RESUMO
Circulating tumor cells (CTCs) are precursors of cancer in the blood and provide an attractive source for dynamic monitoring of disease progression and tumor heterogeneity. However, the scarcity of CTCs in the bloodstream has limited their use in clinical practice. In this study, we present a workflow for easy detection of CTCs by cytokeratin staining using the FDA-cleared Parsortix device for size-based microfluidic enrichment. To minimize sample handling, the isolated cells are stained inside the separation cassette and harvested for subsequent single cell isolation and whole genome copy-number analysis. We validated the workflow on a panel of four prostate cancer cell lines spiked into healthy donor blood collected in CellRescue or EDTA tubes, resulting in mean recoveries of 42% (16-69%). Furthermore, we evaluated the clinical utility in a cohort of 12 metastatic prostate cancer patients and found CTCs in 67% of patients ranging from 0 to 1172 CTCs in 10 mL blood. Additionally, we isolated single patient-derived CTCs and identified genomic aberrations associated with treatment response and clinical outcome. Thus, this workflow provides a readily scalable strategy for analysis of single CTCs, applicable for use in monitoring studies to identify genomic variations important for guiding clinical therapy decision.
Assuntos
Células Neoplásicas Circulantes , Neoplasias da Próstata , Análise de Célula Única , Fluxo de Trabalho , Humanos , Masculino , Células Neoplásicas Circulantes/patologia , Células Neoplásicas Circulantes/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/sangue , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Metástase Neoplásica , Separação Celular/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Coloração e Rotulagem/métodos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangueRESUMO
In Escherichia coli, an increase in the frequency of chromosome replication is lethal. In order to identify compounds that affect chromosome replication, we screened for molecules capable of restoring the viability of hyper-replicating cells. We made use of two E. coli strains that over-initiate DNA replication by keeping the DnaA initiator protein in its active ATP bound state. While viable under anaerobic growth or when grown on poor media, these strains become inviable when grown in rich media. Extracts from actinomycetes strains were screened, leading to the identification of deferoxamine (DFO) as the active compound in one of them. We show that DFO does not affect chromosomal replication initiation and suggest that it was identified due to its ability to chelate cellular iron. This limits the formation of reactive oxygen species, reduce oxidative DNA damage and promote processivity of DNA replication. We argue that the benzazepine derivate (±)-6-Chloro-PB hydrobromide acts in a similar manner.