B1.14 Available online at www.sciencedirect.com
Biochemical and Biophysical Research Communications 368 (2008) 930–936 www.elsevier.com/locate/ybbrc
MDR1 expression identifies human melanoma stem cells Gilmor I. Keshet a,*, Itamar Goldstein a, Orit Itzhaki b, Karen Cesarkas a, Liraz Shenhav a, Arkadi Yakirevitch b, Avraham J. Treves b, Jacob Schachter b, Ninette Amariglio a, Gideon Rechavi a a
Cancer Research Center, Sheba Medical Center, Tel Hashomer and Sackler School of Medicine, Tel Aviv University, Tel Aviv, 52621, Israel b Ella Institute for Melanoma Research and Treatment, Sheba Medical Center, Tel Hashomer and Sackler School of Medicine, Tel Aviv University, Tel Aviv 52621, Israel Received 26 January 2008 Available online 13 February 2008
Abstract ABC transporters are often found to be inherently expressed in a wide variety of stem cells, where they provide improved protection from toxins. We found a subpopulation of human melanoma cells expressing multidrug-resistance gene product 1 (MDR1). This fraction co-expresses the ABC transporters, ABCB5 and ABCC2 in addition to the stem cell markers, nanog and human telomerase reverse transcriptase (hTERT). The clonogenicity and self-renewal capacity of MDR1+ melanoma cells were investigated in single cell settings using the limiting dilution assay. We found that the MDR1+ cells, isolated by FACS sorting, demonstrated a higher self-renewal capacity than the MDR1 fraction, a key stem cell feature. Moreover, MDR1+ cells had higher ability to form spheres in low attachment conditions, a hallmark of cancer. In conclusion, these novel findings imply that the MDR1+ cells represent melanoma stem cells and thus should be considered as a unique cellular target for future anti-melanoma therapies. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Multidrug resistance; Melanoma; Cancer stem cells; Self-renewal; Anchorage independence
There are an estimated 132,000 new melanoma skin cancer cases worldwide each year (WHO). In its early stages malignant melanoma can be cured by surgical resection, but once it has progressed to the metastatic stage it is extremely difficult to treat and is resistant to most current therapies. Reports on cancer stem cells in various tumors (reviewed in [1]) raise the possibility that this long-lived tumor subpopulation is resistant to chemotherapy. Normal stem cells are thought to achieve their longevity by several mechanisms among which are slow divisions, anti apoptotic mechanisms, and expression of efflux pumps that provide protection from toxins [2]. Various types of ATP-binding cassette (ABC) transporters including multidrug-resistant (MDR) gene prod*
Corresponding author. Address: Cancer Research Center, Sheba Medical Center, Tel-Hashomer, 52621, Israel. Fax: +972 3 5305351. E-mail address:
[email protected] (G.I. Keshet). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.02.022
uct 1, contribute to drug resistance in many cancers by removing cytotoxic drugs from the cell [2]. Some of these transporters actively exclude vital Hoechst dye in a verapamil-dependent manner, identifying a negative side-population (SP) by flow cytometry. SP was originally described by Goodell et al. [3] in self-renewing bone marrow cells, and have since then been demonstrated to reflect stem cells in various tissues [4]. MDR1 is constitutively expressed at high levels on stem and progenitor cell populations [5–8]. Similarly, over expression of MDR1 in murine bone marrow cells resulted in a marked increase in SP cell numbers [9]. Several human tumors and cancer cell lines, including three metastatic melanoma cell lines [10], were reported to contain SP cells [11–13]. It is yet unclear whether tumor cells that constitutively express MDR1 are stem cells. Here we show that primary cultures of human melanoma contain MDR1+ cells that are considerably more clonogenic,
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self-renewing, and anchorage independent when compared to the MDR cells. Materials and methods Melanoma primary cultures. Melanoma lesions were obtained from patients that signed an informed consent approved by the Israeli Ministry of Health. Primary cultures were prepared as previously described [14,15] from 15 lymph node, subcutaneous and lung melanoma metastases and one primary tumor (001) (patient information is summarized in supplemental table 1, and images of cultures are shown in supplemental figure 1). To enrich for melanoblasts, the precursors for melanocytes, the cells were plated in MCDB 153 medium (Sigma–Aldrich Corp., St. Louis, MO, USA) containing 10% chelated FBS, 2% FBS, 2 mM glutamine, 10 ng/ml stem cell factor (SCF), and 2.5 ng/ml basic fibroblast growth factor (bFGF) (both from R&D Systems Inc., Minneapolis, MN, USA), 100 nM endothelin 3 (EDN3) (American Peptide, Sunnyvale, CA, USA) [16]. Sphere formation was tested by plating the cells in either 6, or 96 multiwell ultra-low attachment plates (Corning Life Sciences, Wilkes Barre, PA, USA), with medium generally used to culture embryonic bodies from human embryonic cells, which contains knockout DMEM (Gibco-Invitrogen, Paisley, Scotland, UK), FBS defined (HiClone, Logan, UT, USA) 20%, L-glutamine, pen-strep, non-essential amino acids 10%, supplemented with bFGF 100 ng/ml, SCF 10 ng/ml, and EGF 100 ng/ml. All experiments were performed with low passage cultures to avoid clonal selection that may occur after prolonged ex vivo culturing.
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Cell staining and flow cytometry. Cells were detached by EDTA 0.2 M (Gibco-Invitrogen, Paisley, Scotland, UK), blocked, and then stained with primary antibody, or with the isotype-matched control. Cells were washed and stained with secondary antibody when needed, in addition to 7-aminoactinomycin D (7AAD, eBiosciences, San Diego, CA, USA) as a viability dye. The cells were analyzed on a FACSCalibur using the CellQuest software (both from BD Biosciences). Viable single cells were defined by their forward scatter/side scatter characteristics and 7AAD exclusion, thereby, excluding aggregates, debris, and dead cells from analysis. Negative staining was defined by replacing the antibody with its isotype-matched control. Cell sorting was performed by the FACSAria (BD Biosciences) digital cell sorter. Antibodies. The following antibodies were used: purified a-MDR1, clone MM 4.17, and its isotype-matched control, purified mouse IgG2a (Chemicon, Temecula, CA, USA). The MM 4.17 MAb recognizes an epitope localized in the external P-glycoprotein domain of human living MDR cells [17]; both antibody and isotype were detected by a secondary goat a-mouse Alexa fluor 647 or a-mouse Alexa fluor 488 (molecular probes, Invitrogen, Paisley, Scotland, UK); a-CD133:PE, cKit (CD117):APC, MCSP:PE and their isotype-matched controls (Miltenyi Biotech, Bergisch Gladbach, Germany); anti nestin:PE, ABCG2:APC, MRP1:FITC and their isotype controls (R&D Systems Inc., Minneapolis, MN, USA). In vitro limiting dilution analysis. Limiting dilution assay on tumor cells that were sorted for MDR1 expression or lack of MDR1 expression was performed similarly as previously described [18,19]. Briefly, sorted cells were plated in 96-well microwell plates (Greiner Bio-One, Mediscan,
Fig. 1. MDR1+ cell fraction in human melanoma. (A) Flow cytometry analysis of the expression of MDR1 melanoma primary cultures (six representative dot plots). Dead cells, debris, and aggregates were excluded from analysis. Quadrant lines were drawn according to staining of isotype-matched control. (B) MDR1+ cell fraction in human melanoma primary cultures. Some of the primary cultures exhibited a relatively large dim population (% MDR low), in addition to the rare bright MDR population (% MDR high). (C) Flow cytometry analysis of the ABC transporters, ABCG2 and MRP1 showing that they are not widely expressed in the melanoma cultures tested. (D) Flow cytometry analysis of MDR1 expression, showing co-expression with MCSP and cKit.
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Kremsmunster, Austria) in 150 ll of culture media, at 0.3 or 1 cells per well dilution. The number of colonized wells was recorded. To test selfrenewal capacity the cells in each well were replated in a 1:10 dilution into a new 24 well. RNA extraction and quantitative real time PCR (qRT-PCR). Cells were harvested or sorted as indicated and RNA was extracted using RNeasy Micro kit (Qiagen, Hilden, Germany). RNA was transcribed into cDNA using random hexamers and the High Capacity Reverse Transcription kit from Applied Biosystems (Foster city, CA, USA). All qRT-PCR reactions were performed on a 7900HT ABI platform using primers and TaqMan probes (validated assays on demand) from Applied Biosystems in conjunction with the TaqMan Universal Master Mix purchased from the same manufacturer. The endogenous control was GAPDH. For each gene, the relative expression ratio was calculated by the DDCt method. The value gives the expression levels for each gene relative to the expression of the particular gene in the matched MDR1 fraction, unless indicated otherwise.
expressed ABCG2 and MRP1 (Fig. 1C). The size of the subpopulation of cells expressing high levels of MDR1 varied between 1.3% and 9.7% of the viable single cells (Fig. 1B). The MDR1+ cell-subset was found even in patients that did not receive therapy (patients 003, 005, 008, and 010) indicating that it is an inherent chemotherapy-independent feature of melanoma. All MDR1+ cells expressed cKit, the SCF receptor, often expressed in melanoma cells (Fig. 1D). Additionally, 89% of the MDR1+ cells co-expressed the melanoma-associated chondroitin sulfate proteoglycan (MCSP), a melanoma-specific marker (Fig. 1D), indicating that these are tumor cells. In contrast to previous reports most melanoma cultures tested did not express CD133 (supplemental figure 2).
Results
Enrichment of MDR1+ cells in precursor enriching conditions
A subpopulation of MDR1+ cells is present within primary melanoma cell cultures We tested whether the primary cultures contained cells expressing drug resistance proteins such as ABCG2, MRP1, and MDR1 that have been implicated in normal stem cell function. All primary melanoma cultures tested (obtained from 10 patients) contained a fraction of MDR1+ cells when analyzed by flow cytometry (Fig. 1A). In contrast, none of the cell samples tested
Precursor enrichment
We have cultured the melanoma derived melanocytes in conditions that were designed to enrich for melanocyte stem cells [16], in order to test whether an enrichment of the MDR1+ subpopulation occurs. Similarly to the normal melanoblasts, in our system, the melanoma cultures also lost their pigmentation, thus exhibiting a typical de-differentiated phenotype (Fig. 2A). Furthermore, the number of cells expressing MDR1 increased from 4.5±1% of the viable single cells in regular conditions to 14.6±1.9% in
Regular conditions
Relative expression
MDR1 expression in sorted fractions (qRT PCR) 2.75 2.55 2.35 2.15 1.95 1.75 1.55 1.35 1.15 0.95
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Fig. 2. MDR1 cell fraction is enriched in precursor enriching conditions and can be isolated by sorting. (A) The cell pellet of melanoma cells cultured in precursor enriching conditions was unpigmented, indicating an immature phenotype. In contrast, the cells cultured in the regular RPMI media were pigmented indicating differentiated phenotype. (B) Flow cytometry analysis of MDR1 expressing cell fraction shows an increase in the number of MDR+ cells in precursor enriching conditions. (C) Sorting of MDR1+ cells using flow cytometry. (D) Real Time PCR of the sorted fractions with primers for MDR1 indicating the high specificity of the a-MDR1 antibody used for sorting.
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that MDR1+ subpopulation was enriched with self-renewing cells as compared with the MDR1 fraction (Fig. 3A). This enhanced clonogenic as well as self-renewal capacity of the MDR1+ fraction was observed in all the four different primary cultures (from patients 007, 008, 009, and 02) we tested. In addition, anchorage independence, a hallmark of cancer, was tested in ultra-low attachment plates following sorting. The cultures originating from 1000 MDR1+ cells/well gave rise to more spheres compared to cultures originating from the same number of isolated MDR1 cells/well, as shown in Fig. 3C and D, respectively. While 100 MDR1 cells/well did not yield spheres, the same number of MDR1+ cells formed spheres as demonstrated in Fig. 3F and E. When cells were plated after sorting in 96-well plate, either at 1 or 5 cells per well, more spheres were formed from MDR1+ cells, than from MDR1 cells (Fig. 3B). The increased sphere forming ability of MDR1+ melanoma cells represents anchorage independence, a key cancer related function essential to metastasis formation. This phenomenon was observed in all the three different melanoma primary cultures tested (patients 05, 007, and 003). Thus, MDR1+ melanoma cells demonstrate
de-differentiating conditions, as seen in melanoma cultures obtained from four different patients (009, 13, 02, and 001). A representative plot (patient 009) is shown in Fig. 2B. Therefore, MDR expression positively correlates with an immature and unmelanized phenotype of the cells. MDR1+ cells exhibit increased potential for clonogenicity, self-renewal, and anchorage independence We inquired whether the expression of MDR1 marks cancer stem cells, similarly to its expression in normal stem cells. To this aim we tested the ability of sorted melanoma cells to form colonies, self-renew, and form spheres in culture. We isolated MDR1+ and MDR cells by flow cytometry (Fig. 2C). In concert with the FACS data, qRT-PCR validated the enrichment of MDR1 expression in the MDR1+ fraction as compared with the MDR1 cell fraction (Fig. 2D). We used the limiting dilution assay to determine clonogenicity and self-renewal of the different fractions. We found that the MDR1+ fraction contained more clonogenic cells than the MDR1 fraction (Fig. 3A). Serial replating of the colonized wells showed
Sphere forming in low density (1, 5 cells/well)
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C
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Fig. 3. Enhanced clonogenicity, self-renewal capacity, and anchorage independence of MDR+ cells. (A) Limiting dilution assay for clonogenicity and selfrenewal. The sorted fractions were plated as a single cell per well in the limiting dilution assay and the number of colonized wells was recorded, as shown in the first data point, P0, which represents the clonogenicity. In order to test self-renewal, cells in the colonized wells were serially replated. These data were obtained with cells from patients 007, 008, 009, and 02. (B) The number of wells populated with spheres 4 weeks following plating of either single cell per well or 5 cells per well. A representative of three different experiments (Patients 05, 007, and 003). (C–F) 1000 and 100 cells from each fraction (Patient 05) were plated in ultra-low attachment 6-well plates for 14 days.
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higher clonogenicity, self-renewal capacity, and anchorage independence which constitute three essential cancer stem cell functions.
indicating higher survival skills and self-renewal capacity. The functional assay of self-renewal, limiting dilution in a single cell setting and serial replating, demonstrated that MDR1+ cells have unlimited self-renewal capacity. In addition, anchorage independence was shown by the ability of single MDR1+ cells to form spheres in low attachment conditions. Interestingly, the difference between the two cell types became more evident as the serial replating proceeded suggesting that while the MDR1+ fraction contains selfrenewing stem cells, the MDR1 cell fraction contains aside from non-stem cells also progenitors that can form colonies that cannot be further passaged. Thus, not only do MDR1+ cells have the stem cell functions of clonogenicity and self-renewal but they also possess an important malignant function of anchorage independence. Following sorting we verified that two additional ABC transporters, ABCB5 and ABCC2 that are expressed in melanoma, were enriched in the MDR1+ cell fraction. In this context, Schatton et al. recently showed tumor initiating melanoma xenograft cells expressing ABCB5, an ABC transporter [22]. However, the study lacks in vitro demonstration of clonogenicity, self-renewal, and anchorage independence in a setting that is independent of mice microenvironment. In addition, the ABCB5+ fraction was tumorigenic in no less than 100,000 cells although the fraction size was approximately 30%. In contrast, our experiments were performed in a single cell setting and the MDR1+ fraction size is 0.4–3%. Our study provides substantiation for the stem cell properties of cells expressing ABC transporters. Furthermore, we show that MDR+ cells co-express ABCB5 and stem cell markers such as hTERT
Co-expression of ABCB5, ABCC2, hTERT, and nanog in MDR1+ cells ABCB5 was implicated with drug resistance in melanoma [20] and ABCC2 was shown to be expressed in melanoma [21]. Using a qRT-PCR, we found that ABCB5 and ABCC2 are co-expressed with MDR1 (Fig. 4A), thus reinforcing the role of MDR1+ cell population in melanoma. The augmentation of drug resistance gene expression can provide cells with increased survival ability. Self-renewal and telomerase activity are additional mechanisms for cell longevity. Indeed, as shown in Fig. 4B the expression of hTERT was higher in MDR1+ cells, thereby increasing their immortality potential. Nanog levels were increased in MDR1+ cells as well (Fig. 4C). The elevation in survival and stemness genes in MDR1+ cells when compared to MDR cells supports a stem-like function of MDR+ cells in melanoma. Discussion Here we demonstrate that the expression of MDR1 marks a melanoma self-renewing population. We found that stem cell enriching culture conditions resulted in enrichment of the MDR1+ cell fraction. We found coexpression of MDR1 with other ABC transporters such as ABCB5, ABCC2, as well as with hTERT and nanog,
1.95 1.85 1.75 1.65 1.55 1.45 1.35 1.25 1.15 1.05 0.95
hTERT expression in MDR1+ cells MDR+ MDR-
ABCB5
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ABCB5 and ABCC2 expression in MDR1+ cells 3.7
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Nanog expression in MDR1 cells Relative expression
3.7
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3.2 2.7 2.2 1.7 1.2 0.7 02
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Fig. 4. (A) Relative levels of ABCB5, ABCC2, (B) hTERT, and (C) nanog in MDR+ cells compared to MDR cells determined by qRT-PCR.
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and nanog. The present finding that an MDR1+ fraction exists in each of the 13 different primary melanoma cultures we analyzed suggests that this marker may be used as a general marker to identify melanoma stem cells. Notably, five of the patients, whose melanoma cells were tested and found to contain a MDR1+ fraction, were not exposed to chemotherapy prior to obtaining the tumor tissue. This indicates that MDR1 expression is an innate characteristic of the resting tumor stem cell. The cancer stem cell expresses constitutive MDR activity, which is independent of chemotherapeutic-drug exposure, and is down-regulated in more differentiated tumor progeny [23]. It should be noted however, that chemotherapy imposes a selection pressure which leads to MDR upregulation in the bulk of the tumor through either gene amplification or rearrangement [24,25]. In this study, we found that expression levels of hTERT were elevated in MDR+ cell fraction from all cultures tested. This is consistent with the work of Ho et al. [26], that showed elevated hTERT levels in the SP of lung cancer, where it has been implicated in malignant transformation and tumor invasion. hTERT stabilizes the shortening chromosomes. In adults, telomerase remains active only in immature germ cells, certain stem, and progenitor cell compartments [27]. In line with the established roles for telomerase in tumor initiation and cellular immortalization [28], the majority of human cancers over express telomerase [29]. Nanog, an embryonic stem cell marker was found here to be increased in MDR1+ cells. Interestingly, Seigel et al. found colocalization of nanog and ABCG2, an ABC transporter, in retinoblastoma. Therefore, nanog may be involved in self-renewal mechanisms in cancer as well as in embryonic stem cells. In conclusion, our study demonstrates the existence of self-renewing melanoma cells marked by the functional marker MDR1. These cells are enriched in precursor promoting conditions and possess higher clonogenic and selfrenewal capacity, in addition to anchorage independence, compared to the MDR1 population. Strikingly, these stem-like cancer cells express higher levels of hTERT, nanog, and ABCB5. Future studies that will further characterize these putative melanoma stem cells may contribute to the development of therapies that specifically target this unique self-renewing cell fraction. Acknowledgments We thank the Kahn Family Foundation and N. and C. Lemelbaum for their support. We thank A. Ovcharenko for excellent technical work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc. 2008.02.022.
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