Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden
© Abubaker et al.; licensee BioMed Central Ltd. 2013
Received: 6 December 2012
Accepted: 19 March 2013
Published: 27 March 2013
Over 80% of women diagnosed with advanced-stage ovarian cancer die as a result of disease recurrence due to failure of chemotherapy treatment. In this study, using two distinct ovarian cancer cell lines (epithelial OVCA 433 and mesenchymal HEY) we demonstrate enrichment in a population of cells with high expression of CSC markers at the protein and mRNA levels in response to cisplatin, paclitaxel and the combination of both. We also demonstrate a significant enhancement in the sphere forming abilities of ovarian cancer cells in response to chemotherapy drugs. The results of these in vitro findings are supported by in vivo mouse xenograft models in which intraperitoneal transplantation of cisplatin or paclitaxel-treated residual HEY cells generated significantly higher tumor burden compared to control untreated cells. Both the treated and untreated cells infiltrated the organs of the abdominal cavity. In addition, immunohistochemical studies on mouse tumors injected with cisplatin or paclitaxel treated residual cells displayed higher staining for the proliferative antigen Ki67, oncogeneic CA125, epithelial E-cadherin as well as cancer stem cell markers such as Oct4 and CD117, compared to mice injected with control untreated cells. These results suggest that a short-term single treatment of chemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential. The novel findings in this study are important in understanding the early molecular mechanisms by which chemoresistance and subsequent relapse may be triggered after the first line of chemotherapy treatment.
KeywordsOvarian carcinoma Cancer stem cell Metastasis Ascites Chemoresistance Recurrence
Epithelial ovarian cancer (EOC) is the fifth most common cancer among women and is the leading cause of death among gynaecological cancers. Over 80% of women with EOC are diagnosed at a late-stage with dissemination of tumor implants throughout the peritoneal cavity . The combination of cisplatin and paclitaxel based chemotherapy was introduced as a first line of treatment for the clinical management of advanced-stage ovarian cancer patients nearly 17 years ago . Cisplatin is a DNA strand cross-linking drug that generates DNA damage leading to the activation of cyclin-dependent kinase inhibitors such as p21 and wee1/mik1, which subsequently arrest cells in either G1 or G2 phase . Resistance to cisplatin has been associated with increased glutathione and metallothionein levels, decreased drug uptake, increased DNA repair (due to enhanced expression of excision repair enzymes) and the tolerance of the formation of platinum-DNA adducts . The status of p53 mutation plays a significant role in DNA repair, proliferative arrest and apoptosis and there is a correlation between cancer cell p53 status and cisplatin sensitivity [5, 6]. Paclitaxel on the other hand, is a mitotic inhibitor that promotes the formation and stabilization of microtubules leading to a cell cycle block at the metaphase to anaphase transition . In contrast to cisplatin, the cytotoxic effect of paclitaxel is independent of p53 status  and alterations in β-tubulin isotypes have been associated with paclitaxel resistance in cancer cells . Both cisplatin and paclitaxel through distinct molecular mechanisms trigger an apoptotic cascade resulting in the death of the majority of ovarian cancer cells. In spite of this, approximately 80% of ovarian cancer patients experience incurable recurrent cancer within 6–20 months post-chemotherapy  as a consequence of the survival of a very small percentage of chemotherapy resistant residual tumor cells which facilitate the development of recurrent progressive disease . Concerted research efforts to tackle the failure of combination chemotherapy have resulted in no effective salvage strategies for the last 17 years . Hence, there is an increasing pressure to seek alternative approaches, which has resulted in the use of combinations of drugs that usually belong to the platinum or taxane families . These alternative drug combinations have provided temporary hope to the patients but have had no clinically effective outcome . To establish an effective treatment protocol for advanced-stage ovarian cancer patients a systematic approach is needed to understand responses of ovarian cancer cells to platinum and taxane-based drugs, individually and in combination. In vivo experiments initially with each drug treatment will result in insights into the molecules that facilitate the evasion of chemotherapy-associated cytotoxicity against each individual drug and the subsequent re-growth of tumour cells as recurrent tumor masses. This is particularly important for a large proportion of chemorefractory ovarian cancer patients who are resistant to platinum-based drugs and are normally prescribed taxane-based treatment. On the other hand, some ovarian cancer patients respond badly towards taxane-based drugs and develop serious side effects, in which case they are prescribed platinum-based treatment.
We and others have recently demonstrated an association between chemoresistance and the acquisition of epithelial mesenchymal transition (EMT) and CSC-like phenotypes in cancer [10–12] and found chemoresistant recurrent ovarian tumors to be enriched in CSCs and stem cell pathway mediators, suggesting that CSCs may contribute to recurrent disease [13, 14]. The first involvement of stem cells in ovarian cancer was reported in the ascites of an ovarian cancer patient, derived from a single cell that could sequentially propagate tumors over several generations . CSCs have also been isolated from ovarian cancer cell lines based on their abilities to differentially efflux the DNA binding dye Hoechst 33342 . This population of cells termed the ‘side population’ (SP) displayed the classical stem cell property in tumorigenicity assays. More recently, a population of normal murine OSE  have been identified to have putative stem cell characteristics indicating that these may be the originators of CSCs in the ovaries. Few other recent reports have shown the presence of CSCs in ovarian tumors as well as in patients’ ascites [18–20]. CSCs in these studies were reported to be resistant to conventional chemotherapy and were able to recapitulate in vivo the original tumor suggesting that these CSCs control self-renewal as well as metastasis and chemoresistance.
In this study, we demonstrate that a short-term single exposure of chemotherapy (cisplatin, paclitaxel or both in combination) treatment induced in surviving ovarian cancer cells a CSC-like profile which was independent of the type of chemotherapy and the associated cytotoxicity. We further demonstrate that chemotherapy surviving residual cells were able to generate tumors with greater capacity (tumor burden) than control untreated cells, and that they retained their inherent CSC-like profile in tumor xenografts. These novel findings emphasize the need to understand the CSC-like phenotype of ovarian tumors which may arise after the first line of chemotherapy treatment and may be crucial in facilitating the aberrant events leading to recurrent disease.
Methods and materials
The human epithelial ovarian cancer line OVCA 433 was derived from the ascites of an ovarian cancer patient and generously provided by Dr Robert Bast Jr. (MD Anderson Cancer Centre, Houston, TX). The cell line was grown as described previously . The human ovarian HEY cell line was derived from a peritoneal deposit of a patient diagnosed with papillary cystadenocarcinoma of the ovary . The cell line was grown as described previously .
Antibodies and reagents
Monoclonal and polyclonal antibodies against CD44, CD24, CD117, CD133, were obtained from Millipore (Melbourne, Australia). Monoclonal antibodies against excision repair complement complex 1 (ERCC1) and β-tubulin isotype III were obtained from Sapphire Biosciences and Sigma Aldrich (Melbourne, Australia). Polyclonal antibody against EpCAM was obtained from Cell Signalling Technology (Beverly, MA, USA). Antibodies against cytokeratin 7 (cyt7), Ki67, CA125, E-cadherin, vimentin, Oct4 and CD117 (c-Kit) used for immunohistochemistry were obtained from Ventana (Roche, Arizona, USA).
Treatment of ovarian cancer cells with cisplatin, paclitaxel and combination of both
Ovarian cancer cell lines OVCA 433 and HEY were treated with cisplatin and paclitaxel concentrations at which 50% growth inhibition was obtained (GI50) for 3–5 days. OVCA 433 cells were treated with cisplatin (5 μg/ml) for five days, paclitaxel (2 ng/ml) and combination (2.5 μg/ml of cisplatin and 1 ng/ml of paclitaxel) for three days. HEY cells were treated with cisplatin (1 μg/ml) five days, paclitaxel (1 ng/ml) and combination (1 μg/ml of cisplatin and 1 ng/ml of paclitaxel) for three days. For combination treatment, samples were screened for response to different combination of drug treatments and the concentration of combination treatment which gave the GI50 value while maintaining the enhancement in resistant phenotype (ERCC1 and β-tubulin expression) and cancer stem cell marker expression was chosen for experiments.
Immunofluorescence analysis of ERCC1 and β-tubulin isotype III was performed as described previously . Images were captured by the photo multiplier tube (PMT) using the Leica TCS SP2 laser, and viewed on a HP workstation using the Leica microsystems TCS SP2 software. The mean fluorescence intensity was quantified using Cell-R software (Olympus Soft Imaging Solutions).
Flow cytometric analysis
Flow cytometry was performed as described previously . Briefly, untreated or chemotherapy treated cells were collected and rinsed twice with phosphate buffered saline (PBS). 106 cells were incubated with primary antibody for 1 hr at 4°C and excess unbound antibody was removed by washing twice with PBS. Cells were stained with secondary antibody conjugated with phycoerythrin for 20 minutes at 4°C, washed twice with PBS and then resuspended in 0.5 ml PBS prior to FACScan analysis. In each assay background staining was detected using an antibody-specific IgG isotype. All data were analysed using Cell Quest software (Becton-Dickinson, Bedford, MA, USA). Results are presented as histogram overlay.
Sphere forming assay
The sphere forming ability of untreated and chemotherapy treated OVCA 433 cells and HEY cells were determined as described previously . The sphere forming ability of the cells was photographed over 21 days using a phase contrast microscope (Axiovert 100, Zeiss, Germany) and assessed with the DeltaPix Viewer software (Denmark). Cellular aggregates with a diameter larger than 50 μm were classified as ‘spheres’.
RNA extraction and Real Time-PCR
RNA extractions were performed using Trizol (Life Technologies, USA) using the Qiashredder and RNeasy kits (QIAGEN, Australia) according to the manufacturer’s instructions. The concentration and purity of RNA was determined using spectrophotometry (Nanodrop ND-1000 spectrophotometer, Thermo Scientific, USA) and 0.5 μg of RNA was used for cDNA synthesis. cDNA synthesis was performed using Superscript VILO (Invitrogen, Australia) according to manufacturer’s instructions. Quantitative determination of mRNA levels of various genes was performed in triplicate using SYBR green (Applied Biosystems, Australia) as described previously . The primers for Oct-4A, Nanog, CD44, CD117, EpCAM have been described previously .
Animal ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of the Laboratory Animals of the National Health and Medical Research Council of Australia. The experimental protocol was approved by the Ludwig/Department of Surgery, Royal Melbourne Hospital and University of Melbourne’s Animal Ethics Committee (Project-006/11), and was endorsed by the Research and Ethics Committee of Royal Women’s Hospital Melbourne, Australia.
Female Balb/c nu/nu mice (age, 6–8 weeks) were obtained from the Animal Resources Centre, Western Australia. Animals were housed in a standard pathogen-free environment with access to food and water.
HEY cells were treated with cisplatin and paclitaxel as described previously. 5x106 residual cisplatin or paclitaxel surviving cells treated for 4 days were injected intraperitoneally (ip) in nude mice. Mice were inspected weekly and tumor progression was monitored based on overall health and body weight until one of the pre-determined endpoints was reached. Endpoint criteria included loss of body weight exceeding 20% of initial body weight, anorexia, general patterns of diminished well-being such as reduced movement and lethargy resulting from lack of interest in daily activities. Mice were euthanized and organs (liver, stomach, lungs, gastrointestinal tract, pancreas, uterus, skeletal muscle, colon, kidney, peritoneum, ovaries and spleen) and solid tumors were collected for further examination. Metastatic development was documented by a Royal Women’s Hospital pathologist according to histological examination (H & E staining) of the organs.
Immunohistochemistry of mouse tumors
For immunohistochemistry, formalin fixed, paraffin embedded 4 μm sections of the xenografts were stained using a Ventana Benchmark Immunostainer (Ventana Medical Systems, Inc, Arizona, USA). Detection was performed using Ventana’s Ultra View DAB detection kit (Roche/Ventana, Arizona, USA) using the method described previously . Briefly, tumor sections were dewaxed with Ventana EZ Prep and endogenous peroxidase activity was blocked using the Ventana’s Universal DAB inhibitor. Primary antibodies against Oct4, Ki67, E-cadherin, vimentin, CA125, cytokeratin 7 and CD117 (c-Kit) were diluted according to the instruction provided by the manufacturer. The sections were counter stained with Ventana Haematoxylin and Blueing Solution. Immunohistochemistry images were taken using Axioskop 2 microscope, captured using a Nikon DXM1200C digital camera and processed using NIS-Elements F3.0 software.
Student’s t-test was used for the statistical analyses of sphere formation and qPCR analysis. Data are presented as mean ± SEM. A probability level of p < 0.05 was adopted throughout to determine statistical significance. Treatment groups were compared with the control group using one way- ANOVA and Dunnett’s Multiple Comparison post-tests.
Chemotherapy induced morphological changes in ovarian cancer cell lines
Morphological changes in response to cisplatin or paclitaxel were dose-dependent (data not shown). Cisplatin-induced morphological changes were evident at concentrations between 1–10 μg/ml (GI50 ~ 5 μg/ml) for OVCA 433 cells. However, HEY cells responded to much lower cisplatin concentration of 0.5-5 μg/ml (GI50 ~ 1 μg/ml) (Figures 1A-B). On the other hand, paclitaxel-induced epithelial morphology was evident at a concentration of 0.5-2.5 ng/ml (GI50 ~ 2 ng/ml) for OVCA 433 cells, and 0.1-2 ng/ml (GI50 ~ 1 ng/ml) for HEY cells. Similar change to epithelial morphology in clones of surviving cells, but to a greater extent than that observed with paclitaxel only, was evident after combination treatment (cisplatin + paclitaxel). Both OVCA 433 and HEY demonstrated discrete epithelial colonies and very few mesenchymal cells which were scattered in between epithelial cells (Figures 1A-B). Different concentrations of combination treatments were tried but as described previously  the drugs concentration at or below the GI50 value were used for further study.
Chemotherapy induces the expression of cisplatin and paclitaxel resistant phenotypes
Chemotherapy enhances the expression of CSC markers
Residual cancer cells after chemotherapy treatments exhibited metastatic and CSC-like features in nude mice
Chemoresistance is a major obstacle towards the successful treatment of ovarian cancer patients. The molecular and the cellular mechanisms of the resistance of ovarian cancer cells to platinum and taxane-based therapies, the two agents used as standard treatment, remains unknown in vitro and in vivo. In this study we have used two very different ovarian cancer cell lines, OVCA 433 (mainly epithelial) and HEY (mainly mesenchymal), treated short-term with cisplatin or paclitaxel or the combination of both to dissect those initial cellular responses that facilitate the survival of residual cells and their subsequent regrowth in an in vivo mouse model. We have demonstrated that cisplatin or paclitaxel or combination treatment of ovarian cancer cell lines, generates in each case a population of residual cells with features of CSC-like cells. An enhanced expression of CSC markers in the residual cancer cells after chemotherapy treatments coincided with an enhanced expression of ERCC1 and/or β-tubulin isotype III, the two proteins commonly associated with resistance of cancer cells to platinum and taxane-based chemotherapies [29, 30]. Enhancement in ERCC1 expression in response to cisplatin was consistent with the enhanced expression of β-tubulin isotype III within the same population of cells after paclitaxel treatment. However, in response to paclitaxel and combination treatments a greater degree of β-tubulin isotype III expression was observed, suggesting that cisplatin resistant cells may be cross resistant to paclitaxel but the reverse may not be the case. ERCC1 has been associated with cisplatin resistance in ovarian tumors and cancer cell lines [25, 29]. Recent clinical trials suggest that patients with low ERCC1 levels benefit preferentially from cisplatin-based chemotherapy compared to patients who have a higher expression of ERCC1 in their tumors . On the other hand, tumors resistant to paclitaxel or cancer cell lines rendered resistant to paclitaxel have substantially enhanced levels of isotypes III or IV β-tubulin [32–34]. Evidence for the enhancement in isotype-specific taxane-resistant tubulin has also been described in the tumors of ovarian cancer patients . Paired samples from advanced-stage ovarian cancer patients who developed clinical paclitaxel resistance showed increases in β-tubulin isotypes I (3.6-fold), III (4.4-fold) and IV (7.6-fold) .
Long-term repeated chemo-treatment approaches have been shown to generate chemoresistant cancer cell lines with features of CSCs [35, 36]. The novelty of the current study is the demonstration of CSC-like features in ovarian cancer cell lines by a single short-term exposure of chemotherapeutic agents. The fact that short-term single exposure of chemotherapeutic agents is capable of modulating the expression of specific chemoresistant genes (ERCC1 and β-tubulin III) and potential CSC genes, suggests that selection of existing chemoresistant CSC-like subpopulation of ovarian cancer cells is embedded within the bulk of the original cancer population. As shown in our previous studies, this pattern of selection of CSC-like cells is not limited to ovarian cancer cell lines but can be displayed in tumor cells isolated from primary ovarian tumors and ascites of ovarian cancer patients . This suggests that in the clinical scenario, CSC enriched residual cells are generated in the host tumor microenvironment after the first round of chemotherapy treatment. Whether these cells further enrich their CSC-like characteristics after consecutive chemotherapy treatments or retain the original CSC-like features to facilitate the re-growth of secondary tumors is not known. However, we have previously demonstrated that the expression level of CSC-like markers in OVCA 433 cells remains unchanged after single or long-term treatments with cisplatin . In this context, few previous studies have demonstrated the existence of CSC-enriched side population of cells [28, 37] or CD44, CD117, CD133, CD24 enriched population of cells in ovarian cancer cell lines or ovarian cancer patient’s samples [38–40]. These CSC-enriched cells have been shown to develop tumors on sequential inoculation in nude mice and retain the original CSC-like phenotype observed in the parental sample.
Recent data suggest that CSCs rely on the presence of a ‘CSC niche’ which controls their self-renewal and differentiation . Current studies have also shown that residual cells after chemotherapy treatment secrete soluble factors that provide a favourable microenvironment to facilitate the growth of residual cells [42, 43]. This close relationship between chemotherapy-surviving cells and their secretory microenvironment represents a potential ‘CSC niche’ that can provide survival signals to residual cells for re-growth into a recurrent cancer. Moreover, CSCs can also be generated by the complex tumor microenvironment composed of diverse stromal cells, including tissue specific fibroblasts, cancer associated fibroblasts, tissue specific and bone marrow-derived mesenchymal stem cells, infiltrating immune cells, endothelial cells and their associated vascular network, soluble and other growth factors and/or extracellular matrix component . Growth of recurrent tumors seems to rely on the permissive microenvironment provided by each component of ‘CSC niche’. The CSCs retain their exclusive abilities to self-renew and give rise to differentiated progenitor cells, while staying in an undifferentiated state themselves .
In the current study we have demonstrated that both the epithelial OVCA 433 and mesenchymal HEY cell lines respond to cisplatin or paclitaxel by enhancing the expression of CD24, CD117, CD133 and EpCAM. However, the enhancement of CD44 in response to cisplatin or paclitaxel treatments differed between the cell lines and may depend on the inherent epithelial or mesenchymal phenotype of the cell line. CD44 is not only a stem cell marker but has been shown to be highly expressed in cells with mesenchymal phenotype. The HEY cell line is inherently mesenchymal, with high endogenous expression of CD44 prior to chemotherapy. On the other hand, OVCA 433 is an epithelial cell line with a minimal expression of CD44. The addition of cisplatin drives both the cell lines to a mesenchymal state . This correlates nicely with a slight increase in the expression of CD44 in both OVCA 433 and HEY cell lines. On the contrary, paclitaxel treatment induced a more epithelial-like morphology in the inherently mesenchymal HEY cell line, which may result in the down regulation of CD44 expression. This holds true only at the protein level. At the mRNA level, the expression of CD44 was elevated with all chemotherapy treatments in both the cell lines. This suggests, an inability of translation of CD44 mRNA in HEY cells. This may occur due to epigenetic changes in CD44 with paclitaxel treatment in HEY cells . However, the disparity of EpCAM expression at the protein and mRNA levels in HEY cells is difficult to explain. One possible explanation can be that DNA damage response initiated by cisplatin has no effect on the transcriptional expression of EpCAM but it may trigger enhanced translation of EpCAM from the existing endogenous EpCAM mRNA.
Tumors generated from control untreated and cisplatin/paclitaxel treated cells were invasive and invaded peritoneal organs such as pancreas and liver. With the small number of tumor xenografts analysed in this study (n = 3) we have demonstrated some differences in the invasion to kidney by chemotherapy treated cells. No pattern of kidney invasion was observed with control untreated mice. However, paclitaxel-treated HEY cells invaded kidney, but the invasion with cisplatin treated cells was not consistent and differed between mice. In two out of the three mice analysed, invasion to kidney was observed, but in one mouse tumor cells surrounded the kidney with no apparent invasion. This variation in the invasion pattern between the control and chemotherapy treated cells may be due to the phenotypic changes induced in the cells by the chemotherapeutic agents or it may be due to the induced ‘CSC-niche’ created by the cells within the tumor microenvironment.
Enhanced CSC-like characteristics observed in ovarian cancer cells after a single dose of chemotherapy treatment were retained in in vivo mouse xenografts (enhanced expression of Oct4 and CD117 in tumors derived from cisplatin and paclitaxel treated cells). Tumor cells within the xenografts of chemotherapy treated cells had a greater proliferative potential as evaluated by enhanced Ki67 staining, and a greater tumor burden within the same time frame as that of the tumors derived from control untreated cells. In addition, tumors derived from chemotherapy treated cells had an enhanced expression of CA125 and were more epithelial in phenotype with enhanced E-cadherin expression compared to tumors generated from control untreated HEY cells. The relative high abundance of epithelial markers (enhanced expression of E-cadherin and CA125) in tumors derived from HEY cells treated with chemotherapy in vitro, compared to untreated control cells, is consistent with our recent observation of ascites tumor cells of recurrent patients which had an enhanced expression of epithelial and CSC-like markers compared to tumor cells of ascites obtained from chemonaive untreated patients . We have previously reported that ovarian cancer cells possess a certain level of epithelial mesenchymal plasticity that allows them to change their phenotype and acquire different functions and properties under the influence of the local tumor environment [12, 45, 46]. Considering that HEY cells have inherent mesenchymal phenotype and very low/no expression of E-cadherin and CA125 in vitro, the expression of E-cadherin and CA125 in vivo control mouse xenografts implies such plasticity. The dynamics of ovarian tumor cell plasticity in relation to tumor cell dissemination and engraftment on secondary site is not well understood but the potential ‘mesenchymal to epithelial transition’ (MET) is assumed to occur in the late phase of ovarian tumor dissemination when the tumor cells adapt to the ascites microenvironment [46–49]. The expression of E-cadherin and CA125 in xenografts obtained from mesenchymal HEY cells, and enhancement of that expression in mouse xenografts derived from residual chemotherapy treated cells, further illustrates plasticity related changes in HEY cells influenced by the in vivo microenvironment which acts as a ‘CSC niche’, and may facilitate the rapid proliferation of chemotherapy-treated CSC-rich residual cells resulting in increased tumor burden. These novel observations are consistent with a recent study that demonstrated the epithelial phenotype of side population cells sorted from ovarian cancer lines and ascites of ovarian cancer patients . These stem-like side population cells exhibited decreased adhesive and invasive potential compared to the more differentiated non-side population cells and were localized on tumor boundary when implanted into nude mice along with non-side population cells . These results suggest that the relationship between malignant potential, CSC phenotype and cellular plasticity in ovarian cancer is a developing field and more research is needed to understand the processes. In this context, the identification of E-cadherin rich metastatic tumors in breast and brain cancers [48, 51, 52], and an association between increased pluripotency and the epithelial subcomponent of human bladder and prostatic carcinoma cells , and normal breast cells  exerts a strong link between epithelial plasticity and CSCs. Perhaps consistent with this is the observation that BRCA1-associated basal breast cancers better resemble aberrant luminal progenitor cells rather than the mesenchymal-like mammary stem cells [55, 56].
The authors wish to thank Royal Women’s Hospital Foundation, Women’s Cancer Foundation, National Health and Medical Research Council of Australia (JKF, RegKey#441101) and the Victorian Government’s Operational Infrastructure Support Program and National Breast Cancer Foundation (EWT) for supporting this work. AL and KA are the recipients of Royal Women’s Hospital Scholarship and Australian Postgraduate Award.
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