Fluorescence- and multispectral optoacoustic imaging for an optimized detection of deeply located tumors in an orthotopic mouse model of pancreatic carcinoma.

A crucial point for the management of pancreatic ductal adenocarcinoma (PDAC) is the decrease of R1 resections. Our aim was to evaluate the combination of multispectral optoacoustic tomography (MSOT) with fluorescence guided surgery (FGS) for diagnosis and perioperative detection of tumor nodules and resection margins in a xenotransplant mouse model of human pancreatic cancer. The peptide cRGD, conjugated with the near infrared fluorescent (NIRF) dye IRDye800CW and with a trans-cyclooctene (TCO) tag for future click chemistry (cRGD-800CW-TCO), was applied to PDAC bearing immunodeficient nude mice; 27 days after orthotopic transplantation of human AsPC-1 cells into the head of the pancreas, mice were injected with cRGD-800CW-TCO and imaged with fluorescence- and optoacoustic devices before and 2, 6 and 24 hr after injection, before they were sacrificed and dissected with a guidance of FGS imaging system. Fluorescence imaging of cRGD-800CW-TCO allowed detection of the tumor area but without information about the depth, whereas MSOT allowed high resolution 3 D identification of the tumor area, in particular of small tumor nodules. Highly sensitive delineation of tumor burden was achieved during FGS in all mice. Imaging of whole-mouse cryosections, histopathological analysis and NIRF microscopy confirmed the localization of cRGD-800CW-TCO within the tumor tissue. In principle, all imaging modalities applied here were able to detect PDAC in vivo. However, the combination of MSOT and FGS provided detailed spatial information of the signal and achieved a complete overview of the distribution and localization of cRGD-800CW-TCO within the tumor before and during surgical intervention.

Characterization of a commercial bioluminescence tomography-guided system for pre-clinical radiation research.

BACKGROUND: Widely used Cone-beam computed tomography (CBCT)-guided irradiators have limitations in localizing soft tissue targets growing in a low-contrast environment. This hinders small animal irradiators achieving precise focal irradiation. PURPOSE: To advance image-guidance for soft tissue targeting, we developed a commercial-grade bioluminescence tomography-guided system (BLT, MuriGlo) for pre-clinical radiation research. We characterized the system performance and demonstrated its capability in target localization. We expect this study can provide a comprehensive guideline for the community in utilizing the BLT system for radiation studies. METHODS: MuriGlo consists of four mirrors, filters, lens, and charge-coupled device (CCD) camera, enabling a compact imaging platform and multi-projection and multi-spectral BLT. A newly developed mouse bed allows animals imaged in MuriGlo and transferred to a small animal radiation research platform (SARRP) for CBCT imaging and BLT-guided irradiation. Methods and tools were developed to evaluate the CCD response linearity, minimal detectable signal, focusing, spatial resolution, distortion, and uniformity. A transparent polycarbonate plate covering the middle of the mouse bed was used to support and image animals from underneath the bed. We investigated its effect on 2D Bioluminescence images and 3D BLT reconstruction accuracy, and studied its dosimetric impact along with the rest of mouse bed. A method based on pinhole camera model was developed to map multi-projection bioluminescence images to the object surface generated from CBCT image. The mapped bioluminescence images were used as the input data for the optical reconstruction. To account for free space light propagation from object surface to optical detector, a spectral derivative (SD) method was implemented for BLT reconstruction. We assessed the use of the SD data (ratio imaging of adjacent wavelength) in mitigating out of focusing and non-uniformity seen in the images. A mouse phantom was used to validate the data mapping. The phantom and an in vivo glioblastoma model were utilized to demonstrate the accuracy of the BLT target localization. RESULTS: The CCD response shows good linearity with < 0.6% residual from a linear fit. The minimal detectable level is 972 counts for 10 × 10 binning. The focal plane position is within the range of 13-18 mm above the mouse bed. The spatial resolution of 2D optical imaging is < 0.3 mm at Rayleigh criterion. Within the region of interest, the image uniformity is within 5% variation, and image shift due to distortion is within 0.3 mm. The transparent plate caused < 6% light attenuation. The use of the SD imaging data can effectively mitigate out of focusing, image non-uniformity, and the plate attenuation, to support accurate multi-spectral BLT reconstruction. There is < 0.5% attenuation on dose delivery caused by the bed. The accuracy of data mapping from the 2D bioluminescence images to CBCT image is within 0.7 mm. Our phantom test shows the BLT system can localize a bioluminescent target within 1 mm with an optimal threshold and only 0.2 mm deviation was observed for the case with and without a transparent plate. The same localization accuracy can be maintained for the in vivo GBM model. CONCLUSIONS: This work is the first systematic study in characterizing the commercial BLT-guided system. The information and methods developed will be useful for the community to utilize the imaging system for image-guided radiation research.

Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype.

A duplex 21 nucleotide small interfering RNA (siRNA) against human Keap1 is described that represents a unique class of cancer chemopreventive agent. This siRNA can knockdown Keap1 mRNA and thereby relieve negative regulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-mediated gene expression. The siRNA lowered endogenous Keap1 mRNA to <30% of control levels between 24 and 72 h after transfection in human HaCaT keratinocyte cells and was capable of blocking ectopic expression of FLAG-tagged human Keap1 protein but not that of ectopic V5-tagged mouse Keap1 protein. Transfection of human HaCaT cells with Keap1 siRNA markedly enhanced endogenous levels of nuclear factor erythroid 2 p45-related factor 2 (Nrf2) protein and increased transcription of an antioxidant response element-driven reporter gene by 2.3-fold. Furthermore, 48 h after transfection of these cells with Keap1 siRNA, expression of aldo-keto reductase 1C1/2 and the glutamate cysteine ligase catalytic and modifier subunits was elevated between 5- and 14-fold. A modest increase of 3-fold in NAD(P)H:quinone oxidoreductase 1 was also observed. The Keap1 siRNA produced a 1.75-fold increase in intracellular glutathione 48 h after transfection. Thus, antagonism of Keap1 by siRNA can be used to preadapt human cells to oxidative stress without the need to expose them to redox stressors.