Quantitative performance characterization of three-dimensional noncontact fluorescence molecular tomography

© 2016 The Authors.Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities. Fluorescence molecular tomography (FMT) detects and reconstructs in three dimensions the distribution of a fluorophore in vivo. Noncontact FMT allows fast scanning of an excitation source and noninvasive measurement of emitted fluorescent light using a virtual array detector operating in free space. Here, a rigorous process is defined that fully characterizes the performance of a custom-built horizontal noncontact FMT setup. Dynamic range, sensitivity, and quantitative accuracy across the visible spectrum were evaluated using fluorophores with emissions between 520 and 660 nm. These results demonstrate that high-performance quantitative three-dimensional visible light FMT allowed the detection of challenging mesenteric lymph nodes in vivo and the comparison of spectrally distinct fluorescent reporters in cell culture

Extracellular vesicles swarm the cancer microenvironment: from tumor–stroma communication to drug intervention

Intercellular communication sets the pace for transformed cells to survive and to thrive. Extracellular vesicles (EVs), such as exosomes, microvesicles and large oncosomes, are involved in this process shuttling reciprocal signals and other molecules between transformed and stromal cells, including fibroblasts, endothelial and immune cells. As a result, these cells are adapted or recruited to a constantly evolving cancer microenvironment. Moreover, EVs take part in the response to anticancer therapeutics not least by promoting drug resistance throughout the targeted tumor. Finally, circulating EVs can also transport important molecules to remote destinations in order to prime metastatic niches in an otherwise healthy tissue. Although the understanding of EV biology remains a major challenge in the field, their characteristics create new opportunities for advances in cancer diagnostics and therapeutics

The novel choline kinase inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth.

The glycerophospholipid phosphatidylcholine is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. Inhibition of choline kinase alpha (CHKA), the first committed step to phosphatidylcholine synthesis, by the selective small-molecule ICL-CCIC-0019, potently suppressed growth of a panel of 60 cancer cell lines with median GI50 of 1.12 μM and inhibited tumor xenograft growth in mice. ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by [18F]-fluoromethyl-[1,2–2H4]-choline positron emission tomography. Herein, we reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased mitochondria function with associated reduction of citrate synthase expression and AMPK activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids

Fluorescence strategies for high-throughput quantification of protein interactions

Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function. Their further characterization requires quantitative experimental strategies that are easy to implement in laboratories without specialized equipment. We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies. These methods have high sensitivity, a broad dynamic range, and can be performed in a high-throughput manner. In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein–DNA and protein–protein interactions. We applied these methods to describe the preference of linker histone H1 for nucleosomes over DNA, the ionic dependence of the DNA repair enzyme PARP1 in DNA binding, and the interaction between the histone chaperone Nap1 and the histone H2A–H2B heterodimer

Michel Goulet

Major contributions of the 2nd annual meeting of the International Society of Cancer Metabolism, held in Venice, September 16-19, 2015, are here described and discussed. Among these, the impact of cancer metabolism on local and systemic aggressiveness was analyzed in the context of interactions between cancer and stroma, microenvironmental changes, epigenetic, and stemness modulation

Targeting tumor-stroma crosstalk: the example of the NT157 inhibitor

Recent clinical research has provided evidence that cancer progression and therapy resistance is driven not only by tumor’s genetic profile but also by complex paracrine interactions within the tumor microenvironment (TME). The role of TME in modulating tumor drug sensitivity is increasingly recognized and targeting TME has been the focus of novel therapeutic approaches. Two recent reports show that a new anti-cancer drug, the inhibitor NT157 has the potential to inhibit IGF-1R and STAT3 signaling pathways in cancer cells and stoma cells of TME leading to a decrease in cancer cell survival