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BACKGROUND: Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy,although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation. METHODOLOGY/FINDINGS: Treatment of lung tumor cells with doxorubicin,cisplatin,or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133,CD117,SSEA-3,TRA1-81,Oct-4,and nuclear beta-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres,maintain self-renewal capacity,and differentiate. Differentiated progenitors lost expression of CD133,gained CK 8/18 and acquired drug sensitivity. In the presence of drugs,differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice,which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF,bFGF,IL-6,IL-8,HGF,PDGF-BB,G-CSF,and SCGF-beta). CSCs also showed elevated levels of expression of human VEGFR2,FGFR2,CXCR1,2 and 4 receptors. Moreover,human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors. CONCLUSIONS/SIGNIFICANCE: These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy. View Publication

PURPOSE: Circulating cell-free DNA in the blood of cancer patients harbors tumor-specific aberrations. Here,we investigated whether this DNA might also reflect the presence of circulating tumor cells (CTC). EXPERIMENTAL DESIGN: To identify the source of cell-free DNA in blood,plasma derived from 81 patients with prostate cancer was examined for CTCs and cell-free DNA. An epithelial immunospot assay was applied for detection of CTCs,and a PCR-based fluorescence microsatellite analysis with a panel of 14 polymorphic markers was used for detection of allelic imbalances (AI). RESULTS: The plasma DNA levels significantly correlated with the diagnosis subgroups of localized (stage M0,n = 69) and metastasized prostate cancer (stage M1,n = 12; P = 0.03) and with the tumor stage of these patients (P textless 0.005). AI was found on cell-free DNA in plasma from 45.0% and 58.5% of M0 and M1 patients,respectively. Detection of CTCs showed that 71.0% or 92.0% of the M0 and M1 patients harbored 1 to 40 CTCs in their blood,respectively. The occurrence of CTCs correlated with tumor stage (P textless 0.03) and increasing Gleason scores (P = 0.04). Notably,significant associations of the number of CTCs with the AI frequencies at the markers D8S137 (P = 0.03),D9S171 (P = 0.04),and D17S855 (P = 0.02) encoding the cytoskeletal protein dematin,the inhibitor of the cyclin-dependent kinase CDKN2/p16 and BRCA1,respectively,were observed. CONCLUSIONS: These findings show,for the first time,a relationship between the occurrence of CTCs and circulating tumor-associated DNA in blood,which,therefore,might become a valuable new source for monitoring metastatic progression in cancer patients. View Publication

BACKGROUND: Malignant melanoma is an exceptionally aggressive,drug-resistant and heterogeneous cancer. Recently it has been shown that melanoma cells with high clonogenic and tumourigenic abilities are common,but markers distinguishing such cells from cells lacking these abilities have not been identified. There is therefore no definite evidence that an exclusive cell subpopulation,i.e. cancer stem cells (CSC),exists in malignant melanoma. Rather,it is suggested that multiple cell populations are implicated in initiation and progression of the disease,making it of importance to identify subpopulations with elevated aggressive properties. METHODS AND FINDINGS: In several other cancer forms,Aldehyde Dehydrogenase (ALDH),which plays a role in stem cell biology and resistance,is a valuable functional marker for identification of cells that show enhanced aggressiveness and drug-resistance. Furthermore,the presence of ALDH(+) cells is linked to poor clinical prognosis in these cancers. By analyzing cell cultures,xenografts and patient biopsies,we showed that aggressive melanoma harboured a large,distinguishable ALDH(+) subpopulation. In vivo,ALDH(+) cells gave rise to ALDH(-) cells,while the opposite conversion was rare,indicating a higher abilities of ALDH(+) cells to reestablish tumour heterogeneity with respect to the ALDH phenotype. However,both ALDH(+) and ALDH(-) cells demonstrated similarly high abilities for clone formation in vitro and tumour initiation in vivo. Furthermore,both subpopulations showed similar sensitivity to the anti-melanoma drugs,dacarbazine and lexatumumab. CONCLUSIONS: These findings suggest that ALDH does not distinguish tumour-initiating and/or therapy-resistant cells,implying that the ALDH phenotype is not associated with more-aggressive subpopulations in malignant melanoma,and arguing against ALDH as a universal" marker. Besides� View Publication

Prostate cancer (PCa) contains a small population of cancer stem cells (CSCs) that contribute to its initiation and progression. The development of specific markers for identification of the CSCs may lead to new diagnostic strategies of PCa. Increased aldehyde dehydrogenase 1A1 (ALDH1A1) activity has been found in the stem cell populations of leukemia and some solid tumors. The aim of the study was to investigate the stem-cell-related function and clinical significance of the ALDH1A1 in human PCa. ALDEFLUOR assay was used to isolate ALDH1A1(+) cells from PCa cell lines. Stem cell characteristics of the ALDH1A1(+) cells were then investigated by in vitro and in vivo approaches. The ALDH1A1 expression was also analyzed by immunohistochemistry in 18 normal prostate and 163 PCa tissues. The ALDH1A1(+) PCa cells showed high clonogenic and tumorigenic capacities,and serially reinitiated transplantable tumors that resembled histopathologic characteristics and heterogeneity of the parental PCa cells in mice. Immunohistochemical analysis of human prostate tissues showed that ALDH1A1(+) cells were sparse and limited to the basal component in normal prostates. However,in tumor specimens,increased ALDH1A1 immunopositivity was found not only in secretory type cancer epithelial cells but also in neuroendocrine tumor populations. Furthermore,the high ALDH1A1 expression in PCa was positively correlated with Gleason score (P=0.01) and pathologic stage (P=0.01),and inversely associated with overall survival and cancer-specific survival of the patients (P=0.00093 and 0.00017,respectively). ALDH1A1 could be a prostate CSC-related marker. Measuring its expression might provide a potential approach to study tumorigenesis of PCa and predict outcome of the disease. View Publication

Transformed,oncogenic precursors,possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours,have been identified in human brain cancers. Here we report that bone morphogenetic proteins (BMPs),amongst which BMP4 elicits the strongest effect,trigger a significant reduction in the stem-like,tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly,in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation,and increased expression of markers of neural differentiation,with no effect on cell viability. The concomitant reduction in clonogenic ability,in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP-BMPR signalling system--which controls the activity of normal brain stem cells--may also act as a key inhibitory regulator of tumour-initiating,stem-like cells from GBMs and the results also identify BMP4 as a novel,non-cytotoxic therapeutic effector,which may be used to prevent growth and recurrence of GBMs in humans. View Publication

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055,an ATP-competitive mTOR inhibitor,by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors,including RMS. Therefore,in the present study,we searched for AZD8055-based combination therapies. Here,we identify a new synergistic lethality of AZD8055 together with ABT-737,a BH3 mimetic that antagonizes Bcl-2,Bcl-xL,and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index textless 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level,as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential,activation of caspases,and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055,the PI3K/mTOR inhibitor NVP-BEZ235,the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis,whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly,molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly,knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737,our findings have important implications for the development of molecular targeted therapies for RMS. View Publication

Metastatic colorectal cancer (CRC) is incurable for most patients. Since mammalian target of rapamycin (mTOR) has been suggested as a crucial modulator of tumor biology,we aimed at evaluating the effectiveness of mTOR targeting for CRC therapy. To this purpose,we analyzed mTOR expression and the effect of mTOR inhibition in cancer stem-like cells isolated from three human metastatic CRCs (CoCSCs). CoCSCs exhibited a strong mTOR complex 2 (mTORC2) expression,and a rare expression of mTOR complex 1 (mTORC1). This latter correlated with differentiation,being expressed in CoCSC-derived xenografts. We indicate Serum/glucocorticoid-regulated kinase 1 (SGK1) as the possible main mTORC2 effector in CoCSCs,as highlighted by the negative effect on cancer properties following its knockdown. mTOR inhibitors affected CoCSCs differently,resulting in proliferation,autophagy as well as apoptosis induction. The apoptosis-inducing mTOR inhibitor Torin-1 hindered growth,motility,invasion,and survival of CoCSCs in vitro,and suppressed tumor growth in vivo with a concomitant reduction in vessel formation. Torin-1 also affected the expression of markers for cell proliferation,angio-/lympho-genesis,and stemness in vivo,including Ki67,DLL1,DLL4,Notch,Lgr5,and CD44. Importantly,Torin-1 did not affect the survival of normal colon stem cells in vivo,suggesting its selectivity towards cancer cells. Thus,we propose Torin-1 as a powerful drug candidate for metastatic CRC therapy. View Publication

Most forms of chemotherapy employ mechanisms involving induction of oxidative stress,a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However,recent studies have shown that relative redox levels in primary tumors can be heterogeneous,suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies,we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First,the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed ROS-low"). Second� View Publication

Tunneling nanotubes (TnTs) are long,non-adherent,actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study,we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48. h; and this effect was most prominent in media conditions (low-serum,hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs,in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs,which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation,and also lipid raft formation as a potential biomarker for TnT-forming cells. textcopyright 2014 Elsevier Inc. View Publication

The novel quinazoline derivative 4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154) exhibited significant cytotoxicity against U373 and U87 human glioblastoma cell lines,causing apoptotic cell death at micromolar concentrations. The in vitro antiglioblastoma activity of WHI-P154 was amplified textgreater 200-fold and rendered selective by conjugation to recombinant human epidermal growth factor (EGF). The EGF-P154 conjugate was able to bind to and enter target glioblastoma cells within 10-30 min via receptor (R)-mediated endocytosis by inducing internalization of the EGF-R molecules. In vitro treatment with EGF-P154 resulted in killing of glioblastoma cells at nanomolar concentrations with an IC50 of 813 +/- 139 nM,whereas no cytotoxicity against EGF-R-negative leukemia cells was observed,even at concentrations as high as 100 microM. The in vivo administration of EGF-P154 resulted in delayed tumor progression and improved tumor-free survival in a severe combined immunodeficient mouse glioblastoma xenograft model. Whereas none of the control mice remained alive tumor-free beyond 33 days (median tumor-free survival,19 days) and all control mice had tumors that rapidly progressed to reach an average size of textgreater 500 mm3 by 58 days,40% of mice treated for 10 consecutive days with 1 mg/kg/day EGF-P154 remained alive and free of detectable tumors for more than 58 days with a median tumor-free survival of 40 days. The tumors developing in the remaining 60% of the mice never reached a size textgreater 50 mm3. Thus,targeting WHI-P154 to the EGF-R may be useful in the treatment of glioblastoma multiforme. View Publication

BACKGROUND: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer,as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence,novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. METHODS: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance,irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover,we identified and isolated CD44(+)CD24(+)ESA(+) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. RESULTS: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore,GLV-1h68 also showed preferential replication in CD44(+)CD24(+)ESA(+) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44(+)CD24(-)ESA(+) cells. CONCLUSIONS: Taken together,our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus,GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors,especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors. View Publication