Research progress and prospects of markers for liver cancer stem cells

Abstract

Liver cancer is a common malignancy and surgery is the main treatment strategy. However, the prognosis is still poor because of high frequencies of postoperative recurrence and metastasis. In recent years, cancer stem cell (CSC) theory has evolved with the concept of stem cells, and has been applied to oncological research. According to cancer stem cell theory, liver cancer can be radically cured only by eradication of liver cancer stem cells (LCSCs). This notion has lead to the isolation and identification of LCSCs, which has become a highly researched area. Analysis of LCSC markers is considered to be the primary method for identification of LCSCs. Here, we provide an overview of the current research progress and prospects of surface markers for LCSCs.

Core tip: Liver cancer is a common malignancy and the eradication of liver cancer stem cells (LCSCs) is proposed as the key to improving the curative effect of treatments. Many surface markers have been reported for LCSCs, but there is still no unified standard. This paper addresses the research progress of markers for LCSCs and discusses the relationship with clinical syndrome in hepatocellular carcinoma.

DEFINITIONS OF CANCER STEM CELLS AND LIVER CANCER STEM CELLS

Liver cancer is the fifth most common and third most deadly cancer in the world and hepatocellular carcinoma (HCC) accounts for 90% of liver cancers. Currently, surgical resection and liver transplantation are the sole curative options for the treatment of HCC. However, the 5-year survival rate depends on the stage of the liver cancer at diagnosis. Most patients in the late stage with postoperative recurrence exhibit low sensitivity to radiotherapy and chemotherapy[1]. Therefore, the exploration of liver cancer treatments is currently a highly researched area. Since cancer stem cell theory was first proposed, new approaches have been suggested for the treatment of HCC. Cancer stem cell theory was put forward by Reya et al[2] based on the previous points. The theory considers that tumor tissue has a small number of cells called cancer stem cells (CSCs) that self-renew indefinitely and have the potential for multi-directional differentiation to produce the heterogeneity of tumor cells. Most CSCs are in the G0 phase of the cell cycle and have certain resistance to radiotherapy and chemotherapy.

Studies have demonstrated CSCs in leukemia and breast cancer. Other studies have shown that CSCs may also exist in liver cancer tissues[3,4]. Liver cancer stem cells (LCSCs) have been highly researched in liver cancer for more than 10 years, and are considered to be a population of cells with certain stem cell-like characteristics in liver cancer tissue. These stem cell-like characteristics include indefinite self-renewal and the potential for multi-directional differentiation that constantly produces liver cancer cell populations in various stages of differentiation with different biological behaviors. In this manner, LCSCs maintain tumor growth. Furthermore, compared with non-LCSCs in liver cancer tissue, LCSCs have a stronger migration ability and tumorigenicity that is closely related to metastasis and recurrence of liver cancer. LCSCs are also resistant to radiotherapy and chemotherapy, which is one of the reasons for the poor efficacy of these treatments for liver cancer patients[5,6].

An increasing number of researchers believe that the key to improving the curative effect of treatments for liver cancer is the eradication of LCSCs. Treatments of liver cancer may simply kill cancer cells and reduce the tumor volume without eradication of LCSCs. In addition, detection of LCSC-specific surface markers can be used for diagnosis, prognosis evaluation, and monitoring after treatment of patients with liver cancer.

CLASSIFICATION OF LCSC MARKERS

In recent years, numerous studies have confirmed that the development and progression of hematological cancers and many kinds of solid tumors, including liver cancer, depend on CSCs[7]. However, compared with most other solid tumors with CSC-specific surface markers, there is no consensus on the surface markers of LCSCs. New LCSC surface markers are constantly being discovered and debated.

Currently, using cell surface markers to isolate CSCs has proved to be feasible[8]. To select LCSC surface markers, stem cell markers are used as a reference because CSCs and normal stem cells have many similar biological characteristics. Major LCSC markers are listed in Table 1.

Table 1.

Reported major liver cancer stem cell markers

Marker	Origin	Minimum tumorigenic cells	Clinical relevant characteristics	Related literature
CD133	HuH7 cell,SMMC-7721 cell	1 × 102-1 × 103	With a poor prognosis;Related with invasiveness and distant metastases;Resistance to chemotherapy drugs	[14,15,17,48,59]
CD90	PLC cell,MHCC-97L,HCC tumor,blood samples of 90% patients with liver cancer	5 × 102	Related with tumorigenic ability and metastasis of liver cancer	[23,24]
EpCAM	HuH1 cell,HuH7 cell	1 × 103	The origin of recurrence and metastasis postoperatively;Patients with vascular metastasis and low overall survival rate when peripheral circulation exists	[31]
OV6	SMMC-7721 cell	5 × 103	Drugs resistance;Strong metastasis and invasion	[39]
CD441	MHCC-LM3 cell,MHCC-97L cell	1 × 102	Chemotherapy drug resistance;Related with portal vein metastasis in liver cancer	[41]
SP (ABCG2)	Huh7 cell,PLC/PRF/5 cell	1 × 103	-	[46,60]

¹Co-expression of CD133+ and CD44+ cell surface markers. SP: Side population; EpCAM: Epithelial cell adhesion molecule.

CD133

Human CD133 is a five-transmembrane single-chain glycoprotein that belongs to the prominin family, which contains two large extracellular and two small intracellular loops[9,10]. The role of CD133 as a CSC marker in liver cancer has been documented in several studies[11-13]. Suetsugu et al[14] reported that CD133+ Huh-7 cells (liver cancer cell line) have a high proliferative capacity in vitro and tumorigenic ability in vivo. Subsequently, Yin et al[15] isolated CD133+ cells from the HCC cell line SMMC -7721, and found that these cells had the highest colony-forming ability in vitro and tumorigenic ability in vivo. Yao et al[16] showed that knockout of the CD133 gene in Huh-7 cells suppresses their tumorigenic ability in vivo. Kohga et al[17] reported a relationship between CD133 and the invasion and distant metastasis of liver cancer. A recent study[12] showed that CD133+ liver cancer cells are resistant to apoptosis induced by radiation and have a stronger proliferative ability in vitro and tumorigenic ability in vivo. Some studies have shown that CD133+ liver cancer cells exhibit stronger abilities for colony formation and proliferation, which result in a poorer prognosis compared with CD133- liver cancer cells[18,19]. These findings indicate that CD133+ liver cancer cells may be LCSCs.

However, Salnikov et al[20] found that CD133+ and CD133- liver cancer cells have no significant differences in terms of migration, and the total number of CD33+ cells has no correlation with the clinical features of liver cancer patients. Therefore, CD133 as a LCSC marker requires further study.

CD90

CD90 (Thy-1) is a 25-30 kDa glycosylphosphatidylinositol-anchored glycoprotein expressed on many cell types, including T cells, thymocytes, neurons, endothelial cells, and fibroblasts. It is an important regulator of cell-cell and cell-matrix interactions with important roles in nerve regeneration, metastasis, inflammation, and fibrosis[21]. A study[22] has reported that CD90 is a surface marker for liver stem cells and hepatic progenitor cells during liver development. Recently, CD90 has also received attention as a CSC marker for various types of tumor cells including hepatic stem cells.

Yang et al[23] reported CD45-CD90+ cells in all liver cancer tissues and 90% of blood samples from liver cancer patients. They also found high expression of CD90 during tumor formation. These findings suggest that CD90 may participate in liver cancer development. Yang et al[24] also found that 4 × 10³ CD45-CD90+ cells from tumor tissues can form liver carcinoma in Beige/SCID mice. In addition, CD90+ cells can differentiate asymmetrically into CD90+ cells and CD90- cells, but CD90- daughter cells are all CD90- cells. These results show that CD90+ cells have strong proliferation, drug resistance and self-renewal abilities[25]. Recently, Michishita et al[26] reported that CD90+CD44+ HCC930599 cells (dog liver cancer cell line) have a stronger proliferation ability in vitro as well as self-renewal and tumorigenic abilities than CD90-CD44+ cells. All of these observations suggest that CD90 can be used as an LCSC marker.

The above studies show that CD90 is a potential marker of LCSCs. However, another study[27] reported that CD90+ liver cancer stem-like cells may participate in the late stage of liver cancer, and only appear in hepatitis B infection-related liver cancer. Therefore, CD90 still requires further research as a biomarker for LCSCs.

EPITHELIAL CELL ADHESION MOLECULE

Epithelial cell adhesion molecule (EpCAM), also known as CD326, is a single transmembrane glycoprotein encoded by the tumor-associated calcium signal transducer 1 gene, which belongs to a family of adhesion molecules. It has a molecular mass of 30-40 kDa and consists of three domains: an extracellular domain, a single transmembrane domain, and an intracellular structure domain. EpCAM has proven to be a marker of mature liver stem cells and progenitor cells, and is also a marker of hepatic oval cells[28,29]. Studies have shown that EpCAM participates in the β-catenin/Wnt signaling cascade, in which activation of proto-oncogenic proteins c-myc and cyclinA/E leads to tumorigenesis[30]. Yamashita et al[31] first reported that EpCAM can serve as a marker for LCSCs. Chen et al[32] found that CD133+EpCAM+ Huh7 cells have strong abilities for multi-directional differentiation, self-renewal, and clonal colony formation. Furthermore, only 500 CD133+EpCAM+ cells are tumorigenic in NOD/SCID mice. In addition, CD133+EpCAM+ cells show high expression of stem cell markers Nanog, Oct4, and Sox2.

In HCC patients, Sun et al[33] showed that EpCAM+ cells in peripheral circulation express other reported LCSC markers, CD133 and ABCG2. In NOD/SCID mice, injection of tumor cells showed that 300 EpCAM+CD45- cells were tumorigenic, whereas 1 × 10⁴ EpCAM-CD45- cells were not tumorigenic. Schulze et al[34] also suggested the existence of EpCAM+ cells in peripheral circulation of patients with liver cancer. Their clinical pathologic features tended to be > 400 ng/mL serum α-fetoprotein (AFP), various degrees of blood vessel metastasis, middle and advanced stage, and an overall low survival rate. Guo et al[35] followed patients with liver cancer after radical surgery, and found that the 1-, 2-, and 3-year survival rates of patients with EpCAM+ specimens were 85.7%, 51.3%, and 85.7%, respectively. Therefore, EpCAM+ cells may be LCSCs and radical surgery cannot completely kill these cells, which is the root cause of postoperative recurrence and metastasis. Therefore, EpCAM+ cell-targeting therapies are needed for the treatment of liver cancer.

OV6

Hepatic oval cells, called hepatic stem/progenitor cells in the liver Herring pipe, can differentiate into hepatocytes and bile duct cells. OV6 is a marker of hepatic oval cells[36]. In liver cancer induced by gene mutation, hepatic oval cells can become abnormal and differentiate into liver cancer cells or bile duct epithelial cells[37]. Thus, liver stem/progenitor cells may be involved in the development and progression of liver cancer. Recently, Jia et al[38] examined various cell surface markers, and found that liver cancer cells were derived from liver stem/progenitor cells. Yang et al[39] showed that OV6+ liver cancer cells have a stronger tumorigenic ability and chemotherapy resistance than OV6- cells. In addition, they found that the proportion of CD133+ cells in an HCC cell line ranged from 0.1% to 75%, while the proportion of OV6+ cells was relatively stable at 0.2%-3%. It is interesting to note the CD133+ cells express OV6, which further shows that OV6 can serve as a marker of LCSCs. Using magnetic bead separation, Yang et al[40] isolated OV6+ cells from HCC cell lines SMMC7721 and Huh7, and found that 103 OV6+ SMMC7721 cells or 104 OV6+ HuH7 cells were tumorigenic in NOD/SCID mice. They also found that OV6+ HCC cells in vivo and in vitro had strong invasion and metastatic abilities. These studies suggest that OV6 may be a potential marker of LCSCs.

CD44

CD44 is a transmembrane glycoprotein that mediates adhesion between cells and the extracellular matrix, lymphocyte activation and homing, and plays an important role in the invasion and metastasis of cancer. Zhu et al[41] reported that CD133+CD44+ cells have a strong tumorigenic ability in nude mice and high expression of the ATP binding cassette (ABC) transporter superfamily members (ABCB1, ABCC1, and ABCG2) that mediate resistance to chemotherapeutic drugs such as doxorubicin and vincristine. Further study found that CD133+CD44+ cells express genes related to stem cells such as β-catenin and Bmi-1. Hou et al[42] showed that CD133+CD44+ cells are the initial cells that produce metastasis to the lung and liver in immunodeficient mice. Analyses of human liver specimens showed that CD133+CD44+ liver cancer cells are associated with metastasis to the liver portal vein. Therefore, CD133 and CD44 can better define LCSCs.

Yang et al[23] first reported that CD90+CD44+ liver cancer cells are more aggressive, and both CD44 and CD90 can better define LCSCs. After irradiation of N1S1 rat liver cancer cells, Thompson et al[43] found a 22-fold increase in CD44+CD90+ cells and the use of a BEZ235 blocker could avoid thermal stress damage of PI3K-Akt-mTOR signaling pathways, in which CD44+ cells increased while CD90+ cells did not change. Further study of immunodeficient mice injected with liver cancer cells revealed CD44+ cells, but not CD90+ cells, on the edge of the thermal ablation and the edge of the liver cancer lesion. Therefore, after thermal ablation, recurrence was associated with a small number of CD44+ cells. Recently, Fernando et al[44] reported that transforming growth factor (TGF)-β treatment of long term-cultured PLC/PRF/5 liver cancer cells can induce resistance to sorafenib. Further analysis found that CD44 expression induced epithelial-to-mesenchymal transition characterized by vimentin protein expression, but the drug resistance was proportional to the number of CD44+ cells. In addition, repeated treatment with sorafenib could enrich CD44+ cells. Therefore, CD44 may be a potential molecular marker of LCSCs.

SIDE POPULATION CELLS

Fluorescence-activated cell sorting can isolate CSCs known as side population (SP) cells from a wide variety of tumor cell lines because the ABCG2 transporter effluxes the fluorescent DNA dye Hoechst 33342. The cell surface protein ABCG2, also called breast cancer resistance protein, is a member of the ABC transporter family, which was first identified in drug-resistant breast cancer cells. Cells expressing ABCG2 will pump out drugs, resulting in multi-drug resistance. LCSCs that are resistant to chemotherapy[45] indicate that ABCG2 is a candidate molecular marker of CSCs, which can be used for cell separation.

Chiba et al[46] found 0.25%-0.80% of SP cells among liver cancer cell lines Huh7 and PLC/PRF/5. Compared with non-SP cells, the SP cells had a higher proliferative capacity and considerable ability to resist apoptosis. In vitro studies also suggest that transplantation of 103 SP cells into immunodeficient NOD/SCID mice can cause tumors. Therefore, separation of SP cells can be useful when CSC markers are unknown. A recent study[47] reported that SP cells from the HAK-1A liver cancer cell line do not express CD90, EpCAM, CD13 or CD133. In the HAK-1B cell line, compared with non-SP cells, the SP cells have a clonal growth ability, strong tumorigenic ability, fast growth rate, and highly express CD13. However, there are no differences in the chemotherapy resistance, colony forming ability, or cell cycle.

TARGETED THERAPIES AGAINST LCSC MARKERS

Because LCSCs are related to drug resistance, metastasis, and recurrence of liver cancer, targeting LCSCs as a cancer treatment has become a promising strategy. Some studies have found that treatment measures targeting certain surface markers of LCSCs can inhibit their self-renewal and tumorigenesis, such as disrupting the expression of LCSC surface markers CD133[48], EpCAM[31,49], CD24[50], and CD13[49]. In addition, neutralizing antibodies against CD44 can effectively induce apoptosis of CD90+CD44+ LCSCs[23]. There is a broad prospect to develop targeted drugs against specific surface markers of LCSCs.

Some key signaling pathways in LCSCs are also therapeutic targets. Single target therapy is limited, but targeting both the LCSCs and surrounding environment may be more effective to inhibit the growth and metastasis of HCC.

Determination of specific markers for LCSCs and development of corresponding diagnostic strategies will be useful to detect LCSCs and monitor whether LCSCs have diffused into the blood and bone marrow. Such approaches might accurately predict metastasis and/or recurrence in HCC patients, and enable more individualized treatment plans.

PROBLEMS AND PROSPECTS OF LCSCS

LCSC sources

CSC theory suggests that tumor growth and progression are maintained by a small population of CSCs in tumor tissue. The number of cells with stem cell properties is maintained by CSC self-renewal, while CSCs constantly produce new tumor cells by differentiation.

In terms of the origin of CSCs (including LCSCs), there are two viewpoints. Most researchers believe that CSCs originate from abnormal proliferation and differentiation of stem cells[51-53]. In chronic liver disease caused by a variety of reasons such as viral hepatitis, fatty hepatitis, and metabolic liver disease, liver stem cells are actively proliferating. Under the influence of carcinogenic factors, actively proliferating liver stem cells might undergo malignant changes. In addition, most recognized LCSC surface markers such as CD133, EpCAM, and CD90 are also surface markers of stem cells. Finally, the development of LCSCs has common molecular signaling pathways or regulatory molecules with liver stem cells, such as Wnt, TGF-β, Notch, Hedgehog, Myc, and Bmi1[6]. However, the derivation of induced pluripotent stem cells by Takahashi et al[54] changed the understanding of the sources of stem cells. Therefore, some researchers consider that CSCs can also be induced from mature tumor cells under the action of various factors[55,56]. Recently, Holczbauer et al[57] reported that liver stem/progenitor cells and mature liver cells can transform into LCSCs by excessive activation of Ras. LCSCs are a dynamic cell population. Therefore, factors in the surrounding environment (such as chemotherapeutic drugs, radiation, oxygen, growth factors, and inflammatory factors) might induce both differentiation of LCSCs to cancer cells and mature cells to LCSCs to maintain the growth and progression of tumors.

FUTURE APPLICATIONS OF LCSC MARKERS

Although many surface markers have been reported for LCSCs, there is still no unified standard. At present, cells isolated by most surface markers have LCSC characteristics, but some markers mutually have less cross expression. Therefore, each marker may represent a subset of LCSCs. For example, Yamashita et al[27] reported that EpCAM+ and CD90+ LCSCs represent different cells with different biological characteristics. In addition, CSCs are considered to be a small population of tumor cells, and some markers, such as CD133, EpCAM, CD44, and CD24, can be expressed by 50% or more cells in HCC cell lines. Whether these markers can fully represent LCSCs is unclear, and their sensitivity and specificity require further study. To improve the specificity of LCSC markers, some researchers have advocated the combined use of multiple markers for LCSCs, such as CD90 and CD44[23,24], CD133 and CD44[41], CD133 and ALDH[11], EpCAM and AFP[31], and CD133 and EpCAM[32]. Considering a consensus is yet to be reached for LCSC markers, these markers may represent heterogeneous cells. Which surface markers or which combination of markers has higher specificity still requires further research.

LCSC markers have not been recognized until now, and the current problem is determination of LCSC-specific markers for identification, separation and cultivation of LCSCs. Different LCSC markers may represent different stages of liver stem cell differentiation[58]. Furthermore, LCSCs of different origins may express different markers[31].