| BACKGROUND & OBJECTIVEStem cells possess the capacity of self-renewal and differentiation, which can differentiate into a variety of functional cells under certain conditions. Stem cells are generally classified as embryonic stem cells (ESCs) and adult stem cells (ASCs). ESCs are pluripotent, precursors of all cells of the organism. Tissue specific ASCs reside in adult tissues, which are capable of self-renewal and multipotent differentiation.With the increasing studies on stem cell, supposing a minority of cells in tumor cell population that named as cancer stem cell, which possessed unlimited self-renewal and differentiated potentiality and resulted in tumor growth, proliferation, metastasis and recurrence. But other tumor cells do not possess the capacities. The hematopoetic cancer stem cells were described first. CD34+/CD38-cells were isolated in acute myelogenous leukemia (AML), if injected into non-diabetic/severe combined immunodeficiency (NOD/SCID) mice, human AML can be transferred to mouse. These cells were named as leukemic initiating cells which are necessary and sufficient to maintain the leukemia. Recently, more and more cancer stem cells in solid tumor have been demonstrated. AI-Hajj et al demonstrated that Lin-ESA+CD44+CD24-/ low subtype cells in breast cancers had stronger tumorigenic capacity than Lin-ESA-CD44+CD24-/low cell. Singh et al discovered that only 100 CD133+brain cancer cells transferred into NOD/SCID mouse could induce tumors, further, the cell phenotype and differentiated degree of tumors were similar to these of primar tumor. But 5-10×104 brain cancer cells xenografted only developed to a single band glial scar in NOD/SCID mice. CSCs were also discovered in other solid tumors, such as melanoma, lung cancer, prostatic carcinoma, ovarian cancer, head and neck squamous cancer and colorectal cancer.NSCs and CSCs are a subset of a very rare, which has caused great difficulties to the relevant research work. That fact hinders the study of stem cells. Therefore, it is very important to enrich stem cells in vitro.Up to now, there are only two articles which are involved in the investigation of nasopharyngeal normal and cancer stem cells. Zhang found long-term BrdU-labeled LRCs in nasopharyngeal epithelia of adult mice and subcutaneous xenografts of human NPC cell lines in nude mice. Wang isolated SP cells in nasopharyngeal carcinoma cell line CNE2 and considered that CK19 was a biomarker of nasopharyngeal stem cells. Although research is rapidly advanced in this field, to our knowledge there are scarce published reports investigating CSC in human NPC.ALDH1 belongs to the aldehyde dehydrogenase superfamily which is responsible for the oxidation of aldehydes to their corresponding carboxylic acids.It is widely expressed during normal tissue development and homeostasis and is also found in immune cells. The human ALDH1 gene expresses in the cytoplasm, and locates in the 9q21 chromosome, which is comprise of the 53×103 base pairs form, including 13 exons and encoding 501 amino acid sequence, without repeats, N'terminal for the methionine M (Met), with a glutamic acid (Glu) and a cysteine (Cys) active site. The gene promoter region contains a ATA box and a CCAAT box, which locate upstream of transcription start sites at 32 bp and 74 bp-respectively. Recently scholars have found ALDH1 can be used as markers of cancer stem cells. ALDH1 was first used as marker of cancer stem cell in hematopoietic cells. In solid tumors, Ginestier et al discovered that ALDHl could be enriched cancer stem cell in breast cancer in 2007. ALDH1+cells possess self-renewal, tumorigenicity, multi-potential differentiation, which accounted for only 5% of the total number of breast cancer cells. They assayed tumorigenic capacity of ALDHl+cells and discovered only 500 ALDH1+cells could induce tumor, while 5000 ALDH1-cells not, it was showed that ALDH1+cells with high tumorigenic capacity than ALDH1-cells. They demonstrated that ALDH1 is a marker of breast stem cell and a predictor of poor clinical outcome. Subsequent studies have shown that ALDH1 can also serve as stem cell markers in head and neck squamous carcinoma, lung cancer, prostate cancer, pancreatic cancer, eye cancer and colorectal cancer. ALDHl have not been found in articles reported at home and abroad in NPC. The aim of this experiment was to study biological function of ALDH1 in vivo and vitro and the relationship between ALDH1 and Clinical outcomes in nasopharyngeal carcinoma patients.Materials and methods1. Biological function of ALDH1+cells of NPC 5-8F cell lines in vivo and vitro.1) Sorting ALDH1+cells and ALDH1-cells of 5-8Fcell line by ALDEFLUOR Reagent.2) The self-renewal and self-proliferation ability in vitro between ALDH1+cells and ALDH1-cells were detected by the plate colony formation test and MTT assay.3) To examine the migration ability of ALDH1+cells and ALDH1-cells we performed in transwell migration test.4) Tumor sphere formation assay ALDHl+cells and ALDH1-cells (1000 cells/ml) were cultured in serum-free DMEM-F12 supplemented with EGF, bFGF, B27, and observed everyday under light microscope.5) Staining of tumor sphere with PKH266) Detecting ALDH1 expression of tumor sphere by Immunofluorescence7) Tumor formation in vivo experiments To address the issue of whether tumorigenic activity differs between ALDH1+cells and ALDH1-cells, various numbers of ALDH1+cells and ALDH1-cells in 5-8F cell line were injected into SCID mice.8) Detection mRNA expression of stem markers in ALDH1+cells and ALDH1-cells by real time PCR2. Analyzing the relationship between ALDH1 and side population cells1) Sorting SP and NSP cells by Flow cytometry2) Detecting ALDH1 expression of SP and NSP cells by Immunofluorescence3) Isolating ALDH1+cells and ALDH1-cells of 5-8F cell line by Flow cytometry4) Detecting ABCG2 expression of ALDH1+cells and ALDHl-cells by Immunofluorescence5) ALDH1 expression of SP and NSP cells and ABCG2 expression of ALDH1+cells and ALDH1-cells were detected by real time PCR.3. Analyzing the relationship between ALDH1 and Clinical outcomes in NPC patientsFirst, we detected ALDH1 expression of NPCs and NP tissue by real time PCR, Second, we detected ALDH1 expression of NPCs tissue by immunohistochemistry, and also analyzed the relationship between ALDH1 and Clinical outcomes in NPC patients. Furthermore, we analyzed the risk of clinical outcomes by COX regression. 4. Analyzing the relationship between ALDH1 and Epithelial-Mesenchymal Transition。1) E-cadherin and vimentin expression of NPCs tissue was detected by immunohistochemistry. Furthermore, analyzing ALDH1 correlated with E-cadherin and vimentin by spearman rank correlation.2) Sorting ALDH1+ cells and ALDH1-cells of 5-8Fcell line by Flow cytometry3) Detecting E-cadherin and vimentin expression of ALDH1+cells and ALDH1-cells by real time PCR4) Detecting E-cadherin and vimentin expression of ALDH1+cells and ALDH1-cells by immunofluorescence.Result1. Biological function of ALDH1 in vivo and vitro1)ALDH1 cells accounted for unsorted 5-8F cells about 1.94%±0.69%2) Cell proliferation testALDH1+cells and ALDH1-cells were harvested from flow cytometer and were seeded in 96-well plate. We analyzed the cell vitality using MTT assay. The result showed that there were a significant difference in proliferation activity between ALDH1+ cells and ALDH1-cells (F=154.433, P=0.000).3) Colony formation assayThe number of the colony was assessed by counting under microscope.Good views were photographed. The ratio of colony formation of ALDH1+cells and ALDH1-cells were (53.500±2.784)% and (27.667±2.843)%.It demonstrated statistical significance (t=11.245, P=0.000). The results strongly indicated that ALDH1+cells possessed higher capability of colonogenicity.4) Transwell migration testThe result showed that the cells that penetrated artificial basement membrane was more per high power field for ALDH1+cells than ALDH1-cells, the difference being significant (t=26.292, P=0.000).5) Tumor sphere formation assayThe tumor sphere formation capacity of ALDH1+cells was significantly higher than ALDH1-cells and unsorted 5-8F cells (P=0.000), furthermore, without separation of 5-8F cells than ALDH1-cells (P= 0.044). It showed that ALDH1+ cells have a stronger self-renewal, proliferation.6) Staining of tumor sphere PKH26Very few cells within tumor sphere retained strong epifluorescence7) Detecting ALDH1 expression of tumor sphere by immunofluorescence.ALDH1 was universal expression in tumor sphere.8) Tumorigenesis of ALDH1+cells and ALDH1-cells in SCID mice by subcutaneous injection. 1×105,1×104,5000,500 ALDH1+cells and ALDH1-cells were injected into SCID mice respectively, as low as 1×104ALDH1+cells injection could initiate tumors. However, the ALDH1-cells injection consistently failed to form tumors. The result suggested that the ALDH1+cells had stronger tumorigenic capacity.9) Expression of stem markersALDH1+cells expressed higher levels of stem markers than ALDH1-cells do. such as OCT4 (t=9.305, P=0.001), BMI1 (t=7.224, P=0.002), KLF4 (t=8.737 P=0.001), SOX2 (t=10.309, P=0.000).2. The relationship between ALDH1+cells and SP cells1) Expression of ALDH1 in the SP was significantly higher than in NSP cells (t= 8.925, P=0.001), similarly, ABCG2 in ALDH1+cells was significantly higher than that of ALDH1-cells (t-11.044, P=0.000).2) ALDH1 was highly expressed in SP cells with low expression in the NSP, similarly, ABCG2 was highly in the expression of ALDH1+cells but in the ALDH1-cells with low expression.3. ALDH1 can be used as a risk factor for clinical prognosis of nasopharyngeal carcinomaThe expression of ALDH1 in nasopharyngeal carcinoma was detected by immunohistochemistry. It suggested that the expression of ALDH1 was weakly positive or negative in NPCs, but the edge of the cancer nests and stroma, particularly at spindle cancer cells were strongly positive. Clinical prognosis of ALDH1 was analyzed by Kaplan-Meier, it showed that the stronger was ALDH1 expression, the worse the prognosis of patients. Also, the rsultes indicated that ALDH1 closely correlated with tumor invasion and metastasis. COX regression analysis showed that, ALDHl was independent risk factor which affected the prognosis of nasopharyngeal carcinoma patients.4. The relationship between ALDH1 and epithelial-mesenchymal transitionThe expression of E-cadherin and Vimentin in 122 cases of NPC was detected by immunohistochemistry. It suggested that the expression of E-cadherin was low in most of NPC tissuess. On the contrary, vimentin was highly expressed. Spearman rank correlation analysis showed that ALDH1 was negatively correlated with E-cadherin (r=-0.264, P=0.003), but ALDH1 was positively correlated with vimentin (r= 0.240, P= 0.008). The result indicated that ALDHl was related with invasion and metastasis of NPC.Conclusion1. In vitro experiments suggested that ALDH1+cells possessed self-renewal, proliferation, Pluripotency, invasion and metastasis. ALDH1+cells had strong tumorigenicity capacity in vivo. ALDH1+cells possessed cancer stem cell-like characteristics, may be used as functional stem cell maker of nasopharyngeal carcinoma. These findings offer an important new tool for the study of nasopharyngeal carcinoma stem cells.2. ALDHl+cells and SP cells existed intersection3. The expression of ALDH1 correlated with clinic outcome of NPC, the result showed that ALDH1 was an independent risk factor for clinic outcome of NPC.4. ALDH1 possessed the properties of epithelial-mesenchymal transition...
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