BackgroundTo improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.Methodology/Principal FindingsWe purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.Conclusions/SignificanceFor the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of ‘liquid biopsies’ to better model drug discovery.

Synthesis of inorganic polyphosphate (poly P) from the terminal phosphate of ATP is catalyzed reversibly by poly P kinase (PPK, now designated PPK1) initially isolated from Escherichia coli. PPK1 is highly conserved in many bacteria, including some of the major pathogens such as Pseudomonas aeruginosa. In a null mutant of P. aeruginosa lacking ppk1, we have discovered a previously uncharacterized PPK activity (designated PPK2) distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn 2؉ over Mg 2؉ , and a stimulation by poly P. The reverse reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the forward reaction, poly P synthesis from GTP. The gene encoding PPK2 (ppk2) was identified from the amino acid sequence of the protein purified near 1,000-fold, to homogeneity. The 5-end is 177 bp upstream of the annotated genome sequence of a ''conserved hypothetical protein''; ppk2 (1,074 bp) encodes a protein of 357 aa with a molecular mass of 40.8 kDa. Sequences homologous to PPK2 are present in two other proteins in P. aeruginosa, in two Archaea, and in 32 other bacteria (almost all with PPK1 as well); these include rhizobia, cyanobacteria, Streptomyces, and several pathogenic species. Distinctive features of the poly P-driven nucleoside diphosphate kinase activity and structural aspects of PPK2 are among the subjects of an accompanying report. Inorganic polyphosphate (poly P) is a polymer of tens or hundreds of phosphate residues found in all cells (bacterial, fungal, plant, and animal) that have been examined (1-3). Among its many functions, poly P is needed for bacterial survival in the face of stress and stringencies and is required for the virulence of some pathogens (4-12).The gene ppk1 encodes poly P kinase (PPK1), the enzyme that converts ATP to poly P among other activities (13). Null mutants of Pseudomonas aeruginosa PAO1 lacking ppk1 are deficient in motility, quorum sensing, biofilm formation, and virulence in mouse models (9,10,12). Despite the lack of detectable PPK1 activity (Ͻ1% of wild type), these mutants still possess as much as 20% of the wild-type levels of poly P (9).The purpose of this study was to determine the source of poly P in the ppk1 mutants of P. aeruginosa. No clue could be obtained from extensive searches for homologies to PPK1 in the available databases. Thus, the putative PPK2 would have to be revealed by novel assays of poly P synthesis in cell-free extracts of the mutants and the subsequent isolation of the responsible enzyme.In this report, we describe the identification and novel features of PPK2, its sequence in the P. aeruginosa genome, and the conservation of this sequence among 35 microorganisms, notably rhizobia, cyanobacteria, Streptomyces, several pathogenic species, and two Archaea.Simultaneously with these studies, we pursued a very potent activity in P. aeruginosa that uses poly P as a donor to convert GDP to GTP (14). This activity, previously designated PNDK (poly P-driven ...

Key Points Germ line variants in TERT, SH2B3, TET2, ATM, CHEK2, PINT, and GFI1B are associated with JAK2 V617F clonal hematopoiesis and MPNs. Age-related JAK2 V617F clonal hematopoiesis is found in ∼2 out of 1000 individuals in the general population.

An enzyme that uses inorganic polyphosphate (poly P) as a donor to convert GDP to GTP has been purified 1,300-fold to homogeneity from lysates of Pseudomonas aeruginosa PAOM5. Poly P chains of 30 -50 residues are optimal; those of 15-700 residues can also serve. GDP is preferred over ADP among nucleoside diphosphate acceptors. This nucleoside diphosphate kinase (NDK) activity resides in the same protein isolated for its synthesis of poly P from GTP and designated PPK2 in an accompanying report. The reaction that synthesizes poly P and the reaction that utilizes poly P differ in their kinetic features. Especially notable is the catalytic potency of the NDK activity, which is 75-fold greater than that of poly P synthesis. PPK2 appears in the stationary phase of growth and reaches NDK levels of 5-10% that of the classic NDK; both kinase activities may figure in the generation of the guanosine precursors in the synthesis of alginate, an exopolysaccharide essential for the virulence of P. aeruginosa.A mong its several functions, inorganic polyphosphate (poly P) serves as a kinase donor to generate ATP and GTP (1-3). Universally distributed in all cells, these chains of phosphate residues, usually hundreds long, linked by phosphoanhydride (metaphosphate) bonds, as in ATP, can be used to phosphorylate glucose, nucleoside diphosphates, and protein (3). In this capacity, poly P was found in extracts of Pseudomonas aeruginosa to be a donor to GDP and a potent source of GTP (4, 5). Of particular physiologic and clinical interest are the sources of GTP needed by P. aeruginosa to provide the intermediates for the synthesis of alginate, its exopolysaccharide associated with mucoidy and virulence (6-8).The present study was undertaken to purify the poly P donor activity designated earlier as PNDK (poly P-driven nucleoside diphosphate kinase; ref. 5) to characterize the enzyme, identify its gene, and, with that, explore the physiologic consequences of overexpression and removal of the gene. At the same time, we were also in pursuit of a putative poly P kinase (PPK2) activity in extracts of P. aeruginosa in addition to the known PPK1 (9, 10) that converts ATP to poly P. The PNDK and PPK2 activities, ostensibly different because of different kinetic features, have proved on isolation of the homogeneous proteins in each case to be the same and to be encoded by the same gene, now designated ppk2 (11). In this report, we describe the isolation and structural properties of PPK2 and its kinetic features based on its activity as a nucleoside diphosphate kinase (NDK). The accompanying report (11) describes the genetic sequence of PPK2 and its conservation in many other bacteria, as well as features of the enzyme in the synthesis of poly P from GTP or ATP. Materials and MethodsStrains and Materials. P. aeruginosa PAOM5 (12), the ⌬ppk::tet derivative of non-mucoid strain PAO1, was used in this study. All of the reagents including poly P 15 were obtained from Sigma. Purified PPK1 was a generous gift from S. Lee (ICOS Corporation, Bothell, ...

To evaluate the application of urinary metabolomics on discovering potential biomarkers for epithelial ovarian cancer (EOC), urine samples from 40 preoperative EOC patients, 62 benign ovarian tumor (BOT) patients, and 54 healthy controls were collected and analyzed with ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). Good separations were obtained for EOC vs BOT, EOC vs healthy controls analyzed by partial least-squares discriminant analysis, or principal component analysis. Twenty-two ascertained metabolomic biomarkers were found to be disturbed in several metabolic pathways among EOC patients, including nucleotide metabolism (pseudouridine, N4-acetylcytidine), histidine metabolism (L-histidine, imidazol-5-yl-pyruvate), tryptophan metabolism (3-indolelactic acid), and mucin metabolism (3'-sialyllactose and 3-sialyl-N-acetyllactosamine). In addition, the concentrations of some urinary metabolites of 18 postoperative EOC patients among the 40 EOC patients changed significantly compared with those of their preoperative condition, and four of them suggested recovery tendency toward normal level after surgical operation, including N4-acetylcytidine, pseudouridine, urate-3-ribonucleoside, and succinic acid. These metabolites would be highly postulated to be associated with EOC. In conclusion, our study demonstrated that urinary metabolomics analysis by UPLC-QTOF/MS, performed in a minimally noninvasive and convenient manner, possessed great potential in biomarker discovery for EOC.