Circulating tumor microemboli: Progress in molecular understanding and enrichment technologies

Circulating tumor cells (CTCs) and their clusters, also known as circulating tumor microemboli (CTM), have emerged as valuable tool that can provide mechanistic insights into the tumor heterogeneity, clonal evolution, and stochastic events within the metastatic cascade. However, recent investigations have hinted that CTM may not be mere aggregates of tumor cells but cells comprising CTM exhibit distinct phenotypic and molecular characteristics in comparison to single CTCs. Moreover, in many cases CTM demonstrated higher metastatic potential and resistance to apoptosis as compared to their single cell counterparts. Thus, their evaluation and enumeration may provide a new dimension to our understanding of cancer biology and metastatic cancer spread as well as offer novel theranostic biomarkers. Most of the existing technologies for isolation of hematogenous tumor cells largely favor single CTCs, hence there is a need to devise new approaches, or re-configure the existing ones, for specific and efficient CTM isolation. Here we review existing knowledge and insights on CTM biology. Furthermore, a critical commentary on current and emerging trends in CTM enrichment and characterization along with recently developed ex-vivo CTC expansion methodologies is presented with the aim to facilitate researchers to identify further avenues of research and development.     

Differential expression and functions of neuronal and glial neurofascin isoforms and splice variants during PNS development

The cell adhesion molecule neurofascin (NF) has a major neuronal isoform (NF186) containing a mucin-like domain followed by a fifth fibronectin type III repeat while these domains are absent from glial NF155. Neuronal NF isoforms lacking one or both of these domains are expressed transiently in embryonic dorsal root ganglia (DRG). These two domains are co-expressed in mature NF186, which peaks in expression prior to birth and then persists almost exclusively at nodes of Ranvier on myelinated axons. In contrast, glial NF155 is only detected postnatally with the onset of myelination. All these forms of NF bound homophilically and to Schwann cells but only the mature NF186 isoform inhibits cell adhesion, and this activity may be important in formation of the node of Ranvier. Schwann cells deficient in NF155 myelinated DRG axons in a delayed manner and they showed significantly decreased clustering of both NF and Caspr in regions where paranodes normally form. The combined results suggest that NF186 is expressed prenatally on DRG neurons and it may modulate their adhesive interactions with Schwann cells, which express NF155 postnatally and require it for development of axon-glial paranodal junctions.     

Functional and pathological roles of the 12- and 15-lipoxygenases

The 12/15-lipoxygenase enzymes react with fatty acids producing active lipid metabolites that are involved in a number of significant disease states. The latter include type 1 and type 2 diabetes (and associated complications), cardiovascular disease, hypertension, renal disease, and the neurological conditions Alzheimer’s disease and Parkinson’s disease. A number of elegant studies over the last thirty years have contributed to unraveling the role that lipoxygenases play in chronic inflammation. The development of animal models with targeted gene deletions has led to a better understanding of the role that lipoxygenases play in various conditions. Selective inhibitors of the different lipoxygenase isoforms are an active area of investigation, and will be both an important research tool and a promising therapeutic target for treating a wide spectrum of human diseases.     

Impairment of survival signaling and efferocytosis in TRPC3-deficient macrophages

We have recently shown that in macrophages proper operation of the survival pathways phosphatidylinositol-3-kinase (PI3K)/AKT and nuclear factor kappa B (NFkB) has an obligatory requirement for constitutive, non-regulated Ca²⁺ influx. In the present work we examined if Transient Receptor Potential Canonical 3 (TRPC3), a member of the TRPC family of Ca²⁺-permeable cation channels, contributes to the constitutive Ca²⁺ influx that supports macrophage survival. We used bone marrow-derived macrophages obtained from TRPC3^−/− mice to determine the activation status of survival signaling pathways, apoptosis and their efferocytic properties. Treatment of TRPC3^+/+ macrophages with the pro-apoptotic cytokine TNFα induced time-dependent phosphorylation of IκBα, AKT and BAD, and this was drastically reduced in TRPC3^−/− macrophages. Compared to TRPC3^+/+ cells TRPC3^−/− macrophages exhibited reduced constitutive cation influx, increased apoptosis and impaired efferocytosis. The present findings suggest that macrophage TRPC3, presumably through its constitutive function, contributes to survival signaling and efferocytic properties.     

The occurrence of crassulacean acid metabolism in Cymbidium (Orchidaceae) and its ecological and evolutionary implications

Crassulacean acid metabolism (CAM) is one of the photosynthetic pathways regarded as adaptations to water stress in land plants. Little is known about correlations among the level of CAM activity, environment of habitat, life form, and phylogenetic relationship of a plant group from an evolutionary perspective. We examined these relationships in 18 species of Cymbidium (Orchidaceae) because the genus shows distinctive diversification of habitats and life forms. The photosynthetic type was classed into three categories, strong CAM, weak CAM, and C₃ on the basis of CAM activity. CAM expression in Cymbidium was confined to the epiphytic and lithophytic species. Especially, all of these species from tropical to subtropical rainforest exhibited CAM activity. On the other hand, the terrestrial species always exhibited C₃ metabolism irrespective of their varied habitats. Regarding the evolution of photosynthetic characters, weak CAM was the ancestral state in Cymbidium and strong CAM and C₃ metabolism occurred subsequently. The evolution of strong CAM likely enabled Cymbidium to extend to exposed sites in tropical lowland where marked water stress exists. Further, different levels of CAM activity characterized each species and such potential plasticity of CAM may realize the radiation of Cymbidium into sites with different environmental conditions.     

Subcellular Localization of Oxaloacetate Decarboxylase and Its Isolation from Maize Leaves

The activity of oxaloacetate decarboxylase was revealed in leaves of a C₄ plant, maize (Zea mays L.). This activity was unrelated to decarboxylase activities of other enzymes, e.g., NAD-malate dehydrogenase (EC 1.1.1.38) or NADP-malate dehydrogenase (EC 1.1.1.40), and was located in chloroplasts (83.1%). Using a four-step purification procedure, an electrophoretically pure enzyme preparation of oxaloacetate decarboxylase was obtained from maize leaves. The specific activity of the enzyme was 3.150 EU/mg protein, the factor of purification was 40.4, and the yield was 11.0%. The enzyme exhibited Michaelis–Menten kinetics with K _m for oxaloacetate 30 ± 5 M and pH optimum 7.1 ± 0.5. The metabolite-mediated regulation of oxaloacetate decarboxylase activity has been investigated. It is found that sodium chloride (1.0 mM) activates the enzyme, whereas ATP inhibits the enzyme activity.     

Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo

The peptide growth factor apelin is the high affinity ligand for the G-protein-coupled receptor APJ. During embryonic development of mouse and frog, APJ receptor is expressed at high levels in endothelial precursor cells and in nascent vascular structures. Characterization of Xenopus apelin shows that the sequence of the bioactive region of the peptide is perfectly conserved between frogs and mammals. Embryonic expression studies indicate that apelin is expressed in, or immediately adjacent to, a subset of the developing vascular structures, particularly the intersegmental vessels. Experimental inhibition of either apelin or APJ expression, using antisense morpholino oligos, results in elimination or disruption of intersegmental vessels in a majority of embryos. In gain of function experiments, apelin peptide is a potent angiogenic factor when tested using two in vivo angiogenesis assays, the frog embryo and the chicken chorioallantoic membrane. Furthermore, studies using the mouse brain microvascular cell line bEnd.3 show that apelin acts as a mitogenic, chemotactic and anti-apoptotic agent for endothelial cells in culture. Finally, we show that, similar to a number of other angiogenic factors, expression of the apelin gene is increased under conditions of hypoxia. Taken together, these studies indicate that apelin is required for normal vascular development in the frog embryo and has properties consistent with a role during normal and pathological angiogenesis.