CT scans and bioluminescence signals of mice bearing orthotopic lung tumors.

<p>(A) 2D CT scan of a mouse bearing a lung tumor, after IV injection with 100μL of a mix with PBS and eXIA 160, a iodinated vascular contrast agent. Delineation of the tumor is visible on both planes with a yellow dashed line. (B) CT scan obtained without contrast agent injection. Delineation of the tumor is visible on both planes with a yellow dashed line. (C) Longitudinal BLI study on a mouse bearing a lung tumor between day 7 and day 35 (Photons/sec/cm²/steradian), after implantation of 1.25x10⁵ tumor cells.</p

<i>In vivo</i> identification and monitoring of orthotopic lung tumors.

<p>(A) & (B) Detection of lung tumors by High Resolution Ultrasound Imaging. (A) 2D B-Mode acquisition on a healthy lung in mouse. Vertical white arrows point out the pleural line. Vertical yellow arrows correspond to A lines, representing reverberations of the pleural line. (B) On 2D B-Mode Ultrasound imaging of a lung bearing an orthotopic NCI-H460luc tumor (2.8mmx2.4mm), vertical red arrows point out the margins of the tumor, highlighted by the typical bright shadow artifact. We also remark white and yellow arrows indicating the pleural line and A lines respectively. (C) From 2D to 3D Ultrasound B-Mode imaging of a lung tumor in mouse. The red area corresponds to the lung tumor in the thoracic cavity of the mouse. The red grid corresponds to the tumor volume obtained by tracing margins on each 2D B-mode slices from the 3D acquisition. (D) Assessing tumor burden with BLI (left) and US (right), data are presented as mean ±SEM and statistically analyzed. A two-way repeated-measure analysis of variance followed by Bonferroni post-tests was used for the data of over time course. Differences were considered significant at p< 0.05. Left: Signal intensity from <i>in vivo</i> longitudinal monitoring of tumor proliferation by BLI following the deposition of 1.5x10⁵ or 2.5x10⁵ tumor cells (Photons/sec). Right: <i>In vivo</i> tumor volumes measured by US imaging using a transducer mounted on a 3D motor, comparing the tumor growth between 2 different tumor burdens (mm³). Results represent mean±SEM (n = 5 animals per groups). (***p<0.001; ****p<0.0001).</p

Molecular imaging of VEGFR2 by Targeted Contrast Enhanced Ultrasound Imaging.

<p>(A) & (C) Parametric images of the spatial distribution of contrast agent bubbles. (A) Isotype control conjugated microbubbles. (C) VEGFR2 conjugated microbubbles (Target Ready Vevo MicroMarker^™). (B) Corresponding B-Mode image of the tumor. (D) Differential Targeted enhancement of VEGFR2 and Isotype control conjugated microbubbles (dTE corresponds to the difference between the echo power from both targeted and free bubbles, and the echo power from free bubbles only). Statistical analysis was performed with the Student's unpaired t test (n = 4 animals per group). (****p<0.001). (E) Corresponding PA image highlighting hypoxic areas where VEGFR2 is mainly expressed.</p

Correlation analysis between different modalities for tumor volume assessment.

<p>Nonlinear regression of data points collected from orthotopic lung tumors (n = 12 animals). These graphs compare the correlation between 3D US imaging <i>in vivo</i> and <i>ex vivo</i>, <i>in vivo</i> 3D CT scans, <i>ex vivo</i> volumetric measurements and tumor weight.</p

Contrast enhanced ultrasound imaging and Photoacoustic imaging of hypoxia on orthotopic lung tumors in mice.

<p>(A) B-mode image of a lung tumor with corresponding contrast image before IV injection of Vevo MicroMarker^™. Maximum Intensity Projection after injection of MicroMarker^™. (B) B-mode image of a hypoxic lung tumor with corresponding OxyHemo photoacoustic images. With the OxyHemo-Mode, red areas indicate well oxygenated parts of the tumor whereas blue and dark areas indicate the presence of hypoxia. Regarding the 3D volumes, the red grid corresponds to the hypoxic region of tumor and green grid corresponds to the entire tumor. (C) B-mode image of a well oxygenated lung tumor with corresponding OxyHemo photoacoustic images showing absence of any hypoxic core.</p

Correlation analysis between different methods for tumor volume assessment.

<p>Nonlinear regression of data points collected from orthotopic subcutaneous tumors (n = 10 animals). The correlation coefficient R squared is provided in the lower right hand of each graph.</p

Western-blot analysis of APAF-1 and active form of caspase-9 expression in the atrophied skeletal muscle tissues.

<p>A) At autopsy of mice (day 20), the tibialis skeletal muscle tissues of 3 mice chosen randomly from the pRILES/23aT (atrophy) group of mice were collected and processed for a western-blot analysis using specific antibodies directed against APAF-1, Caspase 9 and tubulin. As control (control) we collected the tibialis skeletal muscle tissues of 2 naïve mice for which the sciatic nerve was not sectioned. Protein bands were revealed by chemiluminescence and autoradiography. B) Protein band intensities were quantified by densitometry using the image J software and expressed as relative protein expression by normalizing band intensities of APAF-1 and Caspase 9 to the band intensity of tubulin, used as loading control. This experiment was repeated at least twice with equivalent results.</p

Peptide-Conjugated MRI Probe Targeted to Netrin-1, a Novel Metastatic Breast Cancer Biomarker

Despite significant progress in cancer imaging and treatment over the years, early diagnosis and metastasis detection remain a challenge. Molecular magnetic resonance imaging (MRI), with its high resolution, can be well adapted to fulfill this need, requiring the design of contrast agents which target specific tumor biomarkers. Netrin-1 is an extracellular protein overexpressed in metastatic breast cancer and implicated in tumor progression and the appearance of metastasis. This study focuses on the design and preclinical evaluation of a novel Netrin-1-specific peptide-based MRI probe, GdDOTA-KKTHDAVR (Gd–K), to visualize metastatic breast cancer. The targeting peptide sequence was identified based on the X-ray structure of the complex between Netrin-1 and its transmembrane receptor DCC. Molecular docking simulations support the probe design. In vitro studies evidenced submicromolar affinity of Gd–K for Netrin-1 (KD = 0.29 μM) and good MRI efficacy (proton relaxivity, r1 = 4.75 mM–1 s–1 at 9.4 T, 37 °C). In vivo MRI studies in a murine model of triple-negative metastatic breast cancer revealed successful tumor visualization at earlier stages of tumor development (smaller tumor volume). Excellent signal enhancement, 120% at 2 min and 70% up to 35 min post injection, was achieved (0.2 mmol/kg injected dose), representing a reasonable imaging time window and a superior contrast enhancement in the tumor as compared to Dotarem injection

SPECT/CT imaging of miRNA-122 expression in the liver of mice.

<p>(A) A representative fused whole-body SPECT/CT image collected from one mouse injected hydrodynamically with the pRINES/122T plasmid. (B) Representative sagittal, coronal and transverse SPECT/CT images collected from the liver of one mouse from the pRINES and pRINES/122T group of mice. (C) Quantification of ^99mTc0₄^- uptake in ROIs covering the liver of mice of the pRINES and pRINES/122T group of animals. (D). Quantitative RT-PCR analysis of hNIS transcript detected in the liver of 3 mice collected randomly from the pRINES and pRINES/122T group of animals. Error bars in C are the mean ± SEM (n = 5) of one representative experiment performed at least two times. Error bars in D are the mean ± SD (n = 3) of one representative experiment performed at least three times. Statistics by the two-tailed t-test, * P ≤ 0.05 compared with the pRINES control group.</p

Positive radionuclide imaging of miRNA expression using RILES and the human sodium iodide symporter as reporter gene is feasible and supports a protective role of miRNA-23a in response to muscular atrophy

<div><p>MicroRNAs (miRNAs) are key players in many biological processes and are considered as an emerging class of pharmacology drugs for diagnosis and therapy. However to fully exploit the therapeutic potential of miRNAs, it is becoming crucial to monitor their expression pattern using medical imaging modalities. Recently, we developed a method called RILES, for RNAi-Inducible Luciferase Expression System that relies on an engineered regulatable expression system to switch-ON the expression of the luciferase gene when a miRNA of interest is expressed in cells. Here we investigated whether replacing the luciferase reporter gene with the human sodium iodide symporter (hNIS) reporter gene will be also suited to monitor the expression of miRNAs in a clinical setting context. We provide evidence that radionuclide imaging of miRNA expression using hNIS is feasible although it is not as robust as when the luciferase reporter gene is used. However, under appropriate conditions, we monitored the expression of several miRNAs in cells, in the liver and in the tibialis anterior muscle of mice undergoing muscular atrophy. We demonstrated that radiotracer accumulation in transfected cells correlated with the induction of hNIS and with the expression of miRNAs detected by real time PCR. We established the kinetic of miRNA-23a expression in mice and demonstrated that this miRNA follows a biphasic expression pattern characterized by a loss of expression at a late time point of muscular atrophy. At autopsy, we found an opposite expression pattern between miRNA-23a and one of the main transcriptional target of this miRNA, APAF-1, and as downstream target, Caspase 9. Our results report the first positive monitoring of endogenously expressed miRNAs in a nuclear medicine imaging context and support the development of additional work to establish the potential therapeutic value of miRNA-23 to prevent the damaging effects of muscular atrophy.</p></div