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Am J Pathol. Jan 2010; 176(1): 381–392.
PMCID: PMC2797898

Endometrial Cancer Side-Population Cells Show Prominent Migration and Have a Potential to Differentiate into the Mesenchymal Cell Lineage

Abstract

Cancer stem-like cell subpopulations, referred to as “side-population” (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and, uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, α-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into α-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.

Recently, adult stem cells have been identified in several mature tissues, such as the adult intestine,1 skin,2 muscle,3 blood,4 and the nervous system5–7 A stem cell is an undifferentiated cell that is defined by its ability to both self-renew and to produce mature progeny cells.8 Stem cells are classified based on their developmental potential as totipotent, pluripotent, oligopotent, and unipotent. Adult somatic stem cells were originally thought to be tissue specific and only able to give rise to progeny cells corresponding to their tissue of origin. Recent studies, however, have shown that adult mammalian stem cells are able to differentiate across tissue lineage boundaries,9,10 although this “plasticity” of adult somatic stem cells remains controversial.

Stem cell subpopulations (“side-population” (SP) cells) have been identified in many mammals, including humans, based on the ability of these cells to efflux the fluorescent dye Hoechst 33342.11 Recent evidence suggests that the SP phenotype is associated with a high expression level of the ATP-binding cassette transporter protein ABCG2/Bcrp1.12 Most recently, established malignant cell lines, which have been maintained for many years in culture, have also been shown to contain SP cells as a minor subpopulation.13

The human endometrium is a highly dynamic tissue undergoing cycles of growth, differentiation, shedding, and regeneration throughout the reproductive life of women. Endometrial adult stem/progenitor cells are likely responsible for endometrial regeneration.14 Rare populations of human endometrial epithelial and stromal colony-forming cells15 and SP cells16,17 have been identified. Although coexpression of CD146 and PDGFRβ isolates a population of mesenchymal stem like cells from human endometrium,18 specific stem cell markers of endometrium remain unclear. Recently, Gotte et al19 demonstrated that the adult stem cell marker Musashi-1 was coexpressed with Notch-1 in a subpopulation of endometrial cells. Furthermore, they showed that telomerase and Musashi-1-expressing cells were significantly increased in proliferative endometrium, endometriosis, and endometrial carcinoma tissue, compared with secretary endometrium, suggesting the concept of a stem cell origin of endometriosis and endometrial carcinoma.

Recent evidence suggests that cancer stem-like cells exist in several malignant tumors, such as leukemia20,21 breast cancer,22 and brain tumors,23 and that these stem cells express surface markers similar to those expressed by normal stem cells in each tissue.20,24

Development of endometrial carcinoma is associated with a variety of genetic alterations. For example, increased expression and activity of telomerase25,26 and frequent dysregulation of signaling pathways have been observed in endometrial carcinoma. Some of these pathways are important determinants of stem cell activity (Wnt-β-catenin and PTEN).27–29 These suggest a stem cell contribution to endometrial carcinoma development.

Recently, we isolated SP cells from the human endometrium. These SP cells showed long-term proliferating capacity in cultures and produced both gland and stromal-like cells. Additionally, they were able to function as progenitor cells.16 In this study, we isolated and characterized SP cells from human endometrial cancer cells and from rat endometrial cells expressing oncogenic [12Val] human K-Ras protein and demonstrated their cancer stem-like cell phenotypes.

Materials and Methods

Plasmid

pZIP-Neo SV(X)1 containing [12Val] human K-ras 4B cDNA was a gift from Dr. C. Der (University of North Carolina, Chapel Hill, NC).30,31 The pZeo vector was purchased from Invitrogen (Carlsbad, CA). We cut the 1.1-kb fragment containing [12Val] human K-ras 4B cDNA from the pZIP-Neo SX (X)1 construct with BamHI and ligated it to the BamHI site of pZeo vector.

Cell Culture

An endometrial cancer cell line (Hec-1) and a rat endometrial cell line (RENT4) were used in the present study. The Hec-1 cell line was established by Kuramoto et al32 from explants of adenocarcinoma of human endometrium. RENT4 cells were established by Wiehle et al33 and obtained from the European Collection of Cell Cultures. Hec1 cells expressed CD9 and Tie-2 but not CD13 or α-smooth muscle actin (α-SMA). Although Tie-2 is an endothelial cell marker, it has been reported that Tie-2 is expressed in glandular cells of both normal endometrium and endometrial adenocarcinoma.34 These results demonstrated that Hec-1 cells exhibited the phenotype of endometrial glandular cells and not stromal cells. RENT4 cells expressed Tie2, CD9, and CD13. α-SMA were stained weakly in RENT4 cells (Supplemental Figure S1, see http://ajp.amjpathol.org).

RENT4 cells harboring mutant [12Val] versions of K-ras4B were established by transfecting RENT4 cells with pZeo constructs, containing cDNA sequences encoding [12Val] K-Ras using Lipofectoamine (Invitrogen, Tokyo, Japan). Stably transfected cells were selected and isolated in growth medium containing 400 μg/ml Zeocin (Invitrogen) to establish cell lines expressing K-Ras protein, previously described elsewhere.35 Pooled populations were used for the assay. Cells were cultured with Dulbecco's modified Eagle's medium (DMEM) (Nissui, Seika, Japan) supplemented with 20 μg/ml Gly-His-Lys, 2 mmol/L glutamine, 80 IU insulin (Sigma-Aldrich, St. Louis, MO), and 10% fetal calf serum (FCS) (Hyclone, Logan, UT).33

Preparation of Human Endometrial Cancer Cells

Endometrial cancer tissues were obtained from uteri after hysterectomy. This study was approved by the ethical committee of Kyushu University, and preoperative informed consent was obtained from each patient. Cell suspensions of endometrial cancer cells were obtained using enzymatic digestion and mechanical means. The endometrial cancer tissues were diced finely and dissociated in HBSS containing HEPES (25 mmol), penicillin (200 U/ml), streptomycin (200 μg/ml), and collagenase (1 mg/ml, 15 U/mg) (Sigma-Aldrich) for 30 minutes at 37°C with agitation. The dispersed endometrial cancer cells were separated by filtration through a wire sieve.

Isolation of SP Cells

To identify and isolate Hec1 and RK12 V SP cells, the cells were dislodged from the culture dishes with trypsin and EDTA, washed, and suspended at a concentration of 106 cells/ml in DMEM containing 2% FCS. The cells were then labeled in the same medium at 37°C for 90 minutes with 2.5 μg/ml Hoechst 33342 dye (Molecular Probes, Eugene, OR), either alone or in combination with 50 μmol/L verapamil (Sigma-Aldrich). Finally, the cells were counterstained with 1 μg/ml propidium iodide to label dead cells. The cells were then analyzed in a FACS Vantage fluorescence-activated cell sorter (BD Biosciences, San Jose, CA) or the EPICS ALTRA HyPerSort (Beckman Coulter, Fullerton, CA) using dual wavelength analysis (blue, 424–444 nm; red, 675 nm) after excitation with 350 nm of UV light. Propidium iodide-positive dead cells were excluded from the analysis.

The SP cells were separated by FACS from the non-SP (NSP) cells and both fractions were seeded in a mesenchymal stem cell maintenance medium (MF medium) (Toyobo, Osaka, Japan) and 10% FCS on a collagen-coated 24-well plate (2 cm2) (Iwaki, Funabashi, Japan). The cells were cultured for 2 to 4 weeks. The cells were then transferred to collagen-coated plates (60 mm).

Growth Rate Assay

Cells were plated in a mesenchymal stem cell maintenance medium (MF medium; Toyobo). Cells were passaged during the 2 months of cultivation. Viable cells were counted for 2 months. Cell counts were performed using a hemocytometer.

Self-Renewal Assays

SP cells or NSP cells were plated in 24-well collagen-coated dishes (50 cells/cm2). SP cells, but not NSP cells, formed colonies. Cells from individual colonies of SP cells were reseeded at 300 cells/cm2 in triplicate in 100-mm collagen-coated dishes to generate colonies. Colonies were monitored to ensure they were derived from single cells. The secondary colonies were reseeded in a similar manner to generate tertiary colonies. The cloning plates were stained with the crystal violet solution (Sigma-Aldrich).

In Vivo Tumor Formation Assay

We inoculated 1 × 104 cells in Matrigel (BD Matrigel Basement Membrane Matrix High Concentration; BD Biosciences, Bedford, MA) into the s.c. connective tissue of 5-week-old nude mice (Balb nu/nu). After 6 weeks (RK12V- SP and NSP cells) or 3 months (Hec1-SP and -NSP cells), mice were sacrificed and the tumors excised. All mouse experiments were approved by the animal ethics committee of Kyushu University.

Three-Dimensional Cell Culture

Cells (1 × 105) (Hec1-SP or -NSP cells) were seeded in Matrigel in a Cell Culture Insert (0.4 mm) (BD Falcon, Franklin Lakes, NJ) and incubated with either an MF medium, an ordinary growth medium (DMEM containing 10% FCS), or a smooth muscle cell differentiation medium (BD Biosciences). The smooth muscle cell differentiation medium consisted of MCDB 131 basal medium supplemented with insulin, transferrin, selenous acid, linoleic acid, and bovine serum albumin. After 8 weeks, each Matrigel sample was fixed by treatment with 4% paraformaldehyde and analyzed by immunohistochemistry.

Time-Lapse Video Imaging

Hec1-SP and -NSP cells were incubated with mesenchymal stem cell maintenance medium (MF medium; Toyobo) in a collagen-coated 24-well plate. Cells were observed with a ×10 immersion objective every minute for 48 hours under 5% CO2 at 37°C incubation, using a real-time cultured cell monitoring system (Astec, Fukuoka, Japan).

Antibodies

Primary antibodies used in this study were as follows: CD9 polyclonal antibody (H-110), CD13 monoclonal antibody (3D8), vimentin monoclonal antibody (V9) and collagen III polyclonal antibody (H-300), and Tie-2 polyclonal antibody (C-20) (all obtained from Santa Cruz Biotechnology, Santa Cruz, CA). α-SMA monoclonal antibody (1A4) was purchased from MBL (Nagoya, Japan).

Immunohistochemistry and Immunofluorescent Stain

Formalin-fixed histological tumor sections from nude mice, human endometrial cancer tissues, or cultured cells were used. Cultured cells were incubated on glass chamber slides (Lab-Tek; Nalge Nunc International, Naperville, IL) and fixed by treatment with 10% formalin. Sections or cells were rinsed twice in PBS (pH 7.4) for 5 minutes each. Samples were then incubated with 4% blocking horse serum (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature in a humidified chamber, followed by incubation with the primary antibody (200 μg/ml, 1/100 diluted). We also used nonimmune mouse or rabbit IgG as a control for the primary antibody. Staining with the primary or control antibody was performed overnight at 4°C. Bound antibodies were detected with a biotinylated anti-rabbit IgG secondary antibody (1.5 mg/ml) and an avidin-biotin complex linked to horseradish peroxidase (Vectastain; Vector Laboratories), followed by incubation with diaminobenzidine tetrahydrochloride as the substrate. In immunofluorescent stain, bound vimentin antibodies were visualized by green fluorescence from secondary antibody staining with anti-mouse IgG conjugated to Alexa 488 (Molecular Probes). Cell nuclei were counterstained with 4′,6-diamidino-2-phenyl-indole. The level of vimentin in a semiquantitative manner was determined by calculating fluorescent intensity per three different positive or negative areas using a BIOREVO BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).

Microdissection and DNA Extraction

Glass slides with an overlay of 4 mm of thin LM Film (PALM Microlaser Technologies, Bernried, Germany) were prepared. Formalin-fixed, paraffin-embedded tumor tissue was cut into 7-μm sections and placed on the slides. The sections were then deparaffinized and stained with H&E. Using a laser microdissection system (Leica Microsystems, Wetzlar, Germany), tumor cells or stromal cells were isolated into the cap of a 0.5-ml microtube. After retrieval of the cells, 50 μl of proteinase K solution (Pico Pure DNA Extraction kit; Arcturus, Mountain View, CA) was added into the cap. The DNA was extracted by overnight incubation at 65°C. The solution was then boiled for 10 minutes to inactivate the proteinase K.

PCR of the K-ras Gene

To amplify the K-ras gene (human genomic DNA for Hec1-SP tumor cells or cDNA for RK12V-SP tumor), PCR using a T3000 thermal cycler was performed (Biometra, Floral City, FL). The primers used for PCR were as follows: i) 5′-AAAAGGTACTGGTGGAGTATTTGA-3′ (sense), 5′-TTGAAACCCAAGGTACATTTCA-3′ (antisense) for human genomic K-ras DNA; and ii) 5′-GACTGAATATAAACTT-3′ (sense), 5′-CATAATTACACACTTTGTCTT-3′ for human K-ras cDNA. The PCR cycling conditions were as follows: i) preheating for 2 minutes at 94°C, 39 cycles of denaturation for 1 minute at 94°C, annealing for 30 seconds at 59.3°C, and extension for 1 minute at 72°C; ii) preheating for 2 minutes at 94°C, 39 cycles of denaturation for 1 minute at 94°C, annealing for 30 seconds at 59.3°C, and extension for 1 minute at 72°C. After the last cycle, a final extension of 5 minutes at 72°C was added.

Determination of the K-ras Sequence

PCR products were electrophoresed on a 2% agarose gel, and the band corresponding to the desired target was cut from the gel. DNA was extracted using the GFX PCR DNA gel band purification kit (GE Healthcare, Buckingham-Shire, UK). Direct sequencing of the PCR product was then performed using an ABI PRISM Big Dye Termination Ver3.1 Cycle Sequencing Kit according to the manufacturer's instructions and the ABI PRISM 3100 (Applied Biosystems). The primer used for sequencing was as follows: 5′-TTGAAACCCAAGGTACATTTCA-3′ (antisense) for K-ras DNA.

Fluorescence in Situ Hybridization

Fluorescence in situ hybridization (FISH) studies were performed on paraffin-embedded Hec1-SP tumor tissues. Tissue sections were deparaffinized and rehydrated according to standard protocols. After washing with distilled water, the slides were baked in a high-pressure cooker with distilled water for 3 minutes. After washing with distilled water, the slides were digested in pepsin at 37°C for 30 minutes, washed with distilled water, dehydrated with ethanol, and air-dried for 10 minutes, followed by 50°C oven for 10 minutes. A total of 1.4 μl of the FISH probes (DEP X Spectrum Orange, Vysis, Downers Grove, IL; Mouse Pan-centromeric Chromosome Paint, Cambio, Cambridge, UK) was applied onto the slides, and denaturation was performed at 80°C for 30 minutes. The hybridization was performed at 37°C in a humid chamber for 2 days. The excess of the probes was washed with 0.4× standard saline citrate/0.3% IGEPAL at 72°C for 2 minutes, followed by 2× standard saline citrate/0.1% IGEPAL at room temperature for 1 minute and 2× standard saline citrate for 5 minutes. The slides were mounted with antifade solution with 4′,6-diamidino-2-phenyl-indole and checked under fluorescence microscopy.

Data Analysis

Data are represented as the means ± SEM and were analyzed with Student's t-test. A value of P < 0.05 was considered statistically significant.

Results

Isolation of SP Cells from Human Endometrial Cancer Cells

We first analyzed primary endometrial cancer cells freshly isolated from endometrial cancer tissues after 7 days of cultivation (n = 7; Table 1) and a human endometrial cancer cell line, Hec1, by FACS. SP cells were present in both cells (0.20 ± 0.09% in primary endometrial cancer cells from seven cases and 0.63 ± 0.55% in Hec1 cells from 10 independent experiments). Verapamil blocked the dye efflux, increased staining, and made the SP cells undetectable by FACS (Figure 1A). Next, both Hec1-SP cells and -NSP cells were cultured for 2 weeks, stained with Hoechst 33342, and then reanalyzed by FACS. Hec1-SP cell cultures generated both SP and NSP subpopulations. In contrast, NSP cell cultures produced NSP cells but never produced SP cells (Figure 1B).

Figure 1
Isolation of SP cells from human endometrial cancer cells. A: SP cells were present in primary endometrial cancer cells and Hec1 cells (0.20 ± 0.09% in primary endometrial cancer cells from seven cases and 0.63 ± 0.55% ...
Table 1
Patient Characteristics

We previously demonstrated that CD9 is expressed in glandular cells, and CD13 is expressed in both glandular and stromal cells that was stained more intensely in stromal cells than that in glandular cells in the human endometrium.16 Most previously characterized SP cells from human endometrium are negative for both CD9 and CD13.16 We investigated whether this was also the case in the endometrial cancer cell lines by analyzing the expression profiles of CD9 and CD13 in SP and NSP cells isolated from Hec1 cells by immunohistochemistry following 3 days of culture. The expression levels of both CD9 and CD13 were lower in SP cells than in NSP cells (Figure 1C).

SP Cells Demonstrate the Capacity for Both Long-Term Proliferating Capacity of Cell Cultures and Self-Renewal

SP cells and NSP cells derived from Hec1 cells were cultured on collagen-coated plates in mesenchymal stem cell maintenance medium (MF medium). Cell growth rate was analyzed for 2 months. Both cell types grew after 2 weeks of culture. SP cells lost contact inhibition, continued to divide for 2 months, and accumulated in colonies atop the confluent cell layer. In contrast, NSP cells stopped growing following rapid growth for 2 weeks cultures and the total cell number decreased. Finally, the cells became flat and enlarged after 2 months (Figure 2A). These results show that SP cells, but not NSP cells, maintain the capacity for long-term proliferating capacity of the cell culture.

Figure 2
SP cells demonstrated the capacity for long-term proliferation and self-renewal. A: SP cells and NSP cells derived from Hec-1 cells were cultured on collagen-coated plates in mesenchymal stem cell maintenance medium (MF medium) for 2 months. Cell growth ...

When Hec1-SP cells were plated in 24-well collagen-coated dishes (50 cells/cm2), they proliferated and formed colonies (Figure 2Ba). Hec1-NSP cells plated in a similar fashion. The cells were more loosely arranged and did not form well-separated individual colonies (Figure 2Ba). To test the self-renewal capability of SP cells within each colony, we dissociated the primary colonies into single cells and then cultured these cells in 100-mm collagen-coated dishes (300 cells/cm2). A single cell formed a secondary colony (Figure 2B, b and c). The secondary colonies were reseeded in a similar manner to generate tertiary colonies. We showed the stained secondary cloning plates. SP cells produced two types of colonies, large densely packed and small colonies (Figure 2C, upper panel). Large colonies were picked up for the serial cloning. On average, 520 secondary colonies were formed per 2 × 104 seeded single cells (2.6%), and an average of 390 tertiary colonies were formed per 2 × 104 seeded single cells from the secondary colonies (1.9%) (Figure 2C, lower panel). This indicates that the colony-forming cells isolated from existing colonies retain the same colony-forming potential and self-renewal capability of the primary SP cells. NSP cells also produced secondary clones, but there was much overlap. They did not form well-separated individual colonies.

SP Cells Show Lamellipodia and Uropodia Formation and Prominent Migration

Cellular activity was monitored with time-lapse video imaging studies. The frequency of cell division was higher in the Hec1-SP cells than in the Hec1-NSP cells. Interestingly, SP cells showed lamellipodia formation at the leading edge and uropodia formation at the trailing edge (Figure 3) and prominent migration (Supplemental Movie S1, see http://ajp.amjpathol.org). NSP cells did not show podia formation and migration (Supplemental Movie S2, see http://ajp.amjpathol.org).

Figure 3
Hec1-SP cells showed podia formation and prominent migration. In time-lapse video imaging studies, the frequency of cell division was higher in Hec1-SP cells than in Hec1-NSP cells. Hec1-SP cells showed podia formation (lamellipodia at the leading edge, ...

Tumorigenicity Is Enhanced in Hec 1-SP Cells

Hec1-SP cells or -NSP cells were inoculated into the s.c. tissues of nude mice. SP cells and NSP cells formed palpable tumors after 4 and 10 weeks of inoculation, respectively. Tumors generated from the SP cells grew faster than those from the NSP cells, and there is a trend to an increased size of tumors from SP cells compared with NSP cells (Figure 4A, a and b). The tumors were excised after 12 weeks of inoculation (Figure 4Ac). Tumors generated from SP cells (SP tumor) invaded into the surrounding tissues (spine, peritoneum, leg, and bone) and were difficult to resect. In contrast, the tumors from NSP cells (NSP tumor) were encapsulated and clearly separated from the basement membrane of mouse skin (shown in Figure 4B; NSP, ×20). The weights of the SP tumors and the NSP tumors after 12 weeks of inoculation were 1.72 ± 0.54 and 0.18 ± 0.10 g, respectively (Figure 4Ad).

Figure 4
Tumorigenicity was enhanced in Hec1-SP cells. A: SP cells or NSP cells were inoculated into the s.c. tissues of nude mice. Left: There was a trend to an increased size of tumor from SP cells compared with NSP cells, although it was masked by large variation ...

We repeated the experiments three times. The characteristics of tumors from the SP cells and NSP cells were similar in each experiment.

H&E staining showed that the SP tumors were composed of tumor cells, surrounded by stromal-like tissues with a markedly enriched extracellular matrix (ECM) (Figure 4B, left panels). In contrast, most NSP tumors were composed of well-encapsulated tumor cells that were not accompanied by the ECM-enriched stroma (Figure 4B, right panels).

Next, we used a rat endometrial cell line expressing activated [12Val] K-Ras (RK12V cells), which also showed transformed phenotypes.35 SP cells were also present in RK12V cells (1.4 ± 1.7% from four independent experiments) as well as Hec1 cells (Supplemental Figure S2A, see http://ajp.amjpathol.org). Analysis of the cell growth curve over 2 months demonstrated that both RK12V-SP cells and -NSP cells proliferated for long-term cultivation, but SP cells grew faster than NSP cells (Supplemental Figure S2A, see http://ajp.amjpathol.org). The SP cells from RK12V cells generated an SP and an NSP subpopulation (data not shown). Both SP cells and NSP cells were cultured on collagen-coated dishes. SP cells attached to the plate and proliferated. Cell shape remained small and round. In contrast, NSP cells aggregated and formed gland-like structures (Supplemental Figure S2B, see http://ajp.amjpathol.org). Finally, SP cells formed a large solid area containing scant gland-like structures. NSP cells formed large gland-like structures after 2 months in cultures. The expression levels of CD9 and CD13 were lower in SP cells than in NSP cells (Supplemental Figure S2C, see http://ajp.amjpathol.org).

RK12V-SP cells and -NSP cells were inoculated s.c. in nude mice. SP cells formed large, invasive tumors with ECM-enriched stroma-like tissues. In contrast, the tumors generated from NSP cells were small and well encapsulated (Supplemental Figure S3, A and B, see http://ajp.amjpathol.org). These features were similar to those of the Hec1-SP or -NSP cell tumors.

The Developmental Potential of SP Cells to Mesenchymal Cell Lineages

We demonstrated that tumors generated from the SP cells of Hec1 cells and RK12V cells, but not the corresponding NSP cells, contained ECM-enriched, stroma-like tissues (Figure 4B; Supplemental Figure S3, see http://ajp.amjpathol.org). The ECM is thought to be secreted by stromal cells. Therefore, we analyzed the expression profiles of specific mesenchymal cellular markers, vimentin and α-SMA,36 and collagen III as components of ECM (Figure 4C). All vimentin, α-SMA, and collagen III stained more strongly in the Hec1-SP tumors than in the NSP tumors. We determined the level of vimentin staining in a semiquantitative manner using immunofluorescence staining by calculating fluorescent intensity per three independent areas. The mean fluorescence intensity per positive areas with vimentin staining was significantly higher in Hec1-SP tumors than Hec1-NSP tumors (SP: 115 ± 10.9 pixels versus NSP: 73 ± 8.2 pixels) (Supplemental Fig.S4, see http://ajp.amjpathol.org). The expression of proteins specific to mesenchymal cell lineages supported the idea that the ECM was the product from abundant stroma-like tissues in the SP tumors. Because the vimentin monoclonal antibody (V9) used in this study reacted with human and rat vimentin, but not mouse vimentin37 suggesting the possibility that vimentin positive, stromal-like tissues in the tumors originated from the inoculated SP cells.

To further confirm the origin of the stromal-like cells in the Hec1- or RK12V-SP tumors, we microdissected CD9-positive tumor cells and α-SMA-, CD13-positive, and CD9-negative stromal-like cells, respectively, and sequenced exon 1 (codons 27 to 35) in the K-ras gene (Figure 5, A and B). Several bases in this region differ between the human and mouse, enabling the origin determination of the cells. As expected, both tumor cells (data not shown) and the surrounding α-SMA-, CD13-positive, and CD9-negative stromal-like cells contained the human K-ras gene sequences (Figure 5C). Three different regions containing stromal-like cells in Hec1- and RK12V-SP tumors were microdissected. The human K-ras gene sequences were detected in all of them. These results clearly demonstrate that the surrounding stromal-like cells, at least in part, originate from the inoculated SP cells.

Figure 5
The developmental potential of SP cells to mesenchymal cell lineages. A and B: We microdissected CD9-positive tumor cells (b) and α-SMA-, CD13- positive, and CD9-negative surrounding stromal-like cells (a) in Hec1-SP tumors (magnification, ×20, ...

We also performed FISH assay on these two areas of Hec1-SP tumor tissues using the spectrum orange-labeled CEP X (α satellite) DNA probe, which hybridizes to the centromere of human chromosome X and the FITC-labeled DNA probe, which hybridizes to mouse pan-centromeric chromosome (Figure 6). We analyzed the signal numbers in three independent regions of tumor and stromal tissues. Only red signals (human chromosome) were detected in the area of tumor tissues without ECM. Both red signals and green signals (mouse chromosome) were observed in the area of stromal-like cells with enriched ECM. The ratio of cells with red signals was significantly more than that of cells with green signals in stroma-like tissues (red: 76 ± 4%, green: 24 ± 4% from three independent regions). These results clearly demonstrated that most of stromal-like cells were derived from the inoculated Hec1-SP cells.

Figure 6
Most of stromal-like cells were derived from the inoculated Hec1-SP cells. FISH assay was performed on Hec1-SP tumor tissues using the spectrum orange-labeled CEP X (α satellite) DNA probe, which hybridizes to the centromere of human chromosome ...

To further demonstrate the mesenchymal developmental potential of SP cells in vitro, 1 × 105 Hec1-SP or -NSP cells were seeded into Matrigel and incubated with either a mesenchymal stem cell maintenance medium (MF medium), DMEM containing 10% FCS, or a smooth muscle cell differentiation medium. After 8 weeks, the level of α-SMA expression was analyzed in each Matrigel sample (Figure 7). The SP cells cultured with DMEM containing 10% FCS and the smooth muscle cell differentiation medium expressed α-SMA, implying that SP cells differentiated to the smooth muscle cell lineage. Hec1-NSP cells did not proliferate in the smooth muscle cell differentiation medium. The SP cells incubated with MF medium, and the Hec1-NSP cells in MF medium and DMEM failed to express α-SMA protein. These results suggest that SP cells have the potential to develop α-SMA-expressing cells in a smooth muscle differentiation medium.

Figure 7
SP cells differentiated to α-SMA expressing cells. A total of 1 × 105 Hec1-SP or -NSP cells were cultured in Matrigel with a mesenchymal stem cell maintenance medium (MF medium), a standard growth medium (DMEM containing 10% FCS), ...

Discussion

In this study, we demonstrated that an SP subpopulation is present in cultures of human endometrial cancer cells and rat endometrial cells harboring activated [12Val] K-ras gene. The biological characteristics of SP cells were distinct from those of NSP cells. SP cells showed i) a reduction in the expression levels of differentiation markers (CD9 and CD13); ii) long-term proliferating capacity of the cell culture; iii) self-renewal capacity in vitro; iv) enhancement of migration as well as lamellipodia and uropodia formation; v) enhanced tumorigenicity; and vi) bipotent developmental potential (tumor cells and stroma-like cells).

We have recently demonstrated that SP cells exist in the normal human endometrium and function as progenitor cells.16 These SP cells have long-term proliferating capacity. Most SP cells are contained within the CD9CD13 fraction. In the present study, the expression levels of CD9 and CD13 were reduced in SP cells compared with the levels in NSP cells from Hec1 endometrial cancer cells.

SP cells lost contact inhibition and continued divide for 2 months. In turn, NSP cells stopped growing after 2 weeks. These suggest the capacity of SP cells that shows long-term proliferation capacity of the cell culture. Additionally, we demonstrated that Hec1-SP cells generated both an SP and an NSP subpopulation. In contrast, NSP cells produced only NSP cells (Figure 1B). Consistent with a previous report,38 these findings implicate the possibility that endometrial cancer-SP cells undergo asymmetric division and self-renewal capacity, although the accumulation of further data are required to conclude this interpretation.

We evaluated self-renewal capacity by analyzing colony-forming potential derived from a single cell. Hec1-SP cells produced well-separated individual colonies in the serial cloning and retain the same colony-forming potential (Figure 2C). In contrast, Hec1-NSP cells produced secondary clones, which were loosely arranged and not well separated. Because it is difficult to monitor to ensure the secondary clones of Hec1-NSP cells were derived from a single cell, we could not evaluate self-renewal capacity of Hec1-NSP cells.

Time-lapse imaging demonstrated that SP cells had enhanced migration and lamellipodia and uropodia formation. This is the first direct evidence showing prominent podia formation and migration by endometrial cancer SP cells. These characteristics may be associated with enhanced invasiveness and metastatic potential by cancer stem-like cells.

The tumors derived from SP cells of both Hec1 and RK12V cells grew rapidly and became much larger than those derived from NSP cells (Figure 4A; Supplemental Fig. S3, see http://ajp.amjpathol.org). One reason for these data is the greater long-term proliferative capacity of SP cells themselves shown by the in vitro-cultured cells (Figure 2A; Supplemental Fig. S2A, see http://ajp.amjpathol.org). Alternatively, SP cell-induced tumors show higher vascularity than that in NSP tumors, particularly as shown by α-SMA stainings (Figure 4C). Schwab et al18 demonstrated that mesenchymal stem cell-like cells in human endometrium were present near blood vessels, and Tsuji et al17 demonstrated BCRP/ABCG2, known as a marker of SP cells, was strongly expressed in the vascular endothelium. Although the origin of blood vessels in SP tumors remains unknown, a much better blood supply from blood vessels in SP tumors than in NSP tumors may be a factor of the increased size of SP tumors.

The biological natures of tumors formed in nude mice also differed between SP tumors and NSP tumors. In this study, we demonstrated that the tumors generated from SP cells, but not NSP cells, consisted of tumor tissues and an ECM enriched stromal-like components. Evidence exists that stromal cells such as inflammatory cells, vascular cells, and fibroblasts from the bone marrow give rise to a tumor matrix in response to growth factors or cytokines secreted from tumor cells or activated fibroblasts39,40 Alternatively, stromal cells may be derived from tumor cells that have undergone the epithelial mesenchymal transition (EMT).41,42

The stromal-like tissues, which were positively stained with vimentin, CD13, and α-SMA, contained human K-ras DNA sequences. FISH study demonstrated that both human chromosome signals and mouse chromosome signals were detected in the area of these stromal-like tissues with enriched ECM (human 76%, mouse 24%), implicating the possibility that most of these stromal-like cells are derived from inoculated SP cells. Additionally, SP cells had the potential to differentiate into α-SMA-expressing cells when seeded into Matrigel and incubated with a differentiation medium. These results may suggest the mesenchymal transition capability of endometrial cancer SP cells. This feature of endometrial SP cells is putatively involved in the development of endometrial stromal sarcoma or carcinosarcoma of uterus. However, it remains unknown whether this characteristic is specific to endometrial cancer stem-like cells or is common among cancer stem-like cells in other organs.

Recently, Friel et al43 showed that SP cells derived from two endometrial cancer cell lines (AN3CA and Ishikawa), although they did not find SP cells in Hec1 cells. AN3CA had features of cancer stem-like cells including low proliferative activity during 9 days of cultivation, chemoresistance, and enhanced tumorigenicity. They stated that the SP cells showed asymmetrical division and self-renewal, because both SP cells and non-SP cells were readily detected on resorting following 11 passages in culture. Furthermore, the relative proportion of SP cells in the total cell population was similar to that from the initial sort. These characteristics are very similar to those of the Hec1 SP cells in this study. The relatively lower proliferative activity of Hec1-SP cells compared with Hec1-NSP cells following 10 days of cultivation (Figure 2A) positively reflect the SP fraction with a greater percentage of cells in G1 phase, as shown in their study. Friel et al43 found that the cellular phenotype of serially transplanted tumors was histologically identical to the original endometrial carcinoma, and the great majority of cells was positive for Ep-CAM, an epithelial marker. Unlike these results, we showed that tumors derived from SP cells were composed of both tumor cells and ECM-enriched stroma-like cells (Figure 4B). Note, however, that they injected the whole-cell population from primary endometrial cancer, including a small population of SP cells and a large number of NSP cells. In contrast, we compared the histological phenotype of tumors derived from individual Hec1-SP cells and -NSP cells. Thus, it is not possible to compare the results of these two studies.

EMT is an important process during embryonic development as well as cancer progression.44 Mani et al45 have demonstrated that stem-like cells isolated from mammary carcinoma express EMT markers, supporting a direct link between the EMT and the development of an epithelial stem cell population. Most recently, Kabashima et al46 have shown that SP cells of pancreatic cancer cells are more susceptible to transforming growth factor-β-mediated EMT as compared with NSP-derived cells. Now, association EMT with the phenotype of migration enhancement and mesenchymal cell lineage differentiation in endometrial cancer SP cells is under investigation.

Acknowledgments

We thank Dr. Channing Der (University of North Carolina) for generously donating pZIP neo SV(X)1-K-Ras4B(12V) and Miwako Ando and Emiko Hori for technical assistance. We extend thanks to Drs. Nariyuki Saito and Koichi Akashi (Medical and Biosystemic Science, Kyushu University) for technical advice of FISH assay, Drs. Ryota Souzaki, Tatsuro Tajiri, and Tomoaki Taguchi (Department of Pediatric Surgery, Kyushu University) for technical advice of a laser microdissection, and Dr. Hiroyuki Kitao (Department of Molecular Oncology, Kyushu University) for technical advice of immunofluorescent stain.

Footnotes

Supported by Grants-in-aid 17390452 and 18659488 from the Ministry of Education, Culture, Sports, Science, and Technology (Japan).

Supplemental material for this article can be found on http://ajp.amjpathol.org.

Web Extra Material

Supplementary Fig. 1:

Supplementary Fig. 2:

Supplementary Fig. 3:

Supplementary Fig. 4:

Supplementary data legends:

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