The critical role of CD133(+)CD44(+/high) tumor cells in hematogenous metastasis of liver cancers

Metastatic hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. However, the cell population responsible for its metastasis remains largely unknown. Here, we reported that CD133(+)CD44(+/high) defined a subgroup of tumor cells that was responsible for hematogenous metastasis of liver cancers. Immunohistochemical investigation of human HCC specimens revealed that the number of CD133(+) and CD44(+) HCC cells was increased and was associated with portal vein invasion. Purified CD133(+) or CD44(high) HCC cells were superior in clonogenic growth and vascular invasion, respectively. Thus, the combination of CD133 and CD44 was used to define a novel HCC sub-population. CD133(+)CD44(high), but not CD133(+)CD44(low/-), CD133(-)CD44(high) or CD133(-)CD44(low/-) xenografts, produced intrahepatic or lung metastasis in nude mice. Further analysis of human HCC samples by flow cytometry showed that the number of CD133(+)CD44(+) tumor cells was associated with portal vein metastasis. The cDNA microarray analysis of CD133(+)CD44(+) and CD133(+)CD44(-) tumor cells isolated from metastatic HCC patients revealed that these cells comprised of two different populations possessing distinct gene expression profiles. Our results suggest that CD133(+)CD44(+) tumor cells are a particular population responsible for hematogenous metastasis in liver cancers and that these cells might be targets for treatment of HCC metastasis.     

A comparison of CFU-GM, BFU-E and endothelial progenitor cells using ex vivo expansion of selected cord blood CD133(+) and CD34(+) cells

BACKGROUND: CD133 is a newly developed hematopoietic stem cell marker but little is known about its function. Whether CD133(+) cell selection provides any advantage over CD34(+) selection for hematopoietic stem cell isolation and transplantation is unclear. The present study compared colony formation and endothelial cell differentiation of these two cell types from umbilical cord blood (UCB). METHODS: Mononuclear cells from the same UCB samples were used for both CD133(+) and CD34(+) cell selection. Cells with 97.1% purity were incubated in semi-solid culture medium containing stem cell growth factor (SCGF) and G-CSF or erythropoietin (EPO). Purified cells were also cultured in M199 containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-1). RESULTS: CD34(+) and CD133(+) cells produced similar numbers of CFU-GM colonies (median 43.25 and 30.5, respectively; P>0.2). However, a greater than four-fold difference in BFU-E colony formation was observed from CD34(+) cells compared with CD133(+) cells (median 35 and 8, respectively; P<0.04). CD34(+) cells gave rise to endothelial-like cells when stimulated with VEGF, bFGF and IGF-1. CD133(+) cells were unable produce this cell type under the same conditions. DISCUSSION: CD133(+) cells produced smaller BFU-E colonies and were unable to differentiate into mature endothelial cells. CD34(+) cells contained endothelial progenitors that could differentiate into mature cells of this lineage. Based on these data, it appears that CD133 offers no distinct advantage over CD34 as a selective marker for immunoaffinity-based isolation of hematopoietic stem cells and endothelial progenitor cells.     

Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory, CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133(+) HCC CSC population is still heterogeneous, consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach, we compared protein profiles between CD133(+) and CD133(-) subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133(+) subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH(+) to be CD133(+), yet not all CD133(+) HCC cells were ALDH(+). Subsequent studies on purified subpopulations found CD133(+)ALDH(+) cells to be significantly more tumorigenic than their CD133(-)ALDH(+) or CD133(-)ALDH(-) counterparts, both in vitro and in vivo. These data, combined with those from our previous work, reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133(+)ALDH(+) > CD133(+)ALDH(-) > CD133(-)ALDH(-). ALDH, expressed along CD133, can more specifically characterize the tumorigenic liver CSC population.     

Comparative analysis of proliferative potential and clonogenicity of MACS-immunomagnetic isolated CD34+ and CD133+ blood stem cells derived from a single donor

A novel stem cell marker prominin-1 (CD133) has been shown to be expressed on a subpopulation of CD34(+) haematopoietic stem and progenitor cells. The aim of this study was to compare in parallel commercially available CD34(+) and CD133(+) isolation methods based on paramagnetic bead-coupled antibodies using clinical-grade samples of mobilized peripheral blood from 10 individual healthy donors under identical conditions. The CD133 negative fraction from the first selection was used for CD34(+) enrichment to obtain an additional CD34(+)/CD133(-) population. Although no significant difference in total cell expansion between cells isolated from the three procedures was observed in a 7-day cytokine-driven suspension culture, the long-term culture-initiating cell assay demonstrated that cells derived by CD34(+) isolation contain less primitive progenitors than those isolated based on CD133(+) selection. Interestingly, CD34(+)-enriched progenitors, especially the CD34(+)/CD133(-) fraction, contained a significantly higher proportion of erythroid colony-forming cells, whereas the highest content of myeloid colony-forming cells was concentrated in the CD133(+) selected cells. These subtle differences between CD34(+) and CD133(+) immunomagnetic selection will have to be explored for their potential clinical relevance.     

Identification of CD133-positive radioresistant cells in atypical teratoid/rhabdoid tumor

Atypical teratoid/rhabdoid tumor (AT/RT) is an extremely malignant neoplasm in the central nervous system (CNS) which occurs in infancy and childhood. Recent studies suggested that CD133 could be considered a marker for brain cancer stem-like cells (CSCs). However, the role of CD133 in AT/RT has never been investigated. Herein we report the isolation of CD133-positive cells (CD133(+)), found to have the potential to differentiate into three germ layer tissues, from tissues of nine AT/RT patients. The migration/invasion/malignancy and radioresistant capabilities of CD133(+) were significantly augmented when compared to CD133(-). The clinical data showed that the amount of CD133(+) in AT/RTs correlated positively with the degree of resistance to radiation therapy. Using cDNA microarray analysis, the genotoxic-response profiles of CD133(+) and CD133(-) irradiated with 10 Gy ionizing radiation (IR) were analyzed 0.5, 2, 6, 12 and 24 h post-IR. We then validated these microarray data and showed increased phosphorylation after IR of p-ATM, p-RAD17, and p-CHX2 as well as increased expression of BCL-2 protein in CD133(+) compared to CD133(-). Furthermore, we found that CD133(+) can effectively resist IR with cisplatin- and/or TRAIL-induced apoptosis. Immunohistochemical analysis confirmed the up-regulated expression of p-ATM and BCL-2 proteins in IR-treated CD133(+) xenotransgrafts in SCID mice but not in IR-treated CD133(-). Importantly, the effect of IR in CD133(+) transplanted mice can be significantly improved by a combination of BCL-2 siRNA with debromohymenialdisine, an inhibitor of checkpoint kinases. In sum, this is the first report indicating that CD133(+) AT/RT cells demonstrate the characteristics of CSCs. The IR-resistant and anti-apoptotic properties in CD133(+) may reflect the clinical refractory malignancy of AT/RTs and thus the activated p-ATM pathway and BCL-2 expression in CD133(+) could be possible targets to improve future treatment of deadly diseases like AT/RT.     

CD31 (PECAM-1)-bright cells derived from AC133-positive cells in human peripheral blood as endothelial-precursor cells

To clarify the process of endothelial differentiation, we isolated AC133(+) cells and induced the in vitro differentiation of these cells into endothelial cells. AC133(+) cells efficiently differentiated into endothelial cells when the cells were cultured on fibronectin-coated dishes in the presence of vascular endothelial growth factor. Time-course analysis of the alteration of endothelial markers on cultured AC133(+) cells revealed that the expression of CD31 (PECAM-1) on AC133(+) cells was the earliest marker among all of the tested markers. Based on the hypothesis that CD31 is an early indicator during the endothelial differentiation, we examined the relationship between CD31 expression and the ability to differentiate into endothelial cells in cells derived from AC133(+) cells. CD31-bright cells, which were sorted from cultured AC133(+) cells, differentiated more efficiently into endothelial cells than had CD31-positive or CD31-negative cells, suggesting that CD31-bright cells may be precursor cells for endothelial cells. In the present study, we identified CD31(+) cells derived from cultured AC133(+) cells that are able to differentiate to endothelial cells as precursor cells.     

PTEN status is related to cell proliferation and self-renewal independent of CD133 phenotype in the glioma-initiating cells

CD133 is extensively used as a surface marker to identify and isolate glioma-initiating cells (GICs) from malignant brain tumors; however, instances of CD133(-) cells exhibiting similar properties have also been reported. To clarify the availability of CD133 as the GIC marker, we first evaluated the ratio of CD133(+) cells and malignancy of glioma spheroids GIC1 and GIC2, respectively. GIC1, which showed a lower percentage of CD133(+) cells, exhibited a highly aggressive behavior in comparison with GIC2. The following experiments demonstrated that tumor suppressor PTEN was lost in GIC1, resulting in the activation of AKT pathway. Overexpression of recombinant PTEN in GIC1 suppressed its proliferation and self-renew without significant effect on CD133 expression level, indicating that the inconsistence between the ratio of CD133(+) cells and proliferation and self-renewal capacity of GIC1 and GIC2 was caused by PTEN deficiency. To further validate our conclusion, a series of GICs were analyzed and the percentages of CD133(+) cells could not reflect the degrees of cell proliferation and self-renewal characteristics in the PTEN deficient GICs, suggesting that the application of CD133 as the GIC maker was restricted by PTEN loss. Furthermore, down-regulation of PTEN in the PTEN-expressing GICs could break the positive correlation between the ratio of CD133(+) cells and proliferation and self-renewal capacity. Our results demonstrated that PTEN status is related to cell proliferation and self-renewal independent of CD133 phenotype in the glioma-initiating cells, resulting in the limitations of CD133 as a biomarker for PTEN deficient GICs.     

低氧对CD34^＋造血干／祖细胞增殖分化及其对细胞因子依赖性的影响

To elucidate the effects of hypoxia on the proliferation and differentiation of CD34(+) hematopoietic stem/progenitor cells and their response to cytokines, the cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Mononuclear cells (MNC) and CD34(+) cells were incubated in severe hypoxia (1% oxygen) culture system, and the colony forming cells and antigen expression were studied by colony forming assays and FACS analysis. The results showed that incubation in severe hypoxia increased the number of erythroid bursts (BFU-E) (324.8+/-41.4/10(4) cells) generated from CD34(+) cells (191.2+/-34.5/10(4) cells in the control group, P<0.01). Severe hypoxia also enhanced the maintenance and cloning efficiency of BFU-E in a liquid culture system without growth factors, the number of BFU-E (152.4+/-22.6/10(4)cells) was much bigger than that in the control group (74.2+/-9.3/10(4) cells, P<0.01). In cultures incubated in hypoxia, the percentage of CD34(+) cells was significantly higher (2.5+/-1.2-fold, P<0.05) than in those incubated in air. BFU-E cloning efficiency of MNC was not significantly affected by hypoxia. The above results show that hypoxia enhances the maintenance of erythroid progenitor cells generated from CD34(+) hematopoietic stem/progenitor cells, no matter growth factors are present or not. These positive effects of hypoxia did not occur for the other progenitors.     

Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/progenitor cells

The CD133 antigen, identified as a hematopoietic stem cell marker, appears in various human embryonic epithelia including the neural tube, gut, and kidney. We herein investigated whether CD133(+) cells isolated from human hepatocellular carcinoma cell lines possess cancer stem/progenitor cell-like properties. Among the three cell lines studied, the CD133 antigen was found to be expressed only on the surface of Huh-7 cells. CD133(+) cells from Huh-7 performed a higher in vitro proliferative potential and lower mRNA expressions of mature hepatocyte markers, glutamine synthetase and cytochrome P450 3A4, than CD133(-) population of Huh-7 cells. When either CD133(+) or CD133(-) cells were subcutaneously injected into SCID mice, CD133(+) cells formed tumors, whereas CD133(-) cells induced either a very small number of tumors or none at all. Taken together, the identification of CD133(+) cells could thus be a potentially powerful tool to investigate the tumorigenic process in the hepatoma system and to also develop effective therapies targeted against hepatocellular carcinoma.     

Vaccination with CD133+ melanoma induces specific Th17 and Th1 cell�Cmediated antitumor reactivity against parental tumor

Accumulating evidence suggests that cancer cells possess a small subpopulation that survives during potentially lethal stresses, including chemotherapy, radiation treatment, and molecular-targeting therapy. CD133 is a putative marker that distinguishes a minor subpopulation from normal differentiated tumor cells in many cancers. Although it is necessary to eradicate all cancer cells to obtain a cure, effective treatment to eliminate the CD133(+) treatment-tolerant cells has not been elucidated. In this study, we demonstrated that a CD133(+) subpopulation in murine melanoma is immunogenic and that effector T cells specific for the CD133(+) melanoma cells mediated potent antitumor reactivity, curing the mice of the parental melanoma. CD133(+) melanoma antigens preferentially induced type 17 T helper (Th17) cells and Th1 cells but not Th2 cells. CD133(+) melanoma cell-specific CD4(+) T-cell treatment eradicated not only CD133(+) tumor cells but also CD133(-) tumor cells while inducing long-lasting accumulation of lymphocytes and dendritic cells with upregulated MHC class II in tumor tissues. Further, the treatment prevented regulatory T-cell induction. These results indicate that T-cell immunotherapy is a promising treatment option to eradicate CD133(+) drug-tolerant cells to obtain a cure for cancer.     

A Prominin-1-Rich Pediatric Glioblastoma: Biologic Behavior Is Determined by Oxygen Tension-Modulated CD133 Expression but Not Accompanied by Underlying Molecular Profiles

Few studies on the biologic and molecular properties of pediatric glioblastoma have been performed. Until now, differential genomic analysis of CD133(+)ve and CD133(-)ve fractions has not been described in pediatric glioma. We hypothesize not only that the presence of CD133 could be the source of tumor resistance but also that maintenance of this molecule by hypoxia dictates cellular and molecular behavior. From a series of human glioblastoma biopsies investigated, only one, IN699 (from a pediatric glioblastoma), generated greater than 4% of the total cell volume as CD133(+)ve cells. Using this pediatric glioblastoma, containing unprecedented high levels of the putative brain tumor stem cell marker CD133, as a study model, we report biologic and molecular characteristics of the parent culture and of CD133(+)ve and CD133(-)ve populations derived therefrom under atmospheric and hypoxic culture conditions. Immunocytochemistry and flow cytometry were performed with antigenic markers known to characterize neural stem cells and associated glioma behavior. Behavioral analysis was carried out using proliferation, adhesion, migration, and invasion assays. Cell cycle analysis and array comparative genomic hybridization were used to assess copy number profiles for parental cells and CD133(+)ve and CD133(-)ve fractions, respectively. With regard to invasion and proliferation, CD133(+)ve and CD133(-)ve fractions were inversely proportional, with a significant increase in invasive propensity within the CD133(-)ve cells (P < .005) and a significant increase in proliferation within CD133(+)ve cells (P < .005). Our observations indicate identical genomic imbalances between CD133(+)ve and CD133(-)ve fractions. Furthermore, our research documents a direct link between decreasing oxygen tension and CD133 expression.     

miR-130b Promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133? counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133? cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133? cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133? cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.     

Functional expression of chemokine receptor CCR5 on CD4(+) T cells during virus-induced central nervous system disease

Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). CD4(+) T cells are important in amplifying demyelination by attracting macrophages into the central nervous system (CNS) following viral infection; however, the mechanisms governing the entry of these cells into the CNS are poorly understood. The role of chemokine receptor CCR5 in trafficking of virus-specific CD4(+) T cells into the CNS of MHV-infected mice was investigated. CD4(+) T cells from immunized CCR5(+/+) and CCR5(-/-) mice were expanded in the presence of the immunodominant epitope present in the MHV transmembrane (M) protein encompassing amino acids 133 to 147 (M133-147). Adoptive transfer of CCR5(+/+)-derived CD4(+) T cells to MHV-infected RAG1(-/-) mice resulted in CD4(+)-T-cell entry into the CNS and clearance of virus from the brain. These mice also displayed robust demyelination correlating with macrophage accumulation within the CNS. Conversely, CD4(+) T cells from CCR5(-/-) mice displayed an impaired ability to traffic into the CNS of MHV-infected RAG1(-/-) recipients, which correlated with increased viral titers, diminished macrophage accumulation, and limited demyelination. Analysis of chemokine receptor mRNA expression by M133-147-expanded CCR5(-/-)-derived CD4(+) T cells revealed reduced expression of CCR1, CCR2, and CXCR3, indicating that CCR5 signaling is important in increased expression of these receptors, which aid in trafficking of CD4(+) T cells into the CNS. Collectively these results demonstrate that CCR5 signaling is important to migration of CD4(+) T cells to the CNS following MHV infection.     

Human CD34+ CD133+ hematopoietic stem cells cultured with growth factors including Angptl5 efficiently engraft adult NOD-SCID Il2rγ-/- (NSG) mice

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34(+) CD133(+) cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg(-/-) (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34(+) CD133(+) fraction of expanded cells and that CD34(+) CD133(+) cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.     

人喉癌细胞系Hep-2 中CD133 阳性肿瘤干细胞的分离及意义

目的:研究喉癌细胞系Hep-2中CD133的表达;比较CD133~+细胞、未分选细胞、CD133~-细胞的体外增殖、克隆形成能力及其在裸鼠体内的成瘤能力;探讨喉癌肝细胞对化疗药物顺铂(cisplatin,DDP)的抵抗作用。方法:采用流式细胞仪检测CD133在Hep-2细胞系中的表达;免疫磁珠分选技术纯化CD133阳性肿瘤细胞;使用四甲基偶氮唑蓝(MTT)法和平板克隆形成实验检测分选所得各细胞亚群细胞以及未分选细胞的体外增殖能力和克隆形成能力;将CD133阳性肿瘤细胞和CD133阴性肿瘤细胞以一定的数量级注入重症联合免疫缺陷小鼠腹部皮下,比较其成瘤差异性;此外,使用DDP干预分选所得各细胞亚群细胞,检测比较CD133阳性肿瘤细胞和CD133阴性肿瘤细胞的体外增殖能力与体内成瘤能力。结果:流式细胞仪示CD133在Hep-2细胞系中呈微量恒定表达,表达概率为40.12±1.32%;CD133阳性肿瘤细胞的体外增殖能力显著强于CD133阴性肿瘤细胞的增殖能力(P0.05),且其克隆形成能力也强于CD133阴性肿瘤细胞;体内成瘤实验结果显示CD133阳性肿瘤细胞较CD133阴性细胞、未分选细胞在重症联合免疫缺陷小鼠体内具有更强的成瘤性(P0.05);在DDP的干预下,相对于CD133阴性肿瘤细胞,CD133阳性肿瘤细胞表现出更强的抵抗力。结论:喉癌Hep-2细胞系中,CD133阳性癌细胞具有强的体外增殖能力、体内成瘤能力且对化疗药物具有较强的抵抗性,可作为喉癌肿瘤干细胞的标志之一。     

CD133: Enhancement of Bone Healing by Local Transplantation of Peripheral Blood Cells in a Biologically Delayed Rat Osteotomy Model

Sufficient angiogenesis is crucial during tissue regeneration and therefore also pivotal in bone defect healing. Recently, peripheral blood derived progenitor cells have been identified to have in addition to their angiogenic potential also osteogenic characteristics, leading to the hypothesis that bone regeneration could be stimulated by local administration of these cells. The aim of this study was to evaluate the angiogenic potential of locally administered progenitor cells to improve bone defect healing. Cells were separated from the peripheral blood of donor animals using the markers CD34 and CD133. Results of the in vitro experiments confirmed high angiogenic potential in the CD133(+) cell group. CD34(+) and CD133(+) cells were tested in an in vivo rat femoral defect model of delayed healing for their positive effect on the healing outcome. An increased callus formation and higher bone mineral density of callus tissue was found after the CD133(+) cell treatment compared to the group treated with CD34(+) cells and the control group without cells. Histological findings confirmed an increase in vessel formation and mineralization at day 42 in the osteotomy gap after CD133(+) cell transplantation. The higher angiogenic potential of CD133(+) cells from the in vitro experients therefore correlates with the in vivo data. This study demonstrates the suitability of angiogenic precursors to further bone healing and gives an indication that peripheral blood is a promising source for progenitor cells circumventing the problems associated with bone marrow extraction.