Baocun Sun

Recently, tumor initiating cells (TICs), which possess self-renewal and other stem cell properties, are regarded as the cause of tumor initiation, recurrence and metastasis. The isolation and identification of TICs could help to develop novel therapeutic strategies. In this study, we isolated spheroid cells from human renal cell carcinoma (RCC) cell line SN12C in stem cell-conditioned medium. The stemness characteristics of spheroid cells, including tumorigenicity, self-renewal, proliferation and aldehyde dehydrogenase (ALDH) activity were evaluated; the expression levels of stemness genes and related proteins were assessed. Furthermore, study examined the differentiation of TICs into endothelial cells and the relationship between TICs and EMT. Our data demonstrated that spheroid cells cultured in defined serum-free medium possessed TIC properties, such as high tumorigenic capacity, upregulation of TIC-related genes and proteins, persistent self-renewal and extensive proliferation. Furthermore, spheroid cells were more aggressive in growth, invasion, scratch recovery, clonogenic survival and high aldehyde dehydrogenase (ALDH) activity. Interestingly, a marked increase in tumor vascularity compared to adherent tumors in vivo, and spheroid cells can differentiate into functional endothelial-like cells in vitro suggesting a role of tumor initiating cells in tumor angiogenesis. The spheroid cells also demonstrated down-regulated E-cadherin and up-regulated Vimentin expression, which is the typical phenotype of EMT. These results suggest that spheroid cells with tumor initiating cells-like characteristics contributed to tumor generation, progression, high tumorigenicity, pro-angiogenic capability and relationship with EMT. Further experiments using more refined selection criteria such as a combination of two or multiple markers would be useful to specifically identify and purify TICs.

To evaluate erythropoietin (Epo) and erythropoietin receptor (EpoR) expression, its relationship with vasculogenic mimicry (VM) and its prognostic value in human hepatocellular carcinoma (HCC), we examined Epo/EpoR expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-Schiff) double staining on 92 HCC specimens. The correlation between Epo/EpoR expression and VM formation was analyzed using two-tailed Chi-square test and Spearman correlation analysis. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between Epo/EpoR expression and VM formation (P < 0.05). Patients with Epo or EpoR expression exhibited poorer overall survival (OS) than Epo-negative or EpoR-negative patients (P < 0.05). Epo-positive/VM-positive and EpoR-positive/VM-positive patients had the worst OS (P < 0.05). In multivariate survival analysis, age, Epo and EpoR were independent prognostic factors related to OS. These results will provide evidence for further research on HCC microcirculation patterns and also will provide new possible targets for HCC diagnosis and treatment.

Aims: Dickkopf‐1 (Dkk1), an antagonist of the Wnt–β‐catenin signalling pathway, has been reported to play a role in cancer progression. However, little is known about the role of Dkk1 during the colorectal adenoma–carcinoma sequence. This study aimed to elucidate the role of Dkk1 in tumorigenesis and angiogenesis in colorectal cancer.Methods and results: We examined Dkk1 expression immunohistochemically in 476 colorectal tissue samples, including 46 sets of matched specimens. Dkk1 expression was down‐regulated during the colorectal adenoma–carcinoma sequence, both among the 476 samples and in the 46 sets of matched specimens. Dkk1 expression was correlated with decreased microvessel density (P < 0.05) and VEGF expression. In‐vitro 3D coculture experiments showed that Dkk1 overexpression in HCT116 cells inhibited tube‐like structure formation and down‐regulated VEGF expression in human umbilical vein endothelial cells. Xenografts of Dkk1‐overexpressing colorectal cancer cells were smaller, and showed lower microvessel density and VEGF expression levels, than those of control cells.Conclusions: This study is the first to show the roles of Dkk1 during the colorectal adenoma–carcinoma sequence, which may involve suppression of the tumorigenesis and angiogenesis of CRC. Dkk1 could therefore serve as a potential target for tumour therapy.

Physiological vasculogenesis in embryonic tissues share some important features with pathological neoangiogenesis in tumors. Linearly Patterned Programmed Cell Necrosis (LPPCN) and Vasculogenic Mimicry (VM) have been reported in tumors. The term VM refers to the aggressive tumor cells with CD31-negative phenotype to form Periodic Αcid Schiff (PAS)-positive network, that mimics the pattern of embryonic vasculogenic networks. LPPCN had been observed in our laboratory, and served as a spatial infrastructure for VM and endothelium-dependent vessel formation. Studies have been shown that hypoxia-inducible factor-1α (HIF-1α) can induce tumor cells to form vessel-like tubes and express genes associated with VM. Therefore, an analogous investigation has been carried out to determine if these patterns existed in mouse embryonic vasculogenesis. In this essay, the results demonstrated that the number of Linearly Patterned Cell Αpoptosis (LPCA), embryo Vasculogenic Μimicry (embryo VM), endothelium-dependent vessels, and relative-protein of HIF-1α expression all showed time-dependent tendencies on E5.5-E9.5 (p < 0.05). The proteins CD133, VEGF, Twist, E-cadherin, and Vimentin showed local plexus distribution on E6.5-E7.5 (p < 0.05). LPCA and embryo VM existed in embryonic vasculogenesis. The relative protein of HIF-1α regulated the mouse embryonic vasculogenesis.

Purpose. Vasculogenic mimicry (VM) was related to invasion and metastasis of head and neck squamous cell carcinoma (HNSCC) patients. This study was designed to investigate the role of EphA2 in VM formation of HNSCC.Methods. The SiRNA technique was used to knock down the expression of EphA2in vitro. The ability of cell migration and invasion were measured by transwell and wound healing assays; three-dimensional culture was used to detect the ability of channel-like structure formation; Western blot was used to detect the expression of epithelial-mesenchymal transition- (EMT-) related moleculesin vitro. Further semiquantitative real-time RT-PCR assays and immunohistochemistry were used to demonstrate expression of EphA2 and EMT-related molecules according to VM presence or not in human tissue.Results. Knocking down EphA2in vitroleads to disabled channel-like structure formation, reduction of invasion and migration ability, and reverse of EMT-related markers. Both semiquantitative real-time RT-PCR and immunohistochemistry showed that expressions of EphA2, Twist, and Vimentin were higher in the VM-positive group than in the VM-negative group significantly, while expressions of E-cadherin, claudin4, and DSG-3 were reverse.Conclusions. EphA2 played a key role in VM formation of HNSCC through regulation of EMT.

Colon cancer remains one of the lethal malignancies in the world. Aberrant activation of canonical Wnt/β‐catenin signaling pathway has been observed in colon cancer. In contrast, the non‐canonical Wnt signaling functions remain obscure. Wnt5a is a representative non‐canonical Wnt ligand which has gained extensive attention nowadays. Wnt5a has been shown to play an important role in EMT in prostate cancer and melanoma, but its role in colon cancer is still ambiguous. Here we have evaluated Wnt5a expression in a large cohort of 217 colon cancers by immunohistochemistry and analyzed its correlation with clinicopathologic characteristics. We found that expression of Wnt5a was diminished significantly in majority of primary colon cancers and negatively related with EMT biomarkers. To further enlighten the mechanism which Wnt5a regulates EMT in vitro, we established ectopic Wnt5a expression models. Protein analysis demonstrated that Wnt5a inhibited EMT and antagonized canonical Wnt signaling in colon cancer cells. Overexpression of Wnt5a impaired cell motility and invasion and inhibited cell proliferation by manipulating Bax. Moreover, Wnt5a suppressed the tumor growth in nude mice and impaired tumorigenicity in vivo. Wnt5a also induced intracellular calcium and activated non‐canonical Wnt/Ca²⁺ signaling in colon cancer. In summary, although Wnt5a was down‐regulated in majority of colon cancers, enhanced Wnt5a expression predict preferable outcome in colon cancer patients. Our findings indicate that Wnt5a might act as tumor suppressor by inhibiting cell proliferation and attenuating EMT in colon cancer cells. Wnt5a could be used as a novel prognostic marker and/or therapeutic target for colon cancer in the future. J. Cell. Physiol. 229: 1908–1917, 2014.