Slug promoted vasculogenic mimicry in hepatocellular carcinoma

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumour cells to mimic the pattern of embryonic vasculogenic networks. Epithelial–mesenchymal transition (EMT) regulator slug have been implicated in the tumour invasion and metastasis of human hepatocellular carcinoma (HCC). However, the relationship between slug and VM formation is not clear. In the study, we demonstrated that slug expression was associated with EMT and cancer stem cell (CSCs) phenotype in HCC patients. Importantly, slug showed statistically correlation with VM formation. We consistently demonstrated that an overexpression of slug in HCC cells significantly increased CSCs subpopulation that was obvious by the increased clone forming efficiency in soft agar and by flowcytometry analysis. Meantime, the VM formation and VM mediator overexpression were also induced by slug induction. Finally, slug overexpression lead to the maintenance of CSCs phenotype and VM formation was demonstrated in vivo. Therefore, the results of this study indicate that slug induced the increase and maintenance of CSCs subpopulation and contributed to VM formation eventually. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.     

An assay for cancer stem cell-induced angiogenesis on chick chorioallantoic membrane

Angiogenesis is generally involved in tumor growth and metastasis. Cancer stem cells (CSCs) are considered to facilitate the angiogenesis. Therefore, CSCs could be the effective targets to stop angiogenesis. Recently, our group successfully generated CSC models from induced pluripotent stem cells (iPSCs) in the presence of conditioned medium derived from cancer derived cells. These novel model CSCs has been characterized by highly tumorigenic, angiogenic and metastatic potentials in vivo. The angiogenic potential of CSCs has been explained by the expression of both angiogenic factors and their receptors implying the angiogenesis in autocrine manner. In this protocol we optimized the method to evaluate tumor angiogenesis with the CSC model, which was described effective to assess sorafenib as an antiangiogenic drug, on chick chorioallantoic membrane (CAM) assay. Our results demonstrate that CSCs developed from iPSCs and CAM assay are a robust and cost-effective tool to evaluate tumor angiogenesis with CSCs. Collectively, CSCs in CAM assay could serve as a very useful model for the screening of potential therapeutic agents targeting tumor angiogenesis.     

Cancer stem cells and angiogenesis

Cancer stem cells (CSCs) or tumor initiating cells were identified and characterized as a unique subpopulation with stem cell features in many types of cancer. Current CSC studies provide novel insights regarding tumor initiation, progression, angiogenesis, resistance to therapy and interplay with the tumor micro-environment. A cancer stem cell niche has been proposed based on these findings. The niche provides the soil for CSC self-renewal and maintenance, stimulating essential signaling pathways in CSCs and leading to secretion of factors that promote angiogenesis and long term growth of CSCs. We present evidence which has emerged over the past 5 years indicating interaction of CSCs with angiogenesis in the proposed "vascular niche". Based on these findings, targeting the "cancer stem cell niche" by combining an individualized anti-CSC approach with treatment of their microenvironment may represent a novel therapeutic strategy against solid tumor systems.     

Paradoxical role of β8 integrin on angiogenesis and vasculogenic mimicry in glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive and highly vascularized brain tumor with poor prognosis. Endothelial cell-dependent angiogenesis and tumor cell-dependent Vasculogenic mimicry (VM) synergistically contribute to glioma vascularization and progression. However, the mechanism underlying GBM vascularization remains unclear. In this study, GBM stem cells (GSCs) were divided into high and low β8 integrin (ITGB8) subpopulations. Co-culture assays followed by Cell Counting Kit-8 (CCK-8), migration, Matrigel tube formation, and sprouting assays were conducted to assess the proliferative, migratory and angiogenic capacity of GBM cells and human brain microvascular endothelial cells (hBMECs). An intracranial glioma model was constructed to assess the effect of ITGB8 on tumor vascularization in vivo. Our results indicated that ITGB8 expression was elevated in GSCs and positively associated with stem cell markers in glioma tissues, and could be induced by hypoxia and p38 activation. ITGB8 in GSCs inhibited the angiogenesis of hBMECs in vitro, while it promoted the ability of network formation and expression of VM-related proteins. The orthotopic GBM model showed that ITGB8 contributed to decreased angiogenesis, meanwhile enhanced invasiveness and VM formation. Mechanistic studies indicated that ITGB8-TGFβ1 axis modulates VM and epithelial-mesenchymal transition (EMT) process via Smad2/3-RhoA signaling. Together, our findings demonstrated a differential role for ITGB8 in the regulation of angiogenesis and VM formation in GBM, and suggest that pharmacological inhibition of ITGB8 may represent a promising therapeutic strategy for treatment of GBM.Subject terms: Cancer stem cells, CNS cancer     

Traditional Chinese medicine as targeted treatment for epithelial-mesenchymal transition-induced cancer progression

The epithelial-mesenchymal transition (EMT) program, which loosens cell-cell adhesion complexes, endows cells with enhanced migratory and invasive properties. Furthermore, this process facilitates both the development of drug resistance and immunosuppression by tumor cells, which preclude the successful treatment of cancer. Recent research has demonstrated that many signaling pathways are involved in EMT progression. In addition, cancer stem cells (CSCs), vasculogenic mimicry (VM) and the tumor-related immune microenvironment all play important roles in tumor formation. However, there are few reports on the relationships between EMT and these factors. In addition, in recent years, traditional Chinese medicine (TCM) has developed a unique system for treating cancer. In this review, we summarize the crucial signaling pathways associated with the EMT process in cancer patients and discuss the interconnections between EMT and other molecular factors (such as CSCs, VM, and the tumor-related immune microenvironment). We attempt to identify common regulators that might be potential therapeutic targets to thereby optimize tumor treatment. In addition, we outline recent research on TCM approaches that target EMT and thereby provide a foundation for further research on the exact mechanisms by which TCMs affect EMT in cancer.     

VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells

There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.     

A model of cancer stem cells derived from mouse induced pluripotent stem cells

Cancer stem cells (CSCs) are capable of continuous proliferation and self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. CSCs are considered derived from normal stem cells affected by the tumor microenvironment although the mechanism of development is not clear yet. In 2007, Yamanaka's group succeeded in generating Nanog mouse induced pluripotent stem (miPS) cells, in which green fluorescent protein (GFP) has been inserted into the 5'-untranslated region of the Nanog gene. Usually, iPS cells, just like embryonic stem cells, are considered to be induced into progenitor cells, which differentiate into various normal phenotypes depending on the normal niche. We hypothesized that CSCs could be derived from Nanog miPS cells in the conditioned culture medium of cancer cell lines, which is a mimic of carcinoma microenvironment. As a result, the Nanog miPS cells treated with the conditioned medium of mouse Lewis lung carcinoma acquired characteristics of CSCs, in that they formed spheroids expressing GFP in suspension culture, and had a high tumorigenicity in Balb/c nude mice exhibiting angiogenesis in vivo. In addition, these iPS-derived CSCs had a capacity of self-renewal and expressed the marker genes, Nanog, Rex1, Eras, Esg1 and Cripto, associated with stem cell properties and an undifferentiated state. Thus we concluded that a model of CSCs was originally developed from miPS cells and proposed the conditioned culture medium of cancer cell lines might perform as niche for producing CSCs. The model of CSCs and the procedure of their establishment will help study the genetic alterations and the secreted factors in the tumor microenvironment which convert miPS cells to CSCs. Furthermore, the identification of potentially bona fide markers of CSCs, which will help the development of novel anti-cancer therapies, might be possible though the CSC model.     

Direct in vivo evidence for tumor propagation by glioblastoma cancer stem cells

High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC.     

Coaction of spheroid-derived stem-like cells and endothelial progenitor cells promotes development of colon cancer

Although some studies described the characteristics of colon cancer stem cells (CSCs) and the role of endothelial progenitor cells (EPCs) in neovascularization, it is still controversial whether an interaction exists or not between CSCs and EPCs. In the present study, HCT116 and HT29 sphere models, which are known to be the cells enriching CSCs, were established to investigate the roles of this interaction in development and metastasis of colon cancer. Compared with their parental counterparts, spheroid cells demonstrated higher capacity of invasion, higher tumorigenic and metastatic potential. Then the in vitro and in vivo relationship between CSCs and EPCs were studied by using capillary tube formation assay and xenograft models. Our results showed that spheroid cells could promote the proliferation, migration and tube formation of EPCs through secretion of vascular endothelial growth factor (VEGF). Meanwhile, the EPCs could increase tumorigenic capacity of spheroid cells through angiogenesis. Furthermore, higher microvessel density was detected in the area enriching cancer stem cells in human colon cancer tissue. Our findings indicate that spheroid cells possess the characteristics of cancer stem cells, and the coaction of CSCs and EPCs may play an important role in the development of colon cancer.     

综述: 肿瘤干细胞微环境与靶向干预策略

肿瘤干细胞具有自我更新和可塑性的潜能,能够维持肿瘤生长和异质性的能力．肿瘤干细胞是肿瘤产生、转移、耐药和复发的根源,肿瘤干细胞学说逐渐被肿瘤研究者所接受,因此,对肿瘤干细胞的深入理解有重大的科学和临床意义．肿瘤干细胞的微环境是肿瘤微环境的组成部分,包括细胞-细胞接触、分泌型因子等．肿瘤非干细胞和肿瘤干细胞本身都可以作为肿瘤干细胞的微环境．肿瘤干细胞的微环境可以维持肿瘤干细胞的可塑性,保护肿瘤干细胞免受免疫系统攻击,也可以促进其转移．肿瘤干细胞对其微环境的塑造、肿瘤干细胞的微环境对肿瘤干细胞自我更新的影响,以及针对肿瘤干细胞微环境的靶向干预等问题,已成为肿瘤干细胞研究的前沿问题．本文就肿瘤干细胞的发现、自我更新维持机制、肿瘤干细胞的微环境,及其肿瘤干细胞及微环境的干预策略等研究进展进行了综述．     

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 µM cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.     

MicroRNAs and breast cancer stem cells: Potential role in breast cancer therapy

MicroRNAs (miRNAs) can control cancer and cancer stem cells (CSCs), and this topic has drawn immense attention recently. Stem cells are a tiny population of a bulk of tumor cells that have enormous potential in expansion and metastasis of the tumor. miRNA have a crucial role in the management of the function of stem cells. This role is to either promote or suppress the tumor. In this review, we investigated the function and different characteristics of CSCs and function of the miRNAs that are related to them. We also demonstrated the role and efficacy of these miRNAs in breast cancer and breast cancer stem cells (BCSC). Eventually, we revealed the metastasis, tumor formation, and their role in the apoptosis process. Also, the therapeutic potential of miRNA as an effective method for the treatment of BCSC was described. Extensive research is required to investigate the employment or suppression of these miRNAs for therapeutics approached in different cancers in the future.     

Targeting cancer stem cells expressing an embryonic signature with anti-proteases to decrease their tumor potential

Cancer stem cells (CSCs) are a specific subset of cancer cells that sustain tumor growth and dissemination. They might represent a significant treatment target to reduce malignant progression and prevent tumor recurrence. In solid tumors, several hierarchically organized CSC clones coexist, even within a single tumor. Among them, CSCs displaying an embryonic stem cell ‘stemness'' signature, based on the expression of Oct-4, Nanog and Sox2, are present in distinct high-grade tumor types associated with poor prognosis. We previously designed a model to isolate pure populations of these CSCs from distinct solid tumors and used it to screen for molecules showing selective toxicity for this type of CSC. Here we show that human immunodeficiency virus (HIV)-protease inhibitors (HIV-PIs) specifically target CSCs expressing an embryonic signature derived from tumors with distinct origins. They reduced proliferation in a dose-dependent manner with a higher specificity as compared with the total population of cancer cells and/or healthy stem cells, and they were efficient in inducing cell death. Lopinavir was the most effective HIV-PI among those tested. It reduced self-renewal and induced apoptosis of CSCs, subsequently impairing in vivo CSC-induced allograft formation. Two key pharmacophores in the LPV structure were also identified. They are responsible for the specificity of CSC targeting and also for the overall antitumoral activity. These results contribute to the identification of molecules presenting selective toxicity for CSCs expressing an embryonic stemness signature. This paves the way to promising therapeutic opportunities for patients suffering from solid cancer tumors of poor prognosis.     

Stem-like Cancer Cells Are Inducible by Increasing Genomic Instability in Cancer Cells

The existence of cancer stem cells (CSCs) or stem-like cancer cells (SLCCs) is regarded as the cause of tumor formation and recurrence. However, the origin of such cells remains controversial with two competing hypotheses: CSCs are either transformed from tissue adult stem cells or dedifferentiated from transformed progenitor cells. Compelling evidence has determined the chromosomal aneuploidy to be one of the hallmarks of cancer cells, indicating genome instability plays an important role in tumorigenesis, for which CSCs are believed to be the initiator. To gain direct evidence that genomic instability is involved in the induction of SLCCs, we utilized multiple approaches to enhance genomic instability and monitored the percentage of SLCC in cultured cancer cells. Using side population (SP) cells as a marker for SLCC in human nasopharyngeal carcinoma (NPC) and CD133 for human neuroblastoma cells, we found that DNA damage inducers, UV and mitomycin C were capable of increasing SP cells in NPC CNE-2 and neuroblastoma SKN-SH cells. Likewise, either overexpression of a key regulator of cell cycle, Mad2, or knock down of Aurora B, an important kinase in mitosis, or Cdh1, a key E3 ligase in cell cycle, resulted in a significant increase of SP cells in CNE-2. More interestingly, enrichment of SP cells was observed in recurrent tumor tissues as compared with the primary tumor in the same NPC patients. Our study thus suggested that, beside transformation of tissue stem cells leading to CSC generation, genomic instability could be another potential mechanism resulting in SLCC formation, especially at tumor recurrence stage.     

Cancer stem cells: Road to therapeutic resistance and strategies to overcome resistance

Unlike other normal cells, a subpopulation of cells often termed as “stem cells” are long-lived and generate cellular progeny throughout life. Cancer stem cells (CSCs) are rare immortal cells within a tumor that can both self-renew by dividing and giving rise to many cell types that constitute the tumor. CSCs also have been shown to be involved in fundamental processes of cell proliferation and metastatic dissemination. CSCs are generally resistant to chemotherapy and radiotherapy, a subset of remaining CSCs after therapy can survive and promote cancer relapse and resistance to therapies. Understanding the biological characteristics of CSCs, the pathways leading to their sustainability and proliferation, and the CSCs role in drug resistance is crucial for establishing novel tumor diagnostic and therapeutic strategies. In this review, we address the pathways that regulate CSCs, the role of CSCs in the resistance to therapy, and strategies to overcome therapeutic resistance.     

A New Strategy Using ALDHhigh-CD8+T Cells to Inhibit Tumorigenesis

Background

Currently, many studies suggest that cancer stem cells (CSCs) are responsible for tumor initiation, tumorigenesis, metastasis and recurrence. CSCs have been identified from various human and murine tumors. The identification of CSCs allows us to develop strategies to target the CSCs.

Methods and Results

In this study, we used ALDEFLUOR as a single marker to isolate the CSCs from the human lung cancer cell line H460. We then characterized the CSCs by testing their sphere formation ability and tumorigenicity. Furthermore, we used CSC lysate-pulsed dendritic cells to stimulate CD8+T cells as a treatment strategy. Our study demonstrated that ALDEFLUOR could be used as a single marker to identify CSCs from the human lung cancer cell line H460. The ALDH^high cells could form more spheres and were more tumorigenic than the ALDH^low cells. Further study demonstrated that ALDH^high-CD8+T cells conferred more significant antitumor effects, resulting in the inhibition of tumor growth and prolonged survival. And the ALDH^high-CD8+T cells-mediated anti-tumor immunity might be due to the directly targeting against ALDH^high cancer stem cells (CSCs).

Conclusions

This study shows that ALDH^high-CD8+T cells mediate anti-tumor immunity by selectively targeting cancer stem cells, which result in inhibiting tumor growth and prolonging the survival of tumor-bearing mice, which provides a new strategy using ALDH^high-CD8+T cells to treat tumors.     

Advanced research on vasculogenic mimicry in cancer

Vasculogenic mimicry (VM) is a brand‐new tumour vascular paradigm independent of angiogenesis that describes the specific capacity of aggressive cancer cells to form vessel‐like networks that provide adequate blood supply for tumour growth. A variety of molecule mechanisms and signal pathways participate in VM induction. Additionally, cancer stem cell and epithelial‐mesenchymal transitions are also shown to be implicated in VM formation. As a unique perfusion way, VM is associated with tumour invasion, metastasis and poor cancer patient prognosis. Due to VM's important effects on tumour progression, more VM‐related strategies are being utilized for anticancer treatment. Here, with regard to the above aspects, we make a review of advanced research on VM in cancer.