Characterization And Functional Analyses Of The Side Population Of The Nasopharyngeal Cancer Cell Line 5-8F

BACKGROUNDCancer stem cell theory postulates that within every cancer there exists a specific subset of cells that possess characteristics similar to that of normal stem cells, the ability of unlimited proliferation and the potential of differentiation into various cell types. Cancer stem cells account for a small portion of a tumor mass, but they play an important role in tumorigenesis, development, recurrence and metastasis of tumors. Recently, the cancer stem cells and their potential clinical significance have become the forefront of cancer research area.At present, we have isolated and identified cancer stem cell based primarily on tumor cell surface membrane protein, adhesion molecules and receptor differences, such as CD133, CD44 and so on. However, because most of the tumor still lack specific surface markers, the separation and purification of cancer stem cells have been a long-standing difficult problem.It was reported that the side population (SP) cells stained with Hoechst33342 demonstrated low fluorescence after be excited by the UV light, which were found in the one step separation of bone marrow hematopietic stem cells. It was caused by SP cells effluxing DNA binding fluorescent dye. These cells accounted for only 0.05% of whole bone marrow cells, but most of them were hematopoietic repopulating cells, namely hematopoietic stem cells. Nowadays, the SP cells have been found various tissues of human and animals, such as bone marrow, skeletal muscle, nervous system, etc. Furthermore they also exist in many kinds of solid tumors and cancer cell lines, for example stomach cancer, prostate cancer, lung cancer, glioblastoma, and so on. However, not all cancer tissue, like Wilms tumor, rhabdomyosarcoma, osteosarcoma, liver cancer cell line HepG2 and in Huh6, exist SP cells.Studies have shown that SP cells have stem cell characteristics and are enriched in the stem cell population, which have self-renewal, multilineage differentiation potential and repair tissue functions. Specifically, SP cells in the tumor cell lines have a strong ability for tumorigenicity and drug resistance, which may be the root causes of tumor recurrence and drug resistance. SP cells can express higher ABC transporter (ATP-binding cassette transporter) family members, ABCG2/Bcrpl (ATP-binding cassette superfamily G member2/breast cancer resistance protein 1) and stem cell marker. Due to a "pump" function of ABCG2/BCRP1 located in the cell membrane, the SP cells can pump out cytotoxic drug and give rise to tumor resistance to chemotherapeutic drugs. Increasing the expression of ABCG-2/BCRP-1 using transgenic technology could raise the proportion of SP cells. Meanwhile, SP cells could show strong tumorigenic capacity after injection into NOD/SCID mouse. Therefore, due to the absence of useful molecular markers, SP cell might be used as an important means to study cancer stem cells.According to these knowledge, we detected whether there were SP cells in nasopharyngeal carcinoma cell line 5-8F by flow cytometry and to analyze whether they possessed the characteristics of cancer stem cells by various in vivo and in vitro biological experiments. We also tried to examine the expression of ABC transport proteins and other stem cell markers in the side population of nasopharyngeal cancer (NPC) cells and to investigate if any other protein molecules could correlate with the SP phenotype. In addition, the existence and function of cancer stem cell in clinical specimens were to further verify. The aim of this project was to develop a methodology to detect and isolate nasopharyngeal carcinoma stem cells by analyzing SP cells using fluorescent dye Hoechst33342. And more importantly, it may lay a foundation to further investigate the mechanisms underlying the occurrence and development, metastasis and recurrence of NPC.METHODS1. Optimization of the staining condition of SP cells in cell line 5-8FDifferent staining conditions using Hoechst33342, including optimal incubated time, appropriate final concentration, were investigated by flow cytometry and inverted fluorescence microscopy, respectively. The influence of cell growth density and incubation environment on the proportion of SP cells was estimated.2. Measurement of expression of ABCG2, CD 133 in SP cells by flow cytometry.When the cells were co-stained by Fluorescent antibody and Hoechst 33342, flow cytometry was employed to investigate their relationships.3. Identification of cell purityThe cell purity of SP and MP cells was identified by the inverted fluorescence microscopy and flow cytometry, respectively.4. Detection of expression of ABC transporters and sternness markersAfter RNAs were extracted from SP and MP cells, their purity and concentration were measurement. The gene expression of good quality RNA was assayed by qRT-PCR.5. Characterization of SP cells5.1 The self-renewal and self-proliferation ability in vitro of SP and MP cells were detected by the plate colony formation test and MTT growth curve, respectively. Drug resistance was also examined by MTT growth curve.5.2 Measurement of differentiation potential of SP cells SP cells and MP cells were separated by flow cytometer and then cultured in vitro. After 6 days, the proportion of the SP cells was analyzed by flow cytometry.5.3 Matrigel invasion assay and transwell migration test To investigate the invasion and migration ability of SP cells and MP cells, in vitro matrigel invasion assay and transwell migration test were performed.5.4 Cell cycle analyses of SP and MP cell by flow cytometry.5.5 Tumorigenesis of SP and MP cells in mice by subcutaneous injection. To estimate whether tumorigenic activity of SP cells differed from MP cells, various numbers of SP and MP cells in 5-8F cell line were injected into nude mice.RESULTS1. Optimization of the staining condition of SP cells in cell line 5-8F1.1 The incubated time of cells with Hoechst33342 affected the detection of SP cell in cell line 5-8F.When the cells with Hoechst33342 were incubated for 70 min, the most optimized SP cells were screened.1.2 The incubated concentration of cells with the fluorescent dye Hoechst33342 affected the detection of SP cell in 5-8F cell line.The appropriate incubated concentration of the cells with Hoechst33342 was 3 mg/L.1.3 The cell density affected the proportion of SP cellDifferent cell density or confluent may influent on the proportion of SP cells. To some extent, with the decreasing of cell density, the proportion of SP cells may be decreased. Compared to 100% of the cells, the proportion of SP cells reduced from 2.3% to 0.8% in 70% of the cells. Menwhile, SP cells can be inhibited by Verapamil. 1.4 Different incubated environment affected proportion of SP cellsCompared with 37℃water bath, the proportion of SP cells was higher in the 37℃CO2-cell incubator.2. Identification of cell purityIn fluorescence microscope, MP cell nuclei showed strong blue fluorescence while SP cell nucleus was weak, which could be explained by dye resisted or dyed pale cells (UV excitation). The purity of cells was above 98% by flow cytometry.3. Assay of ABCG2, CD133 expression in SP cells by flow cytometry.SP cells have high expression of ABCG2 protein, about 85.5%, while ABCG2 protein in MP cells expresses only 9.2%. But there was no significant difference of CD 133 protein expression between SP and MP cells.4. Detection expression of ABC transporters and sternness markersCompared with MP cells, SP cells express higher levels of ABC transporters and sternness marker, such as ABCG2 (t=-12.775, P= 0.000), MDR1 (t=-6.366, P= 0.003), ABCC2(t=-6.058, P=0.004), OCT4 (t=-8.242, P=0.001), SOX2 (t=-3.560, P=0.024),BMI1(t=-5.049, P=0.007),Klf4(t=-5.479, P=0.005), C-Myc(t=-4.283, P=0.013), CD44 (t=-5.860, P=0.004). However, there were no significant statistical difference with CD133 (t=-1.037, P=0.358), ABCA5 (t=-2.838, P=0.098) between SP and MP cells.5. Characterization of SP cells5.1 Colony formation assay and cell proliferation test5.1.1 Colony formation assayThe number of the colony formation was counted under microscope. The ratio of colony formation of SP and MP cells were (49.111±2.912)% and (23.111±2.009)%, respectively. It demonstrated statistical significance (t=12.729, P=0.000). The results strongly indicated that SP cells possessed higher colonogenicity capability than that of MP cells.5.1.2 Cell proliferation testAfter sorting by flow cytometry, SP cells and MP cells were seeded in 96-well plate. The cell viability was measured by MTT assay. The result showed that proliferation activity of SP cells were significantly different from that of MP cells (F=683.216, P=0.000). SP cells exhibited stronger ability for drug resistance than that of MP cells (F=362.120, P=0.000).5.2 Differentiation potential measurementAfter culturing for 6 days, the side population of in SP cells and MP cells was reanalyzed by flow cytometry. The data showed that SP cells still contain 1% Hoechst 33342 dull cells, whereas MP cells only contain 0.1%, and these may have arisen from residual SP cells from the last sorting. This indicated that SP cells can differentiate into MP cells, but MP cannot differentiate into SP cells. These findings showed that SP cells may undergo asymmetrical division to self-renew and generate heterogeneous phenotypes of low-tumorigenic cells, like MP cells that form the bulk of the tumor. In contrast, MP cannot differentiate into SP cells under normal circumstances.5.3 Matrigel invasion assay and transwell migration testThe result showed that the number of cells penetrating artificial basement membrane in SP cells was more than that of MP cells in per high power field. The invasion and migration ability of SP cells was significantly different from that of MP cells (t=15.826, P=0.000),(t=15.313, P=0.000).5.4 Cell cycle analyses of SP and MP cells by flow cytometryCell cycle analysis revealed there was statistical difference in Go/G1(t=3.795, P=0.019), G2/M(t=-3.686, P=0.021), PI (t=-3.795, P=0.019) between SP and MP cells. The results suggested that the SP cells might be in a relative resting phase. 5.5 Tumorigenesis of SP and MP cells in mice by subcutaneous injection.The experiment demonstrated that 5000 number of SP cell could successfully initiate tumors. However, the same number of MP cells consistently failed to form tumors. The result suggested that the SP cell exhibited stronger tumorigenic capacity compared with that of MP cells.Conclusion1. Sorting conditions of SP cells by flow cytometry:the optimal incubated time of the cells with fluorescent dye Hoechst33342 was 70 min and the optimal dye concentration was 3 mg/L. The side proportion of 5-8F cells was about 2.3%, which was density-dependent. The incubated environment could affect the SP detection.2. High purity of SP cells (98%) could be obtained by flow cytometry.3. Compared with the MP cells, SP cells displayed stem cell-like biological properties of self-renewal and differentiation in vitro and in vivo.4. SP cells could express higher levels of ABC transporters and sternness marker than that of MP cells.5. The result showed that CD 133 was not enriched in SP cells, It may be explained that CD 133 was not the surface markers of the NPC stem cell. The mechanism remains to be further investigated.