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Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.

Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours.

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.

Radiopharmaceuticals labeled with short-lived positron-emitting or gamma-emitting isotopes are injected into patients just prior to performing positron emission tomography (PET) or single photon emission tomography (SPECT) scans, respectively. These imaging modalities are widely used in clinical care, as well as in the development and evaluation of new therapies in clinical research. Prior to injection, these radiopharmaceuticals (tracers) must undergo quality control (QC) testing to ensure product purity, identity, and safety for human use. Quality tests can be broadly categorized as (i) pharmaceutical tests, needed to ensure molecular identity, physiological compatibility and that no microbiological, pyrogenic, chemical, or particulate contamination is present in the final preparation; and (ii) radioactive tests, needed to ensure proper dosing and that there are no radiochemical and radionuclidic impurities that could interfere with the biodistribution or imaging. Performing the required QC tests is cumbersome and time-consuming, and requires an array of expensive analytical chemistry equipment and significant dedicated lab space. Calibrations, day of use tests, and documentation create an additional burden. Furthermore, in contrast to ordinary pharmaceuticals, each batch of short-lived radiopharmaceuticals must be manufactured and tested within a short period of time to avoid significant losses due to radioactive decay. To meet these challenges, several efforts are underway to develop integrated QC testing instruments that automatically perform and document all of the required tests. More recently, microfluidic quality control systems have been gaining increasing attention due to vastly reduced sample and reagent consumption, shorter analysis times, higher detection sensitivity, increased multiplexing, and reduced instrumentation size. In this review, we describe each of the required QC tests and conventional testing methods, followed by a discussion of efforts to directly miniaturize the test or examples in the literature that could be implemented for miniaturized QC testing.

New radiolabeled probes for positron-emission tomography (PET) are providing an ever-increasing ability to answer diverse research and clinical questions and to facilitate the discovery, development, and clinical use of drugs in patient care. Despite the high equipment and facility costs to produce PET probes, many radiopharmacies and radiochemistry laboratories use a dedicated radiosynthesizer to produce each probe, even if the equipment is idle much of the time, to avoid the challenges of reconfiguring the system fluidics to switch from one probe to another. To meet growing demand, more cost-efficient approaches are being developed, such as radiosynthesizers based on disposable "cassettes," that do not require reconfiguration to switch among probes. However, most cassette-based systems make sacrifices in synthesis complexity or tolerated reaction conditions, and some do not support custom programming, thereby limiting their generality. In contrast, the design of the ELIXYS FLEX/CHEM cassette-based synthesizer supports higher temperatures and pressures than other systems while also facilitating flexible synthesis development. In this paper, the syntheses of 24 known PET probes are adapted to this system to explore the possibility of using a single radiosynthesizer and hot cell for production of a diverse array of compounds with wide-ranging synthesis requirements, alongside synthesis development efforts. Most probes were produced with yields and synthesis times comparable to literature reports, and because hardware modification was unnecessary, it was convenient to frequently switch among probes based on demand. Although our facility supplies probes for preclinical imaging, the same workflow would be applicable in a clinical setting.

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 epidermal growth factor receptor (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 AMP-activated protein kinase (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 treatment with CB-839 + erlotinib. 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-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]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, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]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 [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]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 [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

We have developed an efficient, streamlined, cost-effective approach to obtain Investigational New Drug (IND) approvals from the Food and Drug Administration (FDA) for positron emission tomography (PET) imaging probes (while the FDA uses the terminology PET drugs, we are using "PET imaging probes," "PET probes," or "probes" as the descriptive terms). The required application and supporting data for the INDs were collected in a collaborative effort involving appropriate scientific disciplines. This path to INDs was successfully used to translate three [(18) F]fluoro-arabinofuranosylcytosine (FAC) analog PET probes to phase 1 clinical trials. In doing this, a mechanism has been established to fulfill the FDA regulatory requirements for translating promising PET imaging probes from preclinical research into human clinical trials in an efficient and cost-effective manner.

Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.

Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest, and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using (18)F-L-1-(2'-deoxy-2'-FluoroArabinofuranosyl) Cytosine ((18)F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC50 = ∼1-12 nM) using the (18)F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy.

Organic chemistry applications on digital microfluidic devices often involve reagents that are volatile or sensitive and must be introduced to the chip immediately before use. We present a new technique for automated, on-demand loading of ~1 μL droplets from large (~1 mL), sealed, off-chip reservoirs to a digital microfluidic chip in order to address this challenge. Unlike aqueous liquids which generally are non-wetting to the hydrophobic surface and must be actively drawn into the electrowetting-on-dielectric (EWOD) chip by electrode activation, organic liquids tend to be wetting and can spontaneously flood the chip, and hence require a retracting force for controlled liquid delivery. Using a combination of compressed inert gas and gravity to exert driving and retracting forces on the liquid, the simple loading technique enables precise loading of droplets of both wetting and non-wetting liquids in a reliable manner. A key feature from a practical point of view is that all of the wetted parts are inexpensive and potentially disposable, thus avoiding cross-contamination in chemical and biochemical applications. We provide a theoretical treatment of the underlying physics, discuss the effect of geometry and liquid properties on its performance, and show repeatable reagent loading using the technique. Its versatility is demonstrated with the loading of several aqueous and non-aqueous liquids on an EWOD digital microfluidic device.

Digital microfluidic chips provide a new platform for manipulating chemicals for multi-step chemical synthesis or assays at the microscale. The organic solvents and reagents needed for these applications are often volatile, sensitive to contamination, and wetting, i.e. have contact angles of

We demonstrate a new approach to impedance measurement on digital microfluidics chips for the purpose of simple, sensitive, and accurate volume and liquid composition measurement. Adding only a single series resistor to existing AC droplet actuation circuits, the platform is simple to implement and has negligible effect on actuation voltage. To accurately measure the complex voltage across the resistor (and hence current through the device and droplet), the designed system is based on software-implemented lock-in amplification detection of the voltage drop across the resistor which filters out noise, enabling high-resolution and low-limit signal recovery. We observe picoliter sensitivity with linear correlation of voltage to volume extending to the microliter volumes that can be handled by digital microfluidic devices. Due to the minimal hardware, the system is robust and measurements are highly repeatable. The detection technique provides both phase and magnitude information of the real-time current flowing through the droplet for a full impedance measurement. The sensitivity and resolution of this platform enables it to distinguish between various liquids which, as demonstrated in this paper, could potentially be extended to quantify solute concentrations, liquid mixtures, and presence of analytes.

Background: Cancer stem cells play an important role in driving tumor growth and treatment resistance, which makes them a promising therapeutic target to prevent cancer recurrence. Emerging cancer stem cell-targeted therapies would benefit from companion diagnostic imaging probes to aid in patient selection and monitoring response to therapy. To this end, zirconium-89-radiolabeled immunoPET probes that target the cancer stem cell-antigen CD133 were developed using fully human antibody and antibody scFv-Fc scaffolds. Results: ImmunoPET probes [89Zr]-DFO-RW03IgG (CA = 0.7 ± 0.1), [89Zr]-DFO-RW03IgG (CA = 3.0 ± 0.3), and [89Zr]-DFO-RW03scFv − Fc (CA = 2.9 ± 0.3) were radiolabeled with zirconium-89 (radiochemical yield 42 ± 5%, 97 ± 2%, 86 ± 12%, respectively) and each was isolated in > 97% radiochemical purity with specific activities of 120 ± 30, 270 ± 90, and 200 ± 60 MBq/mg, respectively. In vitro binding assays showed a low-nanomolar binding affinity of 0.6 to 1.1 nM (95% CI) for DFO-RW03IgG (CA = 0.7 ± 0.1), 0.3 to 1.9 nM (95% CI) for DFO-RW03IgG (CA = 3.0 ± 0.3), and 1.5 to 3.3 nM (95% CI) for DFO-RW03scFv − Fc (C/A = 0.3). Biodistribution studies found that [89Zr]-DFO-RW03scFv − Fc (CA = 2.9 ± 0.3) exhibited the highest tumor uptake (23 ± 4, 21 ± 2, and 23 ± 4%ID/g at 24, 48, and 72 h, respectively) and showed low uptake (< 6%ID/g) in all off-target organs at each timepoint (24, 48, and 72 h). Comparatively, [89Zr]-DFO-RW03IgG (CA = 0.7 ± 0.1) and [89Zr]-DFO-RW03IgG (CA = 3.0 ± 0.3) both reached maximum tumor uptake (16 ± 3%ID/g and 16 ± 2%ID/g, respectively) at 96 h p.i. and showed higher liver uptake (10.2 ± 3%ID/g and 15 ± 3%ID/g, respectively) at that timepoint. Region of interest analysis to assess PET images of mice administered [89Zr]-DFO-RW03scFv − Fc (CA = 2.9 ± 0.3) showed that this probe reached a maximum tumor uptake of 22 ± 1%ID/cc at 96 h, providing a tumor-to-liver ratio that exceeded 1:1 at 48 h p.i. Antibody-antigen mediated tumor uptake was demonstrated through biodistribution and PET imaging studies, where for each probe, co-injection of excess unlabeled RW03IgG resulted in > 60% reduced tumor uptake. Conclusions: Fully human CD133-targeted immunoPET probes [89Zr]-DFO-RW03IgG and [89Zr]-DFO-RW03scFv − Fc accumulate in CD133-expressing tumors to enable their delineation through PET imaging. Having identified [89Zr]-DFO-RW03scFv − Fc (CA = 2.9 ± 0.3) as the most attractive construct for CD133-expressing tumor delineation, the next step is to evaluate this probe using patient-derived tumor models to test its detection limit prior to clinical translation. [ABSTRACT FROM AUTHOR]

The construction supply chain is complex. This complexity causes problems and errors. In Iran, due to an anarchic atmosphere, it is very common for problems and errors to occur. The Sustainable Antifragile Fragile Lean (SAAL) supply chain was designed to deal with these conditions. One of the widely used tools in this supply chain is the use of information systems such as the control tower. But these systems may also have errors. The main purpose of Poka-yoke is to make mistakes impossible. Therefore, in this research, the main dimensions of Poka-yoke solutions are categorised based on previous research and then experts' opinions are collected with the help of a questionnaire tool. In order to prioritise the factors based on control tower systems, the best-worst method was used. The results showed factors such as proper planning of quality management programs, building information modeling and proper implementation of the productivity cycle is the most important. [ABSTRACT FROM AUTHOR]

In the Chaos/Anarchy business environment of the construction industry of Iran, the Sustainable Antifragile Agile Lean (SAAL) supply chain strategy was proposed as antifragile and value-creating approach. In this strategy, just-in-time data management and analysis are very important to make decisions. The control tower as a controlling unit based on data is the core of this supply chain. The purpose of this article is to investigate the implementation of the control tower in the supply chain of the construction industry. For this purpose, two goals should be followed. The first step is to determine the main indicators to focus on for the initial implementation of the control tower. The second step is to measure the considered indicators using the available data from before the implementation, and after the implementation to compare the performance. To achieve the first goal and to identify the categories of information these categories were divided into three main categories: upstream, midstream and downstream. According to experts’ opinion was considered the middle part of the supply chain, which is carried out on the site by an executive team. At this stage, among the available indicators, the most important indicators were considered using the best-worst method according to previous projects’ data to compare the data. The results of the implementation of a control tower approach showed that it provides the possibility of managing manufacturing operations. This system can guide the managers of the organisation to identify the best strategies and improve risk management in response to the needs, requirements, guidelines, and changing opinions of the stakeholders and external global conditions. [ABSTRACT FROM AUTHOR]

SAAL supply chain management is a comprehensive approach of combining sustainable, Antifragile, agile, and lean strategies. The aim of the current research is to evaluate the SAAL supply chain management strategies using the gap analysis approach in the construction industry. The statistical sample includes 246 of the industry's activists who were randomly selected. First, it is calculated the gap between the existing and ideal situation of each of the indicators and requirements of SAAL supply chain management strategies. Then, the weight of indicators and requirements is determined using the HF-BWM, and based on that, the weighted gap of each is calculated and analyzed. The results of the research show that based on the GWA obtained, Antifragile and Sustainable strategies are the most critical SAAL supply chain management strategies for evaluating the supply chain management performance of Iran's construction industry. [ABSTRACT FROM AUTHOR]

In the challenging environment of the construction industry and especially after the Corona pandemic crisis in Iran, supply chains (SCs) needed to adopt a new management model to continue the activity. Among the investigated approaches, the four sustainable, anti-fragile, agile and lean (SAAL) approaches were given more attention. The main objective of this paper is to draw a conceptual model for SAAL SCs with the aim of improving their operational, economic and environmental performance. For this purpose, several performance measures have been proposed. This concept is drawn based on the literature review of four supply chain management (SCM) approaches and their performance measurement system. Among the measures proposed by SAAL, which affect SC performance, are just-in-time relationships as well as supplier relationships (modular suppliers) and creating anti-fragile infrastructure. [ABSTRACT FROM AUTHOR]