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Background Glioblastomas stem-like cells (GSCs) by invading the brain parenchyma, remains after resection and radiotherapy and the tumoral microenvironment become stiffer. GSC invasion is reported as stiffness sensitive and associated with altered N-glycosylation pattern. Glycocalyx thickness modulates integrins mechanosensing, but details remain elusive and glycosylation enzymes involved are unknown. Here, we studied the association between matrix stiffness modulation, GSC migration and MGAT5 induced N-glycosylation in fibrillar 3D context. Method To mimic the extracellular matrix fibrillar microenvironments, we designed 3D-ex-polyacrylonitrile nanofibers scaffolds (NFS) with adjustable stiffnesses by loading multiwall carbon nanotubes (MWCNT). GSCs neurosphere were plated on NFSs, allowing GSCs migration and MGAT5 was deleted using CRISPR-Cas9. Results We found that migration of GSCs was maximum at 166 kPa. Migration rate was correlated with cell shape, expression and maturation of focal adhesion (FA), Epithelial to Mesenchymal Transition (EMT) proteins and (β1,6) branched N-glycan binding, galectin-3. Mutation of MGAT5 in GSC inhibited N-glycans (β1–6) branching, suppressed the stiffness dependence of migration on 166 kPa NFS as well as the associated FA and EMT protein expression. Conclusion MGAT5 catalysing multibranched N-glycans is a critical regulators of stiffness induced invasion and GSCs mechanotransduction, underpinning MGAT5 as a serious target to treat cancer.

Glioblastoma (GBM) recurrences are inevitable, and mainly originate from residual tumor cells and the presence of glioma stem cells (GSC) around the resection cavity borders. We previously showed that the local treatment of GBM with nanomedicine-based Lauroyl-gemcitabine lipid nanocapsules (GemC12-LNC) hydrogel delayed tumor onset in various preclinical models and can be used as a scaffold to deliver multiple drugs. However, it does not inhibit tumor relapse in the long-term. In this work, we aim at encapsulating an anti-GSC molecule in the GemC12-LNC hydrogel to eliminate both GBM cells and GSC. We performed a screening on GBM cell lines (GL261 and U-87 MG) and patient-derived GSC (GBM9) to select the anti-GSC molecule that could act synergically with GemC12. Based on our results, salinomycin (Sal) and curcumin (Cur) were selected for further development. Both GemC12-Sal-LNC and GemC12-Cur LNC showed similar size (55 nm), zeta potential (- 2 mV) and viscoelastic properties compared to the GemC12-LNC hydrogel. Encapsulation efficiency was above 95 %. Moreover, the GemC12-Sal-LNC hydrogel was stable for at least 6 months and released both drugs over 30 days in vitro. Both hydrogels inhibited the growth of GL261 and U-87 MG spheroids. Flow cytometry analysis showed that Sal reduced the GSC population in GL261 and U-87 MG cells. Our results show that the co-encapsulation of Sal in the GemC12-LNC hydrogel can reduce both GBM cells and GSC, and therefore might be promising to avoid the onset of GBM recurrences.

Taniguchi et al . (Research Articles, 17 July 2020, p. 269) claim that the cytokine interleukin-33 induces accumulation of tumor-associated macrophages expressing the immunoglobulin E receptor FcεRI. Although these findings hold great therapeutic promise, we provide evidence that the anti-FcεRI antibody used in this study is not specific for FcεRI on macrophages, which raises concerns about the validity of some of the conclusions.

International audience; Recently, a broader role of inhibitor of apoptosis (IAP) proteins besides their antiapoptotic functions has been described. Therefore, we investigated the effect of non-toxic concentrations of the small-molecule Smac mimetic BV6, which antagonizes IAP proteins, on differentiation of cancer stem-like cells (CSLCs) derived from primary glioblastoma (GBM) specimens. Here, we identify a novel function of BV6 in regulating differentiation of GBM CSLCs by activating NF-κB. BV6 at non-lethal doses stimulates morphological changes associated with the differentiation of GBM CSLCs. BV6 increases transcriptional activity, mRNA and protein levels of the astrocytic marker GFAP without altering expression of the neuronal marker β-III-tubulin, indicating that BV6 induces astrocytic differentiation of GBM CSLCs. Molecular studies reveal that BV6 triggers processing of the NF-κB subunit p100 to p52, nuclear translocation of p52 and p50 and increased NF-κB DNA-binding. Intriguingly, inhibition of NF-κB by overexpression of dominant-negative IκBα super-repressor (IκBα-SR) blocks the BV6-stimulated increase in GFAP and differentiation. Interestingly, this BV6-stimulated differentiation is associated with reduced expression of stemness markers such as CD133, Nanog and Sox2 in GBM CSLCs. In contrast, BV6 does not alter cell morphology, differentiation and expression of stemness markers in non-malignant neural stem cells. Importantly, BV6 treatment reduces clonogenicity of GBM CSLCs in vitro and in vivo, suppresses their tumorigenicity in orthotopic and subcutaneous mouse models and significantly increases the survival of mice. By identifying a novel role of BV6 in promoting differentiation of GBM CSLCs, these findings provide new insights into Smac mimetic-regulated non-apoptotic functions with important implications for targeting GBM CSLCs.

// Olga Karicheva 1 , Jose Manuel Rodriguez-Vargas 1 , Nadege Wadier 1 , Kathline Martin-Hernandez 1 , Romain Vauchelles 2 , Najat Magroun 1 , Agnes Tissier 3 , Valerie Schreiber 1 , Francoise Dantzer 1 1 Poly(ADP-ribosyl)ation and Genome Integrity, Laboratoire d’Excellence Medalis, UMR7242, Centre National de la Recherche Scientifique/Universite de Strasbourg, Institut de Recherche de l’Ecole de Biotechnologie de Strasbourg, 67412 Illkirch, France 2 Laboratoire de Biophotonique et Pharmacologie, UMR7213, Centre National de la Recherche Scientifique/Universite de Strasbourg, Faculte de Pharmacie, 67401 Illkirch, France 3 EMT and Cancer Cell Plasticity, Laboratoire d’Excellence DevWeCan, Equipe labellisee Ligue Nationale Contre Le Cancer, Centre de Recherche en Cancerologie, UMR INSERM 1052 CNRS 5286, Centre Leon Berard, F-69008 Lyon, France Correspondence to: Francoise Dantzer, email: francoise.dantzer@unistra.fr Keywords: Poly(ADP-ribose) polymerase 3 (PARP3), EMT, TGFβ, ROS, stem cells Received: April 01, 2016 Accepted: August 10, 2016 Published: August 26, 2016 ABSTRACT Several members of the Poly(ADP-ribose) polymerase (PARP) family are essential regulators of genome integrity, actively prospected as drug targets for cancer therapy. Among them, PARP3 is well characterized for its functions in double-strand break repair and mitotis. Here we report that PARP3 also plays an integral role in TGFβ and reactive oxygen species (ROS) dependent epithelial-to-mesenchymal transition (EMT) and stem-like cell properties in human mammary epithelial and breast cancer cells. PARP3 expression is higher in breast cancer cells of the mesenchymal phenotype and correlates with the expression of the mesenchymal marker Vimentin while being in inverse correlation with the epithelial marker E-cadherin. Furthermore, PARP3 expression is significantly upregulated during TGFβ-induced EMT in various human epithelial cells. In line with this observation, PARP3 depletion alters TGFβ-dependent EMT of mammary epithelial cells by preventing the induction of the Snail-E-cadherin axis, the dissolution of cell junctions, the acquisition of cell motility and chemoresistance. PARP3 responds to TGFβ-induced ROS to promote a TG2-Snail-E-cadherin axis during EMT. Considering the link between EMT and cancer stem cells, we show that PARP3 promotes stem-like cell properties in mammary epithelial and breast cancer cells by inducing the expression of the stem cell markers SOX2 and OCT4, by increasing the proportion of tumor initiating CD44 high /CD24 low population and the formation of tumor spheroid bodies, and by promoting stem cell self-renewal. These findings point to a novel role of PARP3 in the control of TGFβ-induced EMT and acquisition of stem-like cell features and further motivate efforts to identify PARP3 specific inhibitors.

International audience; The most relevant therapeutic approaches to treat CML rely on the administration of tyrosine kinase inhibitors (TKIs) like Imatinib, which are able to counteract the activity of Bcr-Abl protein increasing patient's life expectancy and survival. Unfortunately, there are some issues TKIs are not able to address; first of all TKIs are not so effective in increasing survival of patients in blast crisis, second they are not able to eradicate leukemic stem cells (LSC) which represent the major cause of disease relapse, and third patients often develop resistance to TKIs due to mutations in the drug binding site. For all these reasons it's of primary interest to find alternative strategies to treat CML. Literature shows that Hedgehog signaling pathway is involved in LSC maintenance, and pharmacological inhibition of Smoothened (SMO), one of the key molecules of the pathway, has been demonstrated to reduce Bcr-Abl positive bone marrow cells and LSC. Consequently, targeting SMO could be a promising way to develop a new treatment strategy for CML overcoming the limitations of current therapies. In our work we have tested some compounds able to inhibit SMO, and among them MRT92 appears to be a very potent SMO antagonist. We found that almost all our compounds were able to reduce Gli1 protein levels in K-562 and in KU-812 CML cell lines. Furthermore, they were also able to increase Gli1 and SMO RNA levels, and to reduce cell proliferation and induce apoptosis/autophagy in both the tested cell lines. Finally, we demonstrated that our compounds were able to modulate the expression of some miRNAs related to Hedgehog pathway such as miR-324-5p and miR-326. Being Hedgehog pathway deeply implicated in the mechanisms of CML we may conclude that it could be a good therapeutic target for CML and our compounds seem to be promising antagonists of such pathway.

International audience; Intrahepatic malignant tumours include hepatocellular carcinomas (HCC), cholangiocarcinomas (CC) and combined hepatocholangiocarcinomas (cHCC-CC), a group of rare and poorly characterized tumours that exhibit both biliary and hepatocytic differentiation. The aim of the study was to characterize the molecular pathways specifically associated with cHCC-CC pathogenesis. We performed a genome-wide transcriptional analysis of 20 histologically defined cHCC-CC and compared them with a series of typical HCC and of CC. Data were analysed by gene set enrichment and integrative genomics and results were further validated in situ by tissue microarray using an independent series of 152 tumours. We report that cHCC-CC exhibit stem/progenitor features, a down-regulation of the hepatocyte differentiation program and a commitment to the biliary lineage. TGFβ and Wnt/β-catenin were identified as the two major signalling pathways activated in cHCC-CC. A β-catenin signature distinct from that observed in well-differentiated HCC with mutant β-catenin was found in cHCC-CC. This signature was associated with microenvironment remodelling and TGFβ activation. Furthermore, integrative genomics revealed that cHCC-CC share characteristics of poorly differentiated HCC with stem cell traits and poor prognosis. The common traits displayed by CC, cHCC-CC and some HCC suggest that these tumours could originate from stem/progenitor cell(s) and raised the hypothesis of a potential continuum between intrahepatic CC, cHCC-CC and poorly differentiated HCC.

International audience; BACKGROUND: Cellular self-renewal capacity in glioblastomas is heterogeneous, with only stem-like cells having this property. These cells generate a specific tumor phenotype, but no link with tumor location or molecular characteristics has ever been made. METHODS: Two cells lines, established from cell-dissociated glioblastomas and A2B5+ magnetic cell sorting, were used to decipher the mechanisms of cell migration in glioblastomas. GBM6 was derived from a glioblastoma close to the subventricular zone, whereas GBM9 was derived from a cortical glioblastoma and contained a high number of CD133(+) cells. RESULTS: Orthotopic injections in both the subventricular zone and the cortex of nude mice showed that GBM6 and GBM9 cells had a differential pattern of migration that mirrored that of adult and fetal normal neural stem cells, respectively. GBM6 demonstrated higher tumorigenicity than GBM9, and whichever cell line was injected, subventricular zone-implanted tumors were larger than cortical ones. In vitro, GBM6 and GBM9 displayed high autorenewal and proliferation rates, and their expression profiles and genomic status showed that they had distinctive molecular signatures: GBM6 was classified as a mesenchymal glioblastoma and GBM9 as a proneural glioblastoma. CONCLUSIONS: Altogether, our findings suggest that tumor location in addition to molecular signature influence tumor growth and migration pattern.

International audience; Therapeutic resistance of acute myeloid leukemia stem cells, enriched in the CD34(+)38(-)123(+) progenitor population, is supported by extrinsic factors such as the bone marrow niche. Here, we report that when adherent onto fibronectin or osteoblast components, CD34(+)38(-)123(+) progenitors survive through an integrin-dependent activation of glycogen synthase kinase 3β (GSK3β) by serine 9-dephosphorylation. Strikingly, GSK3β-mediated survival was restricted to leukemic progenitors from female patients. GSK3β inhibition restored sensitivity to etoposide, and impaired the clonogenic capacities of adherent leukemic progenitors from female patients. In leukemic progenitors from female but not male patients, the scaffolding protein RACK1, activated downstream of α(5)β(1)-integrin engagement, was specifically upregulated and controlled GSK3β activation through the phosphatase protein phosphatase 2A (PP2A). In a mirrored manner, survival of adherent progenitors (CD34(+)38(-)) from male but not female healthy donors was partially dependent on this pathway. We conclude that the GSK3β-dependent survival pathway might be sex-specific in normal immature population and flip-flopped upon leukemogenesis. Taken together, our results strengthen GSK3β as a promising target for leukemic stem cell therapy and reveal gender differences as a new parameter in anti-leukemia therapy.

International audience; Growing evidence supports the existence of a subpopulation of cancer cells with stem cell characteristics within breast tumors. In spite of its potential clinical implications, an understanding of the mechanisms responsible for retaining the stem cell characteristics in these cells is still lacking. Here, we used the mammosphere model combined with DNA methylation bead arrays and quantitative gene expression to characterize the epigenetic mechanisms involved in the regulation of developmental pathways in putative breast cancer stem cells. Our results revealed that MCF7-derived mammospheres exhibit distinct CpG promoter methylation profiles in a specific set of genes, including those involved in Jak-STAT signaling pathway. Hypomethylation of several gene components of the Jak-STAT pathway was correlated with an increased expression in mammospheres relative to parental cells. Remarkably, cell sorting of the cells with a putative cancer stem cell phenotype (CD44+/CD24 low) suggests a constitutive activation of Jak-STAT pathway in these cells. These results show that Jak-STAT activation may represent a characteristic of putative breast cancer stem cells. In addition, they favor the concept that the expression of cancer stem-like pathways and the establishment and maintenance of defining properties of cancer stem cells are orchestrated by epigenetic mechanisms.

Continuous turnover of epithelia is ensured by the extensive self-renewal capacity of tissue-specific stem cells. Similarly, epithelial tumour maintenance relies on cancer stem cells (CSCs), which co-opt stem cell properties. For most tumours, the cellular origin of these CSCs and regulatory pathways essential for sustaining stemness have not been identified. In murine skin, follicular morphogenesis is driven by bulge stem cells that specifically express CD34. Here we identify a population of cells in early epidermal tumours characterized by phenotypic and functional similarities to normal bulge skin stem cells. This population contains CSCs, which are the only cells with tumour initiation properties. Transplants derived from these CSCs preserve the hierarchical organization of the primary tumour. We describe β-catenin signalling as being essential in sustaining the CSC phenotype. Ablation of the β-catenin gene results in the loss of CSCs and complete tumour regression. In addition, we provide evidence for the involvement of increased β-catenin signalling in malignant human squamous cell carcinomas. Because Wnt/β-catenin signalling is not essential for normal epidermal homeostasis, such a mechanistic difference may thus be targeted to eliminate CSCs and consequently eradicate squamous cell carcinomas. ©2008 Nature Publishing Group., Work in Madrid was supported in part by the Spanish Ministry of Education and Science to A.C.

The accurate identification and thorough characterization of tumorigenic cells in glioblastomas are essential to enhance our understanding of their malignant behavior and for the design of strategies that target this important cell population. We report here that, in rat brain, the scaffolding protein IQGAP1 is a marker of brain nestin+ amplifying neural progenitor cells. In a rat model of glioma, IQGAP1 also characterizes a subpopulation of nestin+ amplifying tumor cells in glioblastoma-like tumors but not in tumors with oligodendroglioma features. We next confirmed that IQGAP1 represents a new marker that may help to discriminate human glioblastoma from oligodendrogliomas. In human glioblastoma exclusively, IQGAP1 specifies a subpopulation of amplifying nestin+ cancer cells. Neoplastic IQGAP1+ cells from glioblastoma can be expanded in culture and possess all the characteristics of cancer stem-like progenitors. The similarities between amplifying neural progenitors and glioblastoma amplifying cancer cells may have significant implications for understanding the biology of glioblastoma. (Cancer Res 2006; 66(18): 9074-82)

Background Chemoresistance of glioblastoma multiforme (GBM) has been attributed to the presence within the tumor of cancer stem cells (GSCs). The standard therapy for GBM consists of surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ). However, TMZ efficacy is limited by O6-methylguanine-DNA-methyltransferase (MGMT) and Mismatch Repair (MMR) functions. Strategies to counteract TMZ resistance include its combination with poly(ADP-ribose) polymerase inhibitors (PARPi), which hamper the repair of N-methylpurines. PARPi are also investigated as monotherapy for tumors with deficiency of homologous recombination (HR). We have investigated whether PARPi may restore GSC sensitivity to TMZ or may be effective as monotherapy. Methods Ten human GSC lines were assayed for MMR proteins, MGMT and PARP-1 expression/activity, MGMT promoter methylation and sensitivity to TMZ or PARPi, alone and in combination. Since PTEN defects are frequently detected in GBM and may cause HR dysfunction, PTEN expression was also analyzed. The statistical analysis of the differences in drug sensitivity among the cell lines was performed using the ANOVA and Bonferroni’s post-test or the non-parametric Kruskal-Wallis analysis and Dunn’s post-test for multiple comparisons. Synergism between TMZ and PARPi was analyzed by the median-effect method of Chou and Talalay. Correlation analyses were done using the Spearman’s rank test. Results All GSCs were MMR-proficient and resistance to TMZ was mainly associated with high MGMT activity or low proliferation rate. MGMT promoter hypermethylation of GSCs correlated both with low MGMT activity/expression (Spearman’s test, P = 0.004 and P = 0.01) and with longer overall survival of GBM patients (P = 0.02). Sensitivity of each GSC line to PARPi as single agent did not correlate with PARP-1 or PTEN expression. Notably, PARPi and TMZ combination exerted synergistic antitumor effects in eight out of ten GSC lines and the TMZ dose reduction achieved significantly correlated with the sensitivity of each cell line to PARPi as single agent (P = 0.01). Conclusions The combination of TMZ with PARPi may represent a valuable strategy to reverse GSC chemoresistance.

A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.

International audience; Cytarabine combined with an anthracycline or an anthracenedione represents the usual intensive induction therapy for the treatment of AML. However, this protocol induces severe side effects and treatment-related mortality due to the lack of selectivity of these cytotoxic agents. In this paper, we present the study of the first galactosidase-responsive molecular "Trojan Horse" programmed for the delivery of doxorubicin exclusively inside AML blasts over-expressing the folate receptor (FR). This targeting system allows the selective killing of AML blasts without affecting normal endothelial, cardiac or hematologic cells from healthy donors suggesting that FDC could reduce adverse events usually recorded with anthracyclines.

International audience; In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

The incidence of oral tumors is increasing around the world and despite recent advances in early detection and diagnosis, current treatments are still unsatisfactory. Recent data suggest that tumor persistence and recurrence could be due to the presence of a rare cell population called cancer stem cells (CSCs), which are generally spared by traditional treatments. Therefore, identification and characterization of CSCs are extremely important to develop novel and effective treatment strategies for cancer. The aim of this study was to identify and isolate CSCs in an established murine head and neck squamous cell carcinoma (HNSCC) cell line and to investigate the influence of hypoxic conditions on the isolated cell popul-ation. Using the expression of the aldehyde dehydrogenase 1 (ALDH1) enzymatic activity, which is now recognized as a CSC marker in various tumors, we isolated a cell population expressing high levels of ALDH1 (ALDH1high) representing 1±0.6% in the murine SCC-VII cell line. These cells were injected subcutaneously in syngeneic animals to evaluate their tumorigenic properties. For the lowest injected cell dose (250 injected cells), tumor occurrence and median tumor size were higher in ALDH1high injected mice than in ALDH1low injected mice. Following an in vivo passage and culture in serum-free medium, the percentage of ALDH1high cells increased by 3‑fold in SCC-VII CSCs (oral spheres) compared to the SCC-VII cell line. This percentage was further increased when oral spheres were cultured under hypoxic conditions. In conclusion, this study reports for the first time the isolation of HNSCC CSCs in a syngeneic mouse model and the use of hypoxia as a method to further enrich the ALDH1high cell population.

International audience

Tumors invade space through cancer cell proliferation and tumor mass expansion. Tumor growth is maintained over time using the cancer stem cell pool, which is the cell population with self-renewal potential. The work by Conley et al. (1) provided evidence that antiangiogenic agents increase cancer stem cells through the generation of tumor hypoxia. These findings are in accordance with the findings showing that hypoxia induces stem cell markers expression in cancer cells (2, 3) and that antiangiogenic therapy elicits malignant progression of tumors (4). Thus, there is now sufficient evidence to reconsider the role of tumor angiogenesis in cancer progression. Tissues need nutrients and oxygen for growth, and tumors behave as other tissues. This view is the classical view, in which, according to the work by Folkman (5), the role of tumor angiogenesis fulfills the function of normal vessels that is to deliver oxygen and metabolites and remove waste products. However, tumor vessels are abnormal and fail to fulfill these functions. Therefore, tumors have naturally chronic and cyclic hypoxic regions. This paradoxical consequence of tumor angiogenesis is that, because of its deficiencies, it also generates hypoxia. In pathology such as cancer, a defect in a physiological process does not necessarily suppress the function of the process but can generate a novel function required for disease development. Here, the other function of tumor angiogenesis is to maintain tumor growth over time through cancer stem cell increase. This dual nature of tumor angiogenesis ensures a balance between growth over space (by providing oxygen and nutrients; i.e., physiological function) and growth over time (by generating hypoxic regions that increase the cancer stem cell pool; i.e., pathophysiological function). Hence, tumors require both the physiological and pathophysiological functions of tumor angiogenesis. Therefore, the significance of tumor angiogenesis is the generation of a tumor space–time continuum.

International audience; Here we describe a protocol established in our laboratory to quantify early common progenitors/stem cells grown as spheres in non-adherent culture conditions. This protocol is based on the combination of two functional tests: the mammosphere assay to maintain and enrich early mammary progenitors/stem cells, and the epithelial colony-forming cells (E-CFC) assay to identify and quantify further differentiated epithelial progenitors. Primary spheres mainly contain progenitors and rare stem/early common progenitor cells while secondary and tertiary spheres contain progenitor cells derived from the early common progenitor/stem cell population maintained through passages and partially differentiated. Spheres are enzymatically and mechanically dissociated; the derived cells are subsequently plated on irradiated NIH-3T3 fibroblasts for further processing, as in the E-CFC assay. The principle of this assay is to quantify the number of epithelial colonies generated by cells present in the different sequential spheres. This assay has therefore been named ECP-DC, standing for Early Common Progenitor-Derived Colonies assay.

International audience; The similarities between brain tumor stem cells and neural stem cells suggest a possible stem cell origin of tumorigenesis. Recently, cells with features of stem cells have been observed in lesions of adult and pediatric cortical dysplasia (CD). Given the evidence for a close relationship between CD and certain brain tumors, together with the finding that CD neural stem cells/progenitors are abnormally developed, we propose that CD is a possible substrate for brain tumors. The neural stem cells/progenitors in CD have accumulating abnormalities, and these abnormal stem/progenitor cells may be the initiating, transformed cells of brain tumors, when subsequently exposed to a carcinogen.

International audience; Recently, cancer stem cells (CSCs) have been identified in many types of cancers, such as colorectal cancer (CRC). CSCs seem to be involved in initiation, growth, and tumor metastasis, as well as in radio- and chemotherapy failures. CSCs appears as new biological targets for cancer therapy, requiring the development of noninvasive cell sorting methods. In this study, we used sedimentation field flow fractionation (SdFFF) to prepare enriched populations of CSCs from eight cell lines corresponding to different CRC grades. On the basis of phenotypic and functional characterizations, "hyperlayer" elution resulted in a fraction overexpressing CSC markers (CD44, CD166, EpCAM) for all cell lines. CSCs were eluted in the last fraction for seven out of eight cell lines, but in the first for HCT116. These results suggest, according to the literature, that two different pools of CSCs exist, quiescent and activated, which can both be sorted by SdFFF. Moreover, according to CSC properties, enriched fractions are able to form colonies.

International audience; Suicide gene therapy is based on the introduction into tumor cells of a viral or a bacterial gene, which allows the conversion of a non-toxic compound into a lethal drug. Although suicide gene therapy has been successfully used in a large number of in vitro and in vivo studies, its application to cancer patients has not reached the desirable clinical significance. However, recent reports on pre-clinical cancer models demonstrate the huge potential of this strategy when used in combination with new therapeutic approaches. In this review, we summarize the different suicide gene systems and gene delivery vectors addressed to cancer, with particular emphasis on recently developed systems and associated bystander effects. In addition, we review the different strategies that have been used in combination with suicide gene therapy and provide some insights into the future directions of this approach, particularly towards cancer stem cell eradication.

International audience; Chronic myeloid leukemia (CML) tumorigenicity is driven by the oncogenic BCR-ABL tyrosine kinase. Specific tyrosine kinase inhibitors (TKI) have been designed and are now used for the treatment of CML. These TKI induce apoptosis in leukemic cells in a BIM-dependent mechanism. We hypothesized that an increase in BIM activity could sensitize CML cells to TKI. We blocked the anti-apoptotic proteins of the Bcl-2 family by using ABT-737, a Bcl-2 and Bcl-XL inhibitor. ABT-737 modified Bcl-2 protein interactions toward a pro-apoptotic phenotype. Its combination with TKI resulted in a strong synergism in CML cell lines. The association also induced a large decrease in X-linked inhibitor of apoptosis (XIAP), followed by caspase-3 activation. This XIAP decrease was due to post-translational events. The mitochondrial serine protease HtrA2/Omi was identified as being responsible for this off-target effect. Then, ABT-737 and TKI cooperate at several levels to induce apoptosis of CML cells lines, and the benefit of this association was also observed in CML hematopoietic progenitors. Interestingly, a lethal effect was also observed in the more immature CD34(+)CD38(-) TKI-insensitive population. Combination therapy might by an interesting strategy for the treatment of CML patients.

International audience; BACKGROUND: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. CONCLUSION/SIGNIFICANCE: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.

International audience; Primary hepatocellular carcinoma (HCC) is a significant human cancer globally, with poor prognosis. New and efficacious therapy strategies are needed as well as new biomarkers for early detection of at-risk patients. In this review, we discuss select microarray studies of human HCCs, and propose a gene signature that has promise for clinical/translational application. This gene signature combines the proliferation cluster of genes and the hepatic cancer initiating/stem cell gene cluster for identification of HCCs with poor prognosis. Evidence from cell-based assays identifies the existence of a mechanistic link between these two gene clusters, involving the proliferation cluster gene polo-like kinase 1 (PLK1). We propose that PLK1 is a promising therapy target for HCC.

International audience; BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05, chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013, log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers, the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients, TSCs have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors.

International audience; BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05, chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013, log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers, the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients, TSCs have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors.

International audience; BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05, chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013, log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers, the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients, TSCs have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors.

International audience; Gliomas are the most common primary brain tumor affecting human adults and remain a therapeutic challenge because cells of origin are still unknown. Here, we investigated the cellular origin of low-grade gliomas in a rat model based on transplacental exposure to N-ethyl-N-nitrosourea (ENU). Longitudinal magnetic resonance imaging coupled to immunohistological and immunocytochemical analyses were used to further characterize low-grade rat gliomas at different stages of evolution. We showed that early low-grade gliomas have characteristics of oligodendroglioma-like tumors and exclusively contain NG2-expressing slow dividing precursor cells, which express early markers of oligodendroglial lineage. These tumor-derived precursors failed to fully differentiate into oligodendrocytes and exhibited multipotential abilities in vitro. Moreover, a few glioma NG2+ cells are resistant to radiotherapy and may be responsible for tumor recurrence, frequently observed in humans. Overall, these findings suggest that transformed multipotent NG2 glial precursor cell may be a potential cell of origin in the genesis of rat ENU-induced oligodendroglioma-like tumors. This work may open up new perspectives for understanding biology of human gliomas.

Glioblastomas are the most common CNS tumors in adults, and they remain resistant to current treatments. ErbB1 signaling is frequently altered in these tumors, which indicates that the erbB receptor family is a promising target for molecular therapy. However, data on erbB signaling in glioblastomas are still sparse. Therefore, we undertook a comprehensive analysis of erbB receptor and ligand expression profiles in a panel of nine glioblastomas that were compared to non-neoplastic cerebral tissue containing neocortex and corresponding portions of subcortical convolutional white matter and we determined the distribution patterns of erbB receptors among the main neural cell types that are present in these tumors, particularly the putative tumoral stem cell population. Using quantitative RT-PCR and western blot analysis, we showed that erbB1 signaling and erbB2 receptors exhibited highly variable deregulation profiles among tumors, ranging from under- to overexpression, while erbB3 and erbB4 were down-regulated. Immunohistochemistry revealed an important inter- and intra-tumoral heterogeneity in all four erbB expression profiles. However, each receptor exhibited a distinct repartition pattern among the GFAP-, Olig2-, NeuN- and CD133-positive populations. Interestingly, while erbB1 immunoreactivity was only detected in small subsets of CD133-positive putative tumoral stem cells, erbB3 immunoreactivity was prominent in this cell population thus suggesting that erbB3 may represent a new potential target for molecular therapy.

International audience; Centrosomes are essential protagonists during cell division through microtubule nucleation and spindle formation which are key to the harmonious distribution of sister chromatids in the two daughter cells. However, during the past decade, a wealth of new observations has extended their role beyond mitosis, particularly in the asymmetrical partition of cell fate determinants. Remarkably, asymmetric centrosome inheritance per se, through the segregation of differently aged mother -centrioles, seems to regulate the differential behaviour of daughter cells, in part through asynchronous expression of primary cilia, governing the response to environmental signals. It is thus understandable why any quantitative or qualitative dysfunction of centrioles contributes to genomic -instability and thus -tumorigenesis.

Background Multicellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs). CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC) after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. Methods Protein expression in whole spheroids (150 μm in diameter) from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC) and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied. Results We first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express β-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9+ cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively). Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to the luminal cell surface of colorectal cancer acini, with CD133+ cellular debris into glandular lumina. Conclusion The present protocol enables in situ visualization of protein expression in compact three-dimensional models by whole mount confocal imaging, allowing the accurate localization and quantification of cells expressing specific markers. It should prove useful to study rare events like CSCs within tumour spheres.

International audience; Gliomas are the most common primary brain tumor affecting human adults and remain a therapeutic challenge because cells of origin are still unknown. Here, we investigated the cellular origin of low-grade gliomas in a rat model based on transplacental exposure to N-ethyl-N-nitrosourea (ENU). Longitudinal magnetic resonance imaging coupled to immunohistological and immunocytochemical analyses were used to further characterize low-grade rat gliomas at different stages of evolution. We showed that early low-grade gliomas have characteristics of oligodendroglioma-like tumors and exclusively contain NG2-expressing slow dividing precursor cells, which express early markers of oligodendroglial lineage. These tumor-derived precursors failed to fully differentiate into oligodendrocytes and exhibited multipotential abilities in vitro. Moreover, a few glioma NG2+ cells are resistant to radiotherapy and may be responsible for tumor recurrence, frequently observed in humans. Overall, these findings suggest that transformed multipotent NG2 glial precursor cell may be a potential cell of origin in the genesis of rat ENU-induced oligodendroglioma-like tumors. This work may open up new perspectives for understanding biology of human gliomas.

Background Tumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies. Methods We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas. Results A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide. Conclusions Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

International audience; The characterisation of normal stem cells and cancer stem cells uses the same paradigm. These cells are isolated by a fluorescence-activated cell sorting step and their stemness is assayed following implantation into animals. However, differences exist between these two kinds of stem cells. Therefore, the translation of the experimental procedures used for normal stem cell isolation into the research field of cancer stem cells is a potential source of artefacts. In addition, normal stem cell therapy has the objective of regenerating a tissue, while cancer stem cell-centred therapy seeks the destruction of the cancer tissue. Taking these differences into account is critical for anticipating problems that might arise in cancer stem cell-centred therapy and for upgrading the cancer stem cell paradigm accordingly.

International audience; Prominin 1/CD133 is a marker of transplantable cancer stem cells. We have generated anti-peptide antibodies against a N-terminal epitope of CD133 belonging to a ganglioside-binding domain. The labelling of colon cancer cells with these antibodies was inhibited by various gangliosides including GM1 and GD3, but not GT1b. CD133 immunolabelling progressively decreased to undetectable levels in post-confluent cultures, possibly through ganglioside-mediated epitope masking since the staining was partially recovered after chemical disruption of lipid rafts. We suggest that selected gangliosides could modulate the accessibility of CD133 and regulate cell-cell contacts involving CD133(+) stem cells at the earliest steps of tumour development.

International audience; Until the last century, infectious diseases were the leading cause of human mortality. Therefore, our current medical reasoning is profoundly influenced by views that originated from medical microbiology. The notion that cancer growth is sustained by a sub-population of particular cells, the cancer stem cells, is highly reminiscent of the germ theory of disease as exemplified by Koch's postulates in the XIX th century. However, accumulating data underscore the importance of cell-cell interactions and tumor environment. Hence it is essential to critically review the basic tenets of the cancer stem cell concept on the light of their relationships with Koch's postulates. Shifting the pathogenic element from a special cellular entity (cancer stem cell or microorganism) to a " pathogenic field " could be critical for curing both cancer and drug-resistant infectious diseases.

Loss of tumor-suppressive pathways that control cellular senescence is a crucial step in malignant transformation. Spleen tyrosine kinase (Syk) is a cytoplasmic tyrosine kinase that has been recently implicated in tumor suppression of melanoma, a deadly skin cancer derived from pigment-producing melanocytes. However, the mechanism by which Syk suppresses melanoma growth remains unclear. Here, we report that reexpression of Syk in melanoma cells induces a p53-dependent expression of the cyclin-dependent kinase (cdk) inhibitor p21 and a senescence program. We first observed that Syk expression is lost in a subset of melanoma cell lines, primarily by DNA methylation–mediated gene silencing and restored after treatment with the demethylating agent 5-aza-2-deoxycytidine. We analyzed the significance of epigenetic inactivation of Syk and found that reintroduction of Syk in melanoma cells dramatically reduces clonogenic survival and three-dimensional tumor spheroid growth and invasion. Remarkably, melanoma cells reexpressing Syk display hallmarks of senescent cells, including reduction of proliferative activity and DNA synthesis, large and flattened morphology, senescence-associated β-galactosidase activity, and heterochromatic foci. This phenotype is accompanied by hypophosphorylated retinoblastoma protein (Rb) and accumulation of p21, which depends on functional p53. Our results highlight a new role for Syk tyrosine kinase in regulating cellular senescence and identify Syk-mediated senescence as a novel tumor suppressor pathway the inactivation of which may contribute to melanoma tumorigenicity. [Cancer Res 2009;69(7):2748–56]

International audience; Loss of tumor-suppressive pathways that control cellular senescence is a crucial step in malignant transformation. Spleen tyrosine kinase (Syk) is a cytoplasmic tyrosine kinase that has been recently implicated in tumor suppression of melanoma, a deadly skin cancer derived from pigment-producing melanocytes. However, the mechanism by which Syk suppresses melanoma growth remains unclear. Here, we report that reexpression of Syk in melanoma cells induces a p53-dependent expression of the cyclin-dependent kinase (cdk) inhibitor p21 and a senescence program. We first observed that Syk expression is lost in a subset of melanoma cell lines, primarily by DNA methylation-mediated gene silencing and restored after treatment with the demethylating agent 5-aza-2-deoxycytidine. We analyzed the significance of epigenetic inactivation of Syk and found that reintroduction of Syk in melanoma cells dramatically reduces clonogenic survival and three-dimensional tumor spheroid growth and invasion. Remarkably, melanoma cells reexpressing Syk display hallmarks of senescent cells, including reduction of proliferative activity and DNA synthesis, large and flattened morphology, senescence-associated beta-galactosidase activity, and heterochromatic foci. This phenotype is accompanied by hypophosphorylated retinoblastoma protein (Rb) and accumulation of p21, which depends on functional p53. Our results highlight a new role for Syk tyrosine kinase in regulating cellular senescence and identify Syk-mediated senescence as a novel tumor suppressor pathway the inactivation of which may contribute to melanoma tumorigenicity.

International audience; Glioblastoma is the most common primary brain tumor, characterized by its resistance to treatments. To define efficient therapy, the origin of tumor-forming cells needs to be elucidated in order to search for new therapeutic pathways. The objective of this study was to determine the different cell populations constituting a human glioblastoma cell line, U-87 MG and their sensitivity to apoptosis induced through the activation of Fas, a membranous death receptor. By a cell sorting method, the sedimentation field flow fractionation, two major cell subpopulations were identified, a most differentiated cell fraction, containing large and adherent cells, sensitive to Fas-induced apoptosis and another one, characterized by small cells forming aggregates, expressing CD133, a marker of stem cells and more resistant to Fas-activated apoptosis. By using a selective method of culture, adapted for neural stem cell cultures, we have verified that the U-87 MG cell line contained cancer stem cells similar to the immature ones obtained by the cell sorting method. Interestingly, while these tumor stem cells, expressing CD133, were resistant to Fas-induced apoptosis, monomeric form of Fas protein was detected predominantly in these cells. In contrast, the most mature cells, responsive to Fas-activated apoptosis, collected in another cell fraction, contained oligomeric aggregates of Fas protein, a pre-signalling form of the Fas receptor, essential for the initiation of apoptosis through its activation. These results suggest that these immature stem cells in glioma could be an important factor of resistance to chemotherapy requiring apoptosis through Fas signalling system. Indeed, future strategies of treatment, inducing differentiation of these stem cells need to be considered to enhance therapeutic efficiency.

The PTEN/Akt/β-catenin pathway is important for maintaining stem or progenitor cells in normal and cancerous breast tissue and may be a promising target for effective, long-lasting cancer treatment., Recent evidence suggests that many malignancies, including breast cancer, are driven by a cellular subcomponent that displays stem cell-like properties. The protein phosphatase and tensin homolog (PTEN) is inactivated in a wide range of human cancers, an alteration that is associated with a poor prognosis. Because PTEN has been reported to play a role in the maintenance of embryonic and tissue-specific stem cells, we investigated the role of the PTEN/Akt pathway in the regulation of normal and malignant mammary stem/progenitor cell populations. We demonstrate that activation of this pathway, via PTEN knockdown, enriches for normal and malignant human mammary stem/progenitor cells in vitro and in vivo. Knockdown of PTEN in normal human mammary epithelial cells enriches for the stem/progenitor cell compartment, generating atypical hyperplastic lesions in humanized NOD/SCID mice. Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/β-catenin pathway through the phosphorylation of GSK3-β. In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts. These studies demonstrate an important role for the PTEN/PI3-K/Akt/β-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells., Author Summary Healthy adult tissues are maintained through the regulated proliferation of a subset of cells known as tissue stem and progenitor cells. Many cancers, including breast cancer, also are thought to arise from and be maintained by a small population of cells that display stem cell-like properties. These so-called “cancer stem cells” may also contribute to tumor spread (metastasis), resistance to treatment, and disease relapse. Effective, long-lasting cancer treatments likely will need to target and eliminate these cancer stem cells specifically. Regulatory pathways responsible for maintaining cancer stem cells therefore may be promising targets for treatment. Breast cancers in humans frequently display abnormalities in the PTEN/PI3K/Akt pathway. We demonstrate using cell culture and a mouse model of breast cancer that stem or progenitor cells in both normal breast tissue and breast tumors are dependent for their continued growth on this pathway and on the Wnt/β-catenin pathway. We further show that the drug perifosine, which inhibits the kinase Akt, is able specifically to reduce the population of breast cancer stem or progenitor cells growing in mice. Our findings support the idea that drugs that selectively target breast cancer stem cells through the PTEN/PI3K/Akt pathway may reduce tumor growth and metastasis and result in improved patient outcomes.

The cell cycle inhibitor p27Kip1also has cyclin–cyclin-dependent kinase (CDK)-independent functions. To investigate the significance of these functions in vivo, we generated a knock-in mouse in which four amino acid substitutions in thecdkn1bgene product prevent its interaction with cyclins and CDKs (p27CK−). In striking contrast to complete deletion of thecdkn1bgene, which causes spontaneous tumorigenesis only in the pituitary, the p27CK−protein dominantly caused hyperplastic lesions and tumors in multiple organs, including the lung, retina, pituitary, ovary, adrenals, spleen, and lymphomas. Moreover, the high incidence of spontaneous tumors in the lung and retina was associated with amplification of stem/progenitor cell populations. Therefore, independently of its role as a CDK inhibitor, p27Kip1promoted stem cell expansion and functioned as a dominant oncogene in vivo. Thus, the p27CK−mouse unveils a dual role for p27 during tumorigenesis: It is a tumor suppressor by virtue of its cyclin–CDK regulatory function, and also an oncogene through a cyclin–CDK-independent function. This may explain why thecdkn1bgene is rarely inactivated in human tumors, and the p27CK−mouse in which the tumor suppressor function is lost but the cyclin–CDK-independent—oncogenic—function is maintained may represent a more faithful model for the widespread role of p27 misregulation in human cancers than the p27 null.

International audience; Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte-like and biliary-like cells at confluence. If hepatocyte-like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) the reversible differentiation fate of HepaRG cells is related to the absence of p21(CIP1) and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of beta-catenin localization and HNF3beta expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells. CONCLUSION: Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors.

International audience; BACKGROUND AND OBJECTIVES: The residual tumor cells remaining after completion of standard chemotherapy and radiation treatment in B lymphoma patients, may be eradicated by active immunotherapy that stimulates tumor-specific T lymphocytes. Irradiated autologous lymphoma cells expressing tumor-associated antigens (TAA) may serve as a potential tumor vaccine, provided that they are effectively targeted to the antigen-presenting cells (APC). We propose exploiting the natural anti-Gal antibody in order to target vaccinating tumor cells to APC. Anti-Gal constitutes 1% of IgG in human serum and interacts specifically with the alpha-gal epitope (Galalpha1-3Galphalbeta1-4GlcNAc-R). DESIGN AND METHODS: Alpha-gal epitopes were synthesized in vitro on the membrane of primary lymphoma cells by using the recombinant glycosylation enzyme alpha1,3galactosyltransferase (alpha1,3GT). Processed tumor cells were opsonized by purified anti-Gal antibodies and studied for uptake (phagocytosis) by APC including monocyte-derived macrophages and dendritic cells. Cross-presentation of tumor antigens after phagocytosis of processed MHC-I negative lymphoma cells was measured by activation of a tumor-specific CD8+ T-cell line. RESULTS: We demonstrate synthesis of alpha-gal epitopes on freshly isolated B lymphoma cells of various types following the use of the recombinant enzyme alpha1,3GT. The subsequent binding of anti-Gal to the de novo synthesized alphagal epitopes opsonizes these tumor cells for effective uptake by macrophages and dendritic cells, through phagocytosis mediated by FcgammaR1 (CD64). Moreover, anti-Gal-mediated phagocytosis resulted in cross-presentation of TAA by dendritic cells. INTERPRETATION AND CONCLUSIONS: This study suggests that immunization with irradiated autologous lymphoma cells processed to express alpha-gal epitopes will result in anti-Gal-mediated, in vivo targeting of the autologous tumor vaccine to APC.

When differentiated into mature macrophages by the combination of all-trans retinoic acid and 1,25-dihydroxyvitamin D3, the human promonocytic cell lines U937 and THP-1 expressed inducible nitric oxide synthase (iNOS) transcripts. During their differentiation, the cells acquired the capacity to produce not only superoxide anion (O2.-) but also nitric oxide (.NO) in response to IgG (or IgE)-opsonized zymosan. The inhibitors of the iNOS pathway, aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), suppressed the production of .NO and enhanced the steady-state concentration of O2.- determined. Conversely, superoxide dismutase (SOD) scavenged the O2.- released and increased the .NO-derived nitrite concentration detected. These data suggested a possible interaction between O2.- and .NO. In differentiated U937 (or THP-1) cells, IgG or IgE-opsonized zymosan induced a strong time-dependent luminol-dependent chemiluminescence (LDCL), which was abrogated by SOD and partially inhibited by aminoguanidine or L-NMMA. Since the iNOS inhibitors did not directly scavenge O2.-, LDCL determination in the presence or absence of SOD and/or iNOS inhibitors demonstrated a concomitant production of O2.- and .NO. These radicals induced the formation of a .NO-derived product(s), probably peroxynitrite (ONOO-), which was required to elicit maximal LDCL. Finally, LDCL measurement provided a convenient tool to characterize iNOS triggering and demonstrated an interaction between NADPH oxidase and iNOS products in human macrophagic cells phagocytizing opsonized-zymosan. These findings show that in activated macrophages, iNOS activity can be involved in LDCL and support the debated hypothesis of iNOS participation to the microbicidal activity of human macrophages.