Detection of Brain Tumors and Systemic Metastases Using NanoLuc and Fluc for Dual Reporter Imaging

Purpose

Bioluminescence imaging (BLI) is a technique with a low background noise and high sensitivity which is widely used in mice models in oncology. We aimed to assess BLI efficiency of the new luciferase NanoLuc (Nluc) for glioblastoma cell lines and tumors, including for dual reporter applications of deep brain tumors and systemic metastasis when combined with firefly luciferase (Fluc).

Procedures

U87 cells were genetically modified for constitutive production of either Nluc, Fluc, or both and assayed for luciferase activity and BLI on cell lysates, living cells, subcutaneous tumors, brain tumors, and systemic metastases.

Results

In vitro, light production by Nluc activity is higher than Fluc. In vivo, Nluc allows for tumor detection including for deep brain tumors and systemic metastases.

Conclusions

Nluc appears to be a useful tool to combine with Fluc for dual imaging in vivo using bioluminescence, allowing for the detection of distinct events in deep tissues within the same organism.

     

Bioluminescence Imaging of Colonization and Clearance Dynamics of <Emphasis Type="Italic">Brucella Suis</Emphasis> Vaccine Strain S2 in Mice and Guinea Pigs

Purpose

The goal of this study was to develop a plasmid-based lux bio-reporter for use to obtain in vivo images of Brucella suis vaccine strain 2 (B.suis S2) infection with high resolution and good definition.

Procedures

The pBBR-lux (pBBR1MCS-2-lxCDABE) plasmid that carries the luxCDABE operon was introduced into B. suis S2 by electroporation yielding B. suis S2-lux. The spatial and temporal transit of B. suis S2 in mice and guinea pigs was monitored by bioluminescence imaging.

Results

The plasmid pBBR-lux is stable in vivo and does not appear to impact the virulence or growth of bacteria. This sensitive luciferase reporter could represent B. suis S2 survival in real time. B. suis S2 mainly colonized the lungs, liver, spleen, and uterus in mice and guinea pigs as demonstrated by bioluminescence imaging.

Conclusion

The plasmid-based lux bioreporter strategy can be used to obtain high resolution in vivo images of B. suis S2 infection in mice and guinea pigs.

     

<Emphasis Type="Italic">In</Emphasis><Emphasis Type="Italic">Vivo</Emphasis> Bioluminescence Imaging of Transplanted Mesenchymal Stromal Cells and Their Rejection Mediated by Intrahepatic NK Cells

Purpose

Mesenchymal stromal cells (MSCs) hold promise in the treatment of liver disease. However, short survival time of MSCs after intrahepatic transplantation limits their value; therefore, understanding the basis of MSCs survival and rejection may increase their utility. This study was aimed at determining the role of intrahepatic natural killer (NK) cells on MSCs survival and their retention in the liver shortly after transplant.

Procedures

Human MSCs were labeled with the Luc2-mKate2 dual-fusion reporter gene (MSCs-R), and the residence time and survival of MSCs-R xenografts after intrahepatic transplantation were evaluated by in vivo bioluminescence imaging (BLI). Coculture of MSCs and NK cells was performed to assess cytotoxicity. To evaluate the role of NK cells in rejection of the xenografted cells, the fates of transplanted MSCs-R were then assessed in vivo by BLI after activation of intrahepatic NK cells.

Results

We observed a linear correlation between luciferase activity from live MSCs-R and cell number in vitro (R ²?=?0.9956). In vivo, we observed a gradual decline in bioluminescent signals from transplanted MSCs-R over a region corresponding to the liver in both the control group and the NK-activated group. However, the survival time and retention of intrahepatic MSCs-R decreased more rapidly in the NK-activated group of mice compared to the control group. This indicated that activated NK cells accelerate the elimination of transplanted MSCs. Also, we found that the number of hepatic NK cells and the expression of NK activation markers significantly increased after intrahepatic delivery of MSCs. This suggested that resident NK cells, in a resting state, were activated by intrahepatic transplantation of human MSCs. Taken together, the data suggests that activated hepatic NK cells mediate, in part, rejection of the MSCs xenografts. Cytotoxicity assays showed that activated NK cells may inhibit the proliferation of MSCs and, to a certain extent, induce MSCs death.

Conclusion

Human MSCs could be followed dynamically in vivo by BLI, and the role of murine hepatic NK cells, especially activated NK cells, could be inferred from the loss of signals from MSCs. This finding may have practical clinical implications in MSCs transplantation in treating liver disease.

     

<Emphasis Type="Italic">L</Emphasis><Subscript><Emphasis Type="Italic">p</Emphasis></Subscript> Regularization for Bioluminescence Tomography Based on the Split Bregman Method

Purpose

Bioluminescence tomography (BLT) is a promising in vivo optical imaging technique in preclinical research at cellular and molecular levels. The problem of BLT reconstruction is quite ill-posed and ill-conditioned. In order to achieve high accuracy and efficiency for its inverse reconstruction, we proposed a novel approach based on L _p regularization with the Split Bregman method.

Procedures

The diffusion equation was used as the forward model. Then, we defined the objective function of L _p regularization and developed a Split Bregman iteration algorithm to optimize this function. After that, we conducted numerical simulations and in vivo experiments to evaluate the accuracy and efficiency of the proposed method.

Results

The results of the simulations indicated that compared with the conjugate gradient and iterative shrinkage methods, the proposed method is more accurate and faster for multisource reconstructions. Furthermore, in vivo imaging suggested that it could clearly distinguish the viable and apoptotic tumor regions.

Conclusions

The Split Bregman iteration method is able to minimize the L _p regularization problem and achieve fast and accurate reconstruction in BLT.

     

<Emphasis Type="Italic">In Vivo</Emphasis> MR Imaging of Dual MRI Reporter Genes and Deltex-1 Gene-modified Human Mesenchymal Stem Cells in the Treatment of Closed Penile Fracture

Purpose

The purpose of this study was to investigate the feasibility of dual magnetic resonance imaging (MRI) reporter genes, including ferritin heavy subunit (Fth) and transferrin receptor (TfR), which provide sufficient MRI contrast for in vivo MRI tracking, and the Deltex-1 (DTX1) gene, which promotes human mesenchymal stem cell (hMSC) differentiation to smooth muscle cells (SMCs), to treat closed penile fracture (CPF).

Methods

Multi-gene co-expressing hMSCs were generated. The expression of mRNA and proteins was assessed, and the original biological properties of hMSCs were determined and compared. The intracellular uptake of iron was evaluated, and the ability to differentiate into SMCs was detected. Fifty rabbits with CPF were randomly transplanted with PBS, hMSCs, Fth-TfR-hMSCs, DTX1-hMSCs, and Fth-TfR-DTX1-hMSCs. In vivo MRI was performed to detect the distribution and migration of the grafted cells and healing progress of CPF, and the results were correlated with histology.

Results

The mRNA and proteins of the multi-gene were highly expressed. The transgenes could not influence the original biological properties of hMSCs. The dual MRI reporter genes increased the iron accumulation capacity, and the DTX1 gene promoted hMSC differentiation into SMCs. The distribution and migration of the dual MRI reporter gene-modified hMSCs, and the healing state of CPF could be obviously detected by MRI and confirmed by histology.

Conclusion

The dual MRI reporter genes could provide sufficient MRI contrast, and the distribution and migration of MSCs could be detected in vivo. The DTX1 gene can promote MSC differentiation into SMCs for the treatment of CPF and effectively inhibit granulation tissue formation.

     

Detection of Enzyme Activity and Inhibition during Studies in Solution, <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> with CatalyCEST MRI

Purpose

The detection of enzyme activities and evaluation of enzyme inhibitors have been challenging with magnetic resonance imaging (MRI). To address this need, we have developed a diamagnetic, nonmetallic contrast agent and a protocol known as catalyCEST MRI that uses chemical exchange saturation transfer (CEST) to detect enzyme activity as well as enzyme inhibition.

Procedures

We synthesized a diamagnetic MRI contrast agent that has enzyme responsive and enzyme unresponsive CEST signals. We tested the ability of this agent to detect the activity of kallikrein 6 (KLK6) in biochemical solutions, in vitro and in vivo, with and without a KLK6 inhibitor.

Results

The agent detected KLK6 activity in solution and also detected KLK6 inhibition by antithrombin III. KLK6 activity was detected during in vitro studies with HCT116 colon cancer cells, relative to the detection of almost no activity in a KLK6-knockdown HCT116 cell line and HCT116 cells treated with antithrombin III inhibitor. Finally, strong enzyme activity was detected within an in vivo HCT116 tumor model, while lower enzyme activity was detected in a KLK6 knockdown tumor model and in the HCT116 tumor model treated with antithrombin III inhibitor. In all cases, comparisons of the enzyme responsive and enzyme unresponsive CEST signals were critical for the detection of enzyme activity.

Conclusions

This study has established that catalyCEST MRI with an exogenous diaCEST agent can evaluate enzyme activity and inhibition in solution, in vitro and in vivo.

     

Noninvasive Monitoring of the Mitochondrial Function in Mesenchymal Stromal Cells

Purpose

Mitochondria are a gatekeeper of cell survival and mitochondrial function can be used to monitor cell stress. Here we validate a pathway-specific reporter gene to noninvasively image the mitochondrial function of stem cells.

Procedures

We constructed a mitochondrial sensor with the firefly luciferase (Fluc) reporter gene driven by the NQO1 enzyme promoter. The sensor was introduced in stem cells and validated in vitro and in vivo, in a mouse model of myocardial ischemia/reperfusion (IR).

Results

The sensor activity showed an inverse relationship with mitochondrial function (R ²?=??0.975, p?=?0.025) and showed specificity and sensitivity for mitochondrial dysfunction. In vivo, NQO1-Fluc activity was significantly higher in IR animals vs. controls, indicative of mitochondrial dysfunction, and was corroborated by ex vivo luminometry.

Conclusions

Reporter gene imaging allows assessment of the biology of transplanted mesenchymal stromal cells (MSCs), providing important information that can be used to improve the phenotype and survival of transplanted stem cells.

     

Bioluminescence Imaging to Monitor the Effects of the Hsp90 Inhibitor NVP-AUY922 on NF-κB Pathway in Endometrial Cancer

Purpose

In this study, we first aimed to evaluate the effects in vitro and in vivo, of the Hsp90 inhibitor NVP-AUY922, in endometrial cancer (EC). We also aimed to track nuclear factor kappa B (NF-κB) signalling, a key pathway involved in endometrial carcinogenesis and to check whether NVP-AUY922 treatment modulates it both in vitro and in vivo.

Procedures

I n vitro effects of NVP-AUY922 on EC cell growth and the signalling pathways were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenic assays, Western Blot and luciferase assay. NVP-AUY922 effect on Ishikawa (IK) xenograft growth was evaluated in vivo, and NF-κB activity was monitored using bioluminescence imaging.

Results

NVP-AUY922 inhibited the growth of three endometrial cell lines tested in vitro. In vivo, NVP-AUY922 reduced tumour growth of 47 % (p?=?0.042) compared to control condition. Moreover, the bioluminescence signal of the tumours harbouring IK NF-κB-LUC cells was significantly reduced in NVP-AUY922-treated animals compared to untreated ones.

Conclusions

NVP-AUY922 reduced EC tumour growth and NF-κB signalling both in vitro and in vivo. As therapeutic resistance of EC remains a challenge for oncologists nowadays, we think that NVP-AUY922 represents a valid alternative to conventional chemotherapy, and we believe that this approach for assessing and tracking the activation of NF-κB pathway may be of therapeutic benefit.

     

<Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Comparison of Selected Ga-68 and Zr-89 Labelled Siderophores

Purpose

Some [⁶⁸Ga]siderophores show promise in specific and sensitive imaging of infection. Here, we compare the in vitro and in vivo behaviour of selected Ga-68 and Zr-89 labelled siderophores.

Procedures

Radiolabelling was performed in HEPES or sodium acetate buffer systems. Radiochemical purity of labelled siderophores was determined using chromatography. Partition coefficients, in vitro stability and protein binding affinities were determined. Ex vivo biodistribution and animal imaging was studied in mice.

Results

Certain differences among studied siderophores were observed in labelling efficiency. Protein binding and stability tests showed highest stabilities and lowest protein binding affinities for Ga-68 and [⁸⁹Zr]triacetylfusarinine C (TAFC). All studied Ga-68 and [⁸⁹Zr]siderophores exhibited a similar biodistribution and pharmacokinetics in mice with the exception of [⁸⁹Zr]ferrioxamine E (FOXE).

Conclusions

Zr-89 and [⁶⁸Ga]siderophores showed analogous in vitro and in vivo behaviour. Tested [⁸⁹Zr]siderophores could be applied for longitudinal positron emission tomography (PET) studies of fungal infections and especially TAFC for the development of novel bioconjugates.

     

Multimodal Genetic Approach for Molecular Imaging of Vasculature in a Mouse Model of Melanoma

Purpose

In this study, we evaluated a genetic approach for in vivo multimodal molecular imaging of vasculature in a mouse model of melanoma.

Procedures

We used a novel transgenic mouse, Ts-Biotag, that genetically biotinylates vascular endothelial cells. After inoculating these mice with B16 melanoma cells, we selectively targeted endothelial cells with (strept)avidinated contrast agents to achieve multimodal contrast enhancement of Tie2-expressing blood vessels during tumor progression.

Results

This genetic targeting system provided selective labeling of tumor vasculature and showed in vivo binding of avidinated probes with high specificity and sensitivity using microscopy, near infrared, ultrasound, and magnetic resonance imaging. We further demonstrated the feasibility of conducting longitudinal three-dimensional (3D) targeted imaging studies to dynamically assess changes in vascular Tie2 from early to advanced tumor stages.

Conclusions

Our results validated the Ts-Biotag mouse as a multimodal targeted imaging system with the potential to provide spatio-temporal information about dynamic changes in vasculature during tumor progression.

     

Multispectral Photoacoustic Imaging of Tumor Protease Activity with a Gold Nanocage-Based Activatable Probe

Purpose

Tumor proteases have been recognized as significant regulators in the tumor microenvironment, but the current strategies for in vivo protease imaging have tended to focus on the development of a probe design rather than the investigation of a novel imaging strategy by leveraging the imaging technique and probe. Herein, it is the first report to investigate the ability of multispectral photoacoustic imaging (PAI) to estimate the distribution of protease cleavage sites inside living tumor tissue by using an activatable photoacoustic (PA) probe.

Procedures

The protease MMP-2 is selected as the target. In this probe, gold nanocages (GNCs) with an absorption peak at ~?800 nm and fluorescent dye molecules with an absorption peak at ~?680 nm are conjugated via a specific enzymatic peptide substrate. Upon enzymatic activation by MMP-2, the peptide substrate is cleaved and the chromophores are released. Due to the different retention speeds of large GNCs and small dye molecules, the probe alters its intrinsic absorption profile and produces a distinct change in the PA signal. A multispectral PAI technique that can distinguish different chromophores based on intrinsic PA spectral signatures is applied to estimate the signal composition changes and indicate the cleavage interaction sites. Finally, the multispectral PAI technique with the activatable probe is tested in solution, cultured cells, and a subcutaneous tumor model in vivo.

Results

Our experiment in solution with enzyme ± inhibitor, cell culture ± inhibitor, and in vivo tumor model with administration of the developed probe ± inhibitor demonstrated the probe was cleaved by the targeted enzyme. Particularly, the in vivo estimation of the cleavage site distribution was validated with the result of ex vivo immunohistochemistry analysis.

Conclusions

This novel synergy of the multispectral PAI technique and the activatable probe is a potential strategy for the distribution estimation of tumor protease activity in vivo.

     

Imaging of <Emphasis Type="Italic">Sleeping Beauty</Emphasis>-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography

Purpose

We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells.

Procedures

We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19⁺ artificial antigen-presenting cells.

Results

After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19⁺ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2′-deoxy-2′-[¹⁸F]fluoro-5-ethyl-1-β-d-arabinofuranosyl-uracil ([¹⁸F]FEAU).

Conclusion

This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.

     

Using Dual Fluorescence Reporting Genes to Establish an <Emphasis Type="Italic">In Vivo</Emphasis> Imaging Model of Orthotopic Lung Adenocarcinoma in Mice

Purpose

Lung adenocarcinoma is characterized by a poor prognosis and high mortality worldwide. In this study, we purposed to use the live imaging techniques and a reporter gene that generates highly penetrative near-infrared (NIR) fluorescence to establish a preclinical animal model that allows in vivo monitoring of lung cancer development and provides a non-invasive tool for the research on lung cancer pathogenesis and therapeutic efficacy.

Procedures

A human lung adenocarcinoma cell line (A549), which stably expressed the dual fluorescence reporting gene (pCAG-iRFP-2A-Venus), was used to generate subcutaneous or orthotopic lung cancer in nude mice. Cancer development was evaluated by live imaging via the NIR fluorescent signals from iRFP, and the signals were verified ex vivo by the green fluorescence of Venus from the gross lung. The tumor-bearing mice received miR-16 nucleic acid therapy by intranasal administration to demonstrate therapeutic efficacy in this live imaging system.

Results

For the subcutaneous xenografts, the detection of iRFP fluorescent signals revealed delicate changes occurring during tumor growth that are not distinguishable by conventional methods of tumor measurement. For the orthotopic xenografts, the positive correlation between the in vivo iRFP signal from mice chests and the ex vivo green fluorescent signal from gross lung tumors and the results of the suppressed tumorigenesis by miR-16 treatment indicated that lung tumor size can be accurately quantified by the emission of NIR fluorescence. In addition, orthotopic lung tumor localization can be accurately visualized using iRFP fluorescence tomography in vivo, thus revealing the trafficking of lung tumor cells.

Conclusions

We introduced a novel dual fluorescence lung cancer model that provides a non-invasive option for preclinical research via the use of NIR fluorescence in live imaging of lung.

     

Gallium-68 Complexes Conjugated to Pittsburgh Compound B: Radiolabeling and Biological Evaluation

Purpose

The aim of this work is to develop an efficient and fully automated radiosynthesis of three derivatives of the Pittsburgh compound B labeled with gallium-68 for the detection of amyloid plaques.

Procedures

The radiolabeling of the precursors and purification of the radiolabeled agents by high pressure liquid chromatography has been studied prior to their in vitro and in vivo evaluations.

Results

The complete process led, in 50 min, to pure Ga-68 products in a 12–38 % yield and with appreciable specific radioactivity (SRA, 85–168 GBq/μmol) which enabled us to demonstrate a considerable in vivo stability of the products. Unfortunately, this result was associated with a poor blood–brain barrier (BBB) permeability and a limited uptake of our compounds by amyloid deposits was observed by in vitro autoradiography.

Conclusion

Although we have not yet identified a compound able to significantly mark cerebral amyloidosis, this present investigation will likely contribute to the development of more successful Ga-68 radiotracers.

     

Quantitative [<Superscript>18</Superscript>F]FMISO PET Imaging Shows Reduction of Hypoxia Following Trastuzumab in a Murine Model of HER2+ Breast Cancer

Purpose

Evaluation of [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO)-positron emission tomography (PET) imaging as a metric for evaluating early response to trastuzumab therapy with histological validation in a murine model of HER2+ breast cancer.

Procedures

Mice with BT474, HER2+ tumors, were imaged with [¹⁸F]FMISO-PET during trastuzumab therapy. Pimonidazole staining was used to confirm hypoxia from imaging.

Results

[¹⁸F]FMISO-PET indicated significant decreases in hypoxia beginning on day 3 (P?<?0.01) prior to changes in tumor size. These results were confirmed with pimonidazole staining on day 7 (P?<?0.01); additionally, there was a significant positive linear correlation between histology and PET imaging (r ² ?=?0.85).

Conclusions

[¹⁸F]FMISO-PET is a clinically relevant modality which provides the opportunity to (1) predict response to HER2+ therapy before changes in tumor size and (2) identify decreases in hypoxia which has the potential to guide subsequent therapy.

     

Bioluminescence Imaging for Monitoring miR-200c Expression in Breast Cancer Cells and its Effects on Epithelial-Mesenchymal Transition Progress in Living Animals

Purpose

Dysregulation of microRNAs (miRNAs) are not only involved in the formation of malignant tumors but also in the processes of differentiation and aggressiveness. However, current methods for detecting miRNA expression have major disadvantages, such as being invasive and non-reproducible. The epithelial-mesenchymal transition (EMT) has been implicated as a pivotal event in the metastasis, stemness, and chemoresistance of malignant tumors.

Procedures

In our study, we constructed a new reporter gene, Luc2/tdT_miR200c_3TS, to examine the in vitro and in vivo expression of miR-200c, an EMT-associated miRNA. Quantitative real-time PCR was used to measure the expression levels of miR-200c and EMT-related mRNA, and luciferase assay and bioluminescence imaging were used to measure the luciferase activities in vitro and in vivo, respectively.

Results

We found that the expression level of miR-200c was negatively associated with cell migration and invasion. Luciferase activities were regulated by the differential expression levels of miR-200c and EMT process.

Conclusions

Our results demonstrate that Luc2/tdT_miR200c_3TS may be a useful tool for monitoring the expression level of miR-200c at both the cellular level and in living animals, thereby providing a potential high-throughput approach for anticancer drug screening.

     

Tumor-Specific Zr-89 Immuno-PET Imaging in a Human Bladder Cancer Model

Purpose

Tumor-specific molecular imaging is an important tool for assessing disease burden and treatment response. CA19.9 is an important tumor-specific marker in several malignancies, including urothelial carcinoma. [⁸⁹Zr]DFO-HuMab-5B1 (MVT-2163) is a CA19.9-specific antibody-based construct that has been validated in preclinical animal models of lung, colorectal, and pancreatic malignancies for positron emission tomography (PET) imaging and is currently in a phase I trial for pancreatic cancer (NCT02687230). Here, we examine whether [⁸⁹Zr]DFO-HuMab-5B1 may be useful in defining urothelial malignancies.

Procedures

Surface expression of CA19.9 was confirmed in the human bladder cancer line HT 1197. The radioimmunoconjugate [⁸⁹Zr]DFO-HuMab-5B1 was injected into mice bearing HT 1197 xenografts, and followed by PET imaging, ex vivo experiments including biodistribution, histology and autoradiography, and analysis of blood samples for shed antigen levels were performed.

Results

[⁸⁹Zr]DFO-HuMab-5B1 specifically accumulates in HT 1197 engrafted tumors when imaged with PET. Ex vivo biodistribution of organs and autoradiography of engrafted tumors confirm our construct’s specific tumor binding. The target antigen CA19.9 was not found to be shed in vitro or in vivo.

Conclusions

[⁸⁹Zr]DFO-HuMab-5B1 can be used to delineate urothelial carcinomas by PET imaging and may provide tumor-specific information prior to, during, and after systemic therapies.

     

Pre-clinical Evaluation of a Cyanine-Based SPECT Probe for Multimodal Tumor Necrosis Imaging

Purpose

Recently we showed that a number of carboxylated near-infrared fluorescent (NIRF) cyanine dyes possess strong necrosis avid properties in vitro as well as in different mouse models of spontaneous and therapy-induced tumor necrosis, indicating their potential use for cancer diagnostic- and prognostic purposes. In the previous study, the detection of the cyanines was achieved by whole body optical imaging, a technique that, due to the limited penetration of near-infrared light, is not suitable for investigations deeper than 1 cm within the human body. Therefore, in order to facilitate clinical translation, the purpose of the present study was to generate a necrosis avid cyanine-based NIRF probe that could also be used for single photon emission computed tomography (SPECT). For this, the necrosis avid NIRF cyanine HQ4 was radiolabeled with ¹¹¹indium, via the chelate diethylene triamine pentaacetic acid (DTPA).

Procedures

The necrosis avid properties of the radiotracer [¹¹¹In]DTPA-HQ4 were examined in vitro and in vivo in different breast tumor models in mice using SPECT and optical imaging. Moreover, biodistribution studies were performed to examine the pharmacokinetics of the probe in vivo.

Results

Using optical imaging and radioactivity measurements, in vitro, we showed selective accumulation of [¹¹¹In]DTPA-HQ4 in dead cells. Using SPECT and in biodistribution studies, the necrosis avidity of the radiotracer was confirmed in a 4T1 mouse breast cancer model of spontaneous tumor necrosis and in a MCF-7 human breast cancer model of chemotherapy-induced tumor necrosis.

Conclusions

The radiotracer [¹¹¹In]DTPA-HQ4 possessed strong and selective necrosis avidity in vitro and in various mouse models of tumor necrosis in vivo, indicating its potential to be clinically applied for diagnostic purposes and to monitor anti-cancer treatment efficacy.

     

Characterization of Magneto-Endosymbionts as MRI Cell Labeling and Tracking Agents

Purpose

Magneto-endosymbionts (MEs) show promise as living magnetic resonance imaging (MRI) contrast agents for in vivo cell tracking. Here we characterize the biomedical imaging properties of ME contrast agents, in vitro and in vivo.

Procedures

By adapting and engineering magnetotactic bacteria to the intracellular niche, we are creating magneto-endosymbionts (MEs) that offer advantages relative to passive iron-based contrast agents (superparamagnetic iron oxides, SPIOs) for cell tracking. This work presents a biomedical imaging characterization of MEs including: MRI transverse relaxivity (r ₂) for MEs and ME-labeled cells (compared to a commercially available iron oxide nanoparticle); microscopic validation of labeling efficiency and subcellular locations; and in vivo imaging of a MDA-MB-231BR (231BR) human breast cancer cells in a mouse brain.

Results

At 7T, r ₂ relaxivity of bare MEs was higher (250 s^?1 mM^?1) than that of conventional SPIO (178 s^?1 mM^?1). Optimized in vitro loading of MEs into 231BR cells yielded 1–4 pg iron/cell (compared to 5–10 pg iron/cell for conventional SPIO). r ₂ relaxivity dropped by a factor of ~3 upon loading into cells, and was on the same order of magnitude for ME-loaded cells compared to SPIO-loaded cells. In vivo, ME-labeled cells exhibited strong MR contrast, allowing as few as 100 cells to be detected in mice using an optimized 3D SPGR gradient-echo sequence.

Conclusions

Our results demonstrate the potential of magneto-endosymbionts as living MR contrast agents. They have r ₂ relaxivity values comparable to traditional iron oxide nanoparticle contrast agents, and provide strong MR contrast when loaded into cells and implanted in tissue.

     

Hybrid Light Imaging Using Cerenkov Luminescence and Liquid Scintillation for Preclinical Optical Imaging <Emphasis Type="Italic">In Vivo</Emphasis>

Purpose

Cerenkov luminescence imaging (CLI) has recently emerged as a molecular imaging modality for radionuclides emitting β-particles. The aim of this study was to develop a hybrid light imaging (HLI) technique using a liquid scintillator to assist CLI by increasing the optical signal intensity from both β-particle and γ-ray emitting radionuclides located at deep regions in vivo.

Procedures

A commercial optical imaging system was employed to collect all images by HLI and CLI. To investigate the performance characteristics of HLI with a commercially available liquid scintillator (Emulsifier-safe), phantom experiments were conducted for two typical β-particle and γ-ray emitters, sodium iodide (Na[¹³¹I]I) and 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG), respectively. To evaluate the feasibility of HLI for in vivo imaging, HLI was applied to a Na[¹³¹I]I injected nu/nu mouse and an [¹⁸F]FDG injected Balb-c mouse and compared with CLI alone.

Results

Measured HLI wavelength spectra with Emulsifier-safe showed higher signal intensities than for CLI at 500–600 nm. For material preventing light transmission of 12-mm thickness, CLI imaging provided quite low intensity and obscure signals of the source. However, despite degraded spatial resolution, HLI imaging provided sustained visualization of the source shape, with signal intensities 10–14 times higher than for CLI at 10-mm thickness. Furthermore, at 0, 4, and 8-mm material thicknesses, HLI showed a strong correlation between Na[¹³¹I]I or [¹⁸F]FDG radioactivity and signal intensity, as for CLI. In vivo studies also demonstrated that HLI could successfully visualize Na[¹³¹I]I uptake in the mouse thyroid gland in the prone position and [¹⁸F]FDG accumulation in the heart in the supine position, which were not observed with CLI.

Conclusion

Our preliminary studies suggest that HLI can provide enhanced imaging of a β-particle probe emitting together with γ-rays at deep tissue locations. HLI may be a promising imaging technique to assist with preclinical in vivo imaging using CLI.