Accumulating evidence supports the existence of cancer stem cells (CSCs) in human tumors, and the successful certification of CSCs may lead to the identification of therapeutic targets, which are more effective for the treatment of cancer. The use of spherical cancer models has increased in popularity in cancer stem cell investigations. Tumorospheres, which are used as a model of CSCs and are established in serum-free medium supplemented with growth factors under non-adherent conditions, are one of the most commonly used cancer spherical models and are a valuable method for enriching the CSC fraction. To investigate whether this model is applicable in lung cancer (LC), the identification of lung CSCs and their capacities is essential. In the present study, lung CSCs were enriched by sphere-forming culturing and their stem-like properties were assessed. The results indicated that the lung tumorospheres had enhanced proliferation, clonality, invasion and cisplatin-resistance, and showed significantly increased expression levels of CD133 and breast cancer resistance protein (ABCG2). These results, together with findings previously reported in literature, indicated that the sphere-forming culturing of LC cells induced the enrichment of CSCs and that the tumorospheres exhibited stem cell characteristics. In addition, the higher expression levels of CD133 and ABCG2 in the tumorospheres may provide a rationale for therapeutic targets for LC.
Lung cancer (LC) is the leading cause of cancer-associated mortality worldwide. Despite continuous efforts to improve therapeutic outcomes, the overall 5-year survival rate remains <16% (
The isolation and identification of CSCs is an essential premise. Currently, CSCs are isolated primarily using four methods: Detection of CSC-specific biomarkers by flow cytometry, assessment of side population (SP) phenotypes by Hoechst 33342 excretion, floating sphere culturing in serum-free conditions and determination of aldehyde dehydrogenase (ALDH) activity (
Simultaneously, several studies have supported the existence of CSCs in LC, and lung CSCs with enhanced tumorigenicity have been reported as a subset of cells, which exclude Hoechst 33342 dye (SP phenotype), including drug-resistant, CD133+, ALDHhigh and CD44+ (
CD133 is a transmembrane glycoprotein; this protein was initially described as a specific marker of human hematopoietic stem cells, and has been used as the primary marker of putative CSCs (
The present study attempted to generate lung CSCs using a sphere-forming assay, and to analyze the features of lung tumorospheres to examine the correlation between the expression levels of two primary candidate CSC markers, CD133 and ABCG2. The experimental results suggested that the stem-like properties were enriched in the lung tumorospheres, with high expression levels of CD133 and ABCG2. Although the present study did not demonstrate that all the sphere-derived cells were CSCs, the results suggested that CD133+ and ABCG2+ cells may be a small subpopulation, which contributes to the determination of stem-like features in lung tumorospheres.
Mouse monoclonal antibody against ABCG2 BXP-21 (cat. no. ab3380) was purchased from Abcam (Cambridge, MA, USA). Allophycocyanin (APC)-conjugated anti-CD133/1 (cat. no. 130-090-826) antibody and rabbit anti-CD133 (cat. no. MAB4399) antibody were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany) and EMD Millipore (Billerica, MA, USA), respectively. Rabbit monoclonal antibody directed against β-actin (cat. no. 4970) and horseradish-peroxidase-conjugated horse anti-rabbit and anti-mouse (cat. nos. 7074 and 7076) IgG secondary antibodies were purchased from Cell Signalling Technology, Inc. (Danvers, MA, USA). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody (cat. no. #E031210-01) was purchased from EarthOx, LLC (San Francisco, CA, USA). Alexa Fluor® 568-conjugated goat anti-rabbit IgG antibody (cat. no. #A-11011) was purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Reverse transcription (RT) and quantitative polymerase chain reaction (qPCR) kits were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Cisplatin was obtained from Sigma-Aldrich (St. Louis, MO, USA), and dissolved in a 0.15 M NaCl solution. Aliquots were stored at −20°C for up to 3 months and thawed immediately prior to use. All other chemicals and solvents were of the highest analytical grade available.
The A549 human LC cell line was obtained from American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI-1640 medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin at an atmosphere of 5% CO2 at 37°C. Cells in the logarithmic phase of growth were used for all experiments.
Suspended cells were collected from confluent A549 cells and centrifuged at 800 × g for 5 min at room temperature. The pellets were gently dissociated with a pipette and resuspended in serum-free Dulbecco's modified Eagle's medium (DMEM)-F12 medium (Gibco; Thermo Fisher Scientific, Inc.). The cells were plated at a density of 1×103 cells/ml in ultra-low attachment plates (Corning Inc. Acton, MA, USA) supplemented with 20 ng/ml basic fibroblast growth factor (bFGF; PeproTech, Inc., Rocky Hill, NJ, USA), 20 ng/ml epidermal growth factor (PeproTech, Inc.) and 2% B27 (Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO2 humidified incubator at 37°C. The medium was replaced every 2–3 days by carefully removing half the upper layer of medium and replacing it with an equal volume of fresh medium. It has been reported that the more serial passages in the spheres, the more CSCs present (
The total RNA of the A549 attached cells and corresponding spheres were extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized from 1
In total, 1.0×106 viable attached cells and sphere-forming cells were collected. The cells were wasHed twice in phosphate-buffered saline (PBS), following which they were incubated with mouse monoclonal anti-ABCG2 (1:200 dilution) for 10 min at 4°C and then incubated with FITC-conjugated goat anti-mouse IgG (1:200 dilution) for another 10 min at 4°C in the dark. Subsequently, the cells were washed in PBS, resuspended, fixed in 100
The A549 attached cells and sphere-forming cells were seeded on glass coverslips in at a density of 5.0×103 cells/well six-well plates, and cultured in a 5% CO2 incubator at 37°C overnight. The cells were fixed by immersion in ice-cold methanol for 15 min. Following washing of the coverslips twice in PBS and blocking for 30 min in blocking buffer (10% normal goat serum and 0.5% Triton X-100 in PBS), the coverslips were incubated with mouse anti-ABCG2 and rabbit anti-CD133 primary antibodies at a 1:200 dilution for 12 h at 4°C. The coverslips were then washed three times in PBS for 5 min each and incubated with FITC-conjugated goat anti-mouse IgG or Alexa Fluor® 568-conjugated goat anti-rabbit IgG secondary antibody at a 1:200 dilution for 30 min at 37°C. Subsequently, the cells were counterstained with 4′,6-diamidino-2-phenylindole and visualized using an inverted fluorescence microscope (Leica, Mannheim, Germany).
The A549 adherent cells and sphere-derived cells were plated in triplicate at a density of 4,000 cells/well in standard coated 96-well plates and allowed to adhere overnight at 37°C in a 5% CO2 humidified incubator. Wells containing medium only were used as a background negative control. Cell proliferation was measured during the following 4 days using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer's protocol. The spectrometric absorbance was measured at 450 nm using a microplate reader. The growth curves were constructed according to the mean value of absorbance relative to the background. To examine drug sensitivity, the cells were first seeded in a 96-well plate at a density of 5,000 cells/well and cultured in medium containing increasing concentrations of cisplatin (0–25.6
Cell invasion assays were performed using 24-well Transwell plates (8
For cell cycle analysis, the adherent A549 cells, spheres and sphere-forming cells following serum-induced culture were harvested by trypsinization and fixed with ice-cold 70% ethanol overnight at 4°C. The cells were then washed twice in PBS, resuspended in PBS and stained with PI/RNase staining buffer (BD Biosciences) at 37°C for 30 min. The cell cycle profiles were obtained using flow cytometry at 488 nm, and the data were analyzed using CellQuest software (BD Biosciences).
Cell lysates of A549 attached cells and spheres were prepared according to manufacturer's details of the protein extract kit (KEYGEN Biotech, Beijing, China). Protein concentrations were determined using the bicinchoninic acid method. Equal quantities of protein (50
Male (n=20) and female (n=200 athymic BALB/c nude mice (4–6 weeks old) with body weights of 18–22 g were provided by the Experimental Animal Centre of Chongqing Medical University (Chongqing, China). All mice were housed in microinsolator cages in a specific homothermic pathogen-free environment with a 12-h light/dark cycle and access to food and water
All experiments were performed in triplicate. The data are graphically represented as the mean ± standard error of the mean. The values were compared with controls using either Student's t-test or two-way analysis of variance using Prism GraphPad 5.0 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
The A549 attached cells were cultured in serum-free medium, as described above. Under ultra-low attached conditions, the cells grew into floating, tridimensional clusters, termed spheres. The spheres began to form on day 3 or 4, and formation was more substantial on day 7. Between days 9 and 12, the spheres had completely formed. By days 15–16, the spheres had become well-rounded structures, which were composed of abundant, cohesive cells.
To examine the subcellular localization of CD133 and ABCG2 in the sphere-forming cells, immunofluorescence staining of CD133 and ABCG2 was performed. Positive staining was observed, with CD133 and ABCG2 present primarily in the membranes of the spheres. Dual staining for CD133 and ABCG2 indicated that the two candidate CSC markers were colocalized in the spheres (
To clarify the differential gene expression profiles between the spheres and the adherent A549 cells, RT-PCR and qPCR analyses were performed (
The results of the flow cytometric analysis revealed the existence of distinct subpopulations with differential expression of CD133 and ABCG2 in the tumorospheres; a small subpopulation of CD133+/ABGC2+ cells was identified in the A549 cells, whereas the primary cell population was negative for the two CSC markers. To investigate possible functional differences between these subpopulations, the CD133+/ABCG2+ cells were subjected to FACS (
It is well known that there is a close association between cell cycle and cell function, including the ability of proliferation and differentiation, and chemosensitivity (
To examine whether LC tumorospheres possess the hypothesized chemoresistant phenotype of CSCs, the present study assessed the sensi tivity of the sphere-forming cells and differentiated cells to cisplatin, which is commonly used in chemotherapy. The LC sphere-forming cells exhibited an increased IC50 value (11.752±1.937, vs. 1.485±0.121
The cancer stem-like properties were further confirmed in the LC spheres through
The CSC theory has received substantial attention in previous years. Determining whether this theory is applicable to LC may shed light on the initiation and metastasis formation of LC, and assist in developing specific therapies to target them, as current therapeutic strategies are developed to target the bulk of cancer cells and not the rarer CSCs. The first challenge lies in the specific isolation and identification of CSCs from LC.
The culture of CSCs as floating spheres was first described in brain tumors by Singh
The present study provided a systematic evaluation of sphere-forming cells derived from the A549 LC cell line. The successful culture and isolation of lung tumorospheres in serum-free and anchorage-independent conditions were performed (
The expression of stemness genes is often used to identify CSCs (
Chemoresistance remains the major cause of cancer associated-mortality and is also considered a hallmark of CSCs (
As chemotherapeutic drugs are generally more toxic towards rapidly proliferating cells, CSCs may resist the toxicity of cisplatin by remaining quiescent (
However, tumorospheres are not homogeneous structures enriched with only undifferentiated cells; they also contain more differentiated cells. Until now, no reports have accurately determined how many cells within tumorospheres are actually CSCs. The gold standard for evaluating the presence of CSCs is the transplantation of a small number of cells into an immunocompromised mouse and evaluating of the capacity of tumorigenesis
In conclusion, the present study demonstrated that non-adherent tumorospheres of an LC cell line cultured in a defined serum-free medium exhibited the characteristics of multipotent stem cells. The genetic composition of the sphere-derived cells, in terms of the co-expression of CD133 and ABCG2, may represent the determining factor for the stem-like features and is likely to be valuable for future gene and stem cell therapy for LC. However, further investigations are required to elucidate the underlying mechanisms of lung CSCs in detail.
cancer stem cells
tumor initiating cells
side population
lung cancer
breast cancer resistance protein
fluorescein isothiocyanate
fluorescence activated cell sorting
propidium iodide
hematoxylin and eosin
This study was supported by the National Natural Science Foundation of China (grant no. 81302018) and the Oncology National Clinical Key Specialty Construction Project [The Medical Letter of National Health and Family Planning Commission Office (2013) 544].
Phase images of sphere-forming assay in A549 cells. (A) Growth of a single cell was recorded separately on days 1, 3, 7, 9, 12 and 16 (original magnification, ×100). (B) Intracellular localization of CD133 and ABCG2. Immunostaining showed upregulation of CD133 and ABCG2 in the lung cancer spheres (original magnification, ×400). Chromatin was stained with DAPI (blue), CD133, with Alexa Fluor® 568 (red) and ABCG2 with fluorescein isothiocyanate (green). The images shown are representative of three independent experiments. ABCG2, breast cancer resistance protein; DAPI, 4′,6-diamidino-2-phenylindole.
Lung cancer sphere-forming cells overexpress candidate CSC markers. (A) Reverse transcription-PCR and quantitative PCR analyses showed the upregulated expression levels of CD133, ABCG2, Oct-4, Sox-2 and Nanog in the A549 spheres (*P<0.05 and **P<0.01). (B) Flow cytometric analysis demonstrated the increased expression of CD133 and ABCG2 cell surface antigens in the A549 lung tumorospheres (*P<0.05 and **P<0.01). The results are presented as the mean ± standard error of the mean. ABCG2, breast cancer resistance protein; PCR, polymerase chain reaction; APC, allophycocyanin; FITC, fluorescein isothiocyanate; Oct-4, octamer-binding transcription factor 4; Sox-2, sex-determining region Y-box 2.
(A) A small population of CD133+/ABGC2+ cells was separated using flow cytometry at a ratio of 0.12%. (B) A Cell Counting Kit-8 cell proliferation assay was used to compare A549 sphere-forming cells with the A549 control cells at each time point. (C) Representative colony formation assay and quantitative analysis of colony formation. The number of A549 colonies in the control was set to 100%. (D) Matrigel invasion assay. Random fields were scanned (four fields per filter) for the presence of cells on the lower side of the membrane. Original magnification, ×400. (E) Cell cycle was arrested at the G0/G1 phase in the sphere-derived cells. The results are presented as the mean ± standard error of the mean and based on three independent experiments (*P<0.05 and **P<0.01, vs. control). ABCG2, breast cancer resistance protein; APC, allophycocyanin; FITC, fluorescein isothiocyanate.
(A) Dose-response curves obtained from adherent and sphere-forming A549 cells following 48 h of treatment with different concentrations of cisplatin; the IC50 values were compared. (B) Expression levels of CD133 and ABCG2 were examined by immunoblotting. The experiments were performed three times in triplicate. *P<0.05 and **P<0.01. Sphere-derived cells exhibited high tumorigenicity