| Background:Colorectal cancer is one of most common digestive tract cancer in the world. In China, the incidence of colorectal cancer is increasing because of some reasons such as the changes of life style, aging of the population, environmental pollution, and so on. The situation of disease prevention is still very severe. The relapse and metastasis of cancer is the major cause of death.Cancer stem cells(CSCs) are the putative cancer-initiating cells and are considered to be the cause of cancer recurrence and metastasis because of the characteristics such as self-renewal, multipotency and limitless proliferation potential, and drug-resistance. These characteristics promote them play crucial roles in cancer generation and development. Besides, epithelial-to-mesenchymal transition(EMT) is considered to be the crucial way of cancer invasion and metastasis. Recnetly, data of CSCs also suggests the close relationship between CSCs and the acquisition of EMT. EMT confers invasive and metastatic characteristics, resitance to therapies, and CSCs phenotypes on cancer cells in experimental models. This transformation is associated with loss of E-Cadherin in epithelial cells and increased expression of mesenchymal markers, such as Vimentin, Fibronectin, mesenchymal associated transcription factors, such as Snail, Twist. Meanwhile, the changes of EMT correlate with increase cellular motility as well. It is reported that induction of EMT in cancer cells with drugs or overexpression of EMT transcription factors results in acquisition of mesenchymal properties and in expression of stem-cell markers. On the other hand, cancer cells following treatment with anti-cancer drugs, which have been shown to enrich CSCs, manifest the phenotypes and gene expression like EMT. These findings indicate that CSCs can invade and metastasize by EMT. However, as EMT is a quick shift of cells and the available date were acquired from experimental models, the definitive proof of EMT happening in human tumors is lacking so far.CSCs possess intrinsic biological characteristics to form tumor and may invade tissues through EMT. But it is unclear that how they evade immune surveillance for final survival in immunocompetent hosts. Previous reports have suggested inherent connections between immune suppression and EMT, such as that Snail-induced EMT induced regulatory T cells and impaired dendritic cells. Taken together, we hypothesize immunoevasion is important for CSCs that undergo EMT through paraneoplastic inflammation region without immune clearance and then implement invasion and metastasis. B7H1, a ligand of programmed cell death 1(PD-1), has been wellknown as a crucial co-stimulatory molecule and plays an important role in the induction and maintenance of peripheral tolerance. B7H1 is upregulated on considerable kinds of cancer cells which offers negative signals and leads to immunosuppression through PD-1-B7H1 interaction between cancer cells and T cells, resulting in tumor-infiltrating T cells dysfunction and Treg recruitment. Nevertheless, B7H1 expression on CSCs is not known well in colorectal cancer. Thus, we aim to sort and identify the CSCs and detect EMT phenotypes and B7H1 expression style in colorectal CSCs in the current study. Besides, IFN-γhas been added in culture systems to upregulate B7H1 to detect the effect on phenotypes and functions of different subpopulation of cancer cells.Methods:(1) HT29 cells were sorted by CD133 magnetic beads. The CD133+ cells and CD133-cells were tested CSCs-related capability such as sphere formation and tumorigenicity in NOD/SCID mice. Meanwhile, stemness genes Oct-4, Sox-2 and EMT-related genes Snail, Twist, E-cadherin, Vimentin and Fibronectin were tested by RT-q PCR.(2) Colorectal cancer tissues were obtained by surgery. The frozen sections of cancer tissues were made and the expression pattern of CD133 was detected by immunofluorescence staining. CD133 and EMT-related markers E-cadherin and vimentin were co-stained to explore the EMT phenotypes of CD133+ cells directly.(3) B7H1 and CD133 were co-stained in HT29 cells and colorectal cancer tissues by immunofluorescence staining to determine the possible immunoevasion way of CSCs. B7H1 and E-cadherin / vimentin were co-stained to explore the EMT phenotypes of B7H1+ cells.(4) IFN-γwas added in HT29 culture systems to detect the effect on different subsets of HT29 cells. CD133, B7H1 and EMT shape were tested. Further more, tumorigenic assays were used to determine the effect of IFN-γon CD133+/CD133- cells.Results:(1) CD133+ and CD133- HT29 cells were sorted by a modified magnetic activated cell sorting way. The purities of each subpopulations reached 80% or more. CD133+ cells possessed more tumorigenic potential and tumor sphere ability. Besides, CD133+ cells expressed other stem cell-associated molecules Oct-4 and Sox-2. Meanwhile, CD133+ cells expressed higher level of Snail, Twist, vimentin, fibronectin but less E-cadherin, manifesting the EMT-like features.(2) CD133 was expressed on cancer cells in colorectal cancer tissue and the expression levels were associated with tumor TNM stage. In addition, some CD133+ cells displayed EMT phenotypes, which were expressed vimentin but not E-cadherin in tissues.(3) Co-expression of B7H1 and CD133 were detected in HT29 cells and colorectal cancer tissues. Though the distribution of B7H1 was complex in cancer tissues, B7H1 was major distributed on cancer cells of 13/20 cases and co-expressed with CD133 in these tissues. What’s more, some B7H1 expressing cells displayed EMT phenotypes in these cases. Besides, B7H1 mainly expressed on stromal cells in 6/20 cases.(4) After treated with IFN-γ, the expression of B7H1 in HT29 cells was upregulated. Meanwhile, the shapes of cells showed EMT changes. But the expression of CD133 was decrease. Nevertheless, the treated of IFN-γ didn’t sharply effect the tumorigenicity of CD133+ cells. However, the tumorigenic capability of CD133- cells was increased. Q-PCR test showed that IFNgR1 and PCNA were increased in CD133- cells after IFN-γtreated, which might be the reasons of the tumorigenic capability changes of CD133- cells.Conclusion:(1) The sorted CD133+ cells had CSCs-like characteristics by showing capability of sphere forming and tumorigenicity, expressing higher levels of stem cell markers and manifesting EMT gene expression profile.(2) CD133 was expressed on cancer cells in colorectal cancer tissues and the expression levels were associated with tumor TNM stage. Some CD133+ cells displayed EMT phenotypes.(3) B7H1 co-expressed with CD133 in HT29 cells and some colorectal cancer cells in tissues and manifested EMT phenotypes, indicating a possible way of immunoevasion of CSCs-like cells of colorectal cancer.(4) IFN-γ could upregulate the expression of B7H1 in HT29 cells and promote the changes of EMT. The expression of CD133 was decreased but the tumorigenicity of CD133+ cells didn’t change. However, the tumorigenic capability of CD133- cells was increased, which might be associated with IFNgR1 distribution and the function of promoting proliferation of IFN-γ. |
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