primary and metastatic tumor growth of XL-2 and XL-2sci cells. All these procedures were conducted as previously described (32,33). For in vivo bioluminescence imaging (in vivo BLI), the animals were given D-luciferin (150 mg/kg body weight in PBS) (Promega, San Luis Obispo, CA, USA) by intra-peritoneal injection and anesthetized with 1% pentobarbital sodium (10 ml/kg body weight) (Sigma-Aldrich). Imaging was completed 15 min after the injection. In monitoring pulmonary lesions, mice were euthanized, and their lungs were excised because signals from the s.c. tumor would disturb the observation. Ex vivo biofluorescence imaging (ex vivo BFI) was adopted in this stage. The exposure time was 20 and 5 sec, respectively, for in vivo BLI and ex vivo BFI.

Micro-computed tomography (micro-CT) scanning

To provide three-dimensional non-destructive detection of the pulmonary lesions, micro-CT (Skyscan1076, Kontich, Belgium) was performed with the following parameters: 50 kV, 200 μA, 130-ms exposure, 0.7 degree at angle of increment and 360 views. A normal mouse was also scanned as a negative control. The mice were anesthetized with 1% pentobarbital sodium (10 ml/kg body weight) prior to the scanning. After being placed on its back on the animal bed and banded across the chest area, each mouse was imaged. Images of the chest were reconstructed.

Migration and Matrigel invasion assays

Analyses assessing the migration and invasion of cells were performed in 24-well Transwell plates (8-μm pore size, Corning). For the invasion assay, we first prepared Matrigel-coated Transwell chambers. Subsequently, 1×105 cells were added into the upper chamber of each well. For the migration assay, the number of cells seeded into upper chamber lined with a non-coated membrane was 5×104. Cells for both assays were resuspended with serum-free culture medium. To induce cell invasion, 800 μl of 10% FBS medium was added to the lower chambers. The incubation lasted 16 h for migration assays and 24 h for invasion assays. Cells that had moved to the basal surface of the chamber were fixed, stained with 0.1% crystal violet and imaged with a CKX41 microscope (Olympus). Images of ten random fields from three replicate wells were obtained. The number of cells were counted, and the differences were analyzed. Assays were conducted three independent times.

In vitro growth kinetics

For growth kinetics, subconfluent cells were seeded at 2,000 cells/well in 96-well plates (Corning). After being incubated for 24 h, the cells were counted at a regular time every day for one week. A mixture of 10 μl of CCK8 (Dojindo, Kumamoto, Japan) and 100 μl of DMEM was added to each well, which were then incubated for 2 h. Three corresponding wells were measured together, and the average number of cells were calculated based on the absorbance at 450 nm of the triplicate wells. The cell doubling time (Td) was calculated using the formula: Td = T × lg2/(lgNt - lgN0), where N0 represents the number of cells at the beginning, Nt means the number of total living cells at the time of counting and T represents the culturing time at the counting. The experiments were independently repeated three times.

Cell cycle analysis

Both XL-2 and XL-2sci cells were fixed with 70% ethanol at −20°C overnight. After fixation, cells were rinsed twice with PBS, and then stained according to the manufacturer's protocol (cell cycle detection kit, KeyGena FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Results were processed using ModFit software (BD Biosciences). Every cell line was independently analyzed three times.

Soft agar colony formation assays

For the soft agar colony formation assay, we used a two-layer technique as described elsewhere (34) with some modifications, which included a base layer consisting of 1% agar (Sigma-Aldrich) (0.25 ml/well) and a second layer containing XL-2 or XL-2sci cells (1,000 cells/well) with 0.6% agar in a 24-well plate. The plate was subsequently cultured for 14 days at 37°C under 5% CO2. Finally, colonies formed were observed using an Axioskop 2 microscope (Carl Zeiss, Oberkochen, Germany) and a DP70 Imaging system (Olympus). This experiment was independently performed three times.

ITRAQ labeled quantitative proteomics analysis of XL-2 and XL-2sci cells

To comprehensively compare the proteome profile of highly metastatic XL-2sci cells and its parental XL-2 cells, iTRAQ quantitative proteomics approach was introduced (17). The first step was to prepare proteins as previously described (17). Second, the iTRAQ 4-Plex reagents (Applied Biosystems, Foster City, CA, USA) were applied to label the samples. Cell lysates of XL-2 and XL-2sci cells were, respectively, labeled with iTRAQ labeling reagents 114 and 115. Briefly, comparable amount of each lysate (100 μg) go through reducing, alkylating, digestion, plus desalination. Then, peptides were separated by means of Agilent HPLC with a high PH RP column (Durashell, C18, 250 mm × 4.6 mm, 5 μm). All these procedures were conducted as previously reported by our lab (35). Third, a NanoLC system (NanoLC-2D Ultra, Eksigent) was introduced to separate fractions and Triple TOF 5600 mass spectrometer (AB SCIEX, USA) was used for analysis. ProteinPilot4.1 software (AB SCIEX), was used for identification and quantification of protein was performed via database searching. The difference of the protein expression levels was considered significant if iTRAQ ratio (115:114) was >1.5 or <0.67. Finally, CapitalBio MAS 3.0 Molecule Annotation System (CapitalBio Corp., Beijing, http://bioinfo.capitalbio.com/mas3/) was used in Gene Ontology (GO) analysis of the upregulated and down-regulated proteins. The analysis was performed mainly in three aspects: cellular component (CC), biological processes (BP) and molecular functions (MF).

Statistical analysis

Quantitative variables were represented as the mean ± SD and analyzed using Student's t-test. Qualitative variables were compared by χ2 test. The significance level was set at <0.05. All the analyses were performed with SPSS 19.0 software (IBM Corp., New York, NY, USA).

Results

Cell morphology and phenotyping

A new lung cancer cell line designated XL-2 was generated from ascites fluid. Cells were regularly certified as free of bacteria or mycoplasma contamination. Determining the morphology revealed that monolayer, polygonal, epithelial-like cells adhered tightly to the bottom of the cell culture flasks (Fig. 1A). The epithelial origin was confirmed by positive immunoreactivity for epithelial marker (pan-cytokeratin) in primary cells (Fig. 1B) and IHC analyses of the s.c. tumor slides (Fig. 1C). XL-2 cell line engrafted well and gave rise to growth in vivo. Tumorigenicity rate was 100% in ten mice (data not shown). H&E staining of the primary s.c. tumor is shown in Fig. 1D.

Chromosomal instability

Chromosome structural and numerical aberrations are critical for the initiation of tumorigenesis (36) and are crucial features of cancer cells versus normal ones. We therefore performed a karyotype analysis of XL-2 cell line. As shown in Fig. 1E (lower panel), the 50 randomly selected cells in metaphase contained between 44 and 55 chromosomes, with 51 and 53 being the predominant numbers. Both gains and loss of chromosomes were frequent in this cell line (Fig. 1E, upper panel). der(1)(?::q11→q11::qter) and der(14)(14pter→q11.2::1q12→qter) were the two significant chromosomal translocations identified. In summary, XL-2 cell line displayed chromosomal instability and abnormal mitosis, which are hallmarks of cancer cells. Additionally, the distinction between the karyotype of XL-2 and that of a mouse, which includes 40 acrocentric chromosomes, confirmed its human cell origin at the same time.

The characteristics of lung adenocarcinoma

Because the clinicopathologic type of the tumor is lung adenocarcinoma, to determine whether XL-2 possesses the corresponding biological features, we first needed to prove that XL-2 is a lung cancer cell line. Immunofluorescence staining and western blotting were used to detect the expression of SP-C, which is produced by AEC-II. As shown in Fig. 2A, the cells were positive for SP-C by immunofluorescence detection. Western blot analysis (Fig. 2B) indicated that the XL-2 cells had a similar secretion level to that of the A549 cells (derived from AEC-II, a positive control). As CEA (37,38) and TTf-1 (3941) are common markers used for identification of lung adeno-carcinoma, we further performed IHC analysis of these two proteins using slides collected from the s.c. tumors of mice in the tumorigenicity assay. Samples derived from the A549 s.c. tumors was used as positive control. Remarkably, the tissues were positive for both CEA and TTf-1, with cytoplasmic and nuclear immunostaining, respectively (Fig. 2C), just as the control group (Fig. 2D). This immunoprofile was compatible with the features of adenocarcinoma. Thus, the XL-2 cell line showed consistency with the clinicopathologic diagnosis.

Drug susceptibility

Doxorubicin (42,43) and docetaxel (4447), which are commonly used as chemotherapy regimens for advanced non-small cell lung cancer (NSCLC), were introduced in the drug-sensitive of Xl-2 cells. As shown in Fig. 3A, in the three subgroups of XL-2 cells, docetaxel was apparently effective in inhibiting tumor volume, compared with the rapid tumor growth of PBS subgroup at day 21, and the difference was statistically significant (Fig. 4). Same conclusion could also be drawn in the A549 positive group, with docetaxel almost completely inhibiting the tumor growth (Fig. 3B). Because some tumors disappeared in A549 group, we can not depict its tumor growth curves. With regard to the sensitivity to doxorubicin, XL-2 and A549 cells exhibited different responses. In the subgroup of A549, the majority of s.c. tumors exhibited a smaller volume than the PBS subgroup, and the tumors of two mice even disappeared (Fig. 3B). However, the antitumor efficiency of doxorubicin in XL-2 subgroup was not so evident, and there was not a statistical difference between the tumor volume of doxorubicin subgroup and the PBS subgroup (Fig. 4).

Highly metastatic XL-2sci cell line obtained from in vivo selection

At the fifth round of screening, the lung metastases were most obvious, and we obtained a highly metastatic subline of XL-2 (designated XL-2sci). Additionally, we representatively present the data in the first, third and fifth rounds of selection to prove the escalating trend of the lung metastatic rate (Table I).

Table I

Lung metastatic rate and selection periods in the first, third and fifth round of in vivo metastasis selection.

Table I

Lung metastatic rate and selection periods in the first, third and fifth round of in vivo metastasis selection.

RoundLung metastatic rateaSelection periods (days)b
First3/1099
Third6/1078
Fifth9/1063

a The number of mice with lung metastasis identified by pathological sections/the total number of mice

b the average time consumed for the appearance of the lung metastasis.

Karyotype analysis of XL-2sci was also performed, and results showed that in the 50 randomly selected cells in meta-phase, the number of chromosomes ranged from 45 to 58, with 49 and 51 being the predominant numbers. The two chromosomal translocations identified in XL-2 cells were also observed in XL-2sci cells. However, der(9)(9pter→p11::1q11→qter) was the third translocation unique to this cell line. Furthermore, there was a marker chromosome in XL-2sci cells (data shown in Fig. 5).

Metastatic lesions were more apparent in mice injected with XL-2sci cells (Fig. 6A), which were further identified by histological analysis (Fig. 6B). Mice in XL-2sci group had significantly more pulmonary metastatic spots than those in XL-2 group (P<0.01) (Fig. 6C). The metastasis rates were 9/10 and 3/10 (P<0.05) in XL-2sci and XL-2 tumor-bearing mice, respectively (Fig. 6D). In brief, the two cell lines differed in metastatic ability, with XL-2sci being superior to XL-2.

Figure 6

A new cell line (XL-2sci) with enhanced metastatic competence was acquired after five rounds of sequential in vivo selection. (A) Corresponding representative images of lungs from the XL-2 and XL-2sci groups. Compared to XL-2 group, massive metastatic nodules to the lungs emerged in the XL-2sci group nine weeks after s.c injection of tumor cells. (B) H&E staining of lungs isolated from the mice. Microphotography showed that massive tumor cells replaced the lung tissues in the XL-2sci group. Magnification, (a and c) ×2; (b and d) ×20. (C) Quantification of microscopic nodules in the lungs in each group (XL-2 versus XL-2sci) (n=10). (D) A table depicting the incidence of lung metastasis in the two groups. *P<0.05, **P<0.01; arrows show the metastatic nodules. (E) Micro-CT scanning of lung tumors. Upper panel, axial transverse sections obtained from the mice revealed that recipients in XL-2sci group had more metastatic nodules. A normal mouse was also scanned as a negative control for more convenient observations. Tumors observed in the lungs are shown by arrows. We also observed in the images that the thorax of the mice had already deformed due to the compression of s.c. tumors. Lower panel, three-dimensional images of the lungs. Mice in XL-2 group also exhibited limited lung lesions, consistent with clinical features of the disease in the patient suffering from extensive metastasis. (F) Representative ex vivo BFI images of lungs harvested at the end of the experiment. The color scale indicates the photon flux emitted from the lungs of each xenografted mouse.

Comparison of the metastatic ability in vivo with optical imaging and micro-CT scanning

To provide a more intuitive way to estimate the in vivo characteristics of these two cell lines, we subcutaneously injected GFP- and luciferase-labeled XL-2 and XL-2sci cells as described above, which permitted us to monitor tumor growth and metastasis non-invasively and semi-quantitatively. When mice displayed signs of being moribund (9 weeks later), lung metastases were monitored by in vivo BLI. However, the positive BLI signals from primary s.c. tumors were so strong that they interfered with the identification of pulmonary nodules. Micro-CT imaging was then introduced. As shown in Fig. 6E, obvious tumor nodules were noted in XL-2sci group. Changes in XL-2 group also appeared but were minor. Significant differences with regard to tumor burden were observed between the two groups. In the subsequent disposal, ex vivo BFI imaging was applied to the evaluation of pulmonary metastasis. A good accordance was demonstrated between ex vivo BFI and micro-CT data from the same time-point. As shown in Fig. 6F, light-emitting foci were mostly localized in the XL-2sci group because only a low level of photons was emitted from the recipients in the XL-2 group. In short, XL-2sci cells were more prone to metastasize in vivo.

Comparison of cellular migration and invasive capability in vitro

Considerable differences were observed in the migration and invasive properties of these two cell lines, as evaluated by in vitro Transwell migration and invasion assays (Fig. 7A). Regarding the invasive potential, significantly more XL-2sci cells passed through the Matrigel than did XL-2 cells. The parallel migration assay yielded similar results. These data suggest that the XL-2sci cells are markedly more aggressive than their parental cells. Consequently, we developed a subpopulation of XL-2 cells (XL-2sci) with enhanced metastatic competence.

Comparison of proliferation ability

The proliferation ability of XL-2 and XL-2sci cells was compared using growth curves and soft agar colony formation assay. The doubling time of XL-2 and XL-2sci cells was 31.92±2.73 and 37.10±4.90 h, respectively (P<0.05, Fig. 7B), indicating that the proliferation rate of XL-2sci was significantly lower than that of XL-2. We achieved similar results as to the growth rate of s.c. tumors (Fig. 7C). In subsequent cell cycle analysis (Fig. 7D), data showed that 35.28% of XL-2 cells were in S phase, in contrast to 28.15% of XL-2sci cells. The number of XL-2sci cells in G2/M phase (18.79%) was approximately two times higher than that of XL-2 cells, indicating a more proliferative phenotype in XL-2 compared with XL-2sci. In the soft agar colony formation assay, both cell lines produced colonies but had significantly different colony-formation ability (Fig. 7E-a). Both the number and size of XL-2 colonies increased when compared with those of the XL-2sci colonies (Fig. 7E-b). The two cell lines showed a significant difference (P<0.01) in the colony formation rate (Fig. 7E-c).

Differentially expressed proteins between XL-2sci and Xl-2

After one technical replicate, we respectively identified a total of 3347/3330 proteins (global FDR <1%) using ProteinPilot 4.1. Limited by the screening condition of unused protein score >1.3 and a number of peptides ≥2, 2148/2373 proteins were identified. There were 18 upregulated (iTRAQ ratio 115:114 >1.5) and 32 downregulated (iTRAQ ratio 115:114 <0.67) proteins in XL-2sci cells, compared with XL-2 cells (Tables II and III).

Table II

List of upregulated proteins.

Table II

List of upregulated proteins.

Accession no.aProtein nameUnused score (mean ± SD)b% cov (95) (mean ± SD)cPeptides (95%) (mean ± SD)diTRAQ rate (mean ± SD)e
P00918CAH2 7.8±0.282842712 28.45±8.6974134096±1 2.49565±0.259578899
P02787TRFE 2.34±0.89095454412.5±03±1 1.9729±0.332481609
P04083ANXA1 47.845±3.217335854 65.6±2.82842712559±4 1.933±0.088105505
P05787K2C8 83.885±4.207285348 71.7±0.14142135675±4 1.6534±0.096873629
P09525ANXA4 18.435±8.195367594 52.2±6.92964645612±4 2.67675±0.485428805
P10768ESTD 7.16±1.060660172 28.2±0.2828427126±1 2.4864±1.081166268
Q04446GLGB 119.205±6.75286976 74.55±1.060660172123±1 1.7236±0.257245447
Q16658FSCN1 19.53±1.824335495 35.7±7.77817459314±0 2.2537±0.205202388
Q16719KYNU 30.67±1.555634919 47.95±2.75771644719±1 1.83485±0.332693741
Q32MZ4LRRF1 29.275±0.36062445846±022±1 1.89915±0.135976634
Q92597NDRG1 17.18±1.187939392 28.9±0.42426406910±0 2.2508±0.747977553
Q96FQ6S10AG 4.02±0.028284271 8.65±2.8991378032±0 2.0106±0.222172951
Q99584S10AD 4.88±3.57796031342.7±03±1 3.37905±1.226335291
Q9H2J7S6A15 3.365±1.85969083525±6.5053823873±1 1.6524±0.053740115
Q9H2J7S6A15 2.84±1.484924248±1.2727922063±1 1.91475±0.062296107
B2WTI4B2WTI4 7.135±1.364716088 23.55±1.6263455976±1 1.60065±0.08336789
E7EU05E7EU05 9.18±0.339411255 21.25±2.616295097±0 3.11955±1.429699201
G3V1S6G3V1S6 17.255±0.091923882 32.75±1.4849242410±2 3.9979±1.49949064

a Accession no. is the Swiss-Prot accession

b unused protein score >1.3

c % cov (95) means proteins total confidence >95%

d peptides ≥2

e iTRAQ rate means the average of 115:114 in the two runs, and the rate >1.5.

Table III

List of downregulated proteins.

Table III

List of downregulated proteins.

Accession no.aProtein nameUnused score (mean ± SD)b% cov (95) (mean ± SD)cPeptides (95%) (mean ± SD)diTRAQ rate (mean ± SD)e
O14965AURKA 4.415±2.057680733 23.3±9.4752308683±1 0.4295±0.107904495
O43175SERA 20.53±1.569777054 32.65±4.03050865312±0 0.31645±0.016475588
O75643U520 62.62±2.884995667 31.9±4.10121933141±7 0.63135±0.032880465
O95433AHSA1 20.09±1.329360749 53.25±2.05060966516±3 0.538±0.045537677
P04818TYSY 11.505±0.728319985 30.2±3.8183766186.5±1 0.42345±0.25335636
P05120PAI2 23.4±0.353553391 55.15±6.15182899614.5±1 0.34675±0.047305444
P09874PARP1 32.07±5.246732316 32.9±0.28284271221.5±5 0.4633±0.086974134
P11216PYGB 51.085±0.091923882 52.1±1.41421356236±1 0.52305±0.160866793
P12277KCRB 21.635±2.312239174 46.05±7.28319984614±3 0.24005±0.01251579
P17931LEG3 16.34±5.628569978 45.6±7.91959594916.5±1 0.23475±0.095105862
P18669PGAM131±5.868986284 63.15±4.73761543422.5±2 0.5916±0.080610173
P23246SFPQ 16.28±1.796051224 29.95±0.49497474710.5±1 0.421±0.02192031
P23381SYWC 19.145±2.877924599 36.95±1.76776695314±0 0.34825±0.065407377
P23526SAHH 28.685±0.16263456 42.85±0.63639610322.5±1 0.2939±0.01145513
P25815S100P 8.58±2.008183259 44.75±3.747665945±0 0.4637±0.066185195
P27695APEX1 18.65±0.707106781 43.25±2.89913780313±0 0.4232±0.03026417
P43490NAMPT 70.375±2.679934701 73.65±2.47487373458±4 0.60645±0.051265242
P46821MAP1B 57.255±6.130615793 28.5±2.54558441234±3 0.54755±0.109813683
P62805H4 19.71±2.870853532 60.2±1.41421356218.5±2 0.037±0.010040916
Q05639EF1A2 10.905±3.712310601 59.55±2.89913780333.5±1 0.2535±0.159664711
Q14432PDE3A 3.575±0.827314934 10.15±1.7677669532±0 0.61095±0.004030509
Q15020SART3 11.395±1.803122292 20.95±2.3334523787±1 0.44965±0.040941483
Q16555DPYL2 46.505±6.13061579358.4±031.5±2 0.48235±0.124238661
Q6EMK4VASN 3.53±0.381837662 10.65±5.1618795032.5±1 0.47635±0.110662211
Q6FI13H2A2A2±0 51.9±3.81837661812.5±1 0.3077±0.09277241
Q9NR30DDX21 29.19±0.537401154 37.65±8.83883476517.5±1 0.38045±0.019869701
Q9Y2J2-2E41L3 12.555±2.453660531 21.35±0.4949747478.5±1 0.2993±0.090226825
D6RD18D6RD18 10.985±0.784888527 36.9±3.2526911938±1 0.42375±0.071347074
F8W1S7F8W1S7 35.69±2.771858582 70.75±1.06066017241±6 0.55985±0.15125014
F8W6L6F8W6L6 37.705±8.20950973 32.5±1.55563491932±10 0.2693±0.012303658
J3QSV6J3QSV6 8.94±0.721248917 29.05±0.6363961034.5±1 0.46375±0.236527218
Q4JM47Q4JM47 11.21±3.012274888 44.65±2.1920310228±0 0.1281±0.108894444

a–e Same as in Table II.

GO analysis

GO analysis of the dysregulated proteins was performed via CapitalBio MAS 3.0. In terms of cellular component (CC), the differentially expressed proteins mainly focused on intracellular non-membrane-bound organelle, non-membrane-bound organelle and cytosol; regarding molecular functions (MF), the dysregulated proteins mainly centered on protein binding, catalytic activity and nucleotide binding; as for biological processes (BP), the top three of the focused aspects are negative regulation of apoptosis, negative regulation of programmed cell death and negative regulation of cell death (Fig. 8).

Discussion

Well-characterized primary tumor cell lines can be invaluable tools for better understanding cancer biology (48) and exploring new treatment modalities. Nevertheless, many cell lines have lost their original biological characteristics during long-term culture in vitro (49,50). Thus, the establishment of novel cell lines that reliably reflect clinical features is apparently needed. In general, the availability of newly established lung cancer cell lines is relatively more limited compared with other types of epithelia-originating cell lines. Not direct ascites fluid incubation, but gradient centrifugation was introduced in our study. The advances in cell culture methods enabled us to develop a permanently growing lung cancer cell line.

Through cellular and histological techniques, XL-2 was finally identified as a lung adenocarcinoma cell line. The structural and numerical aberrations in karyotype analysis illustrated its genomic abnormalities. Drug trail showed that it was sensitive to docetaxel and resistant to doxorubicin, which might be attributed to the patient's multiple chemotherapy with doxorubicin. Hence, XL-2 cell line may serve as a cell line model of drug resistance for identifying mechanisms involved in the resistance to doxorubicin. To sum up, this newly isolated cell line should provide a beneficial addition to the cell lines currently available for investigating the biological characteristics of lung cancer.

Although metastasis is the foremost cause of lung cancer recurrence and mortality, we have limited access to the mechanisms. Searching for metastasis-associated genes and proteins will contribute greatly to the inhibition of tumor development and progression. This spontaneous metastatic model derived from s.c. injection provided us with a new cell line with much increased metastatic properties, XL-2sci. Its enhanced metastatic capacity versus XL-2 was identified by optical imaging, micro-CT scanning in vivo and Transwell assays in vitro. Besides, by means of ITRAQ labeled proteomics profiling study, we also found 50 dysregulated proteins in XL-2sci, some of which played an important role in tumor metastasis. For example, FSCN1 has been reported to be relevant with NSCLC metastasis, and G3V1S6 was said to be involved in the distant metastasis of breast cancer (51), gastric cancer (52) plus liver cancer (53). In addition, NDRG1 was connected with colorectal cancer and liver cancer, whereas, it was highly expressed in lung cancer. Hence, in a subsequent study, we can choose some of these metastasis-associated proteins having been reported in other types of tumors to further explore their role in lung cancer metastasis.

Proliferation ability of the two cell lines were also evaluated in our study. Not only the doubling time and soft agar colony formation assays in vitro but also the growth rate of s.c. tumors in vivo suggested that XL-2sci cell line had a decreased proliferation ability, indicating its enhanced invasion and metastatic activity was not facilitated by a faster growth rate. An inverse correlation between proliferation and metastasis was demonstrated in our study, which was consistent with previous reports (31,54).

In conclusion, we triumphantly established a lung adenocarcinoma cell line (XL-2) from clinical specimens and characterized it at cellular and histological levels. By means of XL-2 cells, we developed a metastatic model via s.c. injection and harvested a novel cell line with enhanced metastatic competence (XL-2sci). Retaining the clinical features of original tumor, this metastatic model will offer a reliable platform for in-depth investigation of lung cancer.

Acknowledgements

This study was supported by Natural Science Foundation of China (81472176), Shanghai Science and Technology Developing Program (13140900502), State Key Laboratory of Oncogenes and Related Genes Research Fund (91-14-15). We would like to thank Li-Xing Zhang, Li-Xia Wu and Yu Zhang for their kind suggestions and excellent technical assistance. We also thank the Nature Publishing Group Language Editing for revising the English used in this manuscript.

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September 2015
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APA
Cui, Y., Geng, Q., Yu, T., Zhang, F., Lin, H., Li, J. ... Yan, M. (2015). Establishment of a highly metastatic model with a newly isolated lung adenocarcinoma cell line. International Journal of Oncology, 47, 927-940. https://doi.org/10.3892/ijo.2015.3065
MLA
Cui, Y., Geng, Q., Yu, T., Zhang, F., Lin, H., Li, J., Zhu, M., Liu, L., Yao, M., Yan, M."Establishment of a highly metastatic model with a newly isolated lung adenocarcinoma cell line". International Journal of Oncology 47.3 (2015): 927-940.
Chicago
Cui, Y., Geng, Q., Yu, T., Zhang, F., Lin, H., Li, J., Zhu, M., Liu, L., Yao, M., Yan, M."Establishment of a highly metastatic model with a newly isolated lung adenocarcinoma cell line". International Journal of Oncology 47, no. 3 (2015): 927-940. https://doi.org/10.3892/ijo.2015.3065