1,721,008 research outputs found
Cancer Stem Cells in Human Osteosarcoma: Tracking Stemness to Target Chemoresistance
No full textTese de doutoramento em Engenharia Biomédica, apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraOsteosarcoma is the most common primary malignant bone tumor, afflicting mainly young patients. Since the inception of chemotherapy, survival rates augmented significantly, but are stagnated over the past decades, due to the absence of improved therapy. Most patients succumb to metastatic disease, which can occur even after apparent successful histological response to chemotherapy that precedes the surgical removal of the primary tumor.
Recently, the cancer stem cell (CSC) model has been receiving attention and suggests that stem-like cells exist in many tumor types, form the clonogenic core of the tumor and actively contribute to chemoresistance. This theory was originally postulated as a somewhat rigid hierarchical model of tumor development, in which a self-renewing CSC originates differentiated progeny and is responsible for feeding the bulk tumor mass. However, evidence suggests that diverse pools of CSCs might exist, contributing to the intratumoral heterogeneity of solid tumors, including osteosarcoma. Besides, extrinsic factors, e.g. drug exposure, may also constitute a source of stemness within the tumors.
In this thesis we performed a molecular and functional characterization of CSCs in a panel of cell lines representative of two histological subtypes of high-grade osteosarcoma, and investigated the central role of the regulatory Wnt/β-catenin pathway in the stemness properties and survival advantages of CSCs.
In the first part, we demonstrated that different CSCs populations may co-exist in osteosarcoma cell lines exhibiting distinct functional properties. CSCs isolated from fibroblastic tumors are slowly-proliferating populations overexpressing Sox2 and Klf4 pluripotency-related markers, have enhanced tumorigenic potential and specific activation of the self-renewal-related Wnt/β-catenin pathway, as assessed by nuclear β-catenin positivity, AXIN2 expression and TCF/LEF transcriptional activity. The Aldefluor+ populations detected in the two histological osteosarcoma subtypes are SOX2+, but KLF4-, whereas the side-population subset phenotype is correlated with ABCG2 drug-efflux transporter expression. Altogether, these results suggest that distinct functional methods identify CSCs with dissimilar characteristics, which may have implications on the design of CSC-targeted therapies.
In the second part, we evaluated the therapeutic potential of inhibiting Wnt/β-catenin against chemoresistant CSCs. The Wnt signaling antagonist IWR-1 was selectively cytotoxic for CSCs, by decreasing cell viability, proliferation and cell cycle progression. IWR-1 induced apoptosis of osteosarcoma CSCs and in combination with doxorubicin treatment elicited synergistic cytotoxicity, reversing CSCs intrinsic resistance to this drug. IWR-1 impaired CSC’ self-renewal capacity by compromising landmark steps of the canonical Wnt signaling, namely nuclear β-catenin translocation and subsequent TCF/LEF activation and expression of downstream targets. Wnt inhibition also hampered Aldefluor activity and expression of key pluripotency-related genes. We observed a remarkable anti-tumor effect of IWR-1 in osteosarcoma-xenografted models that potentiated the anti-tumor efficacy of doxorubicin, accompanied by down-regulation of TCF/LEF transcriptional activity, Sox2 and AXIN2 expression and nuclear β-catenin.
In the third part, we explored the striking hypothesis that drugs used in osteosarcoma treatment induce stemness properties in differentiated cells. Doxorubicin, cisplatin and methotrexate induced a phenotypic stem-like cell transition, by increasing Aldefluor activity and ALDH, ABC transporters and pluripotency markers expression. Doxorubicin up-regulated stemness markers via Wnt/β-catenin activation, but co-treatment with IWR-1 prevented the drug-induced phenotype. Altogether, these results are consistent with the ability of doxorubicin to kill rapidly-dividing cancer cells, and of IWR-1 to eliminate chemoresistant CSCs populations and overcome acquired resistance.
Translational significance of this study was conveyed by the pluripotency mRNA signature found in uncultured osteosarcoma patient samples and by the correlation of stemness-related markers expression with a worst prognosis in osteosarcoma patients who responded poorly to chemotherapy (EuroBonet dataset of whole genome expression).
To conclude, our results suggest the existence of phenotypic heterogeneity in osteosarcoma CSCs, and revealed the Wnt/β-catenin as a key determinant of the stemness and chemoresistant profile of osteosarcoma. Targeting the Wnt pathway may simultaneously circumvent chemoresistance and the phenotypic differentiated-to-stem like cell transition induced by chemotherapeutics, and thus contribute to reduce chemotherapy doses and ameliorate the prognostic outcomes of osteosarcoma patients.O osteossarcoma é o tumor ósseo primário maligno mais comum e afecta sobretudo jovens adolescentes. Com a introdução da quimioterapia, as taxas de sobrevivência aumentaram significativamente, mas estagnaram nas últimas décadas, devido à falta de terapias mais eficazes. Muitos doentes desenvolvem metástases e sucumbem à doença, mesmo após boa resposta histológica à quimioterapia que antecede a remoção cirúrgica do tumor primário.
Recentemente, o modelo das células estaminais cancerígenas (CSCs) sugere a existência de células com propriedades estaminais que formam o núcleo clonogénico tumoral e contribuem para a quimio-resistência. Esta teoria foi inicialmente postulada como um modelo hierárquico rígido, no qual CSCs com capacidade de auto-renovação e de diferenciação sustentam o crescimento do tumor e dão origem à população heterogénea de células diferenciadas. No entanto, há evidências que sugerem a existência de diferentes subpopulações de CSCs, podendo assim contribuir para a marcada heterogeneidade intratumoral típica dos tumores sólidos, incluindo o osteossarcoma. Além disso, factores extrínsecos, como por exemplo a exposição à quimioterapia, podem também contribuir para a aquisição de um fenótipo estaminal nos tumores.
No âmbito desta tese foi realizada uma caracterização molecular e funcional de CSCs num painel de linhas celulares representativas de dois subtipos histológicos de osteossarcoma de alto-grau, tendo-se investigado o papel central da via Wnt/β-catenina na regulação da estaminalidade e na sobrevivência de CSCs.
Numa primeira parte, demonstrámos a existência de diferentes populações de CSCs no osteossarcoma, com propriedades funcionais distintas. As CSCs isoladas a partir de tumores fibroblásticos, caracterizam-se por uma baixa taxa de proliferação, expressão de marcadores de pluripotência (Sox2 e Klf4), elevado potencial tumorigénico e ativação da via Wnt/β-catenina, evidenciada pela localização nuclear de β-catenina, expressão da AXIN2 e actividade de transcrição do TCF/LEF. As populações Aldefluor+ detectadas nos dois subtipos histológicos de osteossarcoma, são SOX2+, mas KLF4-, enquanto o fenótipo side-population se correlaciona com a expressão do transportador de efluxo ABCG2. Estes resultados sugerem que diferentes métodos funcionais identificam CSCs com características distintas, o que pode ter implicações no desenho de terapias dirigidas às CSCs.
Numa segunda parte, avaliámos o potencial terapêutico de inibição da via Wnt/β-catenina em CSCs quimio-resistentes. O inibidor IWR-1 mostrou seletividade citotóxica para as CSCs, como demonstrado pela diminuição na viabilidade, proliferação e progressão do ciclo celular e indução da apoptose. Demonstrou ainda sinergia em combinação com a doxorrubicina, revertendo a resistência intrínseca das CSCs a este fármaco. A inibição da via Wnt, demonstrada pela diminuição da translocação nuclear da β-catenina e repressão da atividade de transcrição do complexo TCF/LEF, comprometeu a capacidade de auto-renovação das CSCs e diminuiu a actividade do Aldefluor, assim como a expressão de genes de pluripotência. No modelo in vivo de osteossarcoma, o tratamento com o inibidor IWR 1 mostrou um efeito anti-tumoral pronunciado, acompanhado por uma diminuição da atividade de transcrição do TCF/LEF e da expressão de AXIN2, Sox2 e β-catenina nuclear.
Na terceira parte, avaliámos os efeitos de agentes de quimioterapia utilizados no tratamento de osteossarcoma, nomeadamente a doxorrubicina, cisplatina e o metotrexato na aquisição de um fenótipo estaminal. Observou-se um aumento na atividade e expressão de ALDH, bem como da expressão de transportadores ABC e dos marcadores de pluripotência nas células expostas à quimioterapia, efeito que foi mediado pela ativação da via de sinalização Wnt/β-catenina. A inibição desta via com o IWR 1 preveniu a aquisição de um fenótipo estaminal induzido pela doxorrubicina, o que demonstra a capacidade do IWR 1 em eliminar CSCs e prevenir o desenvolvimento de resistência adquirida.
O potencial translacional desta tese assenta na observação de uma potencial assinatura genética definida pela expressão de marcadores de pluripotência em amostras clínicas de osteossarcoma, e da correlação da expressão destes marcadores com um mau prognóstico, em doentes não-responsivos à quimioterapia, de acordo com a base de dados EuroBonet.
Em conclusão, este trabalho sugere a existência de heterogeneidade fenotípica em CSCs de osteossarcoma e demonstrou que a via da Wnt/β-catenina constitui um fator chave associado à estaminalidade e à quimio-resistência das CSCs. O desenvolvimento de novas terapias dirigidas às CSCs, tendo como alvo a via da Wnt/β-catenina poderá contribuir para contornar a quimio-resistência intrínseca das CSCs e prevenir a aquisição de um fenótipo estaminal induzido pela exposição a fármacos. Esta abordagem poderá contribuir para reduzir as doses de quimioterapia e melhorar o prognóstico de doentes com osteossarcoma.Centro de Investigação em Meio Ambiente, Genética e Oncobiologia da
Faculdade de Medicina da Universidade de Coimbra” (NRC-LPCC/CIMAGO Grant 2014
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Bone: Conventional Osteosarcoma
Get PDFReview on Osteosarcoma, with data on clinics, and the genes involved
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Identification of markers to characterize and sort human articular chondrocytes with enhanced in vitro chondrogenic capacity
Get PDFObjective. To identify markers associated with the chondrogenic capacity of expanded human articular chondrocytes and to use these markers for sorting of more highly chondrogenic subpopulations. Methods. The chondrogenic capacity of chondrocyte populations derived from different donors (n = 21) or different clonal strains from the same cartilage biopsy specimen (n = 21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analyzed by microarray and flow cytometry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential chondrogenic capacity and again assessed in pellet cultures. Results. Significance Analysis of Microarrays indicated that chondrocytes with low chondrogenic capacity expressed higher levels of insulin-like growth factor I and of catabolic genes (e.g., matrix metalloproteinase 2, aggrecanase 2), while chondrocytes with high chondrogenic capacity expressed higher levels of genes involved in cell-cell or cell-matrix interactions (e.g., CD49c, CD49f). Flow cytometry analysis showed that CD44, CD151, and CD49c were expressed at significantly higher levels in chondrocytes with higher chondrogenic capacity. Flow cytometry analysis of clonal chondrocyte strains indicated that CD44 and CD151 could also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher levels of GAG per DNA (up to 1.4-fold) and type 11 collagen messenger RNA (up to 3.4-fold) than did unsorted cells. Conclusion. We identified markers that allow characterization of the capacity of monolayer-expanded chondrocytes to form in vitro cartilaginous tissue and enable enrichment for subpopulations with higher chondrogenic capacity. These markers might be used as a means to predict and possibly improve the outcome of cell-based cartilage repair techniques
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