Sorting And Identification Of Side Population Cells In Human Hepatocellular Carcinoma

Hepatocellular Carcinoma (HCC) is one of the commonest malignancy tumors in our country. It has become the second cancer killer in China since 1990s. Unfortunately, despite great progress has been made in clinical research of HCC in the past decades and a part of early stage HCC patients have survived for a long time, the overall survival is still unsatisfactory. Most of the patients were diagnosed at a very late stage with extremely poor prognosis. The major reason is the metastatic recurrence after HCC resection. Little has been discovered about the mechanism of metastasis.Therefore,it is urgent to understand the origin of the metastatic recurrence in HCC.Cancer stem cells is the cancer cell that has the potential to self-renew,forming additional tumorigenic cancer cell of similar phenotype and to give rise to diverse cancer cells with limited proliferative potential .Eradication of the stem cell compartment of tumor also may be essential to achieve stable,long-lating remission,and even a cure of cancer.Advances in our knowledge of the properties of stem cells have made specific targeting and eradication of cancer stem cells a topic of considerable interest. The concept was first advised in the later of 19^th century.However, it was not accepted for shorting of evidence. It developed very slow because of several factors such as the limition of technique and experimental condition. Until recent years, as the discover of acute myeloid leukemia stem cell, breast cancer stem cell and brain tumor stem cell, the cancer stem has been accepted by more and more researchers.In 1996, Goodell detected a distinct side population of Hoechst33342 dye-excluding cells when he analysed adult bone marrow cells using flow cytometer. He named these cells side population cells(SP cells).Hoechst33342 is a fluorescent dye that binds to DNA and is taken up and effluxed by the cells in an active biological process.This differential efflux can be measured and used to separate varies cell types by FACS analysis using a UV laser. This SP fraction is highly enriched for stem cell activity and contains the majority of cells that label with hematopoietic stem cell markers. Further studies shows that the SP cells is sensitive to the drug verapamil.Verapamil can prevent the efflux of the Hoechst dye 33342 by blocking multi-drug resistance channel proteins there by allowing cells to retain Hoechst33342 dye and effectively obscuring the SP population. Recently, Zhou and colleagues have shown that the ABC-binding cassette transporter ABCG2/BCRP1 is expressed in SP cells isolated from bone marrow cells, and that transduction of bone marrow cells with a vector expressing ABCG2/BCRP1 results in an increase in the SP population from 0.05% to 62.5%. Moreover, further study show that the loss of ABCG2/ BCRP1 expression lead to a significant reduction in the number of SP cells in the bone marrow. They have demonstrated that Mdr1-type gene products are not required for the SP phenotype by showing that mice deficient for Mdr1a/1b genes have normal numbers of SP cells in bone marrow. These SP cells were shown to express Bcrp1 mRNA, which alone is sufficient to confer the SP phenotype in bone marrow cells.It was known that a solid tumor mass is composed of tumor cell clones of different biological behavior. We name this phenomenon tumor heterogeneity.It means that tumor cells produce a great varity of cells with different degrees of differentiation and phenotype due to their unstable natural and the selective pressure of living surroundings.According to the cancer stem cell theory, only a small subset of cancer cells is capable of extensive proliferation. We speculate that there are cancer stem cells in HCC. However, we don't know the liver cancer stem cell for shorting of specific marker. SP technique has allowed us to pursue the identification of stem cells without knowing the specific marker.As a result, adult stem cell have been isolated from a variety of tissue sources on the basis of differential Hoechst33342 dye-exclusion, including bone marrow,skeletal muscle, nervous tissue and pancreas.The majority of patients who develop recurrence , drug-resistance are due to the ability of cancer stem cells to escape the drugs.ABCG2, otherwise known as the BCRP1 transporter, perhaps confers the ablity to define the drug resistence associated efflux of many chemotherapeutic agents.MHCC97-H/L cell lines are the cell line with high metastatic potential and low metastatic potential cell line established by our institute.For SP cells have the stem-like properties in some cancer cells, the purpose of our study is to examine whether SP cells exist in MHCC97H/L cell lines and fresh specimens of human HCC and observe the its basic biological features. 1. Sorting of side population in MHCC97-H/L cell linesThis study sought to identify the side population (SP) cells in hepatocellular carcinoma MHCC97-H/L cell lines and examine the relationship between ABCG2 and SP phenotype.Hepatocellular carcinoma MHCC97-H/L cells suspension was stained with Hoechst33342 and PI in the absence or presence of verapamil. Then MHCC97-H and MHHCC97-L cells were analyzed. MHCC97-H cells was sorted in the fluorescence-activated cell sorter.We observed the expression of ABCG2 in SP cells using cell immunochemistry, western blot and RT-PCR.The result showed that side population cells exist in human MHCC97-H/L cell lines. The percentage of SP cell in MHCC97-H, HCC cell line with high metastatic potential was significantly higher than MHCC97-L, HCC cell line with low metastatic potential. SP cell maybe associated with the metastatic potential of HCC.ABCG2 is correlated with the SPphenotype.2. The cellular biological characteristics of SP cellsThe major objectives were to sort SP and non-SP cells from MHCC97-H and study their respective cellular biological characteristics.1. Morphologic characteristics:Compare with non-SP cells, SP cells are smaller and SP cell's variability is higher under phase microscope and electron microscope.There are fewer Golgi body, Free ribosome mitochondrion and rough endoplasmic reticulum in SP cells than non-SP cells.2. Clony formation assayClony formation assay was carried out in 6-well plate.Plates were maintained at 37°C in humidified incubator and were fed every 3 days with DMEM medium.After 12 days, the number of the colony formation was assessed by counting under microscope.Good views were photographed.The ratio of clony formation of SP cells and non-SP is (28.6±3.8) %and (8.6±2.8) % .It demonstrated statistical significance. The results strongly indicated that SP cells possessed higher capability of clonogenicity. 3. Cell proliferation testSP cells and SP cells were harvested from flow cytometer and were seeded in 96-well plate.We analyed the cell variability using MTT assay.The result showed that there are a big difference in proliferation activity in SP and non-SP cells.Cell variability in SP cells was consistently higher than in non-SP cells..4. Gelatin zymography assayBecause matrix-degrading proteinases have been implicated as playing an important role in HCC invasion and metastasis, we speculated that the increased invasiveness might be related to MMP activity. To test this hypothesis, we performed a gelatin zymography assay. Increased activity of MMP9 was observed in SP cells, but not in non-SP cells.5. Matrigel invasion assayTo examine the invasion ability of SP cells and non-SP cells,we performed the in vitro matrigel invasion assay.The result showed that the cells that penetrated artificial basement membrane was 4.5+1.0 cells per high power field for SP cells and 16.5±3.0 per high power field for non-SP cells, the difference being significant(P<0.05, t test.)6. Tumor initiation caused by side population injectionTo address the issue of whether tumorigenic activity differs between SP and non-SP cells, various numbers of SP and non-SP cells in MHCC97-H cell line were injected into NOD/SCID mice. Subcutaneous tumor formation required at least 1×10⁶ unsorted MHCC97-H cell injection. As low as 1×10⁴ SP cell injection could initiate tumors in 5 of 6 mice. However, 1×10⁶ non-SP cells injection consistently failed to form tumors in all mice injected. These SP cells could be enriched about 100-fold for tumorigenic cells.7. Growth inhibition by 5-FU in Vitro5-FU has been shown to inhibit tumor cells in vitro and in vivo.Thus, we investigated whether 5-FU inhibits MHCC97-H SP and/or non-SP cells in vitro MTT assay was used to assess proliferation inhibition.The result demonstrated that the chemotherapy drug 5-FU can inhibit the Growth of non-SP cell strongly,whereas not SP cells. 3. Analysis of SP cells in the primary HCC cellsThis study sought to identify the side population (SP) cells in primary hepatocellular carcinoma cells.The procedure of SP analysis is similar to the first part.The result showed that side population cells indeed exist in some of primary HCC tissues. Despite the sample is few, we can see that SP is correleated with the malignant fetures of tumor.Conclusions1. SP cells exist in human MHCC97-H/L cell lines and primary HCC tissues.2. SP cells have some stem cell properties such as high tumorigenic and drug resistence.3. SP cell maybe related to the metastatic potential of HCC.The potential application of this work1. Side population cells correlates with HCC invasion and metastasis, which may provide some insight into the mechanism of HCC metastasis.2. SP phenotype may be a useful marker in the isolation of cancer stem cells and one early detection marker of metastasis and target of chemotherapeutic agent.Originalities of this workTo our knowledge, this is the first discovery that SP cells do exist in human MHCC97-H/L cell lines and primary HCC tissues and maybe correlated with the metastasis of HCC.