Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ.

Canonical non-homologous end-joining (cNHEJ) is the prominent mammalian DNA double-strand breaks (DSBs) repair pathway operative throughout the cell cycle. Phosphorylation of Ku70 at ser27-ser33 (pKu70) is induced by DNA DSBs and has been shown to regulate cNHEJ activity, but the underlying mechanism remained unknown. Here, we established that following DNA damage induction, Ku70 moves from nucleoli to the sites of damage, and once linked to DNA, it is phosphorylated. Notably, the novel emanating functions of pKu70 are evidenced through the recruitment of RNA Pol II and concomitant formation of phospho-53BP1 foci. Phosphorylation is also a prerequisite for the dynamic release of Ku70 from the repair complex through neddylation-dependent ubiquitylation. Although the non-phosphorylable ala-Ku70 form does not compromise the formation of the NHEJ core complex per se, cells expressing this form displayed constitutive and stress-inducible chromosomal instability. Consistently, upon targeted induction of DSBs by the I-SceI meganuclease into an intrachromosomal reporter substrate, cells expressing pKu70, rather than ala-Ku70, are protected against the joining of distal DNA ends. Collectively, our results underpin the essential role of pKu70 in the orchestration of DNA repair execution in living cells and substantiated the way it paves the maintenance of genome stability.

Increase in lamin B1 promotes telomere instability by disrupting the shelterin complex in human cells.

Telomere maintenance is essential to preserve genomic stability and involves telomere-specific proteins, DNA replication and repair proteins. Lamins are key components of the nuclear envelope and play numerous roles, including maintenance of the nuclear integrity, regulation of transcription, and DNA replication. Elevated levels of lamin B1, one of the major lamins, have been observed in some human pathologies and several cancers. Yet, the effect of lamin B1 dysregulation on telomere maintenance remains unknown. Here, we unveil that lamin B1 overexpression drives telomere instability through the disruption of the shelterin complex. Indeed, lamin B1 dysregulation leads to an increase in telomere dysfunction-induced foci, telomeric fusions and telomere losses in human cells. Telomere aberrations were preceded by mislocalizations of TRF2 and its binding partner RAP1. Interestingly, we identified new interactions between lamin B1 and these shelterin proteins, which are strongly enhanced at the nuclear periphery upon lamin B1 overexpression. Importantly, chromosomal fusions induced by lamin B1 in excess were rescued by TRF2 overexpression. These data indicated that lamin B1 overexpression triggers telomere instability through a mislocalization of TRF2. Altogether our results point to lamin B1 as a new interacting partner of TRF2, that is involved in telomere stability.

Lamin B1 sequesters 53BP1 to control its recruitment to DNA damage.

Double-strand breaks (DSBs) are harmful lesions and a major cause of genome instability. Studies have suggested a link between the nuclear envelope and the DNA damage response. Here, we show that lamin B1, a major component of the nuclear envelope, interacts directly with 53BP1 protein, which plays a pivotal role in the DSB repair. This interaction is dissociated after DNA damage. Lamin B1 overexpression impedes 53BP1 recruitment to DNA damage sites and leads to a persistence of DNA damage, a defect in nonhomologous end joining and an increased sensitivity to DSBs. The identification of interactions domains between lamin B1 and 53BP1 allows us to demonstrate that the defect of 53BP1 recruitment and the DSB persistence upon lamin B1 overexpression are due to sequestration of 53BP1 by lamin B1. This study highlights lamin B1 as a factor controlling the recruitment of 53BP1 to DNA damage sites upon injury.

[Aging: A matter of DNA damage, nuclear envelope alterations and inflammation?] / Le vieillissement - Une histoire de dommages de l'ADN, d'enveloppe nucléaire altérée et d'inflammation ?

The accumulation of senescent cells accompanies organismal aging. Senescent cells produce an inflammatory microenvironment that is conducive to the development of many age-related diseases. Here we describe the different situations leading to cellular senescence and show that these situations are frequently associated with DNA damage. We also discuss the intimate link between cell aging and perturbations in the nuclear envelope, namely in nuclear lamins, as seen in progeroid syndromes. Finally, we present evidence that these alterations are associated with DNA repair defects, the persistence of DNA damage, and an inflammatory phenotype.

TITLE: Le vieillissement - Une histoire de dommages de l'ADN, d'enveloppe nucléaire altérée et d'inflammation ? ABSTRACT: Le vieillissement est associé à une accumulation de cellules sénescentes produisant un environnement cellulaire inflammatoire qui pourrait expliquer différentes maladies liées à l'âge. Diverses situations menant à la sénescence sont liées à la présence de dommages de l'ADN. De plus, de nombreux syndromes progéroïdes sont associés à une instabilité du génome ou de la structure nucléaire. Nous discuterons du lien étroit existant entre l'altération des lamines, composants de l'enveloppe nucléaire, et le vieillissement cellulaire. Nous verrons que l'altération de l'enveloppe nucléaire, comme celle observée dans la Progéria, est aussi associée à des défauts de réparation de l'ADN, à une persistance de dommages de l'ADN et à un phénotype inflammatoire.

Combined PET Imaging of the Inflammatory Tumor Microenvironment Identifies Margins of Unique Radiotracer Uptake.

The tumor microenvironment is highly heterogeneous. For gliomas, the tumor-associated inflammatory response is pivotal to support growth and invasion. Factors of glioma growth, inflammation, and invasion, such as the translocator protein (TSPO) and matrix metalloproteinases (MMP), may serve as specific imaging biomarkers of the glioma microenvironment. In this study, noninvasive imaging by PET with [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) was used for the assessment of localization and quantification of the expression of TSPO and MMP. Imaging was performed in addition to established clinical imaging biomarker of active tumor volume ([18F]FET) in conjunction with MRI. We hypothesized that each imaging biomarker revealed distinct areas of the heterogeneous glioma tissue in a mouse model of human glioma. Tracers were found to be increased 1.4- to 1.7-fold, with [18F]FET showing the biggest volume as depicted by a thresholding-based, volumes of interest analysis. Tumor areas, which could not be detected by a single tracer and/or MRI parameter alone, were measured. Specific compartments of [18F]DPA-714 (14%) and [18F]BR-351 (11%) volumes along the tumor rim could be identified. [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) matched with histology. Glioma-associated microglia/macrophages (GAM) were identified as TSPO and MMP sources. Multitracer and multimodal molecular imaging approaches may allow us to gain important insights into glioma-associated inflammation (GAM, MMP). Moreover, this noninvasive technique enables characterization of the glioma microenvironment with respect to the disease-driving cellular compartments at the various disease stages. Cancer Res; 77(8); 1831-41. ©2017 AACR.

Evaluation of TSPO PET imaging, a marker of glial activation, to study the neuroimmune footprints of morphine exposure and withdrawal.

INTRODUCTION: A growing area of research suggests that neuroimmunity may impact the pharmacology of opioids. Microglia is a key component of the brain immunity. Preclinical and clinical studies have demonstrated that microglial modulators may improve morphine-induced analgesia and prevent the development of tolerance and dependence. Positron emission tomography (PET) using translocator protein 18kDa (TSPO) radioligand is a clinically validated strategy for the non-invasive detection of microglial activation. We hypothesized that TSPO PET imaging may be used to study the neuroimmune component of opioid tolerance and withdrawal. METHODS: Healthy rats (n=6 in each group) received either saline or escalating doses of morphine (10-40mg/kg) on five days to achieve tolerance and a withdrawal syndrome after morphine discontinuation. MicroPET imaging with [18F]DPA-714 was performed 60h after morphine withdrawal. Kinetic modeling was performed to estimate [18F]DPA-714 volume of distribution (VT) in several brain regions using dynamic PET images and corresponding metabolite-corrected input functions. Immunohistochemistry (IHC) experiments on striatal brain slices were performed to assess the expression of glial markers (Iba1, GFAP and CD68) during 14days after morphine discontinuation. RESULTS: The baseline binding of [18F]DPA-714 to the brain (VT=0.086±0.009mLcm-3) was not increased by morphine exposure and withdrawal (VT=0.079±0.010mLcm-3) indicating the absence of TSPO overexpression, even at the regional level. Accordingly, expression of glial markers did not increase after morphine discontinuation. CONCLUSIONS: Morphine tolerance and withdrawal did not detectably activate microglia and had no impact on [18F]DPA-714 brain kinetics in vivo.

Exercise Does Not Protect against Peripheral and Central Effects of a High Cholesterol Diet Given Ad libitum in Old ApoE-/- Mice.

Aim: Advanced atherosclerosis increases inflammation and stroke risk in the cerebral vasculature. Exercise is known to improve cardio-metabolic profiles when associated with a caloric restriction, but it remains debated whether it is still beneficial without the dietary control. The aim of this study was to determine both the peripheral and central effects of exercise training combined with a cholesterol-rich diet given ad libitum in old ApoE-/- mice. Methods: Forty-five-weeks old obese ApoE-/- mice fed with a high cholesterol diet ad libitum were divided into Exercise-trained (EX; running wheel free access) and Sedentary (SED) groups. Insulin tolerance and brain imaging were performed before and after the twelve-weeks training. Tissue insulin resistance, oxidative stress, and inflammation markers in plasma, aorta, and brain were then assessed. Results: In EX ApoE-/- mice, no beneficial effect of exercise was observed on weight, abdominal fat, metabolic parameters, oxidative stress, or inflammation compared to SED. Despite the regular exercise training in ApoE-/- EX mice (mean of 12.5 km/week during 12 weeks), brain inflammation imaging score was significantly associated with increased blood brain barrier (BBB) leakage evaluated by imaging follow-up (r2 = 0.87; p = 0.049) with a faster evolution compared to SED ApoE-/-mice. Conclusion: We conclude that in a context of high cardio-metabolic risk, exercise does not provide any protective effect in old ApoE-/- animals under high cholesterol diet given ad libitum. Peripheral (insulin sensitivity and oxidative/inflammatory status) but also central features (BBB preservation and protection against inflammation) did not show any benefits of exercise. Indeed, there was a fast induction of irreversible brain damage that was more pronounced in exercise-trained ApoE-/- mice.

Magnetic resonance imaging biomarkers of exercise-induced improvement of oxidative stress and inflammation in the brain of old high-fat-fed ApoE-/- mice.

KEY POINTS: Vascular brain lesions and atherosclerosis are two similar conditions that are characterized by increased inflammation and oxidative stress. Non-invasive imaging in a murine model of atherosclerosis showed vascular brain damage and peripheral inflammation. In this study, exercise training reduced magnetic resonance imaging-detected abnormalities, insulin resistance and markers of oxidative stress and inflammation in old ApoE-/- mice. Our results demonstrate the protective effect of exercise on neurovascular damage in the ageing brain of ApoE-/- mice. ABSTRACT: Vascular brain lesions, present in advanced atherosclerosis, share pathological hallmarks with peripheral vascular lesions, such as increased inflammation and oxidative stress. Physical activity reduces these peripheral risk factors, but its cerebrovascular effect is less documented, especially by non-invasive imaging. Through a combination of in vivo and post-mortem techniques, we aimed to characterize vascular brain damage in old ApoE-/- mice fed a high-cholesterol (HC) diet with dietary controlled intake. We then sought to determine the beneficial effects of exercise training on oxidative stress and inflammation in the brain as a treatment option in an ageing atherosclerosis mouse model. Using in vivo magnetic resonance imaging (MRI) and biological markers of oxidative stress and inflammation, we evaluated the occurrence of vascular abnormalities in the brain of HC-diet fed ApoE-/- mice >70 weeks old, its association with local and systemic oxidative stress and inflammation, and whether both can be modulated by exercise. Exercise training significantly reduced both MRI-detected abnormalities (present in 71% of untrained vs. 14% of trained mice) and oxidative stress (lipid peroxidation, 9.1 ± 1.4 vs. 5.2 ± 0.9 µmol mg-1 ; P < 0.01) and inflammation (interleukin-1ß, 226.8 ± 27.1 vs. 182.5 ± 21.5 pg mg-1 ; P < 0.05) in the brain, and the mortality rate. Exercise also decreased peripheral insulin resistance, oxidative stress and inflammation, but significant associations were seen only within brain markers. Highly localized vascular brain damage is a frequent finding in this ageing atherosclerosis model, and exercise is able to reduce this outcome and improve lifespan. In vivo MRI evaluated both the neurovascular damage and the protective effect of exercise.

Multiscale Texture Analysis: From 18F-FDG PET Images to Histologic Images.

Characterizing tumor heterogeneity using texture indices derived from PET images has shown promise in predicting treatment response and patient survival in some types of cancer. Yet, the relationship between PET-derived texture indices, precise tracer distribution, and biologic heterogeneity needs to be clarified. We investigated this relationship using PET images, autoradiographic images, and histologic images. METHODS: Three mice bearing orthotopically implanted mammary tumors derived from transgenic MMTV-PyMT mice were scanned with 18F-FDG PET/CT. The tumors were then sliced, and the slices were imaged with autoradiography and stained with hematoxylin and eosin. Six texture indices derived from the PET images, autoradiographic images, and histologic images were compared for their ability to capture heterogeneity on different scales. RESULTS: The PET-derived indices correlated significantly with the autoradiography-derived ones (R = 0.57-0.85), but the values differed in magnitude. The histology-derived indices correlated poorly with the autoradiography- and PET-derived ones (R = 0.06-0.54). All indices were slightly to moderately influenced by the difference in voxel size and spatial resolution in the autoradiographic images. The autoradiography-derived indices differed significantly (P < 0.05) between regions with a high density of cells and regions with a low density and between regions with different spatial arrangements of cells. CONCLUSION: Heterogeneity derived in vivo from PET images accurately reflects the heterogeneity of tracer uptake derived ex vivo from autoradiographic images. Various tumor-cell densities and spatial cell distributions seen on histologic images can be distinguished using texture indices derived from autoradiographic images despite the difference in voxel size and spatial resolution. Yet, tumor texture derived from PET images only coarsely reflects the spatial distribution and density of tumor cells.

In Vitro and In Vivo Imaging of Fluorescent Aptamers.

Fluorescence imaging techniques could be used in different ways to study the interaction of aptamers with biological systems from cell culture to animal models. Here, we present the methods developed in our laboratory for fluorescently labeled aptamers, study their internalization inside living cells using time-lapse microscopy, and monitor their biodistribution in mice bearing subcutaneous xenograft tumors using planar fluorescence imaging and fluorescence diffuse optical tomography (fDOT).

Multimodal In Vivo Imaging of Tumorigenesis and Response to Chemotherapy in a Transgenic Mouse Model of Mammary Cancer.

PURPOSE: Transgenic mice expressing the polyoma middle T oncoprotein (PyMT) in the mammary epithelium were explored by multimodal imaging to monitor longitudinally spontaneous tumor growth and response to chemotherapy. PROCEDURES: Positron emission tomography (PET) with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), single photon emission tomography (SPECT) with [(99m)Tc]TcO4 ([(99m)Tc]TEC), X-ray computed tomography, and fluorescent confocal endomicroscopy (FCE) images were acquired during tumor progression in female PyMT mice. Imaging with [(18)F]FDG and [(99m)Tc]TEC was also performed in untreated, doxorubicin-treated, and docetaxel-treated PyMT mice. Total tumor volumes were quantified. Tumors were collected and macroscopic and histological examinations were performed. RESULTS: All PyMT mice developed multifocal tumors of the mammary epithelium that became palpable at 8 weeks of age (W8). Computed tomography (CT) detected tumors at W14, while a clear tumoral uptake of [(99m)Tc]TEC and [(18)F]FDG was present as early as W6 and W8, respectively. No contrast between mammary tumors and surrounding tissue was observed at any stage with [(18)F]FLT. FCE detected an angiogenic switch at W10. Lung metastases were not clearly evidenced by imaging. Doxorubicin and docetaxel treatments delayed tumor growth, as shown by [(18)F]FDG and [(99m)Tc]TEC, but tumor growth resumed upon treatment discontinuation. Tumor growth fitted an exponential model with time constant rates of 0.315, 0.145, and 0.212 week(-1) in untreated, doxorubicin, and docetaxel groups, respectively. CONCLUSIONS: Molecular imaging of mammary tumors in PyMT is precocious, precise, and predictive. [(18)F]FDG-PET and [(99m)Tc]TEC SPECT monitor tumor response to chemotherapy.

Monitoring therapeutic efficacy of sunitinib using [(18)F]FDG and [(18)F]FMISO PET in an immunocompetent model of luminal B (HER2-positive)-type mammary carcinoma.

BACKGROUND: Clinical studies implying the sunitinib multi-kinase inhibitor have led to disappointing results for breast cancer care but mostly focused on HER2-negative subtypes. Preclinical researches involving this drug mostly concern Triple Negative Breast Cancer (TNBC) murine models. Here, we explored the therapeutic efficacy of sunitinib on a PyMT-derived transplanted model classified as luminal B (HER2-positive) and monitored the response to treatment using both in vivo and ex vivo approaches. METHODS: Tumour-induced animals were treated for 9 (n = 7) or 14 (n = 8) days with sunitinib at 40 mg/kg or with vehicle only. Response to therapy was assessed in vivo by monitoring glucose tumour metabolism and hypoxia using 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) and [(18)F]fluoromisonidazole ([(18)F]FMISO) Positron Emission Tomography (PET). After primary tumour excision, ex vivo digital microscopy was performed on treated and control samples to estimate vascular density (CD31), apoptosis (Tunel), proliferation (Ki-67), Tumour-Associated Macrophage (TAM) infiltration (F4/80), metabolism (GLUT1) and cellular response to hypoxia (HIF1 alpha). The drug impact on the metastasis rate was evaluated by monitoring the PyMT gene expression in the lungs of the treated and control groups. RESULTS: Concomitant with sunitinib-induced tumour size regression, [(18)F]FDG PET imaging showed a stable glycolysis-related metabolism inside tumours undergoing treatment compared to an increased metabolism in untreated tumours, resulting at treatment end in 1.5 less [(18)F]FDG uptake in treated (n = 4) vs control (n = 3) tumours (p < 0.05). With this small sample, [(18)F]FMISO PET showed a non-significant decrease of hypoxia in treated vs control tumours. The drug triggered a 4.9 fold vascular volume regression (p < 0.05), as well as a 17.7 fold induction of tumour cell apoptosis (p < 0.001). The hypoxia induced factor 1 alpha (HIF1 alpha) expression was twice lower in the treated group than in the control group (p < 0.05). Moreover, the occurrence of lung metastases was not reduced by the drug. CONCLUSIONS: [(18)F]FDG and [(18)F]FMISO PET were relevant approaches to study the response to sunitinib in this luminal B (HER2-positive) model. The sunitinib-induced vascular network shrinkage did not significantly increase tumour hypoxia, suggesting that tumour regression was mainly due to the pro-apoptotic properties of the drug. Sunitinib did not inhibit the metastatic process in this PyMT transplanted model.

In vivo evaluation of inflammatory bowel disease with the aid of µPET and the translocator protein 18 kDa radioligand [18F]DPA-714.

PURPOSE: The purpose of the study was to validate [18F]DPA-714, a translocator protein (TSPO) 18 kDa radioligand, as a probe to non-invasively quantify the inflammatory state in inflammatory bowel disease (IBD) animal models. PROCEDURES: Quantitative positron emission tomography (PET) imaging of intestinal inflammation was conducted with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) a glucose metabolism surrogate marker and [18F]DPA-714 a ligand of the 18 kDa TSPO, on two IBD models. The first model was induced using dextran sodium sulfate (DSS), creating global inflammation in the colon. The second model was induced by rectally administering trinitrobenzenesulfonic acid (TNBS), creating local and acute inflammation. RESULTS: The level of inflammation was analyzed using PET imaging on days 7 and 8. The analysis obtained with [18F]DPA-714, yielded a significant difference between the DSS treated (0.50 ± 0.17%ID/cc) and non-treated rats (0.35 ± 0.15%ID/cc). [18F]FDG on the other hand did not yield a significant difference. We did observe a mean glucose consumption in the colon increase from 0.40 ± 0.11 %ID/cc to 0.54 ± 0.17 %ID/cc. In the TNBS model, the uptake level of [18 F]DPA-714 increased significantly from 0.46 ± 0.23%ID/cc for the non-treated group, to 1.30 ± 0.62%ID/cc for those treated. PET signal was correlated with increased TSPO expression at cellular level. CONCLUSIONS: Results indicate that [18F]DPA-714 is suitable for studying inflammation in IBD models. [18F]DPA-714 could be a good molecular probe to non-invasively evaluate the level and localization of inflammation. Moreover, in vivo imaging using this TSPO ligand is potentially a powerful tool to stage and certainly to follow the evolution and therapeutic efficiency at molecular level within this disease family.

From ugly duckling to swan: unexpected identification from cell-SELEX of an anti-Annexin A2 aptamer targeting tumors.

BACKGROUND: Cell-SELEX is now widely used for the selection of aptamers against cell surface biomarkers. However, despite negative selection steps using mock cells, this method sometimes results in aptamers against undesirable targets that are expressed both on mock and targeted cells. Studying these junk aptamers might be useful for further applications than those originally envisaged. METHODOLOGY/PRINCIPAL FINDINGS: Cell-SELEX was performed to identify aptamers against CHO-K1 cells expressing human Endothelin type B receptor (ETBR). CHO-K1 cells were used for negative selection of aptamers. Several aptamers were identified but no one could discriminate between both cell lines. We decided to study one of these aptamers, named ACE4, and we identified that it binds to the Annexin A2, a protein overexpressed in many cancers. Radioactive binding assays and flow cytometry demonstrated that the aptamer was able to bind several cancer cell lines from different origins, particularly the MCF-7 cells. Fluorescence microscopy revealed it could be completely internalized in cells in 2 hours. Finally, the tumor targeting of the aptamer was evaluated in vivo in nude mice xenograft with MCF-7 cells using fluorescence diffuse optical tomography (fDOT) imaging. Three hours after intravenous injection, the aptamer demonstrated a significantly higher uptake in the tumor compared to a scramble sequence. CONCLUSIONS/SIGNIFICANCE: Although aptamers could be selected during cell-SELEX against other targets than those initially intended, they represent a potential source of ligands for basic research, diagnoses and therapy. Here, studying such aptamers, we identify one with high affinity for Annexin A2 that could be a promising tool for biomedical application.

The translocator protein radioligand 18F-DPA-714 monitors antitumor effect of erufosine in a rat 9L intracranial glioma model.

UNLABELLED: On the one hand, the translocator protein (TSPO) radioligand N,N-diethyl-2-(2-(4-(2-(18)F-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ((18)F-DPA-714) has been suggested to serve as an alternative radiotracer to image human glioma, and on the other hand the alkylphosphocholine erufosine (ErPC3) has been reported to induce apoptosis in otherwise highly apoptosis-resistant glioma cell lines. The induction of apoptosis by ErPC3 requires TSPO, a mitochondrial membrane protein highly expressed in malignant gliomas. In this preclinical study, we monitored the effect of ErPC3 treatment in vivo using (18)F-DPA-714 PET. METHODS: In vitro studies investigated the antitumor effect of ErPC3 in 9L rat gliosarcoma cells. In vivo, glioma-bearing rats were imaged with (18)F-DPA-714 for the time of treatment. RESULTS: A significant decrease in 9L cell proliferation and viability and a significant increase in apoptosis and caspase-3 activation were demonstrated on ErPC3 treatment in cell culture. In the rat model, ErPC3 administration resulted in significant changes in (18)F-DPA-714 tumor uptake over the course of the treatment. Immunohistochemistry revealed reduced tumor volume and increased cell death in ErPC3-treated animals accompanied by infiltration of the tumor core by CD11b-positive microglia/macrophages and glial fibrillary acidic protein-positive astrocytes. CONCLUSION: Our findings demonstrate a potent antitumor effect of ErPC3 in vitro, in vivo, and ex vivo. PET imaging of TSPO expression using (18)F-DPA-714 allows effective monitoring and quantification of disease progression and response to ErPC3 therapy in intracranial 9L gliomas.

Synthetic lipid nanoparticles targeting steroid organs.

UNLABELLED: Lipidots are original nanoparticulate lipid delivery vectors for drugs and contrast agents made from materials generally regarded as safe. Here, we characterized the in vivo stability, biodistribution, and pharmacokinetics of lipidots. METHODS: Lipidots 55 nm in diameter and coated with a phospholipid/poly(ethyleneglycol) surfactant shell were triply labeled with (3)H-cholesteryl-hexadecyl-ether, cholesteryl-(14)C-oleate, and the 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine infrared fluorescent dye and injected intravenously into immunocompetent Friend virus B-type mice. The pharmacokinetics and biodistribution of lipidots were analyzed quantitatively in serial samples of blood and tissue and with in vivo optical imaging and were refined by microscopic examination of selected target tissues. RESULTS: The plasmatic half-life of lipidots was approximately 30 min. Radioactive and fluorescent tracers displayed a similar nanoparticle-driven biodistribution, indicative of the lipidots' integrity during the first hours after injection. Lipidots distributed in the liver and, surprisingly, in the steroid-rich organs adrenals and ovaries, but not in the spleen. This tropism was confirmed at the microscopic level by histologic detection of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine. Nanoparticle loading with cholesterol derivatives increased accumulation in ovaries in a dose-dependent manner. CONCLUSION: This previously unreported distribution pattern is specific to lipidots and attributed to their nanometric size and composition, conferring on them a lipoproteinlike behavior. The affinity of lipidots for steroid hormone-rich areas is of interest to address drugs and contrast agents to lipoprotein-receptor-overexpressing cancer cells found in hormone-dependent tumors.

Cellular uptake and trafficking of polydiacetylene micelles.

Polydiacetylene (PDA) micelles coated with either carboxylate-, ammonium-, or methoxy-polyethyleneglycol (PEG) chains were assembled and loaded with a fluorescent dye (DiO). Their interaction with MCF-7 human breast tumor cells was investigated by epi-fluorescence microscopy and fluorescence-activated cell sorting (FACS) to determine their internalization pathway and intracellular fate. It was found that the ionic character of the micelles influenced their internalization kinetics through a caveolae-mediated pathway and that all micelle types behaved somewhat similarly inside cells.

Fusion of [(18)F]FDG PET with fluorescence diffuse optical tomography to improve validation of probes and tumor imaging.

PURPOSE: Given the progress of fluorescence diffuse optical tomography (fDOT) technology, here, we study the additional benefits provided by multimodal PET/fDOT imaging by comparing the biodistribution of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) in tumors with three fluorescent probes: a glucose analog, a protease activatable optical probe, and a ligand of αvß3 integrin. PROCEDURES: Sequential fDOT/PET/computed tomography (CT) imaging of mice was performed with a custom multimodal mouse support that allows the subject to be transferred between the fDOT and the PET/CT scanners. Experiments were performed in xenografted tumor models derived from the human breast cancer line MDA-MB 231 and compared to ex vivo analysis. RESULTS: The three-dimensional signals showed that the fluorescent glucose analog is not colocalized with [(18)F]FDG, raising questions about its use as a surrogate probe of the PET tracer. Fusion of [(18)F]FDG with the other fluorescent probes showed evidence of high variability both for the protease activity and the αvß3 integrin expression during tumor growth. CONCLUSION: The added value of hybrid PET/fDOT over the two modalities was demonstrated for cross-validation of probes and for better characterization of tumor models.

Imaging of perfusion, angiogenesis, and tissue elasticity after stroke.

Blood flow interruption in a cerebral artery causes brain ischemia and induces dramatic changes of perfusion and metabolism in the corresponding territory. We performed in parallel positron emission tomography (PET) with [(15)O]H(2)O, single photon emission computed tomography (SPECT) with [(99m)Tc]hexamethylpropylene-amino-oxime ([(99m)Tc]HMPAO) and ultrasonic ultrafast shear wave imaging (SWI) during, immediately after, and 1, 2, 4, and 7 days after middle cerebral artery occlusion (MCAO) in rats. Positron emission tomography and SPECT showed initial hypoperfusion followed by recovery at immediate reperfusion, hypoperfusion at day 1, and hyperperfusion at days 4 to 7. Hyperperfusion interested the whole brain, including nonischemic areas. Immunohistochemical analysis indicated active angiogenesis at days 2 to 7, strongly suggestive that hyperperfusion was supported by an increase in microvessel density in both brain hemispheres after ischemia. The SWI detected elastic changes of cerebral tissue in the ischemic area as early as day 1 after MCAO appearing as a softening of cerebral tissue whose local internal elasticity decreased continuously from day 1 to 7. Taken together, these results suggest that hyperperfusion after cerebral ischemia is due to formation of neovessels, and indicate that brain softening is an early and continuous process. The SWI is a promising novel imaging method for monitoring the evolution of cerebral ischemia over time in animals.