Background/Aims: Cancer stem cells (CSCs) exhibit enhanced proliferative capacity and resistance to chemotherapy; however, choriocarcinoma CSCs have not yet been reported. In this study the human choriocarcinoma cell line JEG-3 was cultured in serum free media, and the characteristics of suspension and parental adherent JEG-3 cells were compared. Methods: Cell proliferation, colony-formation, soft agar clonogenicity, and transwell invasion assays were performed in vitro, and tumor xenografts in BALB/c nude mice were used to evaluate stem cell properties. Results: In serum-supplemented medium (SSM), JEG-3 cells were 4.51 ± 1.71% CD44+, 7.67 ± 2.67% CD133+, and 13.85 ± 2.95% ABCG2+. In serum-free medium (SFM), the expression of these markers increased to 53.08 ± 3.15%, 47.40 ± 2.67%, and 78.70 ± 7.16%, respectively. Moreover, suspension JEG-3 cells exhibited enhanced colony-formation capability as well as invasive and proliferative ability in vitro, alongside enhanced tumorigenic properties in vivo. Suspension JEG-3 cells also exhibited resistance to the chemotherapeutic drugs methotrexate, fluorouracil and etoposide. When seeded in serum supplemented medium, suspension JEG-3 cells readopted an adherent phenotype and continued to differentiate with no significant difference in the morphology between suspension and parent cells. Conclusion: In this study, choriocarcinoma stem-like cells (CSLCs) were isolated from the human choriocarcinoma JEG-3 cell line by SFM culture and characterized.

Choriocarcinoma is a malignant cancer of the uterus, cervix, ovary, or testis, characterized by abnormal proliferation of trophoblasts. Early metastasis is commonly observed in choriocarcinoma, as trophoblastic cells have an affinity for the haematogenous route, and lung and brain metastasis are common [1,2,3]. Despite its aggressiveness, choriocarcinoma is highly sensitive to chemotherapy, and metastatic choriocarcinoma has a cure rate exceeding 75% [4].

However, the optimal chemotherapeutic regimen remains controversial, and in recent years drug resistance has been reported in choriocarcinoma [5,6]. Therefore, a better understanding of the molecular mechanisms underlying choriocarcinoma invasion and resistance may inform the development of approaches to reduce metastasis or overcome chemoresistance in patients receiving systemic therapy for choriocarcinoma. In 2006, a consensus definition of cancer stem cells (CSCs) was developed at the Cancer Stem Cell Workshop of the AACR. CSC were described as “cells within a tumor that possess the capacity for self-renewal and that can cause the heterogeneous lineages of cancer cells that constitute the tumor” [7]. Tumor cells invasive and resistant to chemotherapy represent a subpopulation of cells from the original tumor that are molecularly and phenotypically distinct. Indeed, initiation, growth, recurrence, and metastasis of cancers are associated with the behavior of CSCs. Currently, it is believed that only this small fraction of cancer cells are able to drive tumor initiation, proliferation, and spread [8,9,10]. Biologically distinct populations of CSCs have been identified in most solid tumors, including ovarian cancer [11], prostate cancer [12] and human glioblastoma [13]. However the role of CSCs in choriocarcinoma has not yet been reported.

Several methods have previously been reported to identify and isolate CSCs, including fluorescence activated cells sorting (FACS), magnetic activated cells sorting (MACS), side population (SP) cell sorting method and serum-free suspension culture [14,15,16,17]. However, both FACS and MACS require knowledge of specific surface markers of CSCs, and widely accepted specific surface markers of CSCs have not yet been defined. Another widely used method, the SP cell sorting method, utilizes the specific expression of transmembrane channels on the stem cell membrane, such as ATP-binding cassette superfamily G member 2 (ABCG2) or breast cancer resistance protein - 1 (BCRP1), which can pump the fluorescent stains out of stem cells that thus appear comparatively weakly stained and can be isolated by flow cytometry. However, as Hoechst is toxic to cells, CSCs identified in this manner may exhibit decreased self-renewal, tumorigenicity, and other CSC properties [18].

In this study, we employed the human placenta choriocarcinoma cell line JEG-3 [19] to investigate the proliferative capacity and chemotherapeutic resistance of choriocarcinoma. JEG-3 cells were dissociated into a single-cell suspension, cultured without serum. The serum-free suspension culture method is based on supplementation of culture medium with growth factors to promote proliferation of undifferentiated cells, concomitantly with the elimination of differentiated cells. In recent years, this approach has been used to isolate many kinds of CSCs including prostate [20], retinal [21], and prostate [22] cancer stem cells. Serum-free suspension culture may allow isolation and culture of choriocarcinoma stemlike cells (CSLCs) from human choriocarcinoma cell lines.

JEG-3 in serum free suspension culture formed suspended cell spheres, the cells of which expressed the putative stem cell markers CD133, CD44 and ABCG2, and were defined as CSLCs. These CSLS exhibited enhanced tumorigenicity in a mouse model.

Cell culture

The human choriocarcinoma cell line JEG-3 was acquired from the American Type Culture Collection (ATCC, USA), and cultured in DMEM (Invitrogen, Australia) supplemented with 10% fetal calf serum (Invitrogen, Australia) in a humidified atmosphere of 5% CO2 and 37°C.

Logarithmic phase JEG-3 cells were digested in 0.25% trypsin, and once cells detached, digestion was terminated with serum free culture media (SFM) composed of DMEM/F12 (1:1) basal medium (Hyclone, USA) supplemented with 20 ng/ml human epidermal growth factor (EGF) (PeproTech, USA), 20 ng/ml basic fibroblast growth factor (bFGF) (PeproTech, USA), 0.4% bovine serum albumin (BSA) (Sigma, USA), 4 μg/ml insulin (Wanbang Biopharmaceuticals, China), 1:50 B27 supplement (Gibco/Invitrogen, Australia) and 100 U/ml penicillin (Beijing Solarbio Science, China). A single cell suspension of 5000-20000 JEG-3 cells was seeded in each well of a low adhesion 6 well plate. The medium was replaced every other day.

Cell sphere-formation was observed by inverted microscopy; when cell spheres expanded a 50-fold or so, the supernatant was collected, centrifuged at 500 rpm for 5 min, and the cell spheres were washed with phosphate-buffer solution (PBS) twice.

Differentiation of cell spheres into choriocarcinoma stem-like cells

Suspended cell spheres were cultured in RPMI supplemented with 10% fetal calf serum in a humidified atmosphere of 5% CO2 and 37°C. Adherent cells in the logarithmic phase were resuspended in SFM.

Colony formation assay

CSLCs were cultured in low adhesion six well plates at either 0.5, 5, or 10 cells per well in 100 μl SFM. 10 μl SFM was added each day and discontinued when colonies were observed.

Soft agar clonogenicity assay

Each well of a six-well culture dish was coated with 2 ml serum-supplemented medium (SSM) or SFM, containing 0.6% (w/v) agar (Sigma-Aldrich). After the bottom layer solidified, 2 ml SSM or SFM containing 0.3% (w/v) agar was added containing 104 JEG-3 cells or CSLCs, and the dishes were incubated for 10-14 days. Plates were stained with 0.005% Crystal Violet, and colonies were counted. Colony forming efficiency was calculated by dividing the number of total colonies by that of inoculated cells.

Flow cytometric analysis

JEG-3 cell surface expression of CD133, CD44 and ABCG2 was assessed by flow cytometry. Cells were suspended in PBE (PBS containing 0.5% BSA and 2 mM EDTA, pH 7.2) and labeled with mouse anti-human CD133/PE (1:200, Miltenyi Biotec, Germany), anti-human CD44/FITC (1:200, Miltenyi Biotec, Germany) and anti-human ABCG2/APC (1:50, Biolegend, USA) antibodies according to the manufacturer's instructions. Samples were analyzed on Beckman Coulter FC 500 MCL/MPL counter fitted with a 488 nm laser.

qRT-PCR

Two micrograms of total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase (Promega, Beijing, China) following the manufacturer's manual.

Quantitative real-time PCR was performed using SYBR Premix ExTaq (TaKaRa Biotechnology Co. Ltd.) on an iQ5 real-time PCR detection system (Bio-Rad Laboratories, Beijing, China). Oct4, SOX2, Nanog, nestin, E-cadherin and N-cadherin mRNA levels in CSLCs and JEG-3 cells were assessed using the following primers: Oct4 (sense: 5'-GGG GTT CTA TTT GGG AAG GTAT-3', antisense: 5'-ACT CGG TTC TCG ATA CTG GTTC-3'); SOX2 (sense: 5'-AGT GGA AAC TTT TGT CGG AGAC-3', antisense: 5'-GTT CAT GTG CGC GTA ACTGT-3'); Nanog (sense: 5'-TTG TGG GCC TGA AGA AAA CTAT-3', antisense: 5'-TCC CTG GTG GTA GGA AGA GTAA-3'); nestin (sense: 5'-ATC CAG GAC TCC CAG GTT CCT TTG-3', antisense: 5'-ATG TCT CTA GAT TAC CTT CAA GAG-3'); E-cadherin (sense: 5'-TGC CCA GAA AAT GAA AAAGG-3', antisense: 5'-GTG TAT GTG GCA ATG CGTTC-3'); N-cadherin (sense: 5'-CCG GAG AAC AGT CTC CAA CTC-3', antisense: 5'-CCC ACA AAG AGC AGC AGTC-3') and β-actin (sense: 5'-CAC GAA ACT ACC TTC AAC TCC-3', antisense: 5'-CAT ACT CCT GCT TGC TGATC-3'). Real-time PCR was performed on the BIO-RAD CFX96 touch q-PCR system, and data were analyzed using the 2ΔCT or 2ΔΔCT method.

Matrigel invasion assay

Matrigel (1.75 μg/μl, BD Biosciences, Israel) diluted 1:2 in serum-free cell-culture media was added to the upper chamber of a 24-well transwell plate, and incubated at 37°C for 3-4 hours for gelling. JEG-3 cells were harvested by trypsinization, washed and resuspended in SSM, and added to upper wells at 1 × 105 cells/well in 100 μL medium, while 600 μL SSM was added to the lower wells. Plates were incubated at 37°C for 24 hours, and the cells remaining on the upper surface of the membrane were removed with a cotton swab; the filters were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The filters were then treated with destaining solution, and optical density (OD) at 570 nm was measured on Thermo Scientific Microplate Reader (Thermo Fisher Scientific, MA, USA), to reflect the cells that invaded the lower surface of the filter.

Trypan blue cell proliferation assay

Proliferation of JEG-3 cells and CSLCs was assessed by trypan blue exclusion assay. In brief, JEG-3 cells, the third and sixth generations of CSLCs (3 CSL cells/well), were cultured in normal or low adhesion 96 well plates for 24 to 120 h. Each day a sample of cells were stained with 0.4% trypan blue (Merck KGaA, Germany) and viable cells were counted using a Neubauer chamber under an Olympus inverted microscope.

Methyl Thiazolyl Tetrazolium (MTT) assay

JEG-3 cells and CSLCs were seeded in 96-well plates at 5x104 cells/well, and incubated with 100 to 0.01 peak plasma concentration (PCC) Methotrexate (MTX), Fluorouracil (5-FU) or Etoposide (VP-16) (Table 1). PPC was calculated according to the formula 50×D×2×103/5000, where D indicates clinical dosage, 2.5 mg/(kg.d) for 5-FU, 1.0 mg/(kg.d) for VP-16, 1.5 mg/(kg.d) for MTX. After 24 h, 5 mg/ml MTT (Sigma) was added to the culture medium, and after a further 4 h the cells were washed with PBS. Purple formazan crystals were solubilized in 100% DMSO, and absorbance at 570 nm was measured on a microplate reader (BIO-Tek ELx800, Winooski, VT, USA). Cell survival rate was calculated from five independent experiments.

Table 1

Drug concentration (bg / ml)

Drug concentration (bg / ml)
Drug concentration (bg / ml)

Xenograft tumorigenicity assay and limiting dilution assay in vivo

Female, six to eight-week-old athymic BALB/c mice (nu/nu) were obtained from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China), and housed under specific pathogen-free (SPF) conditions in a barrier animal facility. All animal procedures were performed with the approval of the Animal Ethics Committee of Nantong University. After one week of adaptation, the animals were used in in vivo studies. For cancer cell xenograft experiments, 2 x 103 to 1 x 106 CSLCs and 1 x 103 to 1 x 107 JEG-3 cells were washed with PBS, resuspended in SFM, and injected subcutaneously into the left flank of mice (n = 5 per group). Tumor formation was monitored after inoculation. Tumors measuring at least 5 mm in diameter were considered to represent model establishment. After eight weeks, mice were euthanized and tumor formation was assessed. Tumor specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick slides. The slides were then stained with hematoxylin and eosin. Diagnosis of xenograft tumor was confirmed by pathologist.

For limiting dilution assay in vivo, female BALB/c-nu mice (4-6 weeks old; 18-22g) were housed in SPF conditions. A total of 20 animals were divided randomly into four groups (n = 5), and subcutaneously inoculated with human choriocarcinoma stem-like JEG-3 cells (CSLCs) at 2 x 103, 1 x 104, 1 x 105 and 1 x 106, respectively, in the left side near the forelimb. Meanwhile, human choriocarcinoma JEG-3 cells at 1 x 104, 1 x 105, 1 x 106 and 1 x 107, respectively, were subcutaneously inoculated in the right side near the forelimb. Then, general condition of the animals as well as tumor formation and size were assessed daily.

A Vernier caliper was used to measure the long (a) and short (b) diameters (in mm) of each tumor, whose volume was derived as V=1/2a×b2 (mm3). Growth curves were generated, with average volume of transplanted tumor in each group as the ordinate and observation duration as the abscissa. When a tumor grew to a certain size, ulceration and bleeding occurred, causing the tumor to shrink, in which case tumor volume was recorded only until ulceration. The various body organs assessed after autopsy for tumor presence.

Statistical analysis

The SPSS software package, version 11.5 (SPSS, Inc., Chicago, IL, USA), was used for statistical analysis. Continuous variables were compared using student's t test; categorical variables were compared by χ2 test. Flow cytometry data were expressed as arithmetic mean. Other data were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.

JEG-3 cell sphere culture

Choriocarcinoma JEG-3 parental cells were seeded in serum-free medium, cultured in suspension, and observed to grow as round or oval accumulations of closely connected cells. The boundaries between cells in these suspended cell spheres could not be distinguished by light microscopy, and the whole cell spheres appeared translucent with a clear boundary. After three to four days, the cell spheres exhibited logarithmic growth and appeared healthy at day five; thereafter, although the spheres continued to grow, the center appeared discolored, suggesting apoptosis of cells without access to adequate nutrition. From day six, the refractive index of spheres decreased, the sphere outline was less clear and dead cell debris began to appear (Fig. 1). Therefore, we used JEG-3 tumor spheres cultured for five days for further experiments.

Fig. 1

Morphology of JEG-3 cell sphere in serum-free medium (SFM) culture. Phase contrast images of cell spheres derived from JEG-3 on days one to eight of culture in serum-free media. (A) day 0; (B) day 1; (C) day 2; (D) day 3; (E) day 4; (F) day 5; (G) day 6; (H) day 7; (I) day 8; (400X).

Fig. 1

Morphology of JEG-3 cell sphere in serum-free medium (SFM) culture. Phase contrast images of cell spheres derived from JEG-3 on days one to eight of culture in serum-free media. (A) day 0; (B) day 1; (C) day 2; (D) day 3; (E) day 4; (F) day 5; (G) day 6; (H) day 7; (I) day 8; (400X).

Close modal

After passage, cells of the stem cell spheres continued to grow in spheres, and up to 21 generations could be established (data not shown). During culture, cell proliferation was faster in high density than low density medium.

Differentiation of CSLCs

When cell spheres were transferred out of specialized culture medium, they readopted an adherent phenotype within 24 h and grew in a monolayer, established by day 11 (Fig. 2). When these adherent cells were returned to serum-free culture medium they readopted a non-adherent phenotype, and formed spheres, as illustrated in Fig. 1. We found that cellular morphology depended on the culture medium used.

Fig. 2

Differentiation of choriocarcinoma stem-like cells (CSLCs) in serum-supplemented medium (SSM). Phase contrast images of choriocarcinoma stem-like cells on days 1 to 9 of differentiation. Cell spheres transferred out of specialized culture medium readopted an adherent phenotype. (A) day 1; (B) day 3; (C) day 5; (D) day 7; (E) day 9; (F) Day 11 (400X).

Fig. 2

Differentiation of choriocarcinoma stem-like cells (CSLCs) in serum-supplemented medium (SSM). Phase contrast images of choriocarcinoma stem-like cells on days 1 to 9 of differentiation. Cell spheres transferred out of specialized culture medium readopted an adherent phenotype. (A) day 1; (B) day 3; (C) day 5; (D) day 7; (E) day 9; (F) Day 11 (400X).

Close modal

Colony formation of single CSLC

When single cells were grown in isolation, a colony was formed within two weeks (Fig. 3A-F). Colony growth was logarithmic from day seven or eight of culture to day 11. As observed in sphere cultures described above (Fig. 1), brown apoptotic cells were found in the center of the colony, which was surrounded by cell fragments.

Fig. 3

Colony formation of JEG-3 and CSLCs. Phase contrast images of a single CSLC on days 1 to 9 of cultured in isolation in serum free media. (A) day 1; (B) day 3; (C) day 5; (D) day 7; (E) day 9; (F) Day 11 (400X). The clonogenic potential of JEG-3 and CSLCs was determined by soft agar assays in vitro (G; *** p < 0.001 in comparison to the JEG-3 group).

Fig. 3

Colony formation of JEG-3 and CSLCs. Phase contrast images of a single CSLC on days 1 to 9 of cultured in isolation in serum free media. (A) day 1; (B) day 3; (C) day 5; (D) day 7; (E) day 9; (F) Day 11 (400X). The clonogenic potential of JEG-3 and CSLCs was determined by soft agar assays in vitro (G; *** p < 0.001 in comparison to the JEG-3 group).

Close modal

Clonogenicity of CSLCs and JEG-3 cells

When cultured on soft agar for two weeks, clone forming efficiency of CSLCs (42.7 ± 2.3%) was about 3-fold greater than that of JEG-3 cells (12.1 ± 3.1%) (P < 0.001), suggesting that the clonogenicity of CSLCs is stronger than that of JEG-3 (Fig. 3G).

Expression of CD133, CD44 and ABCG2 in CSLCs and JEG-3 cells

Both CSLCs and JEG-3 cells expressed CD44, CD133 and ABCG2 (Fig. 4A-B), but CSLCs expressed higher levels of these markers (53.08 ± 3.15%, 47.40 ± 2.67% and 78.70 ± 7.16% positive, respectively) than JEG-3 cells (4.51 ± 1.71%, 7.67 ± 2.67% and 13.85 ± 2.95% positive, respectively).

Fig. 4

Expression of surface markers and pluripotency-associated transcriptional factors in JEG-3 and CSLCs. (A) Representative histograms of JEG-3 and CSLC surface expression of CD133, CD44 and ABCG2, as assessed by flow cytometry. (B) quantitation of A (mean ± SD. *** p < 0.001 compared to JEG-3 group). (C) Real-time RT-PCR was used to assess Oct-4, Sox2 and Nanog mRNA transcript levels in CSLCs and JEG-3 cells (** p < 0.01 in comparison to JEG-3 group).

Fig. 4

Expression of surface markers and pluripotency-associated transcriptional factors in JEG-3 and CSLCs. (A) Representative histograms of JEG-3 and CSLC surface expression of CD133, CD44 and ABCG2, as assessed by flow cytometry. (B) quantitation of A (mean ± SD. *** p < 0.001 compared to JEG-3 group). (C) Real-time RT-PCR was used to assess Oct-4, Sox2 and Nanog mRNA transcript levels in CSLCs and JEG-3 cells (** p < 0.01 in comparison to JEG-3 group).

Close modal

Expression of Oct4, SOX2, Nanog in CSLCs and JEG-3 cells

As shown in Fig. 4C, mRNA levels of Oct4, SOX2, and Nanog, were all higher in CSLCs than JEG-3 cells, as assessed by real-time PCR.

Proliferation ability of CSLCs and JEG-3 cells

CSLCs exhibited significantly stronger proliferative ability than JEG-3 cells, and the number of viable cells observed by trypan blue staining continued to increase over five days of culture (Fig. 5A).

Fig. 5

Proliferation, invasion and drug resistance of JEG3 and CSLCs. (A) the growth curves of JEG-3 and CSLCs were generated with hemocytometer counts after trypan blue staining at days 0 to 5 of culture. (B) invasion was assessed by Matrigel assay for JEG-3 cells and CSLCs in serum-supplemented medium (SSM) and serum-free medium (SFM) media, respectively (200X). (C) Drug resistance: JEG-3 and CSLCs cells were incubated with 5-FU, MTX and VP-16 for 24 hours, and viability was assessed by MTT assay (mean ± SD, n = 3; * p < 0.05 compared to the JEG-3 group).

Fig. 5

Proliferation, invasion and drug resistance of JEG3 and CSLCs. (A) the growth curves of JEG-3 and CSLCs were generated with hemocytometer counts after trypan blue staining at days 0 to 5 of culture. (B) invasion was assessed by Matrigel assay for JEG-3 cells and CSLCs in serum-supplemented medium (SSM) and serum-free medium (SFM) media, respectively (200X). (C) Drug resistance: JEG-3 and CSLCs cells were incubated with 5-FU, MTX and VP-16 for 24 hours, and viability was assessed by MTT assay (mean ± SD, n = 3; * p < 0.05 compared to the JEG-3 group).

Close modal

Invasion of CSLCs and JEG-3 cells

CSLCs induced by either SSM or SFM exhibited a stronger invasion ability than JEG-3 cells (P < 0.05; Table 2, Fig. 5B), however the culture medium did not significantly affect invasion ability of CSLCs or JEG-3 (P > 0.05; Fig. 5B).

Table 2

Invasion of CSLCs and JEG-3 cells (x ±S, n = 9)

Invasion of CSLCs and JEG-3 cells (x ±S, n = 9)
Invasion of CSLCs and JEG-3 cells (x ±S, n = 9)

Drug resistance of CSLCs and JEG3

Incubation with 5-FU, MTX and VP-16 for 24 h reduced the survival rate of both CSLCs and JEG-3 cells in a dose dependent manner; however, CSLC survival rate was less substantially reduced than that of JEG-3 cells (P < 0.05), suggesting that CSLCs are less sensitive to these drugs than JEG-3 cells (Fig. 5C).

In vivo tumorigenicity experiments revealed that as few as 1 × 104 CSLCs were required to establish a tumor when subcutaneously injected into immuno-deficient mice (BALB/c nu/nu) (Fig. 6A). Seven days after the inoculation of CSLCs, a protuberant violet blue nodule with a diameter exceeding 0.5 cm appeared subcutaneously at the injection site. The nodule continued to grow in an expansive and nodular manner. It gradually developed into a violet black cystic-solid tumor with distinct borders from other tissues. Different amounts of human choriocarcinoma JEG-3 cells and human choriocarcinoma JEG-3 stem-like cells were inoculated into Balb/c-nu mice with immunodeficiency. Seven days after inoculating JEG-3 stem-like cells, purple blue protruding nodules with a long diameter > 0.5 cm appeared subcutaneously on tumor cell injection site, with continued expansion and nodular growth, gradually forming purple black cystic tumors with clear boundary. Some tumors would show subcutaneous hemorrhage, penetrating the surrounding skin after growing to a certain degree. Meanwhile, tumors appeared at 20 days after JEG-3 cell inoculation. In addition, only 104 JEG-3 stem-like cells stably formed tumors, while in the same model, at least 107 individuals choriocarcinoma JEG-3 cells were needed for tumor formation, indicating that human choriocarcinoma JEG-3 stem-like cells had a higher tumorigenicity. H&E staining and pathological analyses showed that tumor phenotype heterogeneity of JEG-3 stem-like cells was similar to that of parental cells (Table 3 and Fig. 6). Growth curves were generated, with average tumor volume in each group as the ordinate and observation duration (days) as the abscissa. Because ulceration and bleeding occurred at certain tumor size, the tumor volume was recorded only until ulceration. At 20 days, JEG-3 cell tumor volume was 405 ± 37 mm3, compared with 1088 ± 83 mm3 obtained for CSLCs at 1 x 104 cells; tumor volumes of 2560 ± 216 and 10000 ± 892 mm3 were obtained for 1 x 105 and 1 x 106 CSLCs, respectively. Significant differences in tumor volumes were found among the 4 groups (Table 3 and Fig. 6).

Table 3

Tumorigenicity of JEG-3 and CSLCs in xenotransplanted tumor formation in BALB/c nu/nu mice

Tumorigenicity of JEG-3 and CSLCs in xenotransplanted tumor formation in BALB/c nu/nu mice
Tumorigenicity of JEG-3 and CSLCs in xenotransplanted tumor formation in BALB/c nu/nu mice
Fig. 6

Xenotransplanted tumor formed by the CSLCs. (A) BALB/c nu/nu mice after xenotransplanted tumor formation produced by CSLCs; (B) gross morphology of xenotransplanted tumors produced by JEG-3 cells and CSLCs; (C) H&E staining of xenotransplanted tumor tissues formed by CSLCs; D, H&E stain of xenotransplanted tumor tissues formed by parent JEG-3 cells; (E) volume-time curve of tumors obtained with JEG-3 cell and CSLC (1 x 104, 1 x 105, and 1 x 106 cells) transplants.

Fig. 6

Xenotransplanted tumor formed by the CSLCs. (A) BALB/c nu/nu mice after xenotransplanted tumor formation produced by CSLCs; (B) gross morphology of xenotransplanted tumors produced by JEG-3 cells and CSLCs; (C) H&E staining of xenotransplanted tumor tissues formed by CSLCs; D, H&E stain of xenotransplanted tumor tissues formed by parent JEG-3 cells; (E) volume-time curve of tumors obtained with JEG-3 cell and CSLC (1 x 104, 1 x 105, and 1 x 106 cells) transplants.

Close modal

Biologically distinct populations of CSCs have been identified in many solid tumors, but the role of CSCs in choriocarcinoma has not yet been reported. In this study, we employed the human placenta choriocarcinoma cell line JEG-3 to investigate the proliferative capacity and chemotherapeutic resistance of choriocarcinoma. JEG-3 cells were dissociated into a single-cell suspension, cultured without serum to choriocarcinoma suspended cell spheres. A DMEM/F12 basal medium was supplemented with B27, bFGF, and EGF. Suspended cell spheres derived from JEG-3 cultures were defined as CSLCs, possessed the stemness characteristics of self-renewal and differentiation, and proliferated more rapidly than the parent adherent JEG-3 cells. These CSLCs were also more invasive in vitro and more tumorigenic in vivo, forming larger tumors in immuno-deficient mice more rapidly than the parent JEG-3 cells. In addition, CSLCs were slightly more resistant to the chemotherapeutic agents 5-FU, MTX, and VP16. These findings indicate that distinct CSLCs may represent CSC or tumor initiating cells in choriocarcinoma.

Our findings are in line with previous reports that CD24+/CD44+ cells possess stemness characteristics of self-renewal and differentiation. CD24+/CD44+ cells exhibited higher invasion in vitro and made higher number of colonies in collagen gels than CD24-/CD44+ HNSCC cells. In addition, the CD24+/CD44+ cells were more chemo-resistant to gemcitabine and cisplatin than CD24-/CD44+ cells. In vivo, CD24+/CD44+ cells exhibited a tendency to generate larger tumors in nude mice than CD24-/CD44+ cells. Primary high-grade serous ovarian carcinoma fresh biopsies cultured under serum-free conditions were also reported to produce floating spheres [23]. Those floating spheres also overexpressed stem cell genes (Oct-4, Nanog, Sox-2, Bmi-1, Nestin, CD133, CD44, CD24, ALDH1, CD117, and ABCG2), and proteins (Oct-4, Nanog, and Sox-2). These isolated tumor cells expanded as spheroid colonies for more than 30 passages and flow cytometry analysis revealed increased CSC markers (CD44, CD24, CD117, CD133, ABCG2, and ALDH1) in the spheroid cell population. In addition, floating spheres exhibited higher chemoresistance to cisplatin and paclitaxel than adherent cells. Moreover, subcutaneous injection of sphere-forming cells into NOD/SCID mice gave rise to new tumors similar to the original human tumors, and could be passaged in mice. These results illustrate the strong proliferative and invasive ability of CSCs, as well as their increased resistance to chemotherapeutic agents.

These are precisely the characteristics that cause malignancy and poor outcomes in the clinic, so determining the molecular mechanisms of CSC proliferation, invasion and chemoresistance is crucial to overcoming metastasis and chemo-resistance of tumors. We have isolated and cultured CSLCs, and confirmed that they exhibit characteristics of CSCs. On culture in SSM, CD44+/CD133+/ABCG2+ cells comprised only a minor percentage of the total population. However, these cells formed a higher proportion of SFM cultures. CD133 is a trans-membrane glycoprotein, the cell-surface expression of which is down-regulated during differentiation [24]. It has been used widely as a marker for CSCs in lung [25], colon [26], prostate [27], and endometrial [28] cancers. CD44 is a multifunctional class I transmembrane glycoprotein and specific receptor for hyaluronic acid, promoting migration in normal cells, and is highly expressed in almost every cancer cell in its standard or variant form [29]. It is considered a potential CSC marker in the majority of cancers, including gastric [30], prostate [31], head and neck [32] and lung [33] cancers. ABCG2 is an important member of the ABC transporter superfamily which has been implicated in multidrug resistance in cancer. Its diverse range of substrates includes many common chemotherapeutics such as imatinib, doxorubicin, and mitoxantrone. ABCG2 also is considered to represent a potential CSC marker in malignancies, such as tongue [34], colon [35], liver [36] and gastric [37,38] cancers.

A limitation of this study should be mentioned: only one choriocarcinoma cell line (JEG3) was assessed and does not necessarily recapitulate the situation in all choriocarcinoma. Further studies are therefore warranted to assess other choriocarcinoma cell lines and confirm our findings.

Although we have not identified any novel choriocarcinoma stem cell markers, our results hint that CD44, CD133 and ABCG2 may also represent choriocarcinoma stem cell markers.

This work was supported by the National Natural Science Foundation of China (Grant No. 81101995 and 81472433) and the natural science foundation of Hunan Province (Grant No. 13JJ4120).

The authors declare that they have no conflict of interest.

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J. Cai and T. Peng contributed equally to this work.

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