We have developed an all-electronic digital microfluidic device for microscale chemical synthesis in organic solvents, operated by electrowetting-on-dielectric (EWOD). As an example of the principles, we demonstrate the multistep synthesis of ½ 18 FFDG, the most common radiotracer for positron emission tomography (PET), with high and reliable radio-fluorination efficiency of ½ 18 FFTAG (88 AE 7%, n ¼ 11) and quantitative hydrolysis to ½ 18 FFDG (>95%, n ¼ 11). We furthermore show that batches of purified ½ 18 FFDG can successfully be used for PET imaging in mice and that they pass typical quality control requirements for human use (including radiochemical purity, residual solvents, Kryptofix, chemical purity, and pH). We report statistical repeatability of the radiosynthesis rather than bestcase results, demonstrating the robustness of the EWOD microfluidic platform. Exhibiting high compatibility with organic solvents and the ability to carry out sophisticated actuation and sensing of reaction droplets, EWOD is a unique platform for performing diverse microscale chemical syntheses in small volumes, including multistep processes with intermediate solvent-exchange steps. molecular imaging | PET probes | synthetic chemistry | lab on a chip | on-chip chemistry T he use of micro-reaction technology in chemistry has grown tremendously over the past several years (1), due primarily to the highly precise control of reaction conditions that is possible through rapid mixing and heat transport, leading to improved reaction speeds and selectivity compared to macroscale approaches (2). Additional advantages include straightforward scale-up of production without changing conditions, and increased safety in dangerous syntheses due to the minute amounts of reagents within the reactor at any given time. A further advantage of microfluidics is the ability to perform reactions in extremely small volumes, which is valuable for many applications, especially when working with scarce reagents, such as isolated proteins or natural products, products of long synthetic pathways, or short-lived radiolabeled radioisotopes where the needed mass quantities are extremely low (3).Myriad microfluidic platforms have been explored for chemical reactions that can be classified into three basic formats: (i) flow-through (or continuous flow), (ii) droplet or slug, or (iii) batch. In flow-through systems, streams of two or more reagents are mixed and reacted by flowing through a residence time unit held at a constant temperature or immersed in a fixed microwave field. Continuous liquid-liquid extraction and other processes have been developed to enable multistep reactions where different solvents are required in different steps (4). Droplet and slug systems are a variant of flow-through systems, in which individual droplets or slugs (with volumes down to tens of nanoliters) are separated by an immiscible carrier fluid, each acting as an isolated batch microreactor and enabling vastly reduced reaction volumes. Screening assays and optimization studies...

Altered metabolism is a hallmark of cancer growth, forming the conceptual basis for development of metabolic therapies as cancer treatments. We performed in vivo metabolic profiling and molecular analysis of lung squamous cell carcinoma (SCC) to identify metabolic nodes for therapeutic targeting. Lung SCCs adapt to chronic mTOR inhibition and suppression of glycolysis through the GSK3α/β signaling pathway, which upregulates glutaminolysis. Phospho-GSK3α/β protein levels are predictive of response to single-therapy mTOR inhibition while combinatorial treatment with the glutaminase inhibitor CB-839 effectively overcomes therapy resistance. In addition, we identified a conserved metabolic signature in a broad spectrum of hypermetabolic human tumors that may be predictive of patient outcome and response to combined metabolic therapies targeting mTOR and glutaminase.

A.J. performed transthoracic injections. G.A. performed a portion of the CT scans. M.M. and S.T.B. performed IHC staining and analyzed data. M.M. J.T.L. and A.J. performed in vitro 18 FBnTP uptake assays. M.M. did TMRE staining and western blots. M.C.F is a board-certified anatomic pathologist who performed the pathological analysis. L.S. and O.S. performed and guided respirometry experiments. S.S., C.M.W., A.G. and T.H. performed radio-tracer synthesis. D.D. and C.K. performed biochemical analysis of mitochondria. E.S. and H.C. performed metabolic analysis of lung tumors. S.M.D. contributed resources and critical feedback on the project. Data availability Source data for Western blots are provided with the paper as Supplementary Figure 1. Source data for Figures 1b-d; Figures 2d-g; Figures 3b, 3d, 3e; ED Figures 3c-e; ED Figures 4c, 4d; ED Figure 9a are provided with the paper. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Cancer cells exhibit increased use of nutrients including glucose and glutamine to support the bioenergetic and biosynthetic demands of proliferation. We tested the small molecule inhibitor of glutaminase CB-839 in combination with erlotinib on EGFR mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMPK pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-Glutamine (11C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following CB-839 + erlotinib treatment. Therefore, PET imaging with 18F-FDG and 11C-Gln tracers can be used to non-invasively measure metabolic response to CB-839 and erlotinib combination therapy.

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds—[18F]Clofarabine; 2-chloro-2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-adenine ([18F]CFA) and 2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-guanine ([18F]F-AraG)—for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [18F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [18F]F-AraG is a better substrate for dGK than for dCK. [18F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [18F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [18F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [18F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [18F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [18F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.