High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice: New Perspectives for Onco-Pharmacology.

OBJECTIVES:We have developed a relevant preclinical model associated with a specific imaging protocol dedicated to onco-pharmacology studies in mice. MATERIALS AND METHODS:We optimized both the animal model and an ultrasound imaging procedure to follow up longitudinally the lung tumor growth in mice. Moreover we proposed to measure by photoacoustic imaging the intratumoral hypoxia, which is a crucial parameter responsible for resistance to therapies. Finally, we compared ultrasound data to x-ray micro computed tomography and volumetric measurements to validate the relevance of this approach on the NCI-H460 human orthotopic lung tumor. RESULTS:This study demonstrates the ability of ultrasound imaging to detect and monitor the in vivo orthotopic lung tumor growth by high resolution ultrasound imaging. This approach enabled us to characterize key biological parameters such as oxygenation, perfusion status and vascularization of tumors. CONCLUSION:Such an experimental approach has never been reported previously and it would provide a nonradiative tool for assessment of anticancer therapeutic efficacy in mice. Considering the absence of ultrasound propagation through the lung parenchyma, this strategy requires the implantation of tumors strictly located in the superficial posterior part of the lung

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

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

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

TRPM8 as an Anti–Tumoral Target in Prostate Cancer Growth and Metastasis Dissemination

International audienceIn the fight against prostate cancer (PCa), TRPM8 is one of the most promising clinical targets. Indeed, several studies have highlighted that TRPM8 involvement is key in PCa progression because of its impact on cell proliferation, viability, and migration. However, data from the literature are somewhat contradictory regarding the precise role of TRPM8 in prostatic carcinogenesis and are mostly based on in vitro studies. The purpose of this study was to clarify the role played by TRPM8 in PCa progression. We used a prostate orthotopic xenograft mouse model to show that TRPM8 overexpression dramatically limited tumor growth and metastasis dissemination in vivo. Mechanistically, our in vitro data revealed that TRPM8 inhibited tumor growth by affecting the cell proliferation and clonogenic properties of PCa cells. Moreover, TRPM8 impacted metastatic dissemination mainly by impairing cytoskeleton dynamics and focal adhesion formation through the inhibition of the Cdc42, Rac1, ERK, and FAK pathways. Lastly, we proved the in vivo efficiency of a new tool based on lipid nanocapsules containing WS12 in limiting the TRPM8–positive cells’ dissemination at metastatic sites. Our work strongly supports the protective role of TRPM8 on PCa progression, providing new insights into the potential application of TRPM8 as a therapeutic target in PCa treatment