RESUMO
Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.
Assuntos
Neoplasias Colorretais , Neoplasias Peritoneais , Humanos , Matriz Extracelular Descelularizada , Peritônio , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/terapia , Organoides , Neoplasias Colorretais/metabolismoRESUMO
A reciprocal translocation involving chromosomes 8 and 21 generates the AML1/ETO oncogenic transcription factor that initiates acute myeloid leukemia by recruiting co-repressor complexes to DNA. AML1/ETO interferes with the function of its wild-type counterpart, AML1, by directly targeting AML1 binding sites. However, transcriptional regulation determined by AML1/ETO probably relies on a more complex network, since the fusion protein has been shown to interact with a number of other transcription factors, in particular E-proteins, and may therefore target other sites on DNA. Genome-wide chromatin immunoprecipitation and expression profiling were exploited to identify AML1/ETO-dependent transcriptional regulation. AML1/ETO was found to co-localize with AML1, demonstrating that the fusion protein follows the binding pattern of the wild-type protein but does not function primarily by displacing it. The DNA binding profile of the E-protein HEB was grossly rearranged upon expression of AML1/ETO, and the fusion protein was found to co-localize with both AML1 and HEB on many of its regulated targets. Furthermore, the level of HEB protein was increased in both primary cells and cell lines expressing AML1/ETO. Our results suggest a major role for the functional interaction of AML1/ETO with AML1 and HEB in transcriptional regulation determined by the fusion protein.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Fusão Oncogênica/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromossomos Humanos Par 19/genética , Células HeLa , Humanos , Camundongos , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas , Proteína 1 Parceira de Translocação de RUNX1 , Transcrição Gênica , Células U937RESUMO
Approximately one third of acute myeloid leukemias (AMLs) are characterized by aberrant cytoplasmic localization of nucleophosmin (NPMc+ AML), consequent to mutations in the NPM putative nucleolar localization signal. These events are mutually exclusive with the major AML-associated chromosomal rearrangements, and are frequently associated with normal karyotype, FLT3 mutations, and multilineage involvement. We report the gene expression profiles of 78 de novo AMLs (72 with normal karyotype; 6 without major chromosomal abnormalities) that were characterized for the subcellular localization and mutation status of NPM. Unsupervised clustering clearly separated NPMc+ from NPMc- AMLs, regardless of the presence of FLT3 mutations or non-major chromosomal rearrangements, supporting the concept that NPMc+ AML represents a distinct entity. The molecular signature of NPMc+ AML includes up-regulation of several genes putatively involved in the maintenance of a stem-cell phenotype, suggesting that NPMc+ AML may derive from a multipotent hematopoietic progenitor.
Assuntos
Citoplasma/química , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Proteínas Nucleares/análise , Regulação para Cima/genética , Doença Aguda , Linhagem da Célula , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas , Leucemia Mieloide/classificação , Proteínas de Neoplasias/análise , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Tirosina Quinase 3 Semelhante a fmsRESUMO
UNLABELLED: GenePicker allows efficient analysis of Affymetrix gene expression data performed in replicate, through definition of analysis schemes, data normalization, t-test/ANOVA, Change-Fold Change-analysis and yields lists of differentially expressed genes with high confidence. Comparison of noise and signal analysis schemes allows determining a signal-to-noise ratio in a given experiment. Change Call, Fold Change and Signal mean ratios are used in the analysis. While each parameter alone yields gene lists that contain up to 30% false positives, the combination of these parameters nearly eliminates the false positives as verified by northern blotting, quantitative PCR in numerous independent experiments as well as by the analysis of spike-in data. AVAILABILITY: http://www.ifom-firc.it/RESEARCH/Appl_Bioinfo/tools.html. SUPPLEMENTARY INFORMATION: http://www.ifom-firc.it/RESEARCH/Appl_Bioinfo/tools.html.
