The effectiveness of most chemotherapeutics is limited by their inability to penetrate deep into tumor tissue and their ineffectiveness against quiescent cells. Motile Salmonella typhimurium, which ...are specifically attracted to compounds produced by quiescent cancer cells, could overcome this therapeutic barrier. We hypothesized that individual chemoreceptors target S. typhimurium to specific tumor microenvironments. To test this hypothesis, we used time-lapse fluorescent microscopy and tumor cylindroids to quantify the accumulation of chemotaxis machinery knockouts, including strains lacking individual cell surface chemoreceptors, chemotaxis signal transduction pathway enzymes, and the flagella and motor assemblies. To measure the extent of apoptosis induced by individual bacterial strains, caspase-3 activity was measured as a function of time. Our results showed how chemoreceptors directed bacterial chemotaxis within cylindroids: the aspartate receptor initiated chemotaxis toward cylindroids, the serine receptor initiated penetration, and the ribose/galactose receptor directed S. typhimurium toward necrosis. In addition, strains lacking proper flagella constructs, signal transduction proteins, or active motor function did not chemotax toward tumor cylindroids, indicating that directed chemotaxis is necessary to promote accumulation in tumors. By deleting the ribose/galactose receptor, bacterial accumulation localized to tumor quiescence and had a greater individual effect on inducing apoptosis than wild-type S. typhimurium. This new understanding of the mechanisms of Salmonella migration in tumors will allow for the development of bacterial therapies with improved targeting to therapeutically inaccessible regions of tumors.
The spatial arrangement of cellular metabolism in tumor tissue critically affects the treatment of cancer. However, little is known about how diffusion and cellular uptake relate to intracellular ...metabolism and cell death in three dimensions. To quantify these mechanisms, fluorescent microscopy and multicellular tumor cylindroids were used to measure pH and oxygen profiles, and quantify the distribution of viable, apoptotic and necrotic cells. Spheroid dissociation, enzymatic analysis, and mass spectrometry were used to measure concentration profiles of glucose, lactate and glutamine. A mathematical model was used to integrate these measurements and calculate metabolic rate parameters. It was found that large cylindroids, >500 μm in diameter, contained apoptotic and necrotic cells, whereas small cylindroids contained apoptotic but not necrotic cells. The center of cylindroids was found to be acidic but not hypoxic. From the edge to the center, concentrations of glucose, lactate and glutamine decreased rapidly. Throughout the cell masses lactate was consumed and not produced. These measurements indicate that apoptosis was the primary mechanism of cell death; acidity was not caused by lactic acid; and cell death was caused by depletion of carbon sources and not hypoxia. The mathematical model showed that the transporter enzymes for glucose and lactate were not saturated; oxygen uptake was limited by intracellular metabolism; and oxygen uptake was not limited by membrane-transport or diffusion. Unsaturated transmembrane uptake may be the cause of both proliferative and apoptotic regimes in cancer. These results suggest that transporter enzymes are excellent targets for treating well oxygenated tumors.
The spatial arrangement of cellular metabolism in tumor tissue critically affects the treatment of cancer.
The heterogeneity of cellular microenvironments in tumors severely limits the efficacy of most cancer therapies. We have designed a microfluidic device that mimics the microenvironment gradients ...present in tumors that will enable the development of more effective cancer therapies. Tumor cell masses were formed within micron-scale chambers exposed to medium perfusion on one side to create linear nutrient gradients. The optical accessibility of the PDMS and glass device enables quantitative transmitted and fluorescence microscopy of all regions of the cell masses. Time-lapse microscopy was used to measure the growth rate and show that the device can be used for long-term efficacy studies. Fluorescence microscopy was used to demonstrate that the cell mass contained viable, apoptotic, and acidic regions similar to in vivo tumors. The diffusion coefficient of doxorubicin was accurately measured, and the accumulation of therapeutic bacteria was quantified. The device is simple to construct, and it can easily be reproduced to create an array of in vitro tumors. Because microenvironment gradients and penetration play critical roles controlling drug efficacy, we believe that this microfluidic device will be vital for understanding the behavior of common cancer drugs in solid tumors and designing novel intratumorally targeted therapeutics.
In this study we have designed the NTFR peptide-amphiphile that mimics a fragment of the N-terminus of the fractalkine receptor (CX3CR1) and specifically targets fractalkine, a novel adhesion ...molecule expressed on the surface of inflamed endothelial cells. Bioartificial membranes were constructed from mixtures of NTFR peptide-amphiphiles and DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) phospholipids, and the affinity and specificity of fractalkine for the synthetic NTFR was investigated with an atomic force microscope (AFM). Fractalkine was immobilized onto the AFM tips, and forces were collected between fractalkine and the bioartificial membranes. The adhesive interactions were studied at the collective level, when each adhesion event corresponded to the rupture of multiple biomolecular bonds. Retraction force profiles for the fractalkine−NTFR system exhibited single or multiple peaks and a small percentage of the force curves demonstrated stretching of the fractalkine−NTFR complex. Strong adhesion was measured when both DPPC and NTFR were present, compared to pure NTFR surfaces. This may be due to the fact that the DPPC molecule is shorter, and thus it can provide more space for the peptide headgroup to bend and expose its sequence at the interface. Specificity was demonstrated by comparing the NTFR−fractalkine adhesion to the forces between the α5β1 integrin (an adhesion receptor expressed on the surface of endothelial cells) and other surfaces such as GRGDSP (the specific ligand for α5β1), GRGESP (an inactive sequence), and NTFR.
The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing ...technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
High resolution RNA analysis using next-generation sequencing (RNA-seq) is a rapidly growing application in cancer research. Formalin-fixed paraffin-embedded (FFPE) tissue is a ubiquitous ...resource for clinical studies. However, the ability to generate high quality libraries is confounded by the low quality and yield of RNA extracted from FFPE specimen. To enable optimal interrogation of such a clinically-relevant tissue resource, it is critical to identify RNA-seq library construction workflows that improve performance using challenging and degraded sample types. In this study, the effects of RNA type, quality, and quantity on RNA-seq library construction are assessed, and expectations regarding sequencing data quality are addressed. Included are recommendations for quality control measures and input-specific modifications that may improve performance Three workflows employing either bead-based or enzymatic strategies for the removal of ribosomal RNA content were used to prepare RNA-seq libraries from degraded paired breast normal and tumor samples. Enzymatic depletion followed by library preparation using the KAPA RNA HyperPrep1 workflow identified the highest number of transcripts for each sample, translating to a 4.9-fold increase in number of differentially expressed transcripts detected between normal and tumor samples. Of the transcripts identified as differentially expressed exclusively by Kapa, 174 are protein-coding RNAs, many of which are frequently dysregulated in breast cancer. In contrast, only two protein-coding RNAs were identified exclusively by the alternative workflows. RealTime Ready Custom panels were used for independent quantitative rt-PCR assessment. Of a subset of 71 transcripts identified exclusively by Kapa, up to 69 (97%) were validated as differentially expressed. Additional performance improvements of the KAPA RNA HyperPrep workflow include coverage balance across transcripts, lower duplication rate, technical replicate reproducibility, agreement across input quantities, and gene expression concordance between paired fresh frozen and FFPE samples. 1For Research Use Only. Not for use in diagnostic procedures.
Citation Format: Nancy H. Nabilsi, Drew Cheney, Leendert Cloete, Ida van Jaarsveld, Rachel Kasinskas, Jennifer Pavlica. Demonstrated improvements in tumor profiling with enzymatic ribosomal RNA depletion and streamlined RNA-sequencing library preparation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5411. doi:10.1158/1538-7445.AM2017-5411
The effectiveness of most chemotherapeutics is limited by their inability to penetrate deep into tumor tissue and their ineffectiveness against quiescent cells located far from tumor vasculature. ...Motile Salmonella typhimurium could overcome these therapeutics barriers. We hypothesize that the accumulation of S. typhimurium in tumors is controlled by two mechanisms: (1) chemotaxis towards compounds produced by quiescent cancer cells and (2) preferential growth within tumor tissue. We also hypothesized that individual chemoreceptors target S. typhimurium to specific tumor microenvironments. Using the tumor cylindroid model, which mimics the microenvironment of in vivo tumors, we elucidated the mechanism by which S. typhimurium is attracted to and accumlates in heterogeneous tumor tissue. Time-lapse fluorescent microscopy was used to quantify the accumulation of wild-type and chemotaxis machinery knockouts, including strains lacking individual cell-surface chemoreceptors, chemotaxis signal transduction pathway enzymes, and the flagella and motor assemblies. To measure the extent of apoptosis induced by individual bacterial strains, caspase-3 activity was measured as a function of time. Spatio-temporal profiles of wild-type bacteria were fit to a mathematical model to calculate two parameters that describe bacterial interaction with tumors: bacterial growth, M, and bacterial chemoattraction, K. It was observed that wild-type S. typhimurium are attracted to cylindroids and accumulate at long time points in the central region of large cylindroids. Both bacterial growth and bacterial chemotaxis were significantly greater in large cylindroids, suggesting that quiescent cells secrete bacterial chemoattractants. Results also showed how individual chemoreceptors directed bacterial chemotaxis within cylindroids: the aspartate receptor initiated chemotaxis towards cylindroids, the serine receptor initiated penetration, and the ribose/galactose receptor directed S. typhimurium towards necrosis. In addition, strains lacking proper flagella constructs, signal transduction proteins, or active motor function, did not chemotax towards tumor cylindroids, indicating that attraction to specific compounds and not random motility is necessary to promote accumulation in tumors. By deleting the ribose/galactose receptor, bacterial accumulation localized to tumor quiescence and had a greater individual effect on inducing apoptosis than wild-type. This new understanding of the mechanisms of Salmonella migration in tumors will allow for the development of therapies with improved targeting to therapeutically inaccessible regions of tumors.
The effectiveness of most chemotherapeutics is limited by their inability to penetrate deep into tumor tissue and their ineffectiveness against quiescent cells located far from tumor vasculature. ...Motile Salmonella typhimurium could overcome these therapeutics barriers. We hypothesize that the accumulation of S. typhimurium in tumors is controlled by two mechanisms: (1) chemotaxis towards compounds produced by quiescent cancer cells and (2) preferential growth within tumor tissue. We also hypothesized that individual chemoreceptors target S. typhimurium to specific tumor microenvironments. Using the tumor cylindroid model, which mimics the microenvironment of in vivo tumors, we elucidated the mechanism by which S. typhimurium is attracted to and accumlates in heterogeneous tumor tissue. Time-lapse fluorescent microscopy was used to quantify the accumulation of wild-type and chemotaxis machinery knockouts, including strains lacking individual cell-surface chemoreceptors, chemotaxis signal transduction pathway enzymes, and the flagella and motor assemblies. To measure the extent of apoptosis induced by individual bacterial strains, caspase-3 activity was measured as a function of time. Spatio-temporal profiles of wild-type bacteria were fit to a mathematical model to calculate two parameters that describe bacterial interaction with tumors: bacterial growth, M, and bacterial chemoattraction, K. It was observed that wild-type S. typhimurium are attracted to cylindroids and accumulate at long time points in the central region of large cylindroids. Both bacterial growth and bacterial chemotaxis were significantly greater in large cylindroids, suggesting that quiescent cells secrete bacterial chemoattractants. Results also showed how individual chemoreceptors directed bacterial chemotaxis within cylindroids: the aspartate receptor initiated chemotaxis towards cylindroids, the serine receptor initiated penetration, and the ribose/galactose receptor directed S. typhimurium towards necrosis. In addition, strains lacking proper flagella constructs, signal transduction proteins, or active motor function, did not chemotax towards tumor cylindroids, indicating that attraction to specific compounds and not random motility is necessary to promote accumulation in tumors. By deleting the ribose/galactose receptor, bacterial accumulation localized to tumor quiescence and had a greater individual effect on inducing apoptosis than wild-type. This new understanding of the mechanisms of Salmonella migration in tumors will allow for the development of therapies with improved targeting to therapeutically inaccessible regions of tumors.
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