- Email: [email protected]
Cellular characteristics of head and neck cancer stem cells in type IV collagen-coated adherent cultures
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4– 1 1 11
Available online at www.sciencedirect.com
www.elsevier.com/locate/yexcr
Research Article
Cellular characteristics of head and neck cancer stem cells in type IV collagen-coated adherent cultures Young Chang Lima, 1 , Se-Yeong Ohb, 1 , Hyunggee Kimb,⁎ a
Department of Otorhinolaryngology-Head and Neck Surgery, Research Institute of Medical Science, Konkuk University School of Medicine, Republic of Korea b School of Life Sciences and Biotechnology, Korea University, Republic of Korea
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Although head and neck squamous carcinoma cancer stem cells (HNSC-CSCs) can be enriched in
Received 5 October 2011
serum-free suspension cultures, it is difficult to stably expand HNSC-CSC lines in suspension due
Revised version received
to spontaneous apoptosis and differentiation. Here, we investigated whether HNSC-CSCs can be
27 February 2012
expanded without loss of stem cell properties by adherent culture methods. Cell culture plates
Accepted 29 February 2012
were coated with type IV collagen, laminin, or fibronectin. We examined cancer stem cell traits
Available online 8 March 2012
of adherent HNSC-CSCs grown on these plates using immunocytochemistry for stem cell marker expression and analyses of chemo-resistance and xenograft tumorigenicity. We also assessed
Keywords:
the growth rate, apoptosis rate, and gene transduction efficiency of adherent and suspended
Cancer stem cell
HNSC-CSCs. HNSC-CSCs grew much faster on type IV collagen-coated plates than in suspension.
Head neck cancer
Adherent HNSC-CSCs expressed putative stem cell markers (OCT4 and CD44) and were chemo-
Type IV collagen
resistant to various cytotoxic drugs (cisplatin, fluorouracil, paclitaxel, and docetaxel). Adherent
Adherent cultures
HNSC-CSCs at the limiting dilution (1000 cells) produced tumors in nude mice. Adherent HNSCCSCs also showed less spontaneous apoptotic cell death and were more competent to lentiviral transduction than suspended HNSC-CSCs. In conclusion, compared to suspension cultures, adherence on type IV collagen-coated culture plates provides better experimental conditions for HNSCCSC expansion, which should facilitate various refined cellular studies. © 2012 Elsevier Inc. All rights reserved.
Introduction Recently, it has been hypothesized that a small subpopulation of tumor cells with “stem cell-like” characteristics called cancer stem cells (CSCs), which are distinct from the bulk of the cancer cells in the tumor, are a principal culprit of tumor initiation, invasion, metastasis, and treatment resistance [1,2]. Initially identified in hematopoietic cancers, a number of CSCs have been isolated
from various solid human malignancies, such as brain, lung, breast, colon, ovary, and prostate cancer, and their molecular and cellular features have been elucidated [3–9]. Although a recent study reported isolation of head and neck squamous carcinoma (HNSC)-derived CSCs by fluorescence-activated cell sorting (FACS) using a specific surface marker, CD44 [10], our laboratory has also isolated and characterized HNSC-CSCs using a more effective experimental technique: a stem cell culture-based isolating
⁎ Corresponding author at: School of Life Sciences and Biotechnology, Korea University, Seoul 136–713, Republic of Korea. Fax: + 82 2 953 0737. E-mail address: [email protected] (H. Kim). Abbreviations: HNSC, head and neck squamous carcinoma; CSC, cancer stem cell; ECM, extracellular matrix; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; EMT, epithelial–mesenchymal transition. 1 These authors contributed equally to this work. 0014-4827/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2012.02.038
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4 –1 1 11
method [11]. Therefore, CSCs may drive HNSC tumorigenesis and represent a valuable therapeutic target. A prominent feature of CSCs is their ability to form floating spheroids in serum-free culture conditions [4]. Thus, enrichment of CSCs by sphere-forming suspension cultures has been applied successfully in several human malignancies, including HNSC [6–8,11]. This method, however, has a number of limitations. First, it is difficult to obtain pure CSCs due to proliferation of short-lived progenitor cells within the sphere cultures [12]. Second, spontaneous differentiation and cell death after stem cell divisions in sphere culture condition may occur [13]. Third, fusion of spheres occurs frequently in suspension culture, which makes it challenging to evaluate CSC traits, as evaluation is based solely on sphere number and size [14]. Finally, genetic manipulations, such as transduction of gene constructs for overexpression or knockdown, or precise evaluation of drug efficacy using entire spheroid-type CSCs, are relatively difficult due to low accessibility of gene constructs or drugs to the inner cells of spheroids compared to the outer cells of spheroids [15]. In contrast, most individual cells in adherent culture conditions are uniformly exposed to defined growth factors and oxygen tension, which allows most CSCs to maintain their stemness without spontaneous differentiation and cell death. Furthermore, the aforementioned experimental limitation in conducting gene transduction and drug efficacy tests using spheroid-type CSCs could be easily overcome by using CSCs grown in adherent culture conditions. Extracellular matrix (ECM) of most epithelium, including head and neck tissues, is composed of laminin, collagen, fibronectin, and glycosaminoglycans, and is involved in tumor proliferation and migration [16]. A recent study reported that ECM also plays a crucial role in the maintenance of embryonic stem cell properties [17]. Furthermore, Pollard et al. demonstrated that pure populations of glioma stem cells can be expanded adherently in laminin-coated culture plates [13]. The aim of this study was to investigate whether HNSC-CSCs can be expanded in adherent cultures without loss of stemness traits, specifically on type IV collagen-coated culture plates.
1105
for at least 3 h prior to use. Subsequently, we dissociated CSC spheres into single cells with Accutase (Sigma-Aldrich) and seeded these cells onto ECM protein-coated plates in serum-free culture medium. Every 3 days, serum-free Dulbecco's Modified Eagle's Medium Ham's F-12 (DMEM/F12) containing bFGF and EGF was replaced by fresh medium.
Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) Total RNA was extracted from HNSC-CSCs grown in suspension and adherent culture conditions as well as differentiated HNSCCSCs using TRIzol (Invitrogen, Carlsbad, CA, USA). Complementary DNA was prepared using the Reverse Transcriptase Kit (Fermentas, Burlington, ON, Canada), according to the manufacturer's instructions. Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) analysis was subsequently performed on an iCycler IQ real-time detection system (Bio-Rad, Hercules, CA, USA), using IQ Supermix with SYBR-Green (Bio-Rad). The sequences of human specific primers used were as follows: ABCA2: F 5′-GTGTTCACCAAGATGGAGCA-3′, R 5′-GCTTCTTGGCAAAGTTCACG-3′ ABCB1:F 5′-AGGGAAAGTGCTGCTTGATG-3′, R 5′-GCATGTATGTTGGCCTCCTT-3′ ABCC1:F 5′-ATGAACCTGGACCCATTCAG-3′, R 5′-CCTTCTGCACATTCATGGTC-3′ ABCC2: F 5′-AAATCCTGGTTGATGAAGGC-3′, R 5′-GGAGATCAGCAATTTCAGCA-3′ ABCC3:F 5′-ACAACCTCATCCAGGCTACC-3′, R 5′-GGTTGGCTGGAGAATCAAAT-3 ABCC4:F 5′-TCAGGTTGCCTATGTGCTTC-3′, R 5′-CGGTTACATTTCCTCCTCCA-3′ ABCC5:F 5′-GGGAGCTCTCAATGGAAGAC-3′, R 5′-CAGCTCTTCTTGCCACAGTC-3′ ABCC6:F 5′-CTGGACGAGGCTACTGCTG-3′, R 5′-TTGTCCATGACCAGAACCC-3′ ABCG2:F 5′-CAGTACTTCAGCATTCCACG-3′ R 5′-TTTCCTGTTGCATTGAGTCC-3′
Materials and methods Immunocytochemistry Isolation and culture of HNSC-CSCs HNSC-CSCs were isolated from primary surgical specimens as previously described [10], and their CSC properties were validated by a number of functional assays, including self-renewal capability, stem cell marker expression, chemo-resistance, and in vivo tumorigenicity, as previously reported [11]. HNSC-CSCs expanded in serum-free DMEM/F12 medium supplemented with B27, N2 supplement, 10 ng/ml human recombinant basic fibroblast growth factor (bFGF; R&D Systems, Minneapolis, MN, USA), and 10 ng/ml epidermal growth factor (EGF; R&D Systems). For differentiation, HNSC-CSCs were cultured in DMEM/F12 supplemented with 10% FBS without EGF and bFGF for at least 2 weeks. Laminin, fibronectin, and type IV collagen were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Adherent cell culture with ECM proteins For adherent cell cultures, culture plates were coated with each 10 μg/ml ECM proteins (laminin, type IV collagen, or fibronectin)
Adherent HNSC-CSCs were grown on coverslips in a 24-well plate at a density of 5 × 104 cells per well for 24 h, and then incubated with anti-OCT4, anti-CD44, or anti-involucrin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA; each at 1:50 dilution) overnight at 4 °C. After washing, cells were incubated with Cy3conjugated anti-mouse secondary antibodies. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Images were acquired using a confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
FACS analysis HNSC-CSC sphere cells were dissociated into single cells with Accutase (Sigma-Aldrich), washed twice, and suspended in PBS. Cells were re-suspended in 100 μl binding buffer and annexin VFITC (BD Pharmingen, San Diego, CA, USA), and then incubated at 4 °C for 30 min. After washing twice with PBS and adding 400 μl binding buffer containing 1 μl propidium iodide (PI), the
1106
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4– 1 1 11
cells were then incubated on ice for 15 min. Samples were analyzed by flow cytometry within 1 h using a FACSCalibur machine (BD Biosciences, Franklin Lakes, NJ, USA). An IgG isotype was included as a negative control.
Cell growth analysis HNSC-CSCs were dissociated into single cells with Accutase. Then single-dissociated cells at a density of 1 × 10 4 cells were plated in the type IV collagen-coated plate or standard culture plate. Cells were then incubated at 37 °C with 5% CO2 under stem cell culture conditions. On indicated day (0, 11, 13, 15, 17 days after seeding), cells were dissociated into single cells with Accutase and the total number of cells were calculated with hematocytometer.
Assays for lentiviral vector transduction efficiency of HNSC-CSCs The lentiviral pLL-CMV-EGFP vector and the two packaging plasmids were co-transfected into the human embryonic kidney cell line 293FT cells using Lipofectamine 2000 (Invitrogen) to produce the lentiviral particles. Then, adherent and spheroid-type CSCs were treated with the supernatant containing lentiviral particles produced by 293FT cells. The transfection efficiency was assessed by FACS and fluorescence microscopy.
Statistical analysis Differences in variables were analyzed using Student's t-test. A p-value of less than 0.05 (p < 0.05) was considered to indicate statistical significance.
Western blot analysis
Results Whole-cell extracts were prepared using radioimmunoprecipitation assay lysis buffer containing 1 mmol/L β-glycerophosphate, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L NaF, 1 mmol/L Na3VO4, and protease inhibitor (Roche). Protein in the extracts (30–100 μg) was separated by a 4% to 12% gradient or 10% SDSPAGE NuPAGE gel (Invitrogen) and was then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk and incubated with anti-Smooth muscle actin (Calbiochem, San Diego, CA, USA), anti-FSP1 (Millipore), anti-βACTIN (Sigma-Aldrich). Membranes were then incubated with horseradish peroxidase-conjugated anti-secondary IgG (Pierce, Rockford, IL, USA) antibody and visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce).
Cell viability assays Undifferentiated adherent CSCs in the ECM protein-coated plates were plated in a 96-well plate at a density of 7 × 103 cells per well in serum-free culture conditions. In parallel, differentiated CSCs were also plated in 10% FBS-supplemented culture conditions as described previously. Both adherent undifferentiated CSCs and differentiated CSCs were treated with 4 chemotherapeutic agents (cisplatin, 5-FU, paclitaxel, and docetaxel) currently being used in the clinical setting, and then cultured at 37 °C under a humidified 5% CO2 atmosphere. Twenty-four hours later, 20 μl of 3-(4,4dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml in PBS) was added to each well, and plates were placed at room temperature for 3 h. The absorbance was then measured using a SpectraMax 190 (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 570 nm.
Xenograft transplantation Undifferentiated adherent CSCs (103 cells) grown on type IV collagen-coated plates were suspended in a mixture of serumfree DMEM/F12 medium with growth factor and Matrigel (Sigma-Aldrich; 1:1 by volume). Subsequently, cells were subcutaneously injected into 8-week-old female BALB/c nude mice using 22-gauge needles. Engrafted mice were visually inspected and palpated weekly to monitor for tumor formation until 3 months post-transplantation.
Adherent HNSC-CSCs have a higher growth rate than spheroid-form HNSC-CSCs We evaluated the attachment rates of CSCs floating as a spheroidtype in serum-free medium onto a culture plastic substratum. Nearly 60% of CSCs attached to type IV collagen- or laminincoated plates, whereas less than 20% attached to fibronectincoated plates (Fig. 1a). Next, we assessed the growth rate of CSCs grown adherently on type IV collagen-coated plates or in spheroid form without coating materials. We found that the growth rate of CSCs that adhered to collagen-coated plates was significantly increased from 15 days after cell plating compared to that of spheroid-type CSCs (Fig. 1b). In addition, cell cycle analysis revealed an increase in the proportion of cells in the S and G2/M phases and a reduction in the proportion of cells in the G1 phase under adherent conditions, compared to spheroid-type CSCs (Fig. 1c).
Adherent HNSC-CSCs show a mesenchymal cell-like phenotypic change As evidenced by cell morphology, CSCs maintained on type IV collagen- or laminin-coated plates displayed a spindle-like mesenchymal cell phenotype, whereas CSCs induced to differentiate in medium containing 10% FBS showed distinct polygonal epithelial cell morphology (Fig. 2a). Epithelial–mesenchymal transition (EMT) was shown to confer normal epithelial cells with stem cell traits, including the ability to self-renew and efficiently initiate tumors [18]. Therefore, we assessed whether adherent CSCs expressed mesenchymal markers using quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) and western blot analysis. The results showed that although mRNA levels of fibroblast specific protein-1 (FSP-1) and smooth muscle actin (SMA) were increased in adherent CSCs in collagen-coated plates (Fig. 2b), the protein levels of only SMA, not FSP-1, were dramatically elevated in adherent CSCs compared to differentiated CSCs (Fig. 2b).
Adherent HNSC-CSCs express stem cell marker genes To ascertain whether adherent CSCs on collagen- or laminincoated plates maintain stem cell characteristics, CSCs were cultured in differentiation (10% FBS), in suspension or adherent
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4 –1 1 11
1107
Fig. 1 – Cellular characteristics of HNSC-CSCs grown in adherent and suspension cultures. (a) Gross morphology and attachment rates of HNSC-CSCs grown in suspension and adherent cultures (fibronectin-, laminin-, and type IV collagen-coated plates). ** p < 0.01. Magnification, 40×. HNSC-CSCs were seeded on 60-mm plates coated with the indicated ECM protein, and then incubated for 24 h at 37 °C in 5% CO2. Following incubation, the cells were washed twice with phosphate-buffered saline (PBS), and the remaining cells were harvested with Accutase. The attachment rate was calculated using following formula: (Attachment rate) % = [(Remaining cell number after PBS washing)/(Seeding cell number)] × 100. (b) Growth rates of HNSC-CSCs grown on type IV collagen-coated plates and in suspension cultures. Single-dissociated cells at a density of 1 × 104 cells were plated in the type IV collagen-coated plate or standard culture plate. On indicated day (0, 11, 13, 15, 17 days after seeding), cells were dissociated into single cells with Accutase and the total number of cells were calculated with hematocytometer. * p < 0.05, ** p < 0.01. (c) Cell cycle analysis of HNSC-CSCs grown on type IV collagen-coated plates and in suspension cultures. * p < 0.05.
Fig. 2 – Cellular characteristics of differentiated and adherent HNSC-CSCs. (a) Cell morphological differences between HNSC-CSCs grown in the differentiation (10% FBS) and adherent (type IV collagen-coated plates) cultures. (b) Levels of mRNA of mesenchymal markers in differentiated and adherent HNSC-CSCs. DSP; dentin sialoprotein, FSP-1; fibroblast specific protein 1, VIM; vimentin, SMA; smooth muscle actin. *p < 0.05. (c) Protein expression levels of SMA and FSP-1 in differentiated and adherent HNSC-CSCs. β-actin is used for an equal loading control.
1108
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4– 1 1 11
culture conditions, and Q-RT-PCR was performed to analyze the expression of stem cell markers, including CD44, OCT4, and SOX2, and the keratinocyte differentiation marker, involucrin. The results showed no significant differences in the expression of stem cell and differentiation markers between the HNSC-CSCs grown in suspension and on collagen-coated plates (Fig. 3a). We further examined stem cell and differentiation markers by immunofluorescence and found that OCT4, SOX2, and CD44, but not involucrin, were expressed by most adherent CSCs grown in collagen-coated plates and, to a lesser extent, by CSCs grown on laminin-coated plates. In contrast, nearly all CSCs differentiated by 10% FBS-supplemented culture conditions expressed involucrin, but not OCT4, SOX2, and CD44 (Fig. 3b and c).
Resistance to spontaneous and drug-induced cell death, and tumorigenicity of adherent HNSC-CSCs Because CSCs are known to undergo spontaneous cell death in sphere-forming suspension cultures [14], we compared the cell death rates of HNSC-CSCs grown in sphere-forming suspension cultures to those of adherent HNSC-CSCs on collagen- or laminin-coated plates. FACS analysis revealed that HNSC-CSCs grown on type IV collagen-coated plates are relatively resistant to spontaneous cell death compared to HNSC-CSCs grown in sphere-forming suspension cultures and on laminin-coated plates (Fig. 4a). We also determined the drug-induced cell death rates of HNSC-CSCs grown in four different culture conditions: sphereforming suspension cultures, collagen- or laminin-coated adherent cultures, and 10% FBS-induced differentiation cultures. As shown in Fig. 4b, in contrast to HNSC-CSCs grown in differentiation cultures,
all three stem cell culture conditions allowed HNSC-CSCs to be resistant to chemotherapeutic agents (cisplatin, fluorouracil, paclitaxel, and docetaxel). In addition, we examined the expression of various ATP transporter genes in HNSC-CSCs grown in suspension, on collagen- and laminin-coated plates, and in differentiation culture conditions. The results showed that ABCC2, ABCC5, and ABCC6 expression levels were increased in HNSC-CSCs with stem cell character (Fig. 4c) compared to differentiated CSCs, which might allow HNSC-CSCs to be resistant to chemotherapy. Because a number of cancer stem cell studies have utilized serially diluted cells for xenografting assays [19–22], we performed subcutaneous transplantation of 103 adherent CSCs into nude mice to test the tumorigenic capacity of HNSC-CSCs grown on collagen-coated plates. The result showed that 1000 adherent CSCs are sufficient to give rise to tumors (Fig. 4d).
Gene transduction efficiency and sustained self-renewal of adherent HNSC-CSCs Given that a previous study reported that spheroid-type CSCs display lower gene transduction efficiency compared to adherent cancer cells [11], we compared the lentiviral transduction efficiency of adherent and spheroid-type HNSC-CSCs with a plasmid encoding green fluorescence protein (GFP). As shown in Fig. 5, adherent HNSC-CSCs exhibited rates of transduction 6 times higher than spheroid-type HNSC-CSCs, indicating that adherent HNSCCSCs are more competent for gene delivery than spheroid-type CSCs. To validate whether long-term cultured adherent HNSCCSCs on type IV collagen-coated plates sustained stem cell features, we performed an immunofluorescence assay to examine
Fig. 3 – Adherent HNSC-CSCs express stem cell marker genes. (a) Levels of mRNA of stem cell markers (CD44, OCT4, and SOX2) and a differentiation marker (involucrin) in suspended, adherent (type IV collagen- and laminin-coated plates), and differentiated (10% FBS cultures) HNSC-CSCs. mRNA expression levels were normalized to the levels in the differentiated HNSC-CSCs. (b) Immunofluorescence analysis showed expression of CD44, OCT4, SOX2, and involucrin in the adherent (type IV collagen- and laminin-coated plates) and differentiated (10% FBS cultures) HNSC-CSCs (3rd passages). DAPI was used for nuclear staining. (c) Quantitative analysis of stem cell or differentiation markers expression shown in (b).
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4 –1 1 11
1109
Fig. 4 – Spontaneous and drug-induced cell death and tumorigenicity of adherent HNSC-CSCs. (a) Spontaneous cell death rates of HNSC-CSCs grown in sphere-forming suspension and adherent (type IV collagen- and laminin-coated) cultures were determined by FACS analysis with annexin V-PI staining. (b) Drug-induced cell death rates of HNSC-CSCs grown in sphere-forming suspension, adherent, and differentiation cultures were examined by MTT assay. (c) The mRNA expression levels of ABC transporter genes were determined by Q-RT-PCR analysis. (d) Xenograft tumorigenicity of 1000 adherent HNSC-CSCs grown on type IV collagen-coated plates.
Fig. 5 – Gene transduction efficiency of HNSC-CSCs grown in sphere-forming suspension and on collagen-coated plates. HNSC-CSCs were infected with lentiviral vector encoding the GFP gene, and transduction efficiency was examined by a FACS analysis and fluorescence microscopy.
1110
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4– 1 1 11
Fig. 6 – Expression of stem cell and differentiation markers in long-term cultured HNSC-CSCs. (a) Expression of stem cell markers (CD44 and OCT4) and a differentiation marker (involucrin) in adherent HNSC-CSCs grown over 15 consecutive passages on type IV collagen-coated plates was determined by immunofluorescence analysis. (b) Quantitative analysis of stem cell or differentiation marker expression shown in (a).
the expression of OCT4, CD44, and involucrin. Similar to early passage adherent HNSC-CSCs (Fig. 3), OCT4 and CD44 stem cell markers were maintained in adherent HNSC-CSCs even after 15 passages on type IV collagen-coated plates, whereas the involucrin differentiation marker was rarely detectable in these cells (Fig. 6).
Discussion Much evidence exists to suggest that the microenvironment plays a critical role in tumor development, progression, invasion, and metastasis [23]. In particular, ECM is now recognized as a critical regulator of cancer cell activity, contributing greatly to cancer progression. It is known that major ECM molecules involved in HNSC cell development and progression include collagens, laminin, and fibronectin [16]. Of these, increased expression of collagens, especially type IV collagen, is associated with malignant transformation of keratinocytes and HNSC cell aggressiveness [23]. Furthermore, there is significant correlation between type IV collagen expression and the existence of nodal metastases in laryngeal carcinoma, and the expression pattern of type IV collagen in the basement membranes surrounding nests of carcinoma is an important prognostic factor [24]. A recent study demonstrated that ECM molecules support the establishment and maintenance of human embryonic stem cells [25]. Furthermore, a collagen receptor, α2β1 integrin has been shown to regulate stem cell fate in human colorectal cancer cells [26], and type I collagen inhibits differentiation and promotes maintenance of stem cell-like phenotype in human colorectal carcinoma cells [27]. Therefore, these observations suggest that collagen plays a crucial role in the maintenance of stemness of cancer cells. In this study, we demonstrated that, similar to spheroidtype HNSC-CSCs, adherent HNSC-CSCs grown on type IV collagen-coated plates display a number of stem cell properties: stem cell marker expression, chemo-resistance, and tumorinitiating ability. HNSC-CSCs grown on type IV collagen-coated plates over 15 consecutive passages sustained expression of stem cell markers, expanded at a higher growth rate, and exhibited reduced spontaneous cell death compared to the HNSC-CSCs grown on laminin-coated plates or in sphere-forming suspension cultures. In addition, the lentiviral vector transduction efficiency of
HNSC-CSCs was much higher in collagen adherent cultures than in sphere-forming suspension cultures. Taken together, our results indicate that type IV collagen adherent cultures are more appropriate for the maintenance of HNSC-CSCs than laminin adherent and sphere-forming suspension cultures. Interestingly, adherent HNSC-CSCs grown on collagen-coated plates showed mesenchymal morphological changes with an elevation of mesenchymal cell markers, in contrast to the epithelial cell phenotype observed in differentiation culture conditions. Epithelial–mesenchymal transition (EMT) plays a critical role in normal embryogenesis and tumor progression, including invasion, metastasis, and resistance to apoptosis [28,29]. A recent study demonstrated that several EMT markers are overexpressed in stem-like cells isolated from human mammary gland and mammary carcinomas [18]. In the process of tumor metastasis, which is presumably accompanied by EMT, disseminated cancer cells appear to possess self-renewal capability [30]. Therefore, our results that show mesenchymal cell morphology and increased expression of mesenchymal markers in adherent HNSC-CSCs grown on type IV collagen-coated plates indicate that type IV collagen allows HNSC-CSC to maintain a mesenchymal phenotype in in vitro culture. In conclusion, compared to “sphere” cultures, adherent in vitro expansion of HNSC-CSCs on type IV collagen-coated culture plates may promote significant advancements in cancer stem cell biology.
Conflict of interest None.
Acknowledgments This study was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (No. 2010-0022256 to Y.C. Lim) and from the National R&D Program for Cancer Control, Ministry for Health and Welfare, the Republic of Korea (No. 2008-0058785 to H. Kim).
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4 –1 1 11
REFERENCES
[1] M. Shackleton, E. Quintana, E.R. Fearon, S.J. Morrison, Heterogeneity in cancer: cancer stem cells versus clonal evolution, Cell 138 (2009) 822–829. [2] M. Dean, T. Fojo, S. Bates, Tumour stem cells and drug resistance, Nat. Rev. Cancer 5 (2005) 275–284. [3] S.K. Singh, I.D. Clarke, M. Terasaki, V.E. Bonn, C. Hawkins, J. Squire, P.B. Dirks, Identification of a cancer stem cell in human brain tumors, Cancer Res. 63 (2003) 5821–5828. [4] G. Mor, G. Yin, I. Ghefetz, Y. Yang, A. Alvero, Ovarian cancer stem cells and inflammation, Cancer Biol Ther 11 (2011) 708–713. [5] C.F. Kim, E.L. Jackson, A.E. Woolfenden, S. Lawrence, I. Babar, S. Vogel, D. Crowley, R.T. Bronson, T. Jacks, Identification of bronchioalveolar stem cells in normal lung and lung cancer, Cell 121 (2005) 823–835. [6] A.T. Collins, P.A. Berry, C. Hyde, M.J. Stower, N.J. Maitland, Prospective identification of tumorigenic prostate cancer stem cells, Cancer Res. 65 (2005) 10946–10951. [7] P. Dalerba, S.J. Dylla, I.K. Park, R. Liu, X. Wang, R.W. Cho, T. Hoey, A. Gurney, E.H. Huang, D.M. Simeone, A.A. Shelton, G. Parmiani, Phenotypic characterization of human colorectal cancer stem cells, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 10158–10163. [8] M. Al-Hajj, M.S. Wicha, A. Benito-Hernandez, S.J. Morrison, M.F. Clarke, Prospective identification of tumorigenic breast cancer cells, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 3983–3988. [9] D. Bonnet, J.E. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell, Nat. Med. 3 (1997) 730–737. [10] B. Joshua, MJ. Kaplan, I. Doweck, R. Pai, IL. Weissman, ME. Prince, LE. Ailles, Frequency of cells expressing CD44, a head and neck cancer stem cell marker: correlation with tumor aggressiveness, Head Neck 34 (2012) 42–49. [11] Y.C. Lim, S.Y. Oh, Y.I. Cha, S.H. Kim, X. Jin, H. Kim, Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas, Oral Oncol. 47 (2011) 83–91. [12] B.A. Reynolds, R.L. Rietze, Neural stem cells and neurospheres re-evaluating the relationship, Nat. Methods 2 (2005) 333–336. [13] S.M. Pollard, K. Yoshikawa, I.D. Clarke, D. Danovi, S. Stricker, I.R. Russell, J. Bayani, R. Head, M. Lee, M. Bernstein, J.A. Squire, A. Smith, Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens, Cell Stem Cell 4 (2009) 568–580. [14] I. Singec, R. Knoth, R.P. Meyer, J. Maciaczyk, B. Volk, G. Nikkhah, M. Frotscher, E.Y. Snyder, Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology, Nat. Methods 3 (2006) 801–806. [15] J. Grill, M.L. Lamfers, V.W. van Beusechem, C.M. Dirven, D.S. Pherai, M. Kater, P. Van der Valk, R. Vogels, W.P. Vandertop, H.M. Pinedo, D.T. Curiel, W.R. Gerritsen, The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro, Mol. Ther. 6 (2002) 609–614.
1111
[16] A.F. Ziober, E.M. Falls, B.L. Ziober, The extracellular matrix in oral squamous cell carcinoma: friend or foe? Head Neck 28 (2006) 740–749. [17] H.N. Suh, H.J. Han, Collagen I regulates the self-renewal of mouse embryonic stem cells through α2β1 integrin- and DDR1-dependent Bmi-1. J. Cell. Physiol. 226 (12) (2011) 3422–3432. [18] S.A. Mani, W. Guo, M.J. Liao, E.N. Eaton, A. Ayyanan, A.Y. Zhou, M. Brooks, F. Reinhard, C.C. Zhang, M. Shipitsin, L.L. Campbell, K. Polyak, The epithelial–mesenchymal transition generates cells with properties of stem cells, Cell 133 (2008) 704–715. [19] S. Zhang, C. Balch, M.W. Chan, H.C. Lai, D. Matei, J.M. Schilder, P.S. Yan, T.H. Huang, K.P. Nephew, Identification and characterization of ovarian cancer-initiating cells from primary human tumors, Cancer Res. 68 (2008) 4311–4320. [20] M.E. Prince, R. Sivanandan, A. Kaczorowski, G.T. Wolf, M.J. Kaplan, P. Dalerba, I.L. Weissman, M.F. Clarke, L.E. Ailles, Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 973–978. [21] Y.C. Lim, S.Y. Oh, Y.Y. Cha, S.H. Kim, H. Kim, Cancer stem cell traits in squamouspheres derived from primary head and neck squamous cell carcinomas, Oral Oncol. 47 (2011) 83–91. [22] P. Dalerba, S.J. Dylla, I.K. Park, R. Liu, X. Wang, R.W. Cho, T. Hoey, A. Gurney, E.H. Huang, D.M. Simeone, A.A. Shelton, G. Parmiani, C. Castelli, M.F. Clarke, Phenotypic characterization of human colorectal cancer stem cell, Proc. Natl. Acad. Sci. U. S. A. 12 (2007) 10158–10163. [23] F. Stenback, M.J. Makinen, T. Jussila, S. Kauppila, J. Ristell, L. Talve, L. Risteli, The extracellular matrix in skin development: a morphological study, J. Cutan. Pathol. 26 (1997) 327–338. [24] T. Krecicki, M. Zalesska-Krecicka, M. Jelen, T. Szkudlarek, M. Horobiowska, Expression of type IV collagen and matrix metalloproteinase-2 (type IV collagenase) in relation to nodal status in laryngeal cancer, Clin. Otolaryngol. Allied Sci. 26 (2001) 469–472. [25] A.B. Prowse, F. Chong, P.P. Gray, T.P. Munro, Stem cell integrins: implications for ex-vivo culture and cellular therapies, Stem Cell Res. 6 (2011) 1–12. [26] S.C. Kirkland, H. Ying, Alpha2beta1 integrin regulates lineage commitment in multipotent human colorectal cancer cells, J. Biol. Chem. 283 (2008) 27612–27619. [27] S.C. Kirkland, Type I collagen inhibits differentiation and promotes a stem cell-like phenotype in human colorectal carcinoma cells, Br. J. Cancer 101 (2009) 320–326. [28] H. Peinado, D. Olmeda, A. Cano, Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat. Rev. Cancer 7 (2007) 415–428. [29] S.A. Mani, J. Yang, M. Brooks, G. Schwaninger, A. Zhou, N. Miura, J.L. Kutok, K. Hartwell, A.L. Richardson, R.A. Weinberg, Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 10069–10074. [30] J.P. Thiery, H. Acloque, R.Y. Huang, M.A. Nieto, Epithelial– mesenchymal transitions in development and disease, Cell 139 (2009) 871–890.
Available online at www.sciencedirect.com
www.elsevier.com/locate/yexcr
Research Article
Cellular characteristics of head and neck cancer stem cells in type IV collagen-coated adherent cultures Young Chang Lima, 1 , Se-Yeong Ohb, 1 , Hyunggee Kimb,⁎ a
Department of Otorhinolaryngology-Head and Neck Surgery, Research Institute of Medical Science, Konkuk University School of Medicine, Republic of Korea b School of Life Sciences and Biotechnology, Korea University, Republic of Korea
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Although head and neck squamous carcinoma cancer stem cells (HNSC-CSCs) can be enriched in
Received 5 October 2011
serum-free suspension cultures, it is difficult to stably expand HNSC-CSC lines in suspension due
Revised version received
to spontaneous apoptosis and differentiation. Here, we investigated whether HNSC-CSCs can be
27 February 2012
expanded without loss of stem cell properties by adherent culture methods. Cell culture plates
Accepted 29 February 2012
were coated with type IV collagen, laminin, or fibronectin. We examined cancer stem cell traits
Available online 8 March 2012
of adherent HNSC-CSCs grown on these plates using immunocytochemistry for stem cell marker expression and analyses of chemo-resistance and xenograft tumorigenicity. We also assessed
Keywords:
the growth rate, apoptosis rate, and gene transduction efficiency of adherent and suspended
Cancer stem cell
HNSC-CSCs. HNSC-CSCs grew much faster on type IV collagen-coated plates than in suspension.
Head neck cancer
Adherent HNSC-CSCs expressed putative stem cell markers (OCT4 and CD44) and were chemo-
Type IV collagen
resistant to various cytotoxic drugs (cisplatin, fluorouracil, paclitaxel, and docetaxel). Adherent
Adherent cultures
HNSC-CSCs at the limiting dilution (1000 cells) produced tumors in nude mice. Adherent HNSCCSCs also showed less spontaneous apoptotic cell death and were more competent to lentiviral transduction than suspended HNSC-CSCs. In conclusion, compared to suspension cultures, adherence on type IV collagen-coated culture plates provides better experimental conditions for HNSCCSC expansion, which should facilitate various refined cellular studies. © 2012 Elsevier Inc. All rights reserved.
Introduction Recently, it has been hypothesized that a small subpopulation of tumor cells with “stem cell-like” characteristics called cancer stem cells (CSCs), which are distinct from the bulk of the cancer cells in the tumor, are a principal culprit of tumor initiation, invasion, metastasis, and treatment resistance [1,2]. Initially identified in hematopoietic cancers, a number of CSCs have been isolated
from various solid human malignancies, such as brain, lung, breast, colon, ovary, and prostate cancer, and their molecular and cellular features have been elucidated [3–9]. Although a recent study reported isolation of head and neck squamous carcinoma (HNSC)-derived CSCs by fluorescence-activated cell sorting (FACS) using a specific surface marker, CD44 [10], our laboratory has also isolated and characterized HNSC-CSCs using a more effective experimental technique: a stem cell culture-based isolating
⁎ Corresponding author at: School of Life Sciences and Biotechnology, Korea University, Seoul 136–713, Republic of Korea. Fax: + 82 2 953 0737. E-mail address: [email protected] (H. Kim). Abbreviations: HNSC, head and neck squamous carcinoma; CSC, cancer stem cell; ECM, extracellular matrix; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; EMT, epithelial–mesenchymal transition. 1 These authors contributed equally to this work. 0014-4827/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2012.02.038
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4 –1 1 11
method [11]. Therefore, CSCs may drive HNSC tumorigenesis and represent a valuable therapeutic target. A prominent feature of CSCs is their ability to form floating spheroids in serum-free culture conditions [4]. Thus, enrichment of CSCs by sphere-forming suspension cultures has been applied successfully in several human malignancies, including HNSC [6–8,11]. This method, however, has a number of limitations. First, it is difficult to obtain pure CSCs due to proliferation of short-lived progenitor cells within the sphere cultures [12]. Second, spontaneous differentiation and cell death after stem cell divisions in sphere culture condition may occur [13]. Third, fusion of spheres occurs frequently in suspension culture, which makes it challenging to evaluate CSC traits, as evaluation is based solely on sphere number and size [14]. Finally, genetic manipulations, such as transduction of gene constructs for overexpression or knockdown, or precise evaluation of drug efficacy using entire spheroid-type CSCs, are relatively difficult due to low accessibility of gene constructs or drugs to the inner cells of spheroids compared to the outer cells of spheroids [15]. In contrast, most individual cells in adherent culture conditions are uniformly exposed to defined growth factors and oxygen tension, which allows most CSCs to maintain their stemness without spontaneous differentiation and cell death. Furthermore, the aforementioned experimental limitation in conducting gene transduction and drug efficacy tests using spheroid-type CSCs could be easily overcome by using CSCs grown in adherent culture conditions. Extracellular matrix (ECM) of most epithelium, including head and neck tissues, is composed of laminin, collagen, fibronectin, and glycosaminoglycans, and is involved in tumor proliferation and migration [16]. A recent study reported that ECM also plays a crucial role in the maintenance of embryonic stem cell properties [17]. Furthermore, Pollard et al. demonstrated that pure populations of glioma stem cells can be expanded adherently in laminin-coated culture plates [13]. The aim of this study was to investigate whether HNSC-CSCs can be expanded in adherent cultures without loss of stemness traits, specifically on type IV collagen-coated culture plates.
1105
for at least 3 h prior to use. Subsequently, we dissociated CSC spheres into single cells with Accutase (Sigma-Aldrich) and seeded these cells onto ECM protein-coated plates in serum-free culture medium. Every 3 days, serum-free Dulbecco's Modified Eagle's Medium Ham's F-12 (DMEM/F12) containing bFGF and EGF was replaced by fresh medium.
Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) Total RNA was extracted from HNSC-CSCs grown in suspension and adherent culture conditions as well as differentiated HNSCCSCs using TRIzol (Invitrogen, Carlsbad, CA, USA). Complementary DNA was prepared using the Reverse Transcriptase Kit (Fermentas, Burlington, ON, Canada), according to the manufacturer's instructions. Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) analysis was subsequently performed on an iCycler IQ real-time detection system (Bio-Rad, Hercules, CA, USA), using IQ Supermix with SYBR-Green (Bio-Rad). The sequences of human specific primers used were as follows: ABCA2: F 5′-GTGTTCACCAAGATGGAGCA-3′, R 5′-GCTTCTTGGCAAAGTTCACG-3′ ABCB1:F 5′-AGGGAAAGTGCTGCTTGATG-3′, R 5′-GCATGTATGTTGGCCTCCTT-3′ ABCC1:F 5′-ATGAACCTGGACCCATTCAG-3′, R 5′-CCTTCTGCACATTCATGGTC-3′ ABCC2: F 5′-AAATCCTGGTTGATGAAGGC-3′, R 5′-GGAGATCAGCAATTTCAGCA-3′ ABCC3:F 5′-ACAACCTCATCCAGGCTACC-3′, R 5′-GGTTGGCTGGAGAATCAAAT-3 ABCC4:F 5′-TCAGGTTGCCTATGTGCTTC-3′, R 5′-CGGTTACATTTCCTCCTCCA-3′ ABCC5:F 5′-GGGAGCTCTCAATGGAAGAC-3′, R 5′-CAGCTCTTCTTGCCACAGTC-3′ ABCC6:F 5′-CTGGACGAGGCTACTGCTG-3′, R 5′-TTGTCCATGACCAGAACCC-3′ ABCG2:F 5′-CAGTACTTCAGCATTCCACG-3′ R 5′-TTTCCTGTTGCATTGAGTCC-3′
Materials and methods Immunocytochemistry Isolation and culture of HNSC-CSCs HNSC-CSCs were isolated from primary surgical specimens as previously described [10], and their CSC properties were validated by a number of functional assays, including self-renewal capability, stem cell marker expression, chemo-resistance, and in vivo tumorigenicity, as previously reported [11]. HNSC-CSCs expanded in serum-free DMEM/F12 medium supplemented with B27, N2 supplement, 10 ng/ml human recombinant basic fibroblast growth factor (bFGF; R&D Systems, Minneapolis, MN, USA), and 10 ng/ml epidermal growth factor (EGF; R&D Systems). For differentiation, HNSC-CSCs were cultured in DMEM/F12 supplemented with 10% FBS without EGF and bFGF for at least 2 weeks. Laminin, fibronectin, and type IV collagen were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Adherent cell culture with ECM proteins For adherent cell cultures, culture plates were coated with each 10 μg/ml ECM proteins (laminin, type IV collagen, or fibronectin)
Adherent HNSC-CSCs were grown on coverslips in a 24-well plate at a density of 5 × 104 cells per well for 24 h, and then incubated with anti-OCT4, anti-CD44, or anti-involucrin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA; each at 1:50 dilution) overnight at 4 °C. After washing, cells were incubated with Cy3conjugated anti-mouse secondary antibodies. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Images were acquired using a confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
FACS analysis HNSC-CSC sphere cells were dissociated into single cells with Accutase (Sigma-Aldrich), washed twice, and suspended in PBS. Cells were re-suspended in 100 μl binding buffer and annexin VFITC (BD Pharmingen, San Diego, CA, USA), and then incubated at 4 °C for 30 min. After washing twice with PBS and adding 400 μl binding buffer containing 1 μl propidium iodide (PI), the
1106
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4– 1 1 11
cells were then incubated on ice for 15 min. Samples were analyzed by flow cytometry within 1 h using a FACSCalibur machine (BD Biosciences, Franklin Lakes, NJ, USA). An IgG isotype was included as a negative control.
Cell growth analysis HNSC-CSCs were dissociated into single cells with Accutase. Then single-dissociated cells at a density of 1 × 10 4 cells were plated in the type IV collagen-coated plate or standard culture plate. Cells were then incubated at 37 °C with 5% CO2 under stem cell culture conditions. On indicated day (0, 11, 13, 15, 17 days after seeding), cells were dissociated into single cells with Accutase and the total number of cells were calculated with hematocytometer.
Assays for lentiviral vector transduction efficiency of HNSC-CSCs The lentiviral pLL-CMV-EGFP vector and the two packaging plasmids were co-transfected into the human embryonic kidney cell line 293FT cells using Lipofectamine 2000 (Invitrogen) to produce the lentiviral particles. Then, adherent and spheroid-type CSCs were treated with the supernatant containing lentiviral particles produced by 293FT cells. The transfection efficiency was assessed by FACS and fluorescence microscopy.
Statistical analysis Differences in variables were analyzed using Student's t-test. A p-value of less than 0.05 (p < 0.05) was considered to indicate statistical significance.
Western blot analysis
Results Whole-cell extracts were prepared using radioimmunoprecipitation assay lysis buffer containing 1 mmol/L β-glycerophosphate, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L NaF, 1 mmol/L Na3VO4, and protease inhibitor (Roche). Protein in the extracts (30–100 μg) was separated by a 4% to 12% gradient or 10% SDSPAGE NuPAGE gel (Invitrogen) and was then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk and incubated with anti-Smooth muscle actin (Calbiochem, San Diego, CA, USA), anti-FSP1 (Millipore), anti-βACTIN (Sigma-Aldrich). Membranes were then incubated with horseradish peroxidase-conjugated anti-secondary IgG (Pierce, Rockford, IL, USA) antibody and visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce).
Cell viability assays Undifferentiated adherent CSCs in the ECM protein-coated plates were plated in a 96-well plate at a density of 7 × 103 cells per well in serum-free culture conditions. In parallel, differentiated CSCs were also plated in 10% FBS-supplemented culture conditions as described previously. Both adherent undifferentiated CSCs and differentiated CSCs were treated with 4 chemotherapeutic agents (cisplatin, 5-FU, paclitaxel, and docetaxel) currently being used in the clinical setting, and then cultured at 37 °C under a humidified 5% CO2 atmosphere. Twenty-four hours later, 20 μl of 3-(4,4dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml in PBS) was added to each well, and plates were placed at room temperature for 3 h. The absorbance was then measured using a SpectraMax 190 (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 570 nm.
Xenograft transplantation Undifferentiated adherent CSCs (103 cells) grown on type IV collagen-coated plates were suspended in a mixture of serumfree DMEM/F12 medium with growth factor and Matrigel (Sigma-Aldrich; 1:1 by volume). Subsequently, cells were subcutaneously injected into 8-week-old female BALB/c nude mice using 22-gauge needles. Engrafted mice were visually inspected and palpated weekly to monitor for tumor formation until 3 months post-transplantation.
Adherent HNSC-CSCs have a higher growth rate than spheroid-form HNSC-CSCs We evaluated the attachment rates of CSCs floating as a spheroidtype in serum-free medium onto a culture plastic substratum. Nearly 60% of CSCs attached to type IV collagen- or laminincoated plates, whereas less than 20% attached to fibronectincoated plates (Fig. 1a). Next, we assessed the growth rate of CSCs grown adherently on type IV collagen-coated plates or in spheroid form without coating materials. We found that the growth rate of CSCs that adhered to collagen-coated plates was significantly increased from 15 days after cell plating compared to that of spheroid-type CSCs (Fig. 1b). In addition, cell cycle analysis revealed an increase in the proportion of cells in the S and G2/M phases and a reduction in the proportion of cells in the G1 phase under adherent conditions, compared to spheroid-type CSCs (Fig. 1c).
Adherent HNSC-CSCs show a mesenchymal cell-like phenotypic change As evidenced by cell morphology, CSCs maintained on type IV collagen- or laminin-coated plates displayed a spindle-like mesenchymal cell phenotype, whereas CSCs induced to differentiate in medium containing 10% FBS showed distinct polygonal epithelial cell morphology (Fig. 2a). Epithelial–mesenchymal transition (EMT) was shown to confer normal epithelial cells with stem cell traits, including the ability to self-renew and efficiently initiate tumors [18]. Therefore, we assessed whether adherent CSCs expressed mesenchymal markers using quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) and western blot analysis. The results showed that although mRNA levels of fibroblast specific protein-1 (FSP-1) and smooth muscle actin (SMA) were increased in adherent CSCs in collagen-coated plates (Fig. 2b), the protein levels of only SMA, not FSP-1, were dramatically elevated in adherent CSCs compared to differentiated CSCs (Fig. 2b).
Adherent HNSC-CSCs express stem cell marker genes To ascertain whether adherent CSCs on collagen- or laminincoated plates maintain stem cell characteristics, CSCs were cultured in differentiation (10% FBS), in suspension or adherent
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4 –1 1 11
1107
Fig. 1 – Cellular characteristics of HNSC-CSCs grown in adherent and suspension cultures. (a) Gross morphology and attachment rates of HNSC-CSCs grown in suspension and adherent cultures (fibronectin-, laminin-, and type IV collagen-coated plates). ** p < 0.01. Magnification, 40×. HNSC-CSCs were seeded on 60-mm plates coated with the indicated ECM protein, and then incubated for 24 h at 37 °C in 5% CO2. Following incubation, the cells were washed twice with phosphate-buffered saline (PBS), and the remaining cells were harvested with Accutase. The attachment rate was calculated using following formula: (Attachment rate) % = [(Remaining cell number after PBS washing)/(Seeding cell number)] × 100. (b) Growth rates of HNSC-CSCs grown on type IV collagen-coated plates and in suspension cultures. Single-dissociated cells at a density of 1 × 104 cells were plated in the type IV collagen-coated plate or standard culture plate. On indicated day (0, 11, 13, 15, 17 days after seeding), cells were dissociated into single cells with Accutase and the total number of cells were calculated with hematocytometer. * p < 0.05, ** p < 0.01. (c) Cell cycle analysis of HNSC-CSCs grown on type IV collagen-coated plates and in suspension cultures. * p < 0.05.
Fig. 2 – Cellular characteristics of differentiated and adherent HNSC-CSCs. (a) Cell morphological differences between HNSC-CSCs grown in the differentiation (10% FBS) and adherent (type IV collagen-coated plates) cultures. (b) Levels of mRNA of mesenchymal markers in differentiated and adherent HNSC-CSCs. DSP; dentin sialoprotein, FSP-1; fibroblast specific protein 1, VIM; vimentin, SMA; smooth muscle actin. *p < 0.05. (c) Protein expression levels of SMA and FSP-1 in differentiated and adherent HNSC-CSCs. β-actin is used for an equal loading control.
1108
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4– 1 1 11
culture conditions, and Q-RT-PCR was performed to analyze the expression of stem cell markers, including CD44, OCT4, and SOX2, and the keratinocyte differentiation marker, involucrin. The results showed no significant differences in the expression of stem cell and differentiation markers between the HNSC-CSCs grown in suspension and on collagen-coated plates (Fig. 3a). We further examined stem cell and differentiation markers by immunofluorescence and found that OCT4, SOX2, and CD44, but not involucrin, were expressed by most adherent CSCs grown in collagen-coated plates and, to a lesser extent, by CSCs grown on laminin-coated plates. In contrast, nearly all CSCs differentiated by 10% FBS-supplemented culture conditions expressed involucrin, but not OCT4, SOX2, and CD44 (Fig. 3b and c).
Resistance to spontaneous and drug-induced cell death, and tumorigenicity of adherent HNSC-CSCs Because CSCs are known to undergo spontaneous cell death in sphere-forming suspension cultures [14], we compared the cell death rates of HNSC-CSCs grown in sphere-forming suspension cultures to those of adherent HNSC-CSCs on collagen- or laminin-coated plates. FACS analysis revealed that HNSC-CSCs grown on type IV collagen-coated plates are relatively resistant to spontaneous cell death compared to HNSC-CSCs grown in sphere-forming suspension cultures and on laminin-coated plates (Fig. 4a). We also determined the drug-induced cell death rates of HNSC-CSCs grown in four different culture conditions: sphereforming suspension cultures, collagen- or laminin-coated adherent cultures, and 10% FBS-induced differentiation cultures. As shown in Fig. 4b, in contrast to HNSC-CSCs grown in differentiation cultures,
all three stem cell culture conditions allowed HNSC-CSCs to be resistant to chemotherapeutic agents (cisplatin, fluorouracil, paclitaxel, and docetaxel). In addition, we examined the expression of various ATP transporter genes in HNSC-CSCs grown in suspension, on collagen- and laminin-coated plates, and in differentiation culture conditions. The results showed that ABCC2, ABCC5, and ABCC6 expression levels were increased in HNSC-CSCs with stem cell character (Fig. 4c) compared to differentiated CSCs, which might allow HNSC-CSCs to be resistant to chemotherapy. Because a number of cancer stem cell studies have utilized serially diluted cells for xenografting assays [19–22], we performed subcutaneous transplantation of 103 adherent CSCs into nude mice to test the tumorigenic capacity of HNSC-CSCs grown on collagen-coated plates. The result showed that 1000 adherent CSCs are sufficient to give rise to tumors (Fig. 4d).
Gene transduction efficiency and sustained self-renewal of adherent HNSC-CSCs Given that a previous study reported that spheroid-type CSCs display lower gene transduction efficiency compared to adherent cancer cells [11], we compared the lentiviral transduction efficiency of adherent and spheroid-type HNSC-CSCs with a plasmid encoding green fluorescence protein (GFP). As shown in Fig. 5, adherent HNSC-CSCs exhibited rates of transduction 6 times higher than spheroid-type HNSC-CSCs, indicating that adherent HNSCCSCs are more competent for gene delivery than spheroid-type CSCs. To validate whether long-term cultured adherent HNSCCSCs on type IV collagen-coated plates sustained stem cell features, we performed an immunofluorescence assay to examine
Fig. 3 – Adherent HNSC-CSCs express stem cell marker genes. (a) Levels of mRNA of stem cell markers (CD44, OCT4, and SOX2) and a differentiation marker (involucrin) in suspended, adherent (type IV collagen- and laminin-coated plates), and differentiated (10% FBS cultures) HNSC-CSCs. mRNA expression levels were normalized to the levels in the differentiated HNSC-CSCs. (b) Immunofluorescence analysis showed expression of CD44, OCT4, SOX2, and involucrin in the adherent (type IV collagen- and laminin-coated plates) and differentiated (10% FBS cultures) HNSC-CSCs (3rd passages). DAPI was used for nuclear staining. (c) Quantitative analysis of stem cell or differentiation markers expression shown in (b).
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4 –1 1 11
1109
Fig. 4 – Spontaneous and drug-induced cell death and tumorigenicity of adherent HNSC-CSCs. (a) Spontaneous cell death rates of HNSC-CSCs grown in sphere-forming suspension and adherent (type IV collagen- and laminin-coated) cultures were determined by FACS analysis with annexin V-PI staining. (b) Drug-induced cell death rates of HNSC-CSCs grown in sphere-forming suspension, adherent, and differentiation cultures were examined by MTT assay. (c) The mRNA expression levels of ABC transporter genes were determined by Q-RT-PCR analysis. (d) Xenograft tumorigenicity of 1000 adherent HNSC-CSCs grown on type IV collagen-coated plates.
Fig. 5 – Gene transduction efficiency of HNSC-CSCs grown in sphere-forming suspension and on collagen-coated plates. HNSC-CSCs were infected with lentiviral vector encoding the GFP gene, and transduction efficiency was examined by a FACS analysis and fluorescence microscopy.
1110
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4– 1 1 11
Fig. 6 – Expression of stem cell and differentiation markers in long-term cultured HNSC-CSCs. (a) Expression of stem cell markers (CD44 and OCT4) and a differentiation marker (involucrin) in adherent HNSC-CSCs grown over 15 consecutive passages on type IV collagen-coated plates was determined by immunofluorescence analysis. (b) Quantitative analysis of stem cell or differentiation marker expression shown in (a).
the expression of OCT4, CD44, and involucrin. Similar to early passage adherent HNSC-CSCs (Fig. 3), OCT4 and CD44 stem cell markers were maintained in adherent HNSC-CSCs even after 15 passages on type IV collagen-coated plates, whereas the involucrin differentiation marker was rarely detectable in these cells (Fig. 6).
Discussion Much evidence exists to suggest that the microenvironment plays a critical role in tumor development, progression, invasion, and metastasis [23]. In particular, ECM is now recognized as a critical regulator of cancer cell activity, contributing greatly to cancer progression. It is known that major ECM molecules involved in HNSC cell development and progression include collagens, laminin, and fibronectin [16]. Of these, increased expression of collagens, especially type IV collagen, is associated with malignant transformation of keratinocytes and HNSC cell aggressiveness [23]. Furthermore, there is significant correlation between type IV collagen expression and the existence of nodal metastases in laryngeal carcinoma, and the expression pattern of type IV collagen in the basement membranes surrounding nests of carcinoma is an important prognostic factor [24]. A recent study demonstrated that ECM molecules support the establishment and maintenance of human embryonic stem cells [25]. Furthermore, a collagen receptor, α2β1 integrin has been shown to regulate stem cell fate in human colorectal cancer cells [26], and type I collagen inhibits differentiation and promotes maintenance of stem cell-like phenotype in human colorectal carcinoma cells [27]. Therefore, these observations suggest that collagen plays a crucial role in the maintenance of stemness of cancer cells. In this study, we demonstrated that, similar to spheroidtype HNSC-CSCs, adherent HNSC-CSCs grown on type IV collagen-coated plates display a number of stem cell properties: stem cell marker expression, chemo-resistance, and tumorinitiating ability. HNSC-CSCs grown on type IV collagen-coated plates over 15 consecutive passages sustained expression of stem cell markers, expanded at a higher growth rate, and exhibited reduced spontaneous cell death compared to the HNSC-CSCs grown on laminin-coated plates or in sphere-forming suspension cultures. In addition, the lentiviral vector transduction efficiency of
HNSC-CSCs was much higher in collagen adherent cultures than in sphere-forming suspension cultures. Taken together, our results indicate that type IV collagen adherent cultures are more appropriate for the maintenance of HNSC-CSCs than laminin adherent and sphere-forming suspension cultures. Interestingly, adherent HNSC-CSCs grown on collagen-coated plates showed mesenchymal morphological changes with an elevation of mesenchymal cell markers, in contrast to the epithelial cell phenotype observed in differentiation culture conditions. Epithelial–mesenchymal transition (EMT) plays a critical role in normal embryogenesis and tumor progression, including invasion, metastasis, and resistance to apoptosis [28,29]. A recent study demonstrated that several EMT markers are overexpressed in stem-like cells isolated from human mammary gland and mammary carcinomas [18]. In the process of tumor metastasis, which is presumably accompanied by EMT, disseminated cancer cells appear to possess self-renewal capability [30]. Therefore, our results that show mesenchymal cell morphology and increased expression of mesenchymal markers in adherent HNSC-CSCs grown on type IV collagen-coated plates indicate that type IV collagen allows HNSC-CSC to maintain a mesenchymal phenotype in in vitro culture. In conclusion, compared to “sphere” cultures, adherent in vitro expansion of HNSC-CSCs on type IV collagen-coated culture plates may promote significant advancements in cancer stem cell biology.
Conflict of interest None.
Acknowledgments This study was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (No. 2010-0022256 to Y.C. Lim) and from the National R&D Program for Cancer Control, Ministry for Health and Welfare, the Republic of Korea (No. 2008-0058785 to H. Kim).
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 0 4 –1 1 11
REFERENCES
[1] M. Shackleton, E. Quintana, E.R. Fearon, S.J. Morrison, Heterogeneity in cancer: cancer stem cells versus clonal evolution, Cell 138 (2009) 822–829. [2] M. Dean, T. Fojo, S. Bates, Tumour stem cells and drug resistance, Nat. Rev. Cancer 5 (2005) 275–284. [3] S.K. Singh, I.D. Clarke, M. Terasaki, V.E. Bonn, C. Hawkins, J. Squire, P.B. Dirks, Identification of a cancer stem cell in human brain tumors, Cancer Res. 63 (2003) 5821–5828. [4] G. Mor, G. Yin, I. Ghefetz, Y. Yang, A. Alvero, Ovarian cancer stem cells and inflammation, Cancer Biol Ther 11 (2011) 708–713. [5] C.F. Kim, E.L. Jackson, A.E. Woolfenden, S. Lawrence, I. Babar, S. Vogel, D. Crowley, R.T. Bronson, T. Jacks, Identification of bronchioalveolar stem cells in normal lung and lung cancer, Cell 121 (2005) 823–835. [6] A.T. Collins, P.A. Berry, C. Hyde, M.J. Stower, N.J. Maitland, Prospective identification of tumorigenic prostate cancer stem cells, Cancer Res. 65 (2005) 10946–10951. [7] P. Dalerba, S.J. Dylla, I.K. Park, R. Liu, X. Wang, R.W. Cho, T. Hoey, A. Gurney, E.H. Huang, D.M. Simeone, A.A. Shelton, G. Parmiani, Phenotypic characterization of human colorectal cancer stem cells, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 10158–10163. [8] M. Al-Hajj, M.S. Wicha, A. Benito-Hernandez, S.J. Morrison, M.F. Clarke, Prospective identification of tumorigenic breast cancer cells, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 3983–3988. [9] D. Bonnet, J.E. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell, Nat. Med. 3 (1997) 730–737. [10] B. Joshua, MJ. Kaplan, I. Doweck, R. Pai, IL. Weissman, ME. Prince, LE. Ailles, Frequency of cells expressing CD44, a head and neck cancer stem cell marker: correlation with tumor aggressiveness, Head Neck 34 (2012) 42–49. [11] Y.C. Lim, S.Y. Oh, Y.I. Cha, S.H. Kim, X. Jin, H. Kim, Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas, Oral Oncol. 47 (2011) 83–91. [12] B.A. Reynolds, R.L. Rietze, Neural stem cells and neurospheres re-evaluating the relationship, Nat. Methods 2 (2005) 333–336. [13] S.M. Pollard, K. Yoshikawa, I.D. Clarke, D. Danovi, S. Stricker, I.R. Russell, J. Bayani, R. Head, M. Lee, M. Bernstein, J.A. Squire, A. Smith, Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens, Cell Stem Cell 4 (2009) 568–580. [14] I. Singec, R. Knoth, R.P. Meyer, J. Maciaczyk, B. Volk, G. Nikkhah, M. Frotscher, E.Y. Snyder, Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology, Nat. Methods 3 (2006) 801–806. [15] J. Grill, M.L. Lamfers, V.W. van Beusechem, C.M. Dirven, D.S. Pherai, M. Kater, P. Van der Valk, R. Vogels, W.P. Vandertop, H.M. Pinedo, D.T. Curiel, W.R. Gerritsen, The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro, Mol. Ther. 6 (2002) 609–614.
1111
[16] A.F. Ziober, E.M. Falls, B.L. Ziober, The extracellular matrix in oral squamous cell carcinoma: friend or foe? Head Neck 28 (2006) 740–749. [17] H.N. Suh, H.J. Han, Collagen I regulates the self-renewal of mouse embryonic stem cells through α2β1 integrin- and DDR1-dependent Bmi-1. J. Cell. Physiol. 226 (12) (2011) 3422–3432. [18] S.A. Mani, W. Guo, M.J. Liao, E.N. Eaton, A. Ayyanan, A.Y. Zhou, M. Brooks, F. Reinhard, C.C. Zhang, M. Shipitsin, L.L. Campbell, K. Polyak, The epithelial–mesenchymal transition generates cells with properties of stem cells, Cell 133 (2008) 704–715. [19] S. Zhang, C. Balch, M.W. Chan, H.C. Lai, D. Matei, J.M. Schilder, P.S. Yan, T.H. Huang, K.P. Nephew, Identification and characterization of ovarian cancer-initiating cells from primary human tumors, Cancer Res. 68 (2008) 4311–4320. [20] M.E. Prince, R. Sivanandan, A. Kaczorowski, G.T. Wolf, M.J. Kaplan, P. Dalerba, I.L. Weissman, M.F. Clarke, L.E. Ailles, Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 973–978. [21] Y.C. Lim, S.Y. Oh, Y.Y. Cha, S.H. Kim, H. Kim, Cancer stem cell traits in squamouspheres derived from primary head and neck squamous cell carcinomas, Oral Oncol. 47 (2011) 83–91. [22] P. Dalerba, S.J. Dylla, I.K. Park, R. Liu, X. Wang, R.W. Cho, T. Hoey, A. Gurney, E.H. Huang, D.M. Simeone, A.A. Shelton, G. Parmiani, C. Castelli, M.F. Clarke, Phenotypic characterization of human colorectal cancer stem cell, Proc. Natl. Acad. Sci. U. S. A. 12 (2007) 10158–10163. [23] F. Stenback, M.J. Makinen, T. Jussila, S. Kauppila, J. Ristell, L. Talve, L. Risteli, The extracellular matrix in skin development: a morphological study, J. Cutan. Pathol. 26 (1997) 327–338. [24] T. Krecicki, M. Zalesska-Krecicka, M. Jelen, T. Szkudlarek, M. Horobiowska, Expression of type IV collagen and matrix metalloproteinase-2 (type IV collagenase) in relation to nodal status in laryngeal cancer, Clin. Otolaryngol. Allied Sci. 26 (2001) 469–472. [25] A.B. Prowse, F. Chong, P.P. Gray, T.P. Munro, Stem cell integrins: implications for ex-vivo culture and cellular therapies, Stem Cell Res. 6 (2011) 1–12. [26] S.C. Kirkland, H. Ying, Alpha2beta1 integrin regulates lineage commitment in multipotent human colorectal cancer cells, J. Biol. Chem. 283 (2008) 27612–27619. [27] S.C. Kirkland, Type I collagen inhibits differentiation and promotes a stem cell-like phenotype in human colorectal carcinoma cells, Br. J. Cancer 101 (2009) 320–326. [28] H. Peinado, D. Olmeda, A. Cano, Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat. Rev. Cancer 7 (2007) 415–428. [29] S.A. Mani, J. Yang, M. Brooks, G. Schwaninger, A. Zhou, N. Miura, J.L. Kutok, K. Hartwell, A.L. Richardson, R.A. Weinberg, Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 10069–10074. [30] J.P. Thiery, H. Acloque, R.Y. Huang, M.A. Nieto, Epithelial– mesenchymal transitions in development and disease, Cell 139 (2009) 871–890.