Quantifying metabolic heterogeneity in head and neck tumors in real time: 2-DG uptake is highest in hypoxic tumor regions

Purpose: Intratumoral metabolic heterogeneity may increase the likelihood of treatment failure due to the presence of a subset of resistant tumor cells. Using a head and neck squamous cell carcinoma (HNSCC) xenograft model and a real-time fluorescence imaging approach, we tested the hypothesis that tumors are metabolically heterogeneous, and that tumor hypoxia alters patterns of glucose uptake within the tumor. Experimental Design: Cal33 cells were grown as xenograft tumors (n = 16) in nude mice after identification of this cell line's metabolic response to hypoxia. Tumor uptake of fluorescent markers identifying hypoxia, glucose import, or vascularity was imaged simultaneously using fluorescent molecular tomography. The variability of intratumoral 2-deoxyglucose (IR800-2-DG) concentration was used to assess tumor metabolic heterogeneity, which was further investigated using immunohistochemistry for expression of key metabolic enzymes. HNSCC tumors in patients were assessed for intratumoral variability of 18F-fluorodeoxyglucose (18F-FDG) uptake in clinical PET scans. Results: IR800-2-DG uptake in hypoxic regions of Cal33 tumors was 2.04 times higher compared to the whole tumor (p = 0.0001). IR800-2-DG uptake in tumors containing hypoxic regions was more heterogeneous as compared to tumors lacking a hypoxic signal. Immunohistochemistry staining for HIF-1α, carbonic anhydrase 9, and ATP synthase subunit 5β confirmed xenograft metabolic heterogeneity. We detected heterogeneous 18F-FDG uptake within patient HNSCC tumors, and the degree of heterogeneity varied amongst tumors. Conclusion: Hypoxia is associated with increased intratumoral metabolic heterogeneity. 18F-FDG PET scans may be used to stratify patients according to the metabolic heterogeneity within their tumors, which could be an indicator of prognosis. © 2014 Nakajima et al

Screening chimeric GAA variants in preclinicalstudy results in hematopoietic stem cell genetherapy candidate vectors for Pompe disease

Pompe disease is a rare genetic neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy, the current standard of care, penetrates poorly into the skeletal muscles and the peripheral and central nervous system (CNS), risks recombinant enzyme immunogenicity, and requires high doses and frequent infusions. Lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy was investigated in a Pompe mouse model using a clinically relevant promoter driving nine engineered GAA coding sequences incorporating distinct peptide tags and codon optimizations. Vectors solely including glycosylation-independent lysosomal targeting tags enhanced secretion and improved reduction of glycogen, myofiber, and CNS vacuolation in key tissues, although GAA enzyme activity and protein was consistently lower compared with native GAA. Genetically modified microglial cells in brains were detected at low levels but provided robust phenotypic correction. Furthermore, an amino acid substitution introduced in the tag reduced insulin receptor-mediated signaling with no evidence of an effect on blood glucose levels in Pompe mice. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model, providing promising vector candidates for further investigation

The Halo Occupation Distribution of Black Holes: Dependence on Mass

We investigate the halo occupation distribution (HOD) of black holes within a hydrodynamic cosmological simulation that directly follows black hole growth. Similar to the HOD of galaxies/subhalos, we find that the black hole occupation number can be described by the form N_BH proportional to 1+ (M_Host)^alpha where alpha evolves mildly with redshift indicating that a given mass halo (M_Host) at low redshift tends to host fewer BHs than at high redshift (as expected as a result of galaxy and BH mergers). We further divide the occupation number into contributions from black holes residing in central and satellite galaxies within a halo. The distribution of M_BH within halos tends to consist of a single massive BH (distributed about a peak mass strongly correlated with M_Host), and a collection of relatively low-mass secondary BHs, with weaker correlation with M_Host. We also examine the spatial distribution of BHs within their host halos, and find they typically follow a power-law radial distribution (i.e. much more centrally concentrated than the subhalo distribution). Finally, we characterize the host mass for which BH growth is feedback dominated (e.g. star formation quenched). We show that halos with M_Host > 3 * 10^12 M_sun have primary BHs that are feedback dominated by z~3 with lower mass halos becoming increasingly more affected at lower redshift.Comment: 10 pages, 7 figures, submitted to MNRA

Beware of the Robot: A Highly Interactive and Immersive Virtual Reality Training Application in Robotic Manufacturing Systems

Part 3: Human Factors, Learning and InnovationInternational audienceA highly interactive and immersive Virtual Reality Training System (VRTS) is developed, in terms of an educational serious game that simulates the cooperation between industrial robotic manipulators and humans, executing manufacturing tasks. “BeWare of the robot” application ultimately aims at studying the acceptability of human-robot collaboration, when both human and robot share the same workspace. The initial version of the application was evaluated by a group of users. Experimental results on usability and technical aspects are presented and several remarks about users’ experience and behavior in the virtual world are discussed

Glucose consumption and lactate production increase under hypoxic conditions.

<p>A and B, Cal33 and OSC19 cells consume more glucose after 48 hours of hypoxic culturing than cells grown in normoxic conditions. C and D, a concomitant rise in lactate production was detected in media from Cal33 and OSC19 cells grown in hypoxic conditions for 48 hours. OSC19 cells are more glycolytic than Cal33 cells. Each dot represents one of four replicate dishes used for each time point and culturing condition. The experiment was performed twice with two replicate dishes. P values indicate a significant difference in metabolite concentrations and were calculated using a two-way ANOVA.</p

Tumor characteristics and ¹⁸F-FDG-PET values recorded from ROI-C using MIM software.

<p>SD, standard deviation. CV, coefficient of variance.</p

Cal33 exposure to hypoxia leads to increased expression of HIF-1α, LDH-M, and PDHK1.

<p>Western blot analysis of changes in HIF-1α, LDH-M, and PDHK1 expression in whole cell lysate from Cal33 cultures grown in normoxic (21%), hypoxic (2%), or hypoxic (2%) followed by normoxic (21%) conditions. Cells cultured in 21% oxygen for 16 hours serve as the control for basal protein expression levels. Densitometry values were calculated using ImageJ. Images are representative of duplicate blots.</p

2-DG uptake in Cal33 xenografts is heterogeneous and is associated with HypoxiSense accumulation.

<p>A, Nude mouse with an ulcerated Cal33 xenograft tumor. B, 3D reconstructions of FMT scans capturing HypoxiSense, AngioSense, and IR800-2-DG signal within the xenograft tumor as shown in A. A manually placed ROI measures the tumor volume. C, Anatomic tumor measurement was calculated from MRI slices. Sagital, axial, and coronal MRI slices of the mouse shown in A and B, with 3D tumor reconstruction. D, two dimensional reconstructions of FMT scans at 2 mm depth within the tumor. E, box plots comparing IR800-2-DG concentration in whole tumors and HypoxiSense-concentrated regions within them (n = 8). Boxes represent the interquartile range, the horizontal line indicates the median, the T-bars indicate the range, and individual points are outliers (p, student t-test). F, Tumors were grouped by the presence or absence of HypoxiSense signal within the tumor (n = 9 for HypoxiSense positive, n = 7 for HypoxiSense negative). The coefficient of variance (CV) for IR800-22-DG concentration within the tumor was calculated (p value was generated using a student's t-test). Each point represents a tumor. Bars indicate the average CV value for the HypoxiSense positive or negative group.</p

HIF-1α, ATP5β and CAIX expression have greater heterogeneity in HypoxiSense-positive tumors as compared to HypoxiSense-negative tumors.

<p>Mean, standard deviation (SD), and coefficient of variance (CV) of staining were quantified by measuring positive pixel count in 20 representative regions of each tumor slice using Aperio version 9. Quantification of protein expression in each region was calculated by multiplying the staining intensity by the positive area percentage as described in materials and methods.</p