Kinetic Trajectories of Glucose Uptake in Single Cancer Cells Reveal a Drug-Induced Cell-State Change Within Hours of Drug Treatment.

The development of drug resistance is a nearly universal phenomenon in patients with glioblastoma multiforme (GBM) brain tumors. Upon treatment, GBM cancer cells may initially undergo a drug-induced cell-state change to a drug-tolerant, slow-cycling state. The kinetics of that process are not well understood, in part due to the heterogeneity of GBM tumors and tumor models, which can confound the interpretation of kinetic data. Here, we resolve drug-adaptation kinetics in a patient-derived in vitro GBM tumor model characterized by the epithelial growth factor receptor (EGFR) variant(v)III oncogene treated with an EGFR inhibitor. We use radiolabeled 18F-fluorodeoxyglucose (FDG) to monitor the glucose uptake trajectories of single GBM cancer cells over a 12 h period of drug treatment. Autocorrelation analysis of the single-cell glucose uptake trajectories reveals evidence of a drug-induced cell-state change from a high- to low-glycolytic phenotype after 5-7 h of drug treatment. Information theoretic analysis of a bulk transcriptome kinetic series of the GBM tumor model delineated the underlying molecular mechanisms driving the cellular state change, including a shift from a stem-like mesenchymal state to a more differentiated, slow-cycling astrocyte-like state. Our results demonstrate that complex drug-induced cancer cell-state changes of cancer cells can be captured via measurements of single cell metabolic trajectories and reveal the extremely facile nature of drug adaptation.

Genetic signature of prostate cancer mouse models resistant to optimized hK2 targeted α-particle therapy.

Hu11B6 is a monoclonal antibody that internalizes in cells expressing androgen receptor (AR)-regulated prostate-specific enzyme human kallikrein-related peptidase 2 (hK2; KLK2). In multiple rodent models, Actinium-225-labeled hu11B6-IgG1 ([225Ac]hu11B6-IgG1) has shown promising treatment efficacy. In the present study, we investigated options to enhance and optimize [225Ac]hu11B6 treatment. First, we evaluated the possibility of exploiting IgG3, the IgG subclass with superior activation of complement and ability to mediate FC-γ-receptor binding, for immunotherapeutically enhanced hK2 targeted α-radioimmunotherapy. Second, we compared the therapeutic efficacy of a single high activity vs. fractionated activity. Finally, we used RNA sequencing to analyze the genomic signatures of prostate cancer that progressed after targeted α-therapy. [225Ac]hu11B6-IgG3 was a functionally enhanced alternative to [225Ac]hu11B6-IgG1 but offered no improvement of therapeutic efficacy. Progression-free survival was slightly increased with a single high activity compared to fractionated activity. Tumor-free animals succumbing after treatment revealed no evidence of treatment-associated toxicity. In addition to up-regulation of canonical aggressive prostate cancer genes, such as MMP7, ETV1, NTS, and SCHLAP1, we also noted a significant decrease in both KLK3 (prostate-specific antigen ) and FOLH1 (prostate-specific membrane antigen) but not in AR and KLK2, demonstrating efficacy of sequential [225Ac]hu11B6 in a mouse model.

The GSK3 Signaling Axis Regulates Adaptive Glutamine Metabolism in Lung Squamous Cell Carcinoma.

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/ß signaling pathway, which upregulates glutaminolysis. Phospho-GSK3α/ß 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.

Performing radiosynthesis in microvolumes to maximize molar activity of tracers for positron emission tomography.

Positron emission tomography (PET) is a molecular diagnostic imaging technology to quantitatively visualize biological processes in vivo. For many applications, including imaging of low tissue density targets (e.g. neuroreceptors), imaging in small animals, and evaluation of novel tracers, the injected PET tracer must be produced with high molar activity to ensure low occupancy of biological targets and avoid pharmacologic effects. Additionally, high molar activity is essential for tracers with lengthy syntheses or tracers transported to distant imaging sites. We show that radiosynthesis of PET tracers in microliter volumes instead of conventional milliliter volumes results in substantially increased molar activity, and we identify the most relevant variables affecting this parameter. Furthermore, using the PET tracer [18F]fallypride, we illustrate that molar activity can have a significant impact on biodistribution. With full automation, microdroplet platforms could provide a means for radiochemists to routinely, conveniently, and safely produce PET tracers with high molar activity.

Production of diverse PET probes with limited resources: 24 18F-labeled compounds prepared with a single radiosynthesizer.

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.

Detection of immune responses after immunotherapy in glioblastoma using PET and MRI.

Contrast-enhanced MRI is typically used to follow treatment response and progression in patients with glioblastoma (GBM). However, differentiating tumor progression from pseudoprogression remains a clinical dilemma largely unmitigated by current advances in imaging techniques. Noninvasive imaging techniques capable of distinguishing these two conditions could play an important role in the clinical management of patients with GBM and other brain malignancies. We hypothesized that PET probes for deoxycytidine kinase (dCK) could be used to differentiate immune inflammatory responses from other sources of contrast-enhancement on MRI. Orthotopic malignant gliomas were established in syngeneic immunocompetent mice and then treated with dendritic cell (DC) vaccination and/or PD-1 mAb blockade. Mice were then imaged with [18F]-FAC PET/CT and MRI with i.v. contrast. The ratio of contrast enhancement on MRI to normalized PET probe uptake, which we term the immunotherapeutic response index, delineated specific regions of immune inflammatory activity. On postmortem examination, FACS-based enumeration of intracranial tumor-infiltrating lymphocytes directly correlated with quantitative [18F]-FAC PET probe uptake. Three patients with GBM undergoing treatment with tumor lysate-pulsed DC vaccination and PD-1 mAb blockade were also imaged before and after therapy using MRI and a clinical PET probe for dCK. Unlike in mice, [18F]-FAC is rapidly catabolized in humans; thus, we used another dCK PET probe, [18F]-clofarabine ([18F]-CFA), that may be more clinically relevant. Enhanced [18F]-CFA PET probe accumulation was identified in tumor and secondary lymphoid organs after immunotherapy. Our findings identify a noninvasive modality capable of imaging the host antitumor immune response against intracranial tumors.

[18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

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-ß-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-ß-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.

Development of 2-Deoxy-2-[(18)F]fluororibose for Positron Emission Tomography Imaging Liver Function in Vivo.

Life-threatening acute liver failure can be triggered by a variety of factors, including common drugs such as acetaminophen. Positron emission tomography (PET) is rarely used to monitor liver function, in part because of a lack of specific imaging agents for liver function. Here we report a new PET probe, 2-deoxy-2-[(18)F]fluororibose ([(18)F]-2-DFR), for use in imaging liver function. [(18)F]-2-DFR was synthesized and validated as a competitive substrate for the ribose salvage pathway. [(18)F]-2-DFR was prepared through an efficient late stage radiofluorination. The desired selectivity of fluorination was achieved using an unorthodox protecting group on the precursor, which could withstand harsh SN2 reaction conditions with no side reactions. [(18)F]-2-DFR accumulated preferentially in the liver and was metabolized by the same enzymes as ribose. [(18)F]-2-DFR could distinguish between healthy liver and liver damaged by acetaminophen. [(18)F]-2-DFR is expected to be a useful PET probe for imaging and quantifying liver functions in vivo, with likely significant clinical utility.

Impact of 68Ga-DOTATATE PET/CT on the management of neuroendocrine tumors: the referring physician's perspective.

UNLABELLED: Somatostatin receptor imaging with (68)Ga-DOTATATE PET/CT (DOTATATE) is increasingly used for managing patients with neuroendocrine tumors. The objective of this study was to determine referring physicians' perspectives on the impact of DOTATATE on the management of neuroendocrine tumors. METHODS: A set of 2 questionnaires (pre-PET and post-PET) was sent to the referring physicians of 100 consecutive patients with known or suspected neuroendocrine tumors, who were evaluated with DOTATATE. Questionnaires on 88 patients were returned (response rate, 88%). Referring physicians categorized the DOTATATE findings on the basis of the written PET reports as negative, positive, or equivocal for disease. The likelihood for metastatic disease was scored as low, moderate, or high. The intended management before and changes as a consequence of the PET study were indicated. RESULTS: The indications for PET/CT were initial and subsequent treatment strategy assessments in 14% and 86% of patients, respectively. Referring physicians reported that DOTATATE led to a change in suspicion for metastatic disease in 21 patients (24%; increased and decreased suspicion in 9 [10%] and 12 [14%] patients, respectively). Intended management changes were reported in 53 of 88 (60%) patients. Twenty patients (23%) scheduled to undergo chemotherapy were switched to treatments without chemotherapy, and 6 (7%) were switched from watch-and-wait to other treatment strategies. Conversely, 5 patients (6%) were switched from their initial treatment strategy to watch-and-wait. CONCLUSION: This survey of referring physicians demonstrates a substantial impact of DOTATATE on the intended management of patients with neuroendocrine tumors.

Quantitative assessments of glycolysis from single cells.

The most common positron emission tomography (PET) radio-labeled probe for molecular diagnostics in patient care and research is the glucose analog, 2-deoxy-2-[F-18]fluoro-D-glucose (18F-FDG). We report on an integrated microfluidics-chip/beta particle imaging system for in vitro18F-FDG radioassays of glycolysis with single cell resolution. We investigated the kinetic responses of single glioblastoma cancer cells to targeted inhibitors of receptor tyrosine kinase signaling. Further, we find a weak positive correlation between cell size and rate of glycolysis.

Surprisal analysis characterizes the free energy time course of cancer cells undergoing epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) initiates the invasive and metastatic behavior of many epithelial cancers. Mechanisms underlying EMT are not fully known. Surprisal analysis of mRNA time course data from lung and pancreatic cancer cells stimulated to undergo TGF-ß1-induced EMT identifies two phenotypes. Examination of the time course for these phenotypes reveals that EMT reprogramming is a multistep process characterized by initiation, maturation, and stabilization stages that correlate with changes in cell metabolism. Surprisal analysis characterizes the free energy time course of the expression levels throughout the transition in terms of two state variables. The landscape of the free energy changes during the EMT for the lung cancer cells shows a stable intermediate state. Existing data suggest this is the previously proposed maturation stage. Using a single-cell ATP assay, we demonstrate that the TGF-ß1-induced EMT for lung cancer cells, particularly during the maturation stage, coincides with a metabolic shift resulting in increased cytosolic ATP levels. Surprisal analysis also characterizes the absolute expression levels of the mRNAs and thereby examines the homeostasis of the transcription system during EMT.

Positron emission tomography probe demonstrates a striking concentration of ribose salvage in the liver.

PET is a powerful technique for quantifying and visualizing biochemical pathways in vivo. Here, we develop and validate a novel PET probe, [(18)F]-2-deoxy-2-fluoroarabinose ([(18)F]DFA), for in vivo imaging of ribose salvage. DFA mimics ribose in vivo and accumulates in cells following phosphorylation by ribokinase and further metabolism by transketolase. We use [(18)F]DFA to show that ribose preferentially accumulates in the liver, suggesting a striking tissue specificity for ribose metabolism. We demonstrate that solute carrier family 2, member 2 (also known as GLUT2), a glucose transporter expressed in the liver, is one ribose transporter, but we do not know if others exist. [(18)F]DFA accumulation is attenuated in several mouse models of metabolic syndrome, suggesting an association between ribose salvage and glucose and lipid metabolism. These results describe a tool for studying ribose salvage and suggest that plasma ribose is preferentially metabolized in the liver.

Treatment response evaluation using 18F-FDOPA PET in patients with recurrent malignant glioma on bevacizumab therapy.

PURPOSE: This study compares the value of 3,4-dihydroxy-6-[(18)F]-fluoro-l-phenylalanine ((18)F-FDOPA) positron emission tomography (PET) and MRI in assessing outcome during antiangiogenic treatment in patients with recurrent high-grade gliomas. EXPERIMENTAL DESIGN: Thirty patients were prospectively studied with (18)F-FDOPA PET scans immediately before, and two and six weeks after start of bevacizumab therapy. (18)F-FDOPA metabolic tumor volumes (MTV) as well as max and mean standardized uptake values (SUV) within this MTV were obtained. MRI treatment response was assessed at six weeks. The predictive ability of (18)F-FDOPA PET and MRI response assessment were evaluated with regard to progression-free survival (PFS) and overall survival (OS). RESULTS: A total of 30, 28, and 24 (18)F-FDOPA PET scans at baseline, two weeks, and six weeks, were available for analysis, respectively. (18)F-FDOPA PET SUVs as well as their changes through therapy were not predictive of outcome. However, MTV parameters such as MTV changes were highly prognostic. Interestingly, absolute MTV at the first follow up scan provides the most significant prediction for increased OS (P < 0.0001) as well as PFS (P = 0.001). This surprising result was scrutinized with cross-validation and simulation analysis. Responders based on (18)F-FDOPA PET data survived 3.5 times longer (12.1 months vs. 3.5 months, median OS, P < 0.001) than nonresponders (17 patients vs. 11 patients, respectively). In comparison, responders based on MRI data lived 1.5 times longer (11.4 months vs 7.7 months, P = 0.03) than nonresponders (22 patients vs. 7 patients, respectively). CONCLUSIONS: (18)F-FDOPA PET identifies treatment responders to antiangiogenic therapy as early as two weeks after treatment initiation.

INDs for PET molecular imaging probes-approach by an academic institution.

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.

¹8F-FLT and ¹8F-FDOPA PET kinetics in recurrent brain tumors.

PURPOSE: In this study, kinetic parameters of the cellular proliferation tracer (18)F-3'-deoxy-3'-fluoro-L-thymidine (FLT) and the amino acid probe 3,4-dihydroxy-6-(18)F-fluoro-L-phenylalanine (FDOPA) were measured before and early after the start of therapy, and were used to predict the overall survival (OS) of patients with recurrent malignant glioma using multiple linear regression (MLR) analysis. METHODS: High-grade recurrent brain tumors in 21 patients (11 men and 10 women, age range 26 - 76 years) were investigated. Each patient had three dynamic PET studies with each probe: at baseline and after 2 and 6 weeks from the start of treatment. Treatment consisted of biweekly cycles of bevacizumab (an angiogenesis inhibitor) and irinotecan (a chemotherapeutic agent). For each study, about 3.5 mCi of FLT (or FDOPA) was administered intravenously and dynamic PET images were acquired for 1 h (or 35 min for FDOPA). A total of 126 PET scans were analyzed. A three-compartment, two-tissue model was applied to estimate tumor FLT and FDOPA kinetic rate constants using a metabolite- and partial volume-corrected input function. MLR analysis was used to model OS as a function of FLT and FDOPA kinetic parameters for each of the three studies as well as their relative changes between studies. An exhaustive search of MLR models using three or fewer predictor variables was performed to find the best models. RESULTS: Kinetic parameters from FLT were more predictive of OS than those from FDOPA. The three-predictor MLR model derived using information from both probes (adjusted R(2) = 0.83) fitted the OS data better than that derived using information from FDOPA alone (adjusted R(2) = 0.41), but was only marginally different from that derived using information from FLT alone (adjusted R(2) = 0.82). Standardized uptake values (either from FLT alone, FDOPA alone, or both together) gave inferior predictive results (best adjusted R(2) = 0.25). CONCLUSION: For recurrent malignant glioma treated with bevacizumab and irinotecan, FLT kinetic parameters obtained early after the start of treatment (absolute values and their associated changes) can provide sufficient information to predict OS with reasonable confidence using MLR. The slight increase in accuracy for predicting OS with a combination of FLT and FDOPA PET information may not warrant the additional acquisition of FDOPA PET for therapy monitoring in patients with recurrent glioma.

Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication.

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.

(18)F-FDOPA PET for differentiating recurrent or progressive brain metastatic tumors from late or delayed radiation injury after radiation treatment.

UNLABELLED: Brain metastases are frequently treated with radiation. It is critical to distinguish recurrent or progressive brain metastases (RPBM) from late or delayed radiation injury (LDRI). The purpose of this study was to examine the diagnostic accuracy as well as the prognostic power of 6-(18)F-fluoro-l-dopa ((18)F-FDOPA) PET for differentiating RPBM from LDRI. METHODS: Thirty-two patients who had 83 previously irradiated brain metastases and who underwent (18)F-FDOPA PET because of an MR imaging-based suggestion of RPBM were studied retrospectively. PET studies were analyzed semiquantitatively (lesion-to-striatum and lesion-to-normal brain tissue ratios based on both maximum and mean standardized uptake values) and visually (4-point scale). The diagnostic accuracy of PET was verified by histopathologic analysis (n = 9) or clinical follow-up (n = 74) on a lesion-by-lesion basis. Receiver operating characteristic curve analysis was used to identify the best diagnostic indices. The power of (18)F-FDOPA PET to predict disease progression was evaluated with the Kaplan-Meier and Cox regression methods. RESULTS: The best overall accuracy was achieved by visual scoring, with which a score of 2 or more (lesion uptake greater than or equal to striatum uptake) resulted in a sensitivity of 81.3% and a specificity of 84.3%. Semiquantitative (18)F-FDOPA PET uptake indices based on lesion-to-normal brain tissue ratios were significantly higher for RPBM than for LDRI. Among the various predictors tested, (18)F-FDOPA PET was the strongest predictor of tumor progression (hazard ratio, 6.26; P < 0.001), and the lesion-to-normal brain tissue ratio or visual score was the best discriminator. The mean time to progression was 4.6 times longer for lesions with negative (18)F-FDOPA PET results than for lesions with positive (18)F-FDOPA PET results (76.5 vs. 16.7 mo; P < 0.001). (18)F-FDOPA PET findings tended to predict overall survival. CONCLUSION: Metabolic imaging with (18)F-FDOPA PET was useful for differentiating RPBM from LDRI. Semiquantitative indices, particularly lesion-to-normal uptake ratios, could be used. A visual score comparing tumor (18)F-FDOPA uptake and striatum (18)F-FDOPA uptake provided the highest sensitivity and specificity and was predictive of disease progression.