OV6+ tumor-initiating cells contribute to tumor progression and invasion in human hepatocellular carcinoma

Research Article

OV6+ tumor-initiating cells contribute to tumor progression and invasion in human hepatocellular carcinoma Wen Yang1, , Chao Wang1, , Yan Lin1, , Qiong Liu1, , Le-xing Yu1, Liang Tang1, He-Xin Yan1, Jing Fu1, Yao Chen1, Hui-Lu Zhang1, Liang Tang1, Long-Yi Zheng1, Ya-Qin He1, Yu-Qiong Li1, Fu-Quan Wu1, Shan-Shan Zou1, Zhong Li1, Meng-Chao Wu1, Gen-Sheng Feng1, Hong-Yang Wang1,2,⇑ 1

International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Second Military Medical University, Shanghai 200438, PR China; 2State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200032, China

Background & Aims: Accumulating evidence suggests the involvement of tumor-initiating cells (T-ICs) in cancer genesis, but whether liver T-ICs contribute to HCC invasion and metastasis remains unclear. Methods: OV6+ T-ICs were isolated from SMMC7721 and HuH7 cell lines by magnetic sorting. Characteristics of T-ICs were assessed by in vitro and mouse xenograft assays. Expression of OV6 was determined by immunostaining in specimens from 218 HCC patients, and Kaplan–Meier survival analysis was used to determine the correlation of OV6 expression with prognosis. Results: OV6+ T-ICs isolated from HCC cell lines not only possess a higher capacity to form tumor spheroids in vitro, but also had a greater potential to form tumors when implanted in non-obese diabetic/severe combined immunodeficient mice, suggesting their elevated self-renewal capacity and tumorigenicity. Moreover, OV6+ T-ICs exhibited more invasive and metastatic potentials both in vitro and in vivo. Patients with more OV6+ tumor cells were associated with aggressive clinicopathologic features and poor prognosis. CXCR4 is expressed at higher levels in OV6+ cells. Recombinant stromal cell-derived factor-1 (SDF-1) treatment expanded the OV6+ HCC T-ICs population, by sustaining the stem cell property of OV6+ cells. The SDF-1 effect was blocked by a specific CXCR4 inhibitor, AMD3100, or transfection of siRNA targeting CXCR4.

Keywords: Hepatocellular carcinoma; Tumor-initiating cells; OV6; Metastasis; CXCR4. Received 9 October 2011; received in revised form 2 April 2012; accepted 19 April 2012; available online 19 May 2012 ⇑ Corresponding author. Address: International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, 225 Changhai Road, Shanghai 200438, China. Tel.: +86 21 8187 5361; fax: +86 21 6556 6851. E-mail address: [email protected] (H.-Y. Wang).   These authors contributed equally to this work. Abbreviations: CSCs, cancer stem cells; T-ICs, tumor-initiating cells; HCC, hepatocellular carcinoma; SDF-1, stromal cell-derived factor 1; RT-PCR, reverse transcription-polymerase chain reaction.

Conclusions: OV6+ HCC cells may represent a subpopulation of T-ICs with augmented invasion and metastasis potential, which contribute to progression and metastasis of HCC. The SDF-1/ CXCR4 axis also provides therapeutic targets for elimination of liver T-ICs. Ó 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction As the sixth most common solid cancer and the third leading cause of cancer-related mortality worldwide, HCC causes approximately 700,000 deaths yearly [1]. Despite recent advances in diagnosis and treatment of HCC, the prognosis of HCC remains dismal. The 5-year tumor recurrence rate ranges from 40% to 70%, which results from aggressive invasion and high metastasis rates [2]. Recent advances in the fields of stem cell and cancer biology have shed light on cancer stem cells (or tumor-initiating cells, T-ICs) and origin of many hematological malignancies and solid tumors [3]. It is widely accepted that cancer stem/progenitor cells exhibit self-renewing capacities and are responsible for tumor maintenance and resistance to conventional anticancer therapeutics. Similarly, recent experiments highlight the importance of TICs in HCC. By using CD133 [4–6], EpCAM [7], CD13 [8], CD44 [9], CD90 [10], CD24 [11] as markers of T-ICs, it has been shown that the isolated marker-positive HCC cells possess tumor-initiating capacity and self-renewal ability, generating the phenotypic heterogeneity of the parental tumor, and being more resistant to conventional chemotherapies than other cancer cells. Increasing evidence has shown that T-ICs may not only be associated with tumor initiation and growth, but may also play a crucial role in tumor invasion and metastasis [12,13]. It is therefore essential to deeply understand the liver T-ICs in order to develop more effective therapeutic strategies. In previous studies [14], we showed that OV6, originally classified as a hepatic progenitor cell marker, was also expressed in a subpopulation of cells with greater ability to form tumor in vivo

Journal of Hepatology 2012 vol. 57 j 613–620

Research Article B

C

SMMC7721

**

100

39.35

40 30 20

0

Attach Spheroid SMMC7721

negative

1st passage 2nd passage

*

*

50 0

6.97 2.75

1st passage 2nd passage

60 OV6 - OV6 +

OV6 - OV6 +

HuH7

2.43

Attach

Spheroid HuH7

weak

E

50 40

* *

30

**

40 20 0

OV6- OV6+ OV6- OV6+

10 0

OV6-OV6+

OV6-OV6+

F

p = 0.028

1.0 0.8 0.6 0.4

OV6+HPCslow OV6+HPCshigh

0.2 0.0

moderate

Patient #3 *

20

Cumulative survival

10

**

Number of spheres per 1000 cells

OV6 positive cells (%)

50

D

Number of spheres per 1000 cells

150

Number of spheres per 1000 cells

A

strong

0

20

Cumulative survival

1.0

60 40 DFS p = 0.001

0.8 0.6 0.4 OV6+HPCslow OV6+HPCshigh

0.2 0.0 0

20

40

60

OS Fig. 1. Characterization of OV6+ cells in HCC cell lines and a higher percentage of OV6+ tumor cells indicate a poor prognosis in HCC patients. (A) Spheres established from SMMC7721 and HuH7 cells were dissociated into single cells and the percentage of OV6+ cells was compared with control cells cultured in adherent conditions, by flow cytometry analysis. Experiments were performed in triplicate and data are shown as mean ± SD. (⁄⁄p <0.01). (B) OV6+ SMMC7721 or HuH7 cells generated more spheroids than OV6 cells. (Left panels) Representative phase-contrast images of a HCC spheroid derived from OV6+ SMMC7721 or HuH7 cells. (Scale bar = 100 lm). (Right panels) Total number of spheroids from 1000 sorted OV6+ cells is shown. Experiments were performed in triplicate and data are shown as mean ± SD. (⁄p <0.05). (C) OV6+ tumor cells isolated from freshly resected HCC generate more primary and serially passaged spheroids than OV6 cells. Representative results of patient #3 are shown. (⁄p <0.05; ⁄⁄p <0.01). (D) Immunohistochemical characteristics of OV6 in HCC specimens. Representative staining of OV6 was shown as negative, weak, medium, and strong staining. Scale bar = 50 lm. (E) OV6 positive tumor cells were often located in invasive fronts of tumors. The right panel shows an enlargement of the indicated area. Scale bar = 50 lm. (F) Kaplan–Meier disease free survival (DFS) and overall survival (OS) curve of HCC patients in correlation with percentage of OV6+ tumor cells. The DFS and OS rate significantly decreased in the subgroup of OV6+HPCshigh (green line) compared with OV6+HPCslow (blue line). [This figure appears in color on the web.]

and substantial resistance to standard chemotherapy. However, the role of OV6+ HCC tumor cells in progression and metastasis of HCC remains unclear. In this article, we report that OV6+ HCC cells exhibit highly invasive and tumorigenic properties, and we also show a crucial role of SDF-1/CXCR4 axis in maintenance and migration of liver T-ICs.

Experimental metastasis model

Materials and methods Histopathologic and immunohistochemical evaluation Tissues were cut into 5-lm-thick sections and the following primary antibodies were used: mouse anti-OV6, and mouse anti-CXCR4 (R&D). Vector ABC kit (Vector Laboratories) and DAB reagent (Dako Comp) were employed in the detection

614

procedure. All slides were observed and photographed with an Olympus microscope (IX-70 OLYMPUS, Japan). OV6 staining of the whole tissue sections was semiquantitatively graded for percentage of cells stained as not detectable (negative), <5% (weak), 5% to 30% (moderate), and >30% (strong) of tumor cells per section, evaluating five medium-power fields of each tumor tissue by two independent observers. Similarly, the staining intensities of CXCR4 were also evaluated in each sample and stratified into negative (0–5%) or positive (>5%) category.

Five 6-week-old NOD/SCID mice in each experimental group were injected intravenously with 5  104 OV6+ or OV6 SMMC7721 cells through the tail vein. Eight weeks after injection, the lungs were isolated and analyzed. Macroscopic lung metastasis was detected visually and microscopic metastases were quantified under microscope after paraffin embedment and hematoxylin and eosin (H&E) staining.

Journal of Hepatology 2012 vol. 57 j 613–620

JOURNAL OF HEPATOLOGY Table 1. Limiting dilution and serial transplantation assay of SMMC 7721 and HuH7 cells (mice with tumors/total No. of mice injected).

Cell type SMMC7721 SMMC7721 serial transplantation

HuH7

5 x 102

1 x 103

5 x 103

1 x 104

5 x 104

-

OV6

-

0/3

0/3

0/3

3/3

OV6+

-

2/3

3/3

2/2

3/3

OV6-

0/3

0/3

0/3

1/3

-

OV6+

3/3

3/3

3/3

3/3

-

Phenotype

104

3 x 104

6 x 104

105

OV6-

0/3

0/3

0/3

1/3

OV6+

1/3

3/3

3/3

3/3

Phenotype

Collection of human tissue specimen, cell culture, magnetic sorting of OV6+ HCC cells, flow cytometry, spheroid assays, RNA extraction and real-time polymerase chain reaction, cell migration and invasion assay, in vivo tumorigenicity experiments, siRNA transfections, statistics. Please see Supplementary Materials and methods.

cytometry. As expected, OV6 expression overlapped with that of CD133 and EpCAM (Supplementary Fig. 3), suggesting that cells positive for these three markers share similar characteristics.

Results

Higher percentage of OV6+ tumor cells predicts poor prognosis of HCCs

OV6+ HCC cells possess characteristics of tumor-initiating cells T-ICs are responsible for tumor progression with self-renewal ability and tumorigenicity. Our previous study indicated that OV6+ HCC cells may represent a potential stem/progenitor-like cell population in HCC cell lines that has stemness characteristic and confers chemoresistance [14]. We further determined whether OV6+ HCC cells were liver T-ICs. It has been shown that T-ICs sorted from established cell lines or primary tumors can form spheroids when plated in a non-attached condition, suggesting their ability to self-renew [8]. We first detected percentage of OV6+ cells in spheroids formed after 10 days of culture in ULA plates by flow cytometry analysis. As shown in Fig. 1A, OV6+ cells were enriched in spheroid cells both in SMMC7721 and HuH7. Furthermore, magnetic-sorted OV6+ SMMC7721 or HuH7 cells (Supplementary Fig. 1) were able to generate an increased number of primary and secondary passaged spheroids as compared with OV6 cells (Fig. 1B). OV6+ tumor cells isolated from freshly resected HCC also displayed a higher spheroid formation ability than OV6 cells (Fig. 1C). Quantitative real-time PCR analysis revealed that OV6+ cells isolated from freshly isolated tumors expressed higher levels of stem cell-associated genes (CD133, Oct4, Nanog, and Sox2) as compared with OV6 cells (Supplementary Fig. 2). These data suggested OV6+ cells had higher self-renewal ability. Next, tumor-forming ability of OV6+ SMMC7721 and HuH7 cells was examined in NOD-SCID mice by limiting dilution and serial transplantation assay. As shown in Table 1, OV6+ SMMC7721 or HuH7 cells were more tumorigenic than their OV6 counterparts in vivo. As few as 1000 OV6+ SMMC7721 cells or 10,000 OV6+ HuH7 cells were sufficient to form tumors in NOD/SCID mice. In a serial transplantation assay, OV6+ SMMC7721 cells were able to selfrenew upon serial transplantation and are highly tumorigenic. These data suggest that HCC T-ICs with higher tumorigenicity could be enriched in OV6+ cancer cells. We then compared the expression of OV6 and previously reported liver T-IC markers (CD133 and EpCAM) in SMMC7721 and Huh-7 cells by flow

To determine whether the ratio of OV6+ tumor cells in HCCs is associated with tumor recurrence and poor survival, we examined OV6 expression by immunostaining in specimens from 218 HCC patients. As shown in Fig. 1D, OV6 expression levels varied widely among different HCC specimens. Notably, many of the OV6+ tumor cells were observed in invasive fronts of tumors (Fig. 1E). Patients with negative, weak (<5%), and moderate (5–30%) staining were stratified as the OV6+HPCslow group (n = 135), and those with strong staining (>30%) as the OV6+HPCshigh group (n = 83). Interestingly, patients in the OV6+HPCshigh group were significantly associated with aggressive clinicopathologic features (high serum alpha-fetoprotein level, tumor multiplicity, absence of tumor encapsulation, late TNM stage, gross portal vein thrombosis and distant metastasis of HCC) (Table 2). To further confirm the correlation of OV6+ cells with HCC prognosis, we compared disease-free survival (DFS) and overall survival (OS) times between these two groups. Kaplan–Meier survival analysis revealed that patients in the OV6+HPCshigh group had either shorter DFS or worse OS (Fig. 1F) than the OV6+HPCslow group. To determine whether the higher percentage of OV6+ tumor cells is an independent prognostic factor for HCC, multivariate survival analysis was performed to identify the prognostic factors for OS. As shown in Supplementary Table 1, the higher percentage of OV6+ tumor cells was an independent prognostic factor for HCC. These data suggest that OV6+ tumor cells contribute to HCC progression and invasion. OV6+ cells have highly invasive and metastatic capacities Because the percentage of OV6+ HCC cells correlates with the metastatic potentials (Table 2), we asked whether the OV6+ subpopulation has increased motility. Migration of magnetic-sorted OV6+ and OV6 HCC cells was measured by transwell movement and matrigel invasion assays. As shown in Fig. 2A, transwell migration assay showed that more cells moved through the 8 lm pores in magnetic-sorted OV6+ cells than OV6 cells. Similarly, the invasion assay also showed that OV6+ cells exhibited a significantly higher rate of cell invasion than that of OV6 cells

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Research Article Table 2. Correlation between percentage of OV6+ tumor cells and clinicopathological characteristics in 218 HCCs.

OV6+HPCslow (n = 135)

OV6+HPCshigh (n = 83)

<50

59

45

≥50

76

38

Male

115

71

Female

20

12

Absent

22

15

Present

113

68

<400

89

37

≥400

46

46

Absent

59

32

Present

76

51

Single

112

59

Multiple

23

24

<5

58

25

≥5

77

58

Absent

43

42

Present

92

41

Low (I/II)

21

8

High (III/IV)

114

75

p value

Age (yr)

Sex

CXCR4 is expressed at higher levels in OV6+ cells

HBV infection

<0.01

AFP (ng/ml)

Liver cirrhosis

<0.05

Tumor multiplicity

Maximal tumor size (cm)

<0.01

Tumor encapsulation

Edmondson grade

Pathologic TNM stage 102

44

Late stage (III)

33

39

Portal vein thrombosis

<0.01

Absence

114

56

Gross

21

27

Absent

123

67

Present

12

16

Distant metastasis <0.05

(Fig. 2B). To determine the relationship between OV6+ cells and metastatic capacity in vivo, we injected 5  104 magnetic-sorted OV6+ or OV6 cells into the tail vein of NOD/SCID mice (5 mice per group). Mice were sacrificed 8 weeks after cell injection, and metastatic tumor nodules formed in the lung were examined by gross observation and histological examination. As shown in Fig. 2C, the numbers of macroscopic and microscopic metastatic lesions were significantly higher in the OV6+ cell injection group

616

It has been reported that CXCR4 may be a key regulator of tumor invasiveness leading to local progression and tumor metastasis [15]. To evaluate the potential contribution of CXCR4 to HCC development in the OV6+ subpopulation, we measured CXCR4 expression in SMMC7721 and HuH7 cells by RT-PCR and flow cytometric analysis. As shown in Fig. 3A and B, spheroid cells and magnetic-sorted OV6+ subpopulation expressed elevated levels of CXCR4 in comparison to the adherent counterpart and OV6 fraction. Flow cytometric analysis also demonstrated that the OV6+ population was almost exclusively contained within the CXCR4+ population (Fig. 3C). Furthermore, we detected CXCR4 expression in 173 HCC tissue samples. Consistent with a previous report [16], CXCR4 expression was observed in 73.41% of all specimens, but much higher (85.14%) in the OV6+HPCshigh group (Fig. 3D and Supplementary Table 2). Immunofluorescent staining of frozen HCC specimens further confirmed the overlapping expression of OV6 and CXCR4 (Fig. 3E). In addition, in the OV6+HPCshigh group, CXCR4 expression was significantly associated with absence of tumor encapsulation, late TNM stage and gross portal vein thrombosis (Supplementary Table 3), indicating that CXCR4 was involved in the OV6+ cell-mediated tumor progression and metastasis. The SDF-1/CXCR4 axis promotes OV6+ cell self-renewal and migration

<0.01

Early stage (I-II)

than the OV6 cell injection group. Then, we checked OV6 expression in the lung xenografts, and detected strong OV6 staining cells in lung metastatic foci (Fig. 2D). Interestingly, one of the mice injected with OV6+ cells developed tumor thromboemboli in the lung. Strong OV6 staining was also observed at the edge of thromboemboli, further indicating their higher invasive potential (Fig. 2E). Collectively, these results suggest that the OV6+ subpopulation has more metastatic potentials both in vitro and in vivo.

SDF-1 is the ligand of CXCR4 and the SDF-1/CXCR4 axis has been reported to play a role in cell migration. When used as a chemoattractant, SDF-1 (100 ng/ml) increased the migration ability of OV6+ cells, verifying the chemoattractant role of SDF-1 to liver T-ICs (Fig. 4A). To evaluate whether the SDF-1/ CXCR4 axis is important in stem cell function, we treated SMMC7721 and HuH7 cells with human recombinant SDF-1 and determined its effect on the self-renewal ability of the TICs population, as measured by formation of tumor spheres. As shown in Fig. 4B, SDF-1 increased the formation of tumorspheres in both cell lines. Addition of AMD3100, a specific inhibitor of CXCR4, abrogated the increase in the sphere number after SDF-1 stimulation, and even decreased the sphere number compared to the unstimulated control, suggesting that an autocrine stimulatory loop exists in these cell lines. Similar results were also observed in magnetic-sorted OV6+ cells (Fig. 4C). Next, SMMC7721 and HuH7 cells were transfected with CXCR4 siRNA or control siRNA. The knockdown efficiency of CXCR4 was confirmed by quantitative RT-PCR and Western blot (Supplementary Fig. 4). When CXCR4 expression was reduced, the sphere formation ability of SMMC 7721 cells was abolished (Fig. 4D). To further study the function of SDF-1, magneticsorted OV6+ SMMC7721 cells were cultured in the presence or

Journal of Hepatology 2012 vol. 57 j 613–620

JOURNAL OF HEPATOLOGY A

C

B

D

anti-OV6 H&E

Number of metastatic foci per mouse

10

Number of invading cells per field (200x)

Number of cells migrated per field (400x)

*

150

*

6 4 2

*

100 50 0 OV6-

OV6- OV6+ OV6- OV6+ macromicrometastatic metastatic foci foci

100

*

E

*

80

200x

8

0

200

100x

60 40 20 0

OV6+

OV6-

OV6+

1.5 1.0 0.5 0.0 Attach

D

3 2 1 0

85.14

DAPI

SMMC7721 *

3 2 1 0

-

OV6 OV6

+

OV6

105

SMMC7721

104

* 6 4

103 102 101

2

100 100 101 102 103 104 105 PE-CXCR4

0 OV6

CXCR4

-

+

OV6

105

merge

HuH7

104

73.41 64.65

60 40

103 102 101

20 0

C

HuH7

8

FITC-OV6

CXCR4 positive rate (%)

*

4

4

Attach Spheroid

E

**

100 80

Spheroid

B

HuH7

FITC-OV6

*

2.0

5

Relative mRNA expression (CXCR4 /18S)

SMMC7721

Relative mRNA expression (CXCR4 /18S)

2.5

Relative mRNA expression (CXCR4 /18S)

A

Relative mRNA expression (CXCR4 /18S)

Fig. 2. OV6+ cells have highly invasive and metastatic potential. Transwell migration (A) and matrigel invasion assay (B) show that OV6+ SMMC7721 cells are more invasive than OV6 cells. Examples of cells migrating or invading through the membrane are shown in the top panel. Columns, mean of triplicate experiments. ⁄p <0.05. (C–E) OV6+ cells exhibit highly metastatic ability in vivo. 5  104 magnetic-sorted OV6+ or OV6 cells were injected into the tail vein of NOD/SCID mice (n = 5 per group). (C) The number of macro- or micro-metastatic nodules was quantified in lungs of NOD/SCID mice. Values for individual mice are shown. (D) H&E and IHC stainings with OV6 antibody were performed on serial sections of representative metastatic lung tumors. The asterisk indicates metastatic foci in lung sections. Scale bar = 50 lm. (E) H&E and IHC stainings with OV6 antibody were performed on serial sections of tumor thromboemboli in the lung. Arrows indicate border of tumor thromboemboli in the lung. Scale bar: 50 lm. [This figure appears in color on the web.]

100 100 101 102 103 104 105 PE-CXCR4

All HPCslow HPCshigh patients OV6+

Fig. 3. CXCR4 is expressed at higher levels in OV6+ cells. (A) Spheres established from SMMC7721 and HuH7 cells exhibit increased CXCR4 expression. Experiments were performed in triplicate and data are shown as mean ± SD. (⁄p <0.05). (B) OV6+ cells had higher CXCR4 expression levels. The CXCR4 expression level of magnetic-sorted OV6+ or OV6 cells was determined by real-time PCR. The data represent the mean ± SD of three independent experiments performed in triplicate. (⁄p <0.05). (C) The flow cytometry analysis revealed that CXCR4 is expressed at higher levels on the cell surface of the OV6+ population in HuH7 and SMMC7721 cells. Representative results from three experiments are shown. (D) Higher CXCR4 expression was observed in HCC specimens with more OV6+ tumor cells. (⁄⁄p <0.01). (E) Immunofluorescent staining of OV6 and CXCR4 in frozen HCC specimens. Representative results are shown. [This figure appears in color on the web.]

absence of SDF-1 for 2 days and the percentage of OV6+ cells in the sorted population after treatment was analyzed. As shown in Fig. 4E, SDF-1 treatment also led to the preservation of a much higher percentage of the OV6+ cell population than

control (50.43% vs. 44.04%). These results suggest that the SDF-1/CXCR4 pathway can not only regulate the migration ability of OV6+ cells, but also expand the T-IC subpopulation of HCC and maintain the stem cell property of OV6+ cells.

Journal of Hepatology 2012 vol. 57 j 613–620

617

Research Article

618

* 150 100 50

SDF-1 SDF-1 (-) (100 ng/ml) SMMC7721

Number of spheres per 1000 cells

** 100

50

0 SDF-1 AMD3100

C

-

+ -

*

#

+

+ +

OV6 + SMMC7721 Number of spheres per 1000 cells

200

Number of spheres per 1000 cells

0

150

** 60 40 20 0

80

HuH7

60

*

40

# *

50

20

-

+ -

+

+ +

60 40

+

+ +

SDF-1 (-)

**

20 0

Con CXCR4 SiRNA siRNA-2 SDF-1 (100 ng/ml)

256

256 44.04%

153 102 51 0 100 101 102 103 104 105

204 SCC-height

204

#

**

80

100

+ -

SDF-1 SDF-1 (-) (100 ng/ml)

D

**

-

HuH7

80

0 SDF-1 AMD3100

150

0 SDF-1 AMD3100

E

Number of cells migrated per field (200x)

SMMC7721

200

Number of spheres per 1000 cells

B

SCC-height

In this study, we observed that OV6 was expressed diversely in our cohort of HCC patients, and an increased number of OV6positive tumorigenic progenitor cells negatively affected disease progression. OV6+ cells isolated from cultured HCC cell lines exhibited tumor-initiating ability and mediated tumor invasion in vitro and tumor metastasis in mouse xenografts. Furthermore, SDF-1/CXCR4 axis played an important role in regulating the stem cell characteristics and migration capacity of OV6+ cells. Accumulating evidence indicated that many hematological malignancies and solid tumors [17], including leukemia, breast cancer, brain tumor, prostate, colon cancer and HCC [18], developed from a small population of cancer cells, termed CSCs or T-ICs. There is also growing evidence, coming from HCC studies, that many HCCs arise from dysfunctional liver stem/ progenitor cells [19,20]. Therefore, a thorough understanding of OV6+ HCC cell properties helps clarify their important role in HCC progression and metastasis. Based on our previous observations that OV6+ HCC cells express stemness-related genes and exhibit chemoresistance, we further determined that OV6+ HCC cells were enriched in liver T-ICs, in this study. OV6+ cells displayed higher self-renewal ability than their OV6 counterparts in both cultured HCC cell lines and freshly isolated tumors. The higher tumor-initiating capacity of OV6+ cells was further demonstrated by serial transplantation assay in NOD-SCID mice. To determine whether the percentage of OV6+ T-ICs in HCCs is associated with tumor recurrence and poor survival, we examined OV6 expression by immunostaining in clinical HCC specimens. We found that patients in the OV6+HPCshigh group were significantly associated with aggressive clinicopathologic features, shorter disease-free survival and overall survival time. Therefore, our data clearly showed that the amount of CSCs/TICs could be used as a prognostic factor for HCC patients to predict tumor recurrence after surgery. Because T-ICs possess the characteristics of self-renewal and pluripotency, and are responsible for the initiation and maintenance of tumor growth, it has been predicted that invasive TICs might also be primarily responsible for tumor metastases [21]. These cells have also been termed migrating T-ICs, which combine the two most decisive traits, stemness and mobility [22]. In our cohort of HCC patients, the presence of more OV6+ cells in HCC tissues was statistically associated with portal venous infiltration and distant metastasis, further indicating that OV6+ HCC cells might be responsible for the metastasis of HCCs. Indeed, the magnetic-sorted OV6+ HCC cells also possessed higher migratory and invasive ability in vitro and stronger metastatic potential in vivo. The immunohistochemical findings that the border of tumors and the edge of thromboemboli contained more OV6+ tumor cells also support the view that these OV6+ cells are the major players in tumor invasion and metastasis. Thus, our results provide new evidence for the existence of metastatic T-ICs in HCC and their important role in metastasis. The chemokine receptor CXCR4 and its ligand SDF-1 were originally found involved in trafficking/homing of hematopoietic stem/progenitor cells [23,24], but they were also essential for the homing of various other cell types including normal hepatic progenitor cells [25]. Recent studies have also suggested that

Number of cells migrated per field (200x)

A

Discussion

50.43%

153 102 51 0 100 101 102 103 104 105

PE-OV6 Fig. 4. SDF-1 stimulates migration and self-renewal of OV6+ cells in vitro. (A) Effect of SDF-1 on the migratory ability of OV6+ SMMC 7721 and HuH7 cells. OV6+ SMMC 7721 and HuH7 cells showed increased migratory ability toward 100 ng/ ml SDF-1. Experiments were performed in triplicate and data are shown as mean ± SD. (⁄p <0.05; ⁄⁄p <0.01). (B and C) Role of SDF-1/CXCR4 axis on tumor sphere formation of SMMC7721, HuH7 cells (B) and magnetic-sorted OV6+ SMMC7721 cells (C). SDF-1 (100 ng/ml) treatment increased the formation of tumor spheres. AMD3100 treatment (20 lg/ml) decreased the sphere number compared with the untreated control and abrogated the increase in sphere number after SDF-1 stimulation. Experiments were performed in triplicate and data are shown as mean ± SD. (⁄p <0.05; ⁄⁄p <0.01, compared with the unstimulated control) (#p <0.01, compared with SDF-1 treated alone). (D) Knockdown of CXCR4 expression inhibited tumor sphere formation ability of SMMC7721 cells. The data shown represent the mean ± SD of three independent experiments. (⁄⁄p <0.01). (E) Magnetic-sorted OV6+ SMMC7721 cells were cultured with or without SDF-1 stimulation. Forty-eight hours later, the percentage of OV6+ cells was determined by flow cytometry analysis. Representative results from three independent experiments are shown.

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JOURNAL OF HEPATOLOGY CXCR4 has a large influence on the biology of a variety of cancers [26]. Hermann et al. demonstrated that a subpopulation of migrating CD133+ CXCR4+ cancer stem cells was essential for pancreatic adenocarcinoma metastasis [15]. However, SDF-1/ CXCR4 axis has not previously been reported to play a role in liver T-ICs function. We explored the role of SDF-1/CXCR4 axis in liver T-ICs in three ways. First, using real time RT-PCR, flow cytometric analysis, and HCC specimens, our results showed that CXCR4 was expressed at a higher level on OV6+ cells. Second, recombinant SDF1 stimulation promoted migration capacity and self-renewal ability of OV6+ cells. Third, specific CXCR4 inhibitor AMD3100 or siRNA knockdown of CXCR4 reduced their invasiveness and self-renewal ability. High levels of SDF-1 in organs and tissue structures such as lymph nodes, lungs, and bones are believed to direct the metastasis of CXCR4-expressing tumor cells [27]. Thus, the gradients of SDF-1 may attract these CXCR4-expressing T-ICs and enhance their invasion potential, facilitating their detachment from the primary tumor mass. At the new distant site, high levels of SDF-1 in the environmental niche further promote the expansion and the proliferation of T-ICs to establish a metastatic tumor. Thus, the SDF-1/CXCR4 axis may play a role in mediating interactions between tumor stem cells and the tumor microenvironment. Consequently, strategies aimed at modulating the SDF-1/ CXCR4 axis could have important clinical applications in metastasis inhibition of CXCR4-expressing T-ICs [28]. The CSC/T-ICs model has important implications in the understanding of the basic biology of HCC and the development of new cancer therapeutics. Our results clearly showed the enhanced tumor-initiating capacity, higher self-renewal ability and stronger invasion and metastasis potential of OV6+ HCC cells, which helped deepen the understanding of liver T-IC. The important role of the SDF-1/CXCR4 axis in regulating the function of OV6+ HCC cells also shed new light on the molecular mechanisms regulating liver T-ICs. Future studies should focus on the characterization of liver T-ICs in clinical specimens and the identification of effective therapeutic strategies targeting T-ICs.

Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Financial support This work was supported by National Natural Science Foundation of China (2012CB316503, 2011AA020111, 30921006, 30900770), Chinese National Key Project (2012ZX10002-009, 011), Key Basic Science Foundation of Shanghai (10JC1418500), Project of the State Key Lab of Shanghai Jiaotong University (91-10-02).

Acknowledgments We thank Dong-Ping Hu, Lin-Na Guo, Dan Cao, Shan-Hua Tang, Dan-Dan Huang, Shan-Na Huang for technical assistance.

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2012.04. 024.

References [1] Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology 2004;127:S5–S16. [2] Tung-Ping Poon R, Fan ST, Wong J. Risk factors, prevention, and management of postoperative recurrence after resection of hepatocellular carcinoma. Ann Surg 2000;232:10–24. [3] Jordan CT, Guzman ML, Noble M. Cancer stem cells. N Engl J Med 2006;355:1253–1261. [4] Yin S, Li J, Hu C, Chen X, Yao M, Yan M, et al. CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity. Int J Cancer 2007;120:1444–1450. [5] Ma S, Tang KH, Chan YP, Lee TK, Kwan PS, Castilho A, et al. MiR-130b Promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1. Cell Stem Cell 2010;7:694–707. [6] Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, et al. Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 2007;132:2542–2556. [7] Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, et al. EpCAMpositive hepatocellular carcinoma cells are tumor-initiating cells with stem/ progenitor cell features. Gastroenterology 2009;136:1012–1024. [8] Haraguchi N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, et al. CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest 2010;120:3326–3339. [9] Zhu Z, Hao X, Yan M, Yao M, Ge C, Gu J, et al. Cancer stem/progenitor cells are highly enriched in CD133+CD44+ population in hepatocellular carcinoma. Int J Cancer 2010;126:2067–2078. [10] Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, et al. Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 2008;13:153–166. [11] Lee TK, Castilho A, Cheung VC, Tang KH, Ma S, Ng IO. CD24(+) liver tumorinitiating cells drive self-renewal and tumor initiation through STAT3mediated NANOG regulation. Cell Stem Cell 2011;9:50–63. [12] Diehn M, Majeti R. Metastatic cancer stem cells: an opportunity for improving cancer treatment? Cell Stem Cell 2010;6:502–503. [13] Liu C, Kelnar K, Liu B, Chen X, Calhoun-Davis T, Li H, et al. The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44. Nat Med 2011;17:211–215. [14] Yang W, Yan HX, Chen L, Liu Q, He YQ, Yu LX, et al. Wnt/beta-catenin signaling contributes to activation of normal and tumorigenic liver progenitor cells. Cancer Res 2008;68:4287–4295. [15] Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 2007;1:313–323. [16] Schimanski CC, Bahre R, Gockel I, Muller A, Frerichs K, Horner V, et al. Dissemination of hepatocellular carcinoma is mediated via chemokine receptor CXCR4. Br J Cancer 2006;95:210–217. [17] Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 2008;8:755–768. [18] Sell S, Leffert HL. Liver cancer stem cells. J Clin Oncol 2008;26:2800–2805. [19] Tang Y, Kitisin K, Jogunoori W, Li C, Deng CX, Mueller SC, et al. Progenitor/ stem cells give rise to liver cancer due to aberrant TGF-beta and IL-6 signaling. Proc Natl Acad Sci USA 2008;105:2445–2450. [20] Lee JS, Heo J, Libbrecht L, Chu IS, Kaposi-Novak P, Calvisi DF, et al. A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nat Med 2006;12:410–416. [21] Dalerba P, Clarke MF. Cancer stem cells and tumor metastasis: first steps into uncharted territory. Cell Stem Cell 2007;1:241–242. [22] Shmelkov SV, Butler JM, Hooper AT, Hormigo A, Kushner J, Milde T, et al. CD133 expression is not restricted to stem cells, and both CD133+ and CD133 metastatic colon cancer cells initiate tumors. J Clin Invest 2008;118:2111–2120. [23] Loetscher P, Moser B, Baggiolini M. Chemokines and their receptors in lymphocyte traffic and HIV infection. Adv Immunol 2000;74:127–180. [24] Kucia M, Reca R, Miekus K, Wanzeck J, Wojakowski W, Janowska-Wieczorek A, et al. Trafficking of normal stem cells and metastasis of cancer stem cells

Journal of Hepatology 2012 vol. 57 j 613–620

619

Research Article involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis. Stem Cells 2005;23:879–894. [25] Mavier P, Martin N, Couchie D, Preaux AM, Laperche Y, Zafrani ES. Expression of stromal cell-derived factor-1 and of its receptor CXCR4 in liver regeneration from oval cells in rat. Am J Pathol 2004;165:1969–1977. [26] Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res 2010;16:2927–2931.

620

[27] Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001;410:50–56. [28] Gelmini S, Mangoni M, Serio M, Romagnani P, Lazzeri E. The critical role of SDF-1/CXCR4 axis in cancer and cancer stem cells metastasis. J Endocrinol Invest 2008;31:809–819.

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