Study Of P75^NTR As The Membrane Mark Of Cancer Stem Cells In Esophageal Squamous Cell Carcinoma

The"cancer stem cell"theory has aroused increasing interest in the field of oncology. According to this theory, cancer stem cells (CSCs), which account for a very small proportion of tumor tissue, have the self-renewal property typical of normal stem cells. CSCs rarely divide, but they are able to produce fast-proliferating daughter cells and play improtance role in tumor initiating, progressing and metastasis. People first separated and identified CSCs in leukemia. Previous studies have demonstrated the existence of CSCs in a variety of tumors including breast carcinoma, brain tumors, Prostate carcinoma, head and neck squamous cell carcinoma, colorectal carcinoma and so on. These studies strongly support the cancer stem cell theory.Low-affinity neurotrophin receptor (p75^NTR), a member of tumor necrosis factor superfamily, has been shown to paradoxically mediate neuronal survival and differentiation or apoptotic cell death, depending on the environment of the cells. In previous studies, p75^NTR was used for screening neural crown stem cells. p75^NTR was also used for screening mouse testis peritubular smooth muscle precursors , rat fat multipotential stem cells , and human corneal epithelial progenitor cells . Okumura et al reported that p75^NTR+ esophageal epithelial cells were stem cells due to their capacity of proliferation, self-renewal and multidirectional differentiation.This experiment determine the expression of p75^NTR in esophageal squamous cell carcinoma (ESCC), use flow cytometry to sort esophageal squamous cell carcinoma with p75^NTR as the mark. Then, study the biological characteristics and the differences in gene expression of the p75^NTR+ and p75^NTR- esophageal squamous carcinoma cells. This study can provide clues for studying the pathogenesis of esophageal tumor and looking for new therapeutic targets from the perspective of cancer stem cells theory.Part1: Expression of p75^NTR in ESCCObjective: To determine the expression of p75^NTR in ESCC, and to investigate the feasibility of p75^NTR as the membrane mark of cancer stem cells in ESCC. Methods: Immunohistochemical technique was used to detect the expression of p75^NTR in 100 patients with histopathologically confirmed ESCC. Then combinate the expression of p63 in ESCC to statistical analysis, and to investigate the feasibility of p75^NTR as the membrane mark of cancer stem cells in ESCC.Results: The expression of p75^NTR in ESCC is broadly consistent with the p63. In well differentiated cases (WDC), p75^NTR and p63 were confined to the surrounding basal-like cells which were at some distance from the centers of terminal differentiation expressed involucrin. In moderately differentiated cases (MDC), the number of involucrin + cells decreased and distribution of p75^NTR and p63 became irregular with respect to the centers of terminal differentiation. In poorly differentiated cases (PDC), p75^NTR and p63 were diffusely distributed, expressed involucrin very lowly.Conclusions: p75^NTR was mainly expressed in immature esophageal squamous carcinoma cells, staying away from the well-differentiated cells expressed involucrin. And the distributions of p75^NTR+ and p63+ cells in ESCC were highly consistent. Because the nature of the cancer stem cells of p63 in skin squamous cell carcinoma and cervical carcinoma has been confirmed, this initially indicate that p75^NTR can be the membrane mark of cancer stem cells in ESCC.Part2: Biological characteristics of p75^NTR+ esophageal squamous carcinoma cellsObjective: To study the biological characteristics of p75^NTR+ and p75^NTR- cells that were separated by fluorescence-activated cell sorting, and to investigate whether p75^NTR+ cells have the characteristics of cancer stem cells. Methods: To determine the biological characteristics of p75^NTR+ and p75^NTR- cells that were separated from ESCC by fluorescence-activated cell sorting, including proliferative capacity, self-renewal and varied differentiation capacity:1. Compare the growth characteristics of p75^NTR+ and p75^NTR- cells, and to draw cells growth curve; 2. Study the capacity of cloning of p75^NTR+ and p75^NTR- cells by monoclonal cultivating; 3. In the cultivating process, detect the percentage of p75^NTR+ cells of the progeny cells generated from monoclonal p75^NTR+ and p75^NTR- cells; 4. Compare the tumorigenicity of p75^NTR+ and p75^NTR- cells.Results: Comparing p75^NTR+ cells with p75^NTR- cells from 3 ESCC cell lines (Eca109,SHEC-4,SHEC-5), we find that p75^NTR+ cells possess higher proliferation ability than p75^NTR- cells(P<0.01). p75^NTR+ cells possess higher capacity of cloning than p75^NTR- cells(P<0.01). The percentage of p75^NTR+ cells of the progeny cells generated from monoclonal p75^NTR+ cells decreased progressively during the first five weeks and maintained steady thereafter. The cells derived from p75^NTR+ cells contained both p75^NTR+ and p75^NTR- cells, whereas those from p75^NTR- cells could not generated p75^NTR+ cells, The tumorigenicity of p75^NTR+ and p75^NTR- cells is not parently different, but p75^NTR+ cells have higher speed of tumorigenicity than p75^NTR- cells. Conclusions: Comparing to p75^NTR- cells, p75^NTR+ cells possess higher proliferation ability,self-renewal and varied differentiation capacity. p75^NTR+ cells could generate both p75^NTR+ and p75^NTR- progenies, but p75^NTR- cells could not generated p75^NTR+ cells. These results suggest that p75^NTR+ cells isolated from ESCC have the characteristics of cancer stem cells.Part3: Differences in gene expression of p75^NTR+ and p75^NTR- esophageal squamous carcinoma cellsObjective: To study the differences in gene expression of p75^NTR+ and p75^NTR- esophageal squamous carcinoma cells, in order to investigate stem cell-related gene expression of p75^NTR+ cells and provide clues for studying the pathogenesis of esophageal tumor and looking for new therapeutic targets from the perspective of cancer stem cells theory.Methods: Compare stem cell-related gene expression of p75^NTR+ and p75^NTR- esophageal squamous carcinoma cells by RT-PCR, including Bmi-1and p63, and the differences in gene expression of p75^NTR+ and p75^NTR- cells by Human Genome U133 Plus 2.0 Array (Affymatrix).Results: Comparing to p75^NTR- cells in 3 ESCC cell lines (Eca109,SHEC-4,SHEC-5), p75^NTR+ cells highly express Bmi-1and p63. In Eca109, p75^NTR+ cells express 476 up-regulated genes and 398 down-regulated genes. According to genetic information retrieval, the biological function of 452 up-regulated genes and 382 down-regulated genes has been tentatively determined. We have initially screened 12 genes related with TGF-βand Wnt signaling pathway which closely related with the characteristics of stem cells according to the biological function classification results.Conclusions: Comparing to p75^NTR- cells, p75^NTR+ cells highly express the genes closely related with the characteristics of stem cells, such as Bmi-1, p63, TGF-βand Wnt etc. This further confirme that p75^NTR+ cells isolated from ESCC have the characteristics of cancer stem cells. These results provide clues for looking for targeted therapy of esophageal squamous cell carcinoma stem cells. Conclusively, p75^NTR+ cells that were separated from ESCC by fluorescence-activated cell sorting possess higher proliferation ability ,self-renewal and differentiation capacity. Furthermore, they highly express the genes closely related with the characteristics of stem cells. This study provides an effective method for further studying the esophageal tumor stem cells and can provide clues for studying the pathogenesis of esophageal tumor and looking for new therapeutic targets from the perspective of cancer stem cells theory.