RESUMO
Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.
Assuntos
Biomarcadores Tumorais/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Imunofluorescência , Imunoterapia Adotiva , Neoplasias/metabolismo , Neoplasias/terapia , Fotodegradação , Análise de Célula Única , Antígenos Thy-1/metabolismo , Morte Celular , Citotoxicidade Imunológica , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplanteRESUMO
The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays(1). Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research (2-4). Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors(4) or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable. Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis (5). To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.
Assuntos
Neoplasias , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante HeterólogoRESUMO
INTRODUCTION: Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells. METHODS: We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations. RESULTS: High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial-mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-ß-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy. CONCLUSIONS: In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Antígenos Embrionários Estágio-Específicos/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Humanos , Camundongos , Transplante de NeoplasiasRESUMO
MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.
Assuntos
MicroRNAs/genética , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute proteins to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. The significance of their mitochondrial localization was investigated by characterizing their genomic context, cross-species conservation and instrinsic features such as their size and thermodynamic parameters. Interestingly, the specificities of mitochondrial versus cytosolic miRNAs were underlined by significantly different structural and thermodynamic parameters. Computational targeting analysis of most mitochondrial miRNAs revealed not only nuclear but also mitochondrial-encoded targets. The functional relevance of miRNAs in mitochondria was supported by the finding of Argonaute 2 localization to mitochondria revealed by immunoblotting and confocal microscopy, and further validated by the co-immunoprecipitation of the mitochondrial transcript COX3. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed 'mitomiRs'.
Assuntos
Núcleo Celular/genética , Mitocôndrias/genética , Interferência de RNA , Animais , Proteínas Argonautas , Sequência de Bases , Linhagem Celular , Cromossomos Humanos/genética , Sequência Conservada/genética , Citosol/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma Mitocondrial/genética , Genômica , Humanos , Imunoprecipitação , MicroRNAs/isolamento & purificação , Modelos Biológicos , Transporte Proteico , Transporte de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da EspécieRESUMO
Sinorhizobium meliloti is a symbiotic soil bacterium of the alphaproteobacterial subdivision. Like other rhizobia, S. meliloti induces nitrogen-fixing root nodules on leguminous plants. This is an ecologically and economically important interaction, because plants engaged in symbiosis with rhizobia can grow without exogenous nitrogen fertilizers. The S. meliloti-Medicago truncatula (barrel medic) association is an important symbiosis model. The S. meliloti genome was published in 2001, and the M. truncatula genome currently is being sequenced. Many new resources and data have been made available since the original S. meliloti genome annotation and an update was needed. In June 2008, we submitted our annotation update to the EMBL and NCBI databases. Here we describe this new annotation and a new web-based portal RhizoGATE. About 1000 annotation updates were made; these included assigning functions to 313 putative proteins, assigning EC numbers to 431 proteins, and identifying 86 new putative genes. RhizoGATE incorporates the new annotion with the S. meliloti GenDB project, a platform that allows annotation updates in real time. Locations of transposon insertions, plasmid integrations, and array probe sequences are available in the GenDB project. RhizoGATE employs the EMMA platform for management and analysis of transcriptome data and the IGetDB data warehouse to integrate a variety of heterogeneous external data sources.
Assuntos
Bases de Dados Genéticas , Genoma Bacteriano , Gestão da Informação , Sinorhizobium meliloti/genética , Proteínas de Bactérias/genética , Armazenamento e Recuperação da Informação , Internet , Medicago truncatula , Análise em Microsséries , Interface Usuário-ComputadorRESUMO
Depending on the phosphate concentration encountered in the environment Sinorhizobium meliloti 2011 synthesizes two different exopolysaccharides (EPS). Galactoglucan (EPS II) is produced under phosphate starvation but also in the presence of extra copies of the transcriptional regulator WggR (ExpG) or as a consequence of a mutation in mucR. The galactoglucan biosynthesis gene cluster contains the operons wga (expA), wge (expE), wgd (expD), and wggR (expG). Two promoters, differentially controlled by WggR, PhoB, and MucR, were identified upstream of each of these operons. The proximal promoters of the wga, wge, and wgd transcription units were constitutively active when separated from the upstream regulatory sequences. Promoter activity studies and the positions of predicted PhoB and WggR binding sites suggested that the proximal promoters are cooperatively induced by PhoB and WggR. MucR was shown to strongly inhibit the distal promoters and bound to the DNA in the vicinity of the distal transcription start sites. An additional inhibitory effect on the distal promoter of the structural galactoglucan biosynthesis genes was identified as a new feature of WggR in a mucR mutant. A regulatory model of the fine-tuning of galactoglucan production is proposed.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas Fúngicas/fisiologia , Galactanos/metabolismo , Glucanos/metabolismo , Polissacarídeos Bacterianos/biossíntese , Regiões Promotoras Genéticas/fisiologia , Sinorhizobium meliloti/metabolismo , Transativadores/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reguladores , Óperon , Fosfatos/farmacologia , Polissacarídeos Bacterianos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sinorhizobium meliloti/genética , Transativadores/genética , Transativadores/metabolismo , Transcrição GênicaRESUMO
The establishment of an effective nitrogen-fixing symbiosis between Sinorhizobium meliloti and its legume host alfalfa (Medicago sativa) depends on the timely expression of nodulation genes that are controlled by LysR-type regulators. Ninety putative genes coding for LysR-type transcriptional regulators were identified in the recently sequenced S. meliloti genome. All 90 putative lysR genes were mutagenized using plasmid insertions as a first step toward determining their roles in symbiosis. Two new LysR-type symbiosis regulator genes, lsrA and lsrB, were identified in the screening. Both the lsrA and lsrB genes are expressed in free-living S. meliloti cells, but they are not required for cell growth. An lsrA1 mutant was defective in symbiosis and elicited only white nodules that exhibited no nitrogenase activity. Cells of the lsrA1 mutant were recovered from the white nodules, suggesting that the lsrA1 mutant was blocked early in nodulation. An lsrB1 mutant was deficient in symbiosis and elicited a mixture of pink and white nodules on alfalfa plants. These plants exhibited lower overall nitrogenase activity than plants inoculated with the wild-type strain, which is consistent with the fact that most of the alfalfa plants inoculated with the lsrB1 mutant were short and yellow. Cells of the lsrB1 mutant were recovered from both pink and white nodules, suggesting that lsrB1 mutants could be blocked at multiple points during nodulation. The identification of two new LysR-type symbiosis transcriptional regulators provides two new avenues for understanding the complex S. meliloti-alfalfa interactions which occur during symbiosis.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Medicago sativa/microbiologia , Sinorhizobium meliloti/genética , Fatores de Transcrição/genética , Transcrição Gênica , Sequência de Bases , Dados de Sequência Molecular , Nitrogênio/metabolismo , Receptores de Superfície Celular , Simbiose , Sítio de Iniciação de TranscriçãoRESUMO
The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.
Assuntos
ADP Ribose Transferases/fisiologia , Flagelina/biossíntese , Regulação Bacteriana da Expressão Gênica , Polissacarídeos Bacterianos/biossíntese , Sinorhizobium meliloti/metabolismo , Adaptação Fisiológica , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Toxinas Bacterianas , Flagelos/metabolismo , Perfilação da Expressão Gênica , Medicago sativa/microbiologia , Movimento , Mutagênese Insercional , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genética , Sinorhizobium meliloti/citologia , Sinorhizobium meliloti/genética , Simbiose/genéticaRESUMO
Sinorhizobium meliloti is an alpha-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. As a step toward understanding the physiology of S. meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state. Data acquisition was based on both macro- and microarrays. Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered. The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability. A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified. Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated. However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.
Assuntos
Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Oxigênio/farmacologia , Sinorhizobium meliloti/genética , Simbiose/genética , Adaptação Biológica/genética , Adaptação Biológica/fisiologia , Perfilação da Expressão Gênica/métodos , Fixação de Nitrogênio/genética , Fixação de Nitrogênio/fisiologia , Filogenia , Análise Serial de Proteínas/métodos , Proteoma/genética , Proteoma/metabolismo , Sinorhizobium meliloti/metabolismo , Simbiose/efeitos dos fármacos , Simbiose/fisiologia , Transcrição Gênica/genéticaRESUMO
Based on the complete Sinorhizobium meliloti genome sequence we established DNA microarrays as a comprehensive tool for systematic genome-wide gene expression analysis in S. meliloti 1021. For these PCR fragment-based microarrays, called Sm6kPCR, a collection of probes for the 6207 predicted protein-coding genes consisting of 6046 gene-specific PCR fragments and 161 70 mer oligonucleotides was arrayed in high density on glass slides. To obtain these PCR fragments primer pairs were designed to amplify internal gene-specific DNA fragments of 80-350 bp. Additionally, these primers were characterized by a 5' extension that allowed for reamplification using standard primers after the first amplification employing the specific primers. In order to ascertain the quality of the Sm6kPCR microarrays and to validate gene expression studies in S. meliloti parallel hybridizations based on RNA samples obtained from cells cultured under identical conditions were performed. In addition, gene expression in S. meliloti in response to an osmotic upshift imposed by the addition of 0.38 M NaCl was monitored. 137 genes were identified showing significant changes in gene expression resulting from the osmotic upshift. From these genes 52 were induced and 85 genes were repressed. Among the genes displaying different RNA levels some functional groups could be identified that are particularly remarkable. Repression was observed for 8 genes related to motility and chemotaxis, 7 genes encoding amino acid biosynthesis enzymes and 15 genes involved in iron uptake whereas 14 genes involved in transport of small molecules and 4 genes related to polysaccharide biosynthesis were induced.
Assuntos
Mapeamento Cromossômico/instrumentação , Perfilação da Expressão Gênica/instrumentação , Regulação Bacteriana da Expressão Gênica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Sinorhizobium meliloti/genética , Equilíbrio Hidroeletrolítico/genética , Mapeamento Cromossômico/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Perfilação da Expressão Gênica/métodos , Genoma Bacteriano , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Sinorhizobium meliloti/classificação , Especificidade da EspécieRESUMO
Sinorhizobium meliloti (Rhizobium meliloti) 2011 has the ability to produce the two acidic exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II). EPS I is a branched heteropolysaccharide composed of octasaccharide repeating units, whereas EPS II is a linear heteropolysaccharide consisting of disaccharide subunits. The exo-exs and exp gene clusters are involved in the biosynthesis of EPSI and EPSII, respectively. EPSI and EPSII biosynthesis genes are differentially expressed resulting in a complex regulation of EPS production in S. meliloti. The phosphate concentration was identified as an important factor affecting the expression of exp genes.
Assuntos
Proteínas de Bactérias/genética , Galactanos , Regulação Bacteriana da Expressão Gênica , Glucanos , Polissacarídeos Bacterianos/biossíntese , Sinorhizobium meliloti/metabolismo , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Família Multigênica , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/crescimento & desenvolvimento , Transcrição GênicaRESUMO
The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Cromossomos Bacterianos/genética , Mapeamento de Sequências Contíguas/métodos , Corynebacterium/genética , Cosmídeos/genética , Genoma Bacteriano , Clonagem Molecular , Genes Bacterianos/genética , Biblioteca Genômica , Genômica/métodos , Análise de Sequência de DNA/métodosRESUMO
The soil bacterium Sinorhizobium meliloti (Rhizobium meliloti) has the ability to produce the alternative exopolysaccharide galactoglucan (EPS II) in addition to succinoglycan (EPS I). In the wild-type strain EPS II production is induced by phosphate-limiting conditions or by extra copies of the exp gene cluster. Based on similarities to transcriptional regulators of the MarR family, an additional putative regulatory gene, expG, was identified in the exp gene cluster. Using exp-lacZ transcriptional fusions, a stimulating effect of extra copies of this expG gene on the transcription of all exp complementation groups was determined. Phosphate limitation also resulted in increased expression of the exp-lacZ fusions. This increase was reduced in strains characterized by a deletion of expG. The previously reported high level of exp gene transcription in a mucR mutant was further elevated under phosphate-limiting conditions. The expA, expD, expG and expE promoters contain sequences with similarities to the PHO box known as the PhoB-binding site in phosphate-regulated promoters in Escherichia coli. The S. meliloti phoB gene was required for the activation of exp gene expression under phosphate limitation, but not for induction of exp expression by MucR or ExpG.