Heterogeneity of Functional Properties of Clone 66 Murine Breast Cancer Cells Expressing Various Stem Cell Phenotypes

Introduction

Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous.

Methods

Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis.

Results

The proportion of cells expressing CD44^highCD24^low/neg, side population (SP) cells, ALDH1⁺, CD49f^high, CD133^high, and CD34^high differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1⁺, CD34^low, and CD49f^high suggested properties of transit amplifying cells. Colony formation was higher from ALDH1⁻ and non-SP cells than ALDH1⁺ and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than “non-stem” cells. Fewer SP cells were needed to form tumors than ALDH1⁺ cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined.

Conclusions

These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells.     

A CD44high/EGFRlow Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment

Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to emergence of therapy resistance which results in local and regional recurrences that may have their origin in resistant cancer stem cells (CSCs) or cells with an epithelial-mesenchymal transition (EMT) phenotype. In the present study, we investigate the possibility of using the cell surface expression of CD44 and epidermal growth factor receptor (EGFR), both of which have been used as stem cell markers, to identify subpopulations within HNSCC cell lines that differ with respect to phenotype and treatment sensitivity. Three subpopulations, consisting of CD44^high/EGFR^low, CD44^high/EGFR^high and CD44^low cells, respectively, were collected by fluorescence-activated cell sorting. The CD44^high/EGFR^low population showed a spindle-shaped EMT-like morphology, while the CD44^low population was dominated by cobblestone-shaped cells. The CD44^high/EGFR^low population was enriched with cells in G0/G1 and showed a relatively low proliferation rate and a high plating efficiency. Using a real time PCR array, 27 genes, of which 14 were related to an EMT phenotype and two with stemness, were found to be differentially expressed in CD44^high/EGFR^low cells in comparison to CD44^low cells. Moreover, CD44^high/EGFR^low cells showed a low sensitivity to radiation, cisplatin, cetuximab and gefitinib, and a high sensitivity to dasatinib relative to its CD44^high/EGFR^high and CD44^low counterparts. In conclusion, our results show that the combination of CD44 (high) and EGFR (low) cell surface expression can be used to identify a treatment resistant subpopulation with an EMT phenotype in HNSCC cell lines.     

Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells

Metastatic colorectal cancer remains a serious health concern with poor patient survival. Although 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) is the standard therapy for colorectal cancer, it has met with limited success. Recurrence of the tumor after chemotherapy could partly be explained by the enrichment of the chemo-resistant sub-population of cancer stem cells (CSCs) that possess the ability for self-renewal and differentiation into different lineages in the tumor. Therefore development of therapeutic strategies that target CSCs for successful treatment of this malignancy is warranted. The current investigation was undertaken to examine the effectiveness of the combination therapy of dasatinib (a Src inhibitor) and curcumin (a dietary agent with pleiotropic effect) in inhibiting the growth and other properties of carcinogenesis of chemo-resistant colon cancer cells that are enriched in CSCs sub-population. Remnants of spontaneous adenomas from APC ^{Min +/-} mice treated with dasatinib and/or curcumin were analyzed for several cancer stem cell markers (ALDH, CD44, CD133 and CD166). Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant) and HT-29 (p53 mutant; K-ras wild type) were used to generate FOLFOX resistant (referred to as CR) cells. The effectiveness of the combination therapy in inhibiting growth, invasive potential and stemness was examined in colon cancer CR cells. The residual tumors from APC ^{Min +/-} mice treated with dasatinib and/or curcumin showed 80-90% decrease in the expression of the CSC markers ALDH, CD44, CD133, CD166. The colon cancer CR cells showed a higher expression of CSCs markers, cell invasion potential and ability to form colonospheres, compared to the corresponding parental cells. The combination therapy of dasatinib and curcumin demonstrated synergistic interactions in CR HCT-116 and CR HT-29 cells, as determined by Calcusyn analysis. The combinatorial therapy inhibited cellular growth, invasion and colonosphere formation and also reduced CSC population as evidenced by the decreased expression of CSC specific markers: CD133, CD44, CD166 and ALDH. Our data suggest that the combination therapy of dasatinib and curcumin may be a therapeutic strategy for re-emergence of chemo-resistant colon cancer by targeting CSC sub-population.     

Evaluation of Cancer Stem Cell Markers CD133, CD44, CD24: Association with AKT Isoforms and Radiation Resistance in Colon Cancer Cells

The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms) expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1) expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133^high/CD44^high) were more resistant to gamma radiation than the ten percent with lowest expression (CD133^low/CD44^low). The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2) did not differ. Our results demonstrate that CD133^high/CD44^high expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.     

Hypoxia Induces EMT in Low and Highly Aggressive Pancreatic Tumor Cells but Only Cells with Cancer Stem Cell Characteristics Acquire Pronounced Migratory Potential

Tumor hypoxia induces epithelial-mesenchymal transition (EMT), which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Whether EMT generates CSCs de novo, enhances migration of existing CSCs or both is unclear. We examined patient tissue of pancreatic ductal adenocarcinoma (PDA) along with carcinomas of breast, lung, kidney, prostate and ovary. For in vitro studies, five established PDA cell lines classified as less (CSC^low) and highly aggressive CSC-like cells (CSC^high) were examined by single and double immunofluorescence microscopy, wound-, transwell-, and time-lapse microscopy. HIF-1α and Slug, as well as HIF-2α and CD133 were co-expressed pointing to a putative co-existence of hypoxia, EMT and CSCs in vivo. CSC^high cells exhibited high basal expression of the mesenchymal Vimentin protein but low or absent expression of the epithelial marker E-cadherin, with the opposite result in CSC^low cells. Hypoxia triggered altering of cell morphology from an epithelial to a mesenchymal phenotype, which was more pronounced in CSC^high cells. Concomitantly, E-cadherin expression was reduced and expression of Vimentin, Slug, Twist2 and Zeb1 enhanced. While hypoxia caused migration in all cell lines, velocity along with the percentage of migrating, polarized and pseudopodia-forming cells was significantly higher in CSC^high cells. These data indicate that hypoxia-induced EMT occurs in PDA and several other tumor entities. However although hypoxia-induced EMT signaling occurs in all tumor cell populations, only the stem-like cells acquire high migratory potential and thus may be responsible for invasion and metastasis.     

Effect of Melatonin in Epithelial Mesenchymal Transition Markers and Invasive Properties of Breast Cancer Stem Cells of Canine and Human Cell Lines

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44⁺/CD24^low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44⁺/CD24^low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.     

肿瘤干细胞与胃癌

最近的一项研究报导,采用流式细胞仪分选技术从人胃癌细胞株中分离出CD44^＋胃癌干细胞. 20～30×10³个CD44＋细胞入NOD/SCID 鼠腹部皮下和胃浆膜下能形成胃癌移植瘤, 100×10³个CD44^－的细胞入NOD/SCID 鼠体内不形成肿瘤.采用无血清、无粘附间质的干细胞体外培养方法,发现CD44^＋的细胞能形成肿瘤微球体,具有自我更新能力,而CD44^－的细胞则不形成球形克隆.上述的实验结果说明,在人胃癌细胞株中存在胃癌肿瘤干细胞.据此可以相信,胃癌干细胞是胃癌细胞中具有自我更新及分化潜能的一小群细胞,不能被目前的化疗、放疗等抗癌治疗措施所杀灭,是胃癌术后复发、肿瘤进展扩散转移的根源.胃癌干细胞可能来源于骨髓干细胞.随着对胃癌肿瘤干细胞生物学研究的深入,必将为胃癌的临床诊断和治疗提供新的策略.     

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny¹. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44⁺CD24^low/-)². Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues^3-5.Recently, we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD133^6-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH⁺ and CD44⁺CD24⁺ pancreatic CSCs are similarly tumorigenic, but ALDH⁺ cells are relatively more invasive⁸. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts⁹. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent, a fluorescent substrate of ALDH¹⁰. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.     

CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549

Cancer stem cells (CSCs) are subpopulations of tumor cells that are responsible for tumor initiation, maintenance and metastasis. Recent studies suggested that lung cancer arises from CSCs. In this study, the expression of potential CSC markers in cell line A549 was evaluated. We applied flow cytometry to assess the expression of putative stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), CD24, CD44, CD133 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) Aria II and characterized using their clonogenic and sphere-forming capacity. A549 cells expressed the CSC markers CD44 and CD24 at 68.16% and 54.46%, respectively. The expression of the putative CSC marker ALDH1 was 4.20%, whereas the expression of ABCG2 and CD133 was 0.93%. Double-positive CD44/133 populations were rare. CD44⁺/24⁺ and CD44⁺/CD24^?/low subpopulations respectively exhibited 64% and 27.92% expression. The colony-forming potentials in the CD44⁺/CD24⁺ and CD44⁺/CD24^?/low subpopulations were 84.37 ± 2.86% and 90 ± 3.06%, respectively, while the parental A549 cells yielded 56.65 ± 2.33% using the colony-formation assay. Both isolated subpopulations formed spheres in serumfree medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). CD44 and CD24 cannot be considered potential markers for isolating lung CSCs in cell line A549, but further investigation using in vivo assays is required.     

Pancreatic Cancer Stem-like Cells Display Aggressive Behavior Mediated via Activation of FoxQ1

Subpopulations of cancer stem cells (CSCs) or cancer stem-like cells (CSLCs) have been identified from most tumors, including pancreatic cancer (PC), and the existence of these cells is clinically relevant. Emerging evidence suggests that CSLCs participate in cell growth/proliferation, migration/invasion, metastasis, and chemo-radiotherapy resistance, ultimately contributing to poor clinical outcome. However, the pathogenesis and biological significance of CSLCs in PC has not been well characterized. In the present study, we found that isolated triple-marker-positive (CD44⁺/CD133⁺/EpCAM⁺) cells of human PC MiaPaCa-2 and L3.6pl cells behave as CSLCs. These CSLCs exhibit aggressive behavior, such as increased cell growth, migration, clonogenicity, and self-renewal capacity. The mRNA expression profiling analysis showed that CSLCs (CD44⁺/CD133⁺/EpCAM⁺) exhibit differential expression of more than 1,600 mRNAs, including FoxQ1, compared with the triple-marker-negative (CD44⁻/CD133⁻/EpCAM⁻) cells. The knockdown of FoxQ1 by its siRNA in CSLCs resulted in the inhibition of aggressive behavior, consistent with the inhibition of EpCAM and Snail expression. Mouse xenograft tumor studies showed that CSLCs have a 100-fold higher potential for tumor formation and rapid tumor growth, consistent with overexpression of CSC-associated markers/mediators, including FoxQ1, compared with its parental MiaPaCa-2 cells. The inhibition of FoxQ1 attenuated tumor formation and growth, and expression of CSC markers in the xenograft tumor derived from CSLCs of MiaPaCa-2 cells. These data clearly suggest the role of differentially expressed genes in the regulation of CSLC characteristics, further suggesting that targeting some of these genes could be important for the development of novel therapies for achieving better treatment outcome of PC.     

Evaluation of STAT3 Signaling in ALDH+ and ALDH+/CD44+/CD24? Subpopulations of Breast Cancer Cells

Background

STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH⁺), or cell surface molecule CD44-positive (CD44⁺) but CD24-negative (CD24⁻) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells is unknown.

Methods and Results

We examined STAT3 activation in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH⁺) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH⁻) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH⁺ subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH⁺ subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH⁺ subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH⁺ breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH⁺ cells were further selected for the stem cell markers CD44⁺ and CD24⁻.

Conclusion

These studies demonstrate an important role for STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.     

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.     

Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties

Background

The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential.

Methodology/Principal Finding

The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44⁺ cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44⁺ cells consisted of mixed CD44⁺ and CD44⁻ cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44⁺ and CD44⁺ cells derived from CD44⁺-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44⁻ cells. Furthermore, freshly sorted CD44⁺ cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44⁻ cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas.

Conclusion/Significance

Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify the role of CD44 in tumor cell renewal and cancer propagation in the in vivo environment.     

Circulating stem cell vary with NYHA stage in heart failure patients

We have investigated the blood levels of sub-classes of stem cells (SCs) [mesenchymal stem cells (MSCs), haematopoietic stem cells (HSCs), endothelial progenitor cells/circulating endothelial cells (EPCs/CECs) and tissue-committed stem cells (TCSCs)] in heart failure (HF) patients at different stage of pathology and correlated it with plasmatic levels of proangiogenic cytokines. Peripheral blood level of SCs were analysed in 97 HF patients (24 in NYHA class I, 41 in class II, 17 in class III and 15 in class IV) and in 23 healthy controls. Plasmatic levels of PDGF-BB, bFGF, HGF, vascular endothelial growth factor (VEGF), SDF-1α, TNF-α and NTproBNP were also measured. Compared with healthy individuals, MSC, and in particular the sub-classes CD45⁻CD34⁻CD90⁺, CD45⁻CD34⁻CD105⁺ and CD45⁻CD34⁻CXCR4⁺ were significantly enhanced in NYHA class IV patients (16.8-, 6.4- and 2.7-fold, respectively). Level of CD45⁻CD34⁻CD90⁺CXCR4⁺cells progressively increased from class II to class IV (fold increases compared with controls: 8.5, 12 and 21.5, respectively). A significant involvement of CXCR4⁺ subpopulation of HSC (CD45⁺CD34⁺CD90⁺CXCR4⁺, 1.4 versus 13.3 cells/μl in controls and NYHA class III patients, respectively) and TCSC (CD45⁻CD34⁺CXCR4⁺, 1.5 cells/ μl in controls versus 12.4 and 28.6 cells/μl in NYHA classes II and IV, respectively) were also observed. All tested cytokines were enhanced in HF patients. In particular, for PDGF-BB and SDF-1α we studied specific ligand/receptors pairs. Interestingly, the first one positively correlated with TCSCs expressing PDGFR (r = 0.52, P = 0.001), whereas the second one correlated with TCSCs (r = 0.34, P = 0.005) and with MSCs CD90⁺ expressing CXCR4 (r = 0.39, P = 0.001). HF is characterized by the increase in the circulating levels of different MSC, HSC, EPC and TCSC subsets. Both the entity and kinetic of this process varied in distinct cell subsets. Specifically, differently from HSCs and EPCs/CECs, MSCs and TCSCs significantly increased with the progression of the disease, suggesting a possible distinct role of these cells in the pathophysiology of HF.     

Human cardiac stem cells isolated from atrial appendages stably express c-kit

The in vivo studies of myocardial infarct using c-kit⁺/Lin⁻ cardiac stem cells (CSCs) are still in the early stage with margin or no beneficial effects for cardiac function. One of the potential reasons may be related to the absence of fully understanding the properties of these cells both in vitro and in vivo. In the present study, we aimed to systematically examine how CSCs adapted to in vitro cell processes and whether there is any cell contamination after long-term culture. Human CSCs were enzymatically isolated from the atrial appendages of patients. The fixed tissue sections, freshly isolated or cultured CSCs were then used for identification of c-kit⁺/Lin⁻ cells, detection of cell contamination, or differentiation of cardiac lineages. By specific antibody staining, we demonstrated that tissue sections from atrial appendages contained less than 0.036% c-kit⁺/Lin⁻ cells. For the first time, we noted that without magnetic activated cell sorting (MACS), the percentages of c-kit⁺/Lin⁻ cells gradually increased up to ∼40% during continuously culture between passage 2 to 8, but could not exceed >80% unless c-kit MACS was carried out. The resulting c-kit⁺/Lin⁻ cells were negative for CD34, CD45, CD133, and Lin markers, but positive for KDR and CD31 in few patients after c-kit MACS. Lin depletion seemed unnecessary for enrichment of c-kit⁺/Lin⁻ cell population. Following induced differentiation, c-kit⁺/Lin⁻ CSCs demonstrated strong differentiation towards cardiomyocytes but less towards smooth and endothelial cells. We concluded that by using an enzymatic dissociation method, a large number, or higher percentage, of relative pure human CSCs with stable expression of c-kit⁺ could be obtained from atrial appendage specimens within ∼4 weeks following c-kit MACS without Lin depletion. This simple but cost-effective approach can be used to obtain enough numbers of stably-expressed c-kit⁺/Lin⁻ cells for clinical trials in repairing myocardial infarction.     

An intensified systemic trafficking of bone marrow‐derived stem/progenitor cells in patients with pancreatic cancer

Various experimental studies indicate potential involvement of bone marrow (BM)-derived stem cells (SCs) in malignancy development and progression. In this study, we comprehensively analysed systemic trafficking of various populations of BM-derived SCs (BMSCs), i.e., mesenchymal, haematopoietic, endothelial stem/progenitor cells (MSCs, HSCs, EPCs respectively), and of recently discovered population of very small embryonic/epiblast-like SCs (VSELs) in pancreatic cancer patients. Circulating CD133⁺/Lin⁻/CD45⁻/CD34⁺ cells enriched for HSCs, CD105⁺/STRO-1⁺/CD45⁻ cells enriched for MSCs, CD34⁺/KDR⁺/CD31⁺/CD45⁻ cells enriched for EPCs and small CXCR4⁺CD34⁺CD133⁺ subsets of Lin⁻CD45⁻ cells that correspond to VSELs were enumerated and sorted from blood samples derived from 29 patients with pancreatic cancer, and 19 healthy controls. In addition, plasma levels of stromal-derived factor-1 (SDF-1), growth/inhibitory factors and sphingosine-1-phosphate (S1P; chemoattractants for SCs), as well as, of complement cascade (CC) molecules (C3a, C5a and C5b-9/membrane attack complex – MAC) were measured. Higher numbers of circulating VSELs and MSCs were detected in pancreatic cancer patients (P < 0.05 and 0.01 respectively). This trafficking of BMSCs was associated with significantly elevated C5a (P < 0.05) and C5b-9/MAC (P < 0.005) levels together with S1P concentrations detected in plasma of cancer patients, and seemed to be executed in a SDF-1 independent manner. In conclusion, we demonstrated that in patients with pancreatic cancer, intensified peripheral trafficking of selected populations of BMSCs occurs. This phenomenon seems to correlate with systemic activation of the CC, hepatocyte growth factor and S1P levels. In contrast to previous studies, we demonstrate herein that systemic SDF-1 levels do not seem to be linked with increased mobilization of stem cells in patients with pancreatic cancer.     

ALDH1-High Ovarian Cancer Stem-Like Cells Can Be Isolated from Serous and Clear Cell Adenocarcinoma Cells,and ALDH1 High Expression Is Associated with Poor Prognosis

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cancer cells that have high tumorigenicity. Furthermore, CSCs/CICs are resistant to several cancer therapies, and CSCs/CICs are therefore thought to be responsible for cancer recurrence after treatment and distant metastasis. In epithelial ovarian cancer (EOC) cases, disease recurrence after chemotherapy is frequently observed, suggesting ovarian CSCs/CICs are involved. There are four major histological subtypes in EOC, and serous adenocarcinoma and clear cell adenocarcinoma are high-grade malignancies. We therefore analyzed ovarian CSCs/CICs from ovarian carcinoma cell lines (serous adenocarcinoma and clear cell adenocarcinoma) and primary ovarian cancer cells in this study. We isolated ovarian CSCs/CICs as an aldehyde dehydrogenase 1 high (ALDH1^high) population from 6 EOC cell lines (3 serous adenocarcinomas and 3 clear cell adenocarcinomas) by the ALDEFLUOR assay. ALDH1^high cells showed greater sphere-forming ability, higher tumorigenicity and greater invasive capability, indicating that ovarian CSCs/CICs are enriched in ALDH1^high cells. ALDH1^high cells could also be isolated from 8 of 11 primary ovarian carcinoma samples. Immunohistochemical staining revealed that higher ALDH1 expression levels in ovary cancer cases are related to poorer prognosis in both serous adenocarcinoma cases and clear cell adenocarcinoma cases. Taken together, the results indicate that ALDH1 is a marker for ovarian CSCs/CICs and that the expression level of ALDH1 might be a novel biomarker for prediction of poor prognosis.     

Plasma Membrane Proteomics of Tumor Spheres Identify CD166 as a Novel Marker for Cancer Stem-like Cells in Head and Neck Squamous Cell Carcinoma

Patients with advanced head and neck squamous cell carcinoma (HNSCC) have a poor prognosis with the currently available therapy, and tumor recurrence is frequently observed. The discovery of specific membrane-associated cancer stem cell (CSC) markers is crucial for the development of novel therapeutic strategies to target these CSCs. To address this issue, we established sphere cultures to enrich CSCs and used them for plasma membrane proteomics to identify specific membrane signatures of the HNSCC spheres. Of a dataset that included a total of 376 identified proteins, 200 were bona fide membrane proteins. Among them, 123 proteins were at least 1.5-fold up- or down-regulated in the spheres relative to the adherent cultures. These proteins included cell adhesion molecules, receptors, and transporter proteins. Some of them play key roles in wnt, integrin, and TGFβ signaling pathways. When we compared our dataset with two published hESC membrane protein signatures, we found 18 proteins common to all three of the databases. CD166 and CD44 were two such proteins. Interestingly, the expression of CD166, rather than that of the well-established HNSCC CSC marker CD44, was significantly related to the malignant behavior of HNSCC. Relative to CD166^low HNSCC cells, CD166^high HNSCC cells had a greater sphere-formation ability in vitro and tumor formation ability in vivo. Patients whose tumors expressed high levels of CD166 had a significantly poorer clinical outcome than those whose tumors expressed low levels of CD166 (cohort 1: 96 cases, p = 0.040), whereas the level of CD44 expression had only a marginal influence on the clinical outcome of patients with HNSCC (p = 0.078). The level of CD166 expression in HNSCC tumors was also associated with the tumor recurrence rate (cohort 2: 104 cases, p = 0.016). This study demonstrates that CD166 is a valuable cell surface marker for the enrichment of HNSCC stem cells and that plasma membrane proteomics is a promising biological tool for investigating the membrane proteins of CSCs.Head and neck squamous cell carcinoma (HNSCC)¹ is the sixth most common cancer worldwide. Despite ongoing improvement in traditional treatments, the long-term survival rate of patients with HNSCC has not significantly improved over the past several decades. More than 60% of patients with advanced tumors or localized lymph node metastases die within five years of their diagnosis (1). Tumor recurrence and resistance to therapy are the major causes of death. Recently, newly recognized cancer stem cells (CSCs) or tumor-initiating cells have been associated in a cause-and-effect manner with tumor recurrence and resistance to therapy. The concept of CSCs was established because of the heterogeneous nature of cancer and suggests that CSCs are a subpopulation of cancer cells with stem-cell-like traits and the source of all cells in the cancer. Conventional cancer therapies such as chemotherapy and radiotherapy may destroy only those cells that form the bulk of the tumor, leaving the CSCs intact and able to give rise to tumor recurrence. Based on this theory, researchers are searching for therapies that would destroy CSCs in the hope of finally curing cancer (2). In order to develop strategies that target CSCs, experimental assays are required to determine how to distinguish CSCs from their progeny. Different methods have been used to isolate CSCs from a range of hematopoietic and solid tumors, and some CSC-specific cell surface markers have been found. These markers are primarily selected from the corresponding normal stem-cell markers based on their heterogeneous expression in the pertinent cancers. Despite some controversy, the CD34+CD38- marker signature was chosen to define the CSCs of leukemia (3), the CD44+CD24- signature was chosen to define breast cancer CSCs (4), and the CD44 marker was chosen to define the CSCs of HNSCC (5). Though membrane proteins represent only one-third of the proteins encoded by the human genome, they represent more than two-thirds of the known protein targets of drugs. These cell surface markers are not only useful for enriching CSCs from different tumors, but also of significant interest for drug discovery.However, as more cell surface markers for different cancers have been identified, conflicting results have been reported regarding the usefulness of some of the markers and the reproducibility of some of the marker profiles (6). Quintana et al. examined the expression of 22 common CSC markers in melanoma and found that none of them were exclusively enriched in tumorigenic cells relative to non-tumorigenic cells derived from melanoma (7). CD133 is a widely accepted cell surface marker for glioblastoma CSCs, but Beier et al. found that some glioblastoma CSCs were CD133- (8). CD44 is a CSC marker that is commonly expressed by different malignancies of hematopoietic and epithelial origin, including HNSCC (5). However, increasing data have demonstrated a high level of expression of CD44 in the great majority of cells in head and neck tissues, including normal mucosa and carcinomas, and its subsequent expression could not be used to distinguish normal from benign or malignant epithelia of the head and neck. These observations suggest the need for a comprehensive investigation and greater understanding of the cell surface molecules of CSCs.Many different “omic” technologies have shown promise as means to identify markers for cancer stem cells and tumors (9). Among them, membrane proteomics can directly detect changes in the cell surface content and provide insights into the post-translational regulation of cell surface functions. Therefore, in this study, we chose to use membrane proteomics both to investigate the cell surface molecules of CSCs that were enriched from the HNSCC cell populations based on their ability to form spheres and to relate their expression to that of stem cell traits. Our results may contribute to further clinical applications of CSCs by providing tools for purifying and identifying CSCs.     

Non-small cell lung cancer stem/progenitor cells are enriched in multiple distinct phenotypic subpopulations and exhibit plasticity

Cancer stem cells (CSCs) represent a population of cancer cells that possess unique self-renewal and differentiation characteristics required for tumorigenesis and are resistant to chemotherapy-induced apoptosis. Lung CSCs can be enriched by several markers including drug-resistant side population (SP), CD133^pos and ALDH^high. Using human non-small cell lung adenocarcinoma cell lines and patient-derived primary tumor cells, we demonstrate that SP cells represent a subpopulation distinct from other cancer stem/progenitor cell (CS/PC) populations marked by CD133^pos or ALDH^high. The non-CS/PCs and CS/PCs of each subpopulation are interconvertible. Epithelial-mesenchymal transition (EMT) promotes the formation of CD133^pos and ALDH^high CS/PC subpopulations while suppressing the SP CS/PC subpopulation. Rac1 GTPase activity is significantly increased in cells that have undergone EMT, and targeting Rac1 is effective in inhibiting the dynamic conversion of non-CS/PCs to CS/PCs, as well as the CS/PC activity. These results imply that various subpopulations of CS/PCs and non-CS/PCs may achieve a stochastic equilibrium in a defined microenvironment, and eliminating multiple subpopulations of CS/PCs and effectively blocking non-CS/PC to CS/PC transition, by an approach such as targeting Rac1, can be a more effective therapy.     

Tumor Cells Positive and Negative for the Common Cancer Stem Cell Markers Are Capable of Initiating Tumor Growth and Generating Both Progenies

The cancer stem cell (CSC) model depicts that tumors are hierarchically organized and maintained by CSCs lying at the apex. CSCs have been “identified” in a variety of tumors through the tumor-forming assay, in which tumor cells distinguished by a certain cell surface marker (known as a CSC marker) were separately transplanted into immunodeficient mice. In such assays, tumor cells positive but not negative for the CSC marker (hereby defined as CSC⁺ and CSC⁻ cells, respectively) have the ability of tumor-forming and generating both progenies. However, here we show that CSC⁺ and CSC⁻ cells exhibit similar proliferation in the native states. Using a cell tracing method, we demonstrate that CSC⁻ cells exhibit similar tumorigenesis and proliferation as CSC⁺ cells when they were co-transplanted into immunodeficient mice. Through serial single-cell derived subline construction, we further demonstrated that CSC⁺ and CSC⁻ cells from CSC marker expressing tumors could invariably generate both progenies, and their characteristics are maintained among different generations irrespective of the origins (CSC⁺-derived or CSC⁻-derived). These findings demonstrate that tumorigenic cells cannot be distinguished by common CSC markers alone and we propose that cautions should be taken when using these markers independently to identify cancer stem cells due to the phenotypic plasticity of tumor cells.