Optical Bioluminescence Imaging Research Articles

Abstract 6905: A multi-targeted approach to evaluate therapeutic selection and efficacy in preclinical GBM models

Abstract Glioblastoma (GBM) is a devastating primary brain cancer with approximately 10,000 new US diagnoses annually. The current standard of care (SOC) for GBM includes surgical resection followed by radiation therapy (RT) and temozolomide (TMZ), however, there is near universal recurrence and development of resistance after treatment. Relapse in disease is tightly linked with dynamic changes in gene expression during tumor evolution, highlighting the need for stronger preclinical GBM models. Here, we report developing a pair of patient-derived xenograft (PDX) models from surgical resections obtained from initial GBM incidence in a patient (BarneyOI™ Cancer Model CRT00433) and subsequent recurrence following treatment (BarneyOI Cancer Model CRT00435) to study disease progression and novel treatment strategies. Recent studies have suggested the utilization of combination therapy approaches for GBM patients to address TMZ resistance. To this end, Certis has developed a personalized, AI-based approach to predict and test combination therapies in preclinical cancer models. Accuracy of the predicted sensitivities to treatment was evaluated in vivo using both subcutaneous (SC) and orthotopic (OT) mouse models. Luciferase-tagged CRT00433 and CRT00435 spheroid cell lines were transduced with firefly luciferase and implanted intracranially by stereotactic surgery for OT monitoring. Optical bioluminescence imaging (BLI) and murine-scale MRI from Aspect Imaging were used to assess therapeutic response of AI-predicted combination therapies and SOC treatment. Citation Format: Emily Eastwood, Bianca Carapia, Javier Rodriguez, Kristen Buck, Derrick Gorospe, Elizabeth Valencia, Bridget Corcoran, Raffaella Pippa, Yuan-Hung Chien, Warren Andrews, Long Do, Jonathan K. Nakashima, Jantzen Sperry. A multi-targeted approach to evaluate therapeutic selection and efficacy in preclinical GBM models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6905.

EXTH-61. A MULTI-TARGETED APPROACH TO EVALUATE THERAPEUTIC SELECTION AND EFFICACY IN PRECLINICAL GBM MODELS

Abstract Glioblastoma (GBM) is a devastating primary brain cancer with approximately 10,000 new US diagnoses annually. The current standard of care for GBM includes surgical resection followed by radiation therapy (RT) and temozolomide (TMZ), however there is near universal recurrence and development of resistance after treatment. Relapse in disease is tightly linked with dynamic changes in gene expression during tumor evolution, highlighting the need for stronger preclinical GBM models. Here we report developing a pair of patient-derived xenograft (PDX) models from surgical resections obtained from initial GBM incidence in a patient (CRT00433) and subsequent recurrence following treatment (CRT00435) in order to study disease progression and novel treatment strategies. Recent studies have suggested the utilization of combination therapy approaches for GBM patients to address TMZ resistance. To this end, Certis has developed a personalized, AI-based approach to predict and test combination therapies for CRT00433/435. Accuracy of the predicted sensitivities to treatment was then evaluated in vivo using both subcutaneously and orthotopically engrafted mouse modeling. Luciferase-tagged CRT00433 and CRT00435 spheroid cell lines were transduced with firefly luciferase and implanted intracranially by stereotactic surgery for orthotopic monitoring. Optical bioluminescence imaging (BLI) and MRI were used to assess therapeutic response of AI-predicted combination therapies and standard of care (RT and/or TMZ). Both in-life imaging and terminal histologic analysis of the tumors confirmed high fidelity to several of the therapies tested. In all, these results demonstrate a novel, combinatorial platform with which to assess therapeutic efficacy in GBM and exploit the overall ability of this deadly disease to adapt to an ever-changing molecular and genetic landscape.

Robust reconstruction of fluorescence molecular tomography based on adaptive adversarial learning strategy

Objective. Fluorescence molecular tomography (FMT) is a promising molecular imaging modality for quantifying the three-dimensional (3D) distribution of tumor probes in small animals. However, traditional deep learning reconstruction methods that aim to minimize the mean squared error (MSE) and iterative regularization algorithms that rely on optimal parameters are typically influenced by strong noise, resulting in poor FMT reconstruction robustness. Approach. In this letter, we propose an adaptive adversarial learning strategy (3D-UR-WGAN) to achieve robust FMT reconstructions. Unlike the pixel-based MSE criterion in traditional CNNs or the regularization strategy in iterative solving schemes, the reconstruction strategy can greatly facilitate the performance of the network models through alternating loop training of the generator and the discriminator. Second, the reconstruction strategy combines the adversarial loss in GANs with the L1 loss to significantly enhance the robustness and preserve image details and textual information. Main results. Both numerical simulations and physical phantom experiments demonstrate that the 3D-UR-WGAN method can adaptively eliminate the effects of different noise levels on the reconstruction results, resulting in robust reconstructed images with reduced artifacts and enhanced image contrast. Compared with the state-of-the-art methods, the proposed method achieves better reconstruction performance in terms of target shape recovery and localization accuracy. Significance. This adaptive adversarial learning reconstruction strategy can provide a possible paradigm for robust reconstruction in complex environments, and also has great potential to provide an alternative solution for solving the problem of poor robustness encountered in other optical imaging modalities such as diffuse optical tomography, bioluminescence imaging, and Cherenkov luminescence imaging.

Non-invasive In Vivo Brain Astrogenesis and Astrogliosis Quantification Using a Far-red E2-Crimson Transgenic Reporter Mouse.

Despite the adaptation of major clinical imaging modalities for small animals, optical bioluminescence imaging technology is the main approach readily reporting gene activity. Yet, in vivo bioluminescence monitoring requires the administration and diffusion of a substrate to the tissues of interest, resulting in experimental variability, high reagent cost, long acquisition time, and stress to the animal. In our study, we avoid such issues upon generating a new transgenic mouse (GFAP-E2crimson) expressing the far-red fluorescent protein E2-crimson under the control of the glial fibrillary acidic protein (GFAP) promoter. Using microscopy, we validated the selective expression of the reporter in the astrocyte cell population and by non-invasive in vivo fluorescence imaging its detection through the scalps and skulls of live animals. In addition, we performed a longitudinal study validating by in vivo imaging that the E2-crimson fluorescence signal is up-regulated, in pups during astrogenesis and in adult mice during astrogliosis upon kainic acid administration. Furthermore, upon crossing GFAP-E2crimson transgenic with 5XFAD Alzheimer's disease mice model, we were able to quantify the chronic inflammation triggered by amyloid deposit and aging over 18months. As many diseases and conditions can trigger neuroinflammation, we believe that the GFAP-E2crimson reporter mice model delivers tremendous value for the non-invasive quantification of astrogliosis responses in living animals.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy.

Simple SummaryCancer cell surface–exposed calreticulin (ecto-CRT) is the primitive form of signal during immunogenic cell death (ICD). It is a well-known candidate to allow “eat-me” signal from dying cells, which further contributes to their perception in directing the immune system. Various forms of anticancer agents and ionizing radiation can facilitate the ICD via ecto-CRT exposure. We engineered CRT-specific human fibronectin domain III (FN3) monobodies (FN3-CRT-Rluc8) fused with peptide sequences for CRT imaging. We assessed the theragnostic use of engineered monobodies for ecto-CRT imaging during ICD for early therapeutic prediction response. Our findings demonstrated that engineered monobodies could involve in targeting dying cells via anticancer-related immunogenic chemotherapeutic treatments, and the obtained imaging results could be used to detect pre-apoptotic cells in ICD. Our data provides the novel FN3-based ecto-CRT imaging method and enables the early immuno-therapeutic response predictions, thereby facilitating early determinations in cancer chemotherapies.Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from E. coli by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment. Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein–engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment.

Investigating the blood-spinal cord barrier in preclinical models: a systematic review of in vivo imaging techniques.

This study is a systematic review. To evaluate current in vivo techniques used in the investigation of the blood-spinal cord barrier (BSCB). Search of English language literature for animal studies that investigated the BSCB in vivo. Data extraction included animal model/type, protocol for BSCB evaluation, and study outcomes. Descriptive syntheses are provided. A total of 40 studies were included, which mainly investigated rodent models of experimental autoimmune encephalomyelitis (EAE) or spinal cord injury (SCI). The main techniques used were magnetic resonance imaging (MRI) and intravital microscopy (IVM). MRI served as a reliable tool to longitudinally track BSCB permeability changes with dynamic contrast enhancement (DCE) using gadolinium, or assess inflammatory infiltrations with targeted alternative contrast agents. IVM provided high-resolution visualization of cellular and molecular interactions across the microvasculature, commonly with either epi-fluorescence or two-photon microscopy. MRI and IVM techniques enabled the evaluation of therapeutic interventions and mechanisms that drive spinal cord dysfunction in EAE and SCI. A small number of studies demonstrated the feasibility of DCE-computed tomography, ultrasound, bioluminescent, and fluorescent optical imaging methods to evaluate the BSCB. Technique-specific limitations and multiple protocols for image acquisition and data analyses are described for all techniques. There are few in vivo investigations of the BSCB. Additional studies are needed in less commonly studied spinal cord disorders, and to establish standardized protocols for data acquisition and analysis. Further development of techniques and multimodal approaches could overcome current imaging limitations to the spinal cord. These advancements might promote wider adoption of techniques, and can provide greater potential for clinical translation.

Optimization of a Luciferase-Expressing Non-Invasive Intrapleural Model of Malignant Mesothelioma in Immunocompetent Mice.

Malignant Pleural Mesothelioma (MPM) is an aggressive tumor of the pleural lining that is usually identified at advanced stages and resistant to current therapies. Appropriate pre-clinical mouse tumor models are of pivotal importance to study its biology. Usually, tumor cells have been injected intraperitoneally or subcutaneously. Using three available murine mesothelioma cell lines with different histotypes (sarcomatoid, biphasic, epithelioid), we have set up a simplified model of in vivo growth orthotopically by inoculating tumor cells directly in the thorax with a minimally invasive procedure. Mesothelioma tumors grew along the pleura and spread on the superficial areas of the lungs, but no masses were found outside the thoracic cavity. As observed in human MPM, tumors were highly infiltrated by macrophages and T cells. The luciferase-expressing cells can be visualized in vivo by bioluminescent optical imaging to precisely quantify tumor growth over time. Notably, the bioluminescence signal detected in vivo correctly matched the tumor burden quantified with classical histology. In contrast, the subcutaneous or intraperitoneal growth of these mesothelioma cells was considered either non-representative of the human disease or unreliable to precisely quantify tumor load. Our non-invasive in vivo model of mesothelioma is simple and reproducible, and it reliably recapitulates the human disease.

Silver-coated magnetic nanocomposites induce growth inhibition and protein changes in foodborne bacteria

Cytotoxicity concerns of nanoparticles on animal or human bodies have led to the design of iron oxide core nanocomposites, coated with elemental silver to allow their magnetic removal from bio-mixtures. Although the antimicrobial effect of silver is well-described, the effects of nanoparticles derived from silver on microorganisms remain unfolded. Here, we characterized a customized magnetic silver nanocomposite (Ag-MNP) and evaluated its effects on bacterial growth and protein changes. The Ag-MNP displayed both longitudinal and round shapes under High-Resolution Transmission Electron Microscopy imaging, while the Energy Dispersive X-ray Spectroscopy and X-ray diffraction analysis confirmed the presence of Ag, Fe3O4 (Magnetite) and FeO2 (Goethite). Optical density, bioluminescence imaging, and Colony Forming Unit assessments revealed that the presence of Ag-MNP induced strong dose-dependent bacteria (Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and S. Anatum) growth inhibition. The TEM imaging showed penetration and infiltration of bacteria by Ag-MNP, leading to membrane degeneration and vacuole formation. The presence of Ag-MNP led to fifteen up-regulated and nine down-regulated proteins (P < 0.05) that are involved in cell membrane synthesis, inhibition of protein synthesis, interference with DNA synthesis, and energy metabolism inhibition. This study provides insights to develop alternative antimicrobials to treat foodborne pathogens with antibiotic resistance avoidance.

Preclinical Models and Methodologies for Monitoring Staphylococcus aureus Infections Using Noninvasive Optical Imaging.

In vivo whole-animal optical (bioluminescence and fluorescence) imaging of Staphylococcus aureus infections has provided the opportunity to noninvasively and longitudinally monitor the dynamics of the bacterial burden and ensuing host immune responses in live anesthetized animals. Herein, we describe several different mouse models of S. aureus skin infection, skin inflammation, incisional/excisional wound infections, as well as mouse and rabbit models of orthopedic implant infection, which utilized this imaging technology. These animal models and imaging methodologies provide insights into the pathogenesis of these infections and innate and adaptive immune responses, as well as the preclinical evaluation of diagnostic and treatment modalities. Noninvasive approaches to investigate host-pathogen interactions are extremely important as virulent community-acquired methicillin-resistant S. aureus strains (CA-MRSA) are spreading through the normal human population, becoming more antibiotic resistant and creating a serious threat to public health.

Imaging of Red-Shifted Light From Bioluminescent Tumors Using Fluorescence by Unbound Excitation From Luminescence.

Early detection of tumors is today a major challenge and requires sensitive imaging methodologies coupled with new efficient probes. In vivo optical bioluminescence imaging has been widely used in the field of preclinical oncology to visualize tumors and several cancer cell lines have been genetically modified to provide bioluminescence signals. However, the light emitted by the majority of commonly used luciferases is usually in the blue part of the visible spectrum, where tissue absorption is still very high, making deep tissue imaging non-optimal, and calling for optimized optical imaging methodologies. We have previously shown that red-shifting of bioluminescence signal by Fluorescence Unbound Excitation from Luminescence (FUEL) is a mean to increase bioluminescence signal sensitivity detection in vivo. Here, we applied FUEL to tumor detection in two different subcutaneous tumor models: the auto-luminescent human embryonic kidney (HEK293) cell line and the murine B16-F10 melanoma cell line previously transfected with a plasmid encoding the Luc2 firefly luciferase. Tumor size and bioluminescence were measured over time and tumor vascularization characterized. We then locally injected near infrared emitting Quantum Dots (NIR QDs) in the tumor site and observed a red-shifting of bioluminescence signal by (FUEL) indicating that FUEL could be used to allow deeper tumor detection in mice.

Orthotopic hepatocellular carcinoma: molecular imaging-monitored intratumoral hyperthermia-enhanced direct oncolytic virotherapy

Objective: To validate the feasibility of molecular imaging-monitored intratumoral radiofrequency hyperthermia (RFH) enhanced direct oncolytic virotherapy for hepatocellular carcinoma (HCC).Methods: This study included in vitro experiments using luciferase-labeled rat HCC cells and in vivo validation experiments on rat models with orthotopic HCCs. Both cells and HCCs in four groups (n = 6/group) were treated by: (1) combination therapy of oncolytic virotherapy (T-VEC) plus RFH at 42 °C for 30 min; (2) oncolytic virotherapy alone; (3) RFH alone; and (4) saline. For in vitro confirmation, confocal microscopy and bioluminescence optical imaging were used to evaluate the cell viabilities. For in vivo validation, oncolytic viruses were directly infused into rat HCCs through a multi-functional perfusion-thermal RF electrode, followed by RFH. Ultrasound and optical imaging were used to follow up size and bioluminescence signal changes of tumors overtime, which were correlated with subsequent laboratory examinations.Results: For in vitro experiments, confocal microscopy showed the lowest number of viable cells, as well as a significant decrease of bioluminescence signal intensity of cells with combination therapy group, compared to other three groups (p < .001). For in vivo experiments, ultrasound and optical imaging showed the smallest tumor volume, and significantly decreased bioluminescence signal intensity in combination therapy group compared to other three groups (p < .05), which were well correlated with pathologic analysis.Conclusion: It is feasible of using molecular imaging to guide RFH-enhanced intratumoral oncolytic virotherapy of HCC, which may open new avenues to prevent residual or recurrent disease of thermally ablated intermediate-to-large HCCs.

4:10 PM Abstract No. 50 Intratumoral radiofrequency hyperthermia-enhanced herpes simplex virus thymidine kinase (HSV-TK) gene therapy of orthotopic lung cancer through the augmentation of apoptosis and heat shock protein (HSP70) expression pathway

To investigate the associated biomolecular mechanisms of intratumoral RFH- enhanced local HSV-TK/ ganciclovir (GCV)-mediated suicide gene therapy of rat orthotopic lung cancers. Human lung cancer cells (A549) transduced with Luciferase/mCherry/lentivirus and 24 nude rats with orthotopic lung cancers were divided into four study groups with treatments of (i) intratumoral HSV-TK/GCV gene therapy followed by RFH at 41-42 °C for 30 minutes; (ii) HSV-TK/GCV therapy alone; (iii) RFH alone; (iv) PBS as a control. Flow cytometry, bioluminescence assay and confocal microscopy were used to determine the apoptosis and viability of cells. Bioluminescence optical imaging was used to evaluate the changes of bioluminescent signals among the groups over 2 weeks. The expression of Bcl-2/Bax, Caspase-3 and HSP70 in both cells and tumor tissue were quantified by western blotting (WB) and immunohistochemistical (IHC) staining respectively. Compared with gene therapy alone, RFH alone or PBS, combination therapy induced the lowest cell viability (30.56 ± 9.12% VS 65.47 ± 8.02% VS 89.95 ± 6.33% VS 100% P<0.05), the highest cell apoptosis (20.05 ± 2.12% VS 9.87 ± 2.02% VS 4.31 ± 4.31% VS 3.12 ± 1.03%, P<0.05), and a remarkable decrease of relative bioluminescence signal (16.54 ± 0.12% VS 54.8 ± 0.10% VS 85.01 ± 0.08% VS 100%, P<0.01). Optical imaging showed a markedly decreased bioluminescence signal of the tumor in combination therapy (1.29 ± 0.18 VS 1.76 ± 0.13 VS 2.26 ± 0.17 VS 2.94 ± 0.15, P<0.05). Both WB analysis and IHC staining displayed the significantly decreased expression of Bcl-2 (1.03 ± 0.28 VS 2.15 ± 0.33 VS 3.48 ± 0.12 VS 3.87 ± 0.17×107 IOD, P<0.01), as well as increased expression of Bax (3.30 ± 0.0.36 VS 2.02 ± 0.20 VS 1.48 ± 0.31 VS 1.26 ± 0.07, 107 IOD, P<0.01), Caspase-3 (5.58 ± 0.52 VS 3.36 ± 0.29 VS 1.42 ± 0.34VS 1.21 ± 0.52, 107 IOD, P<0.05) and HSP70 (8.79 ± 0.62 VS 3.97 ± 0.31 VS 4.48 ± 0.42 VS 1.60 ± 0.23, 107 IOD, P<0.01) in cancer tissues of combination therapy group. Intratumoral RFH-enhanced suicide gene therapy on rat orthotopic lung cancers through the mechanisms of augmenting apoptosis and the expression of HSP-70 pathway, which may provide additional insights to further improving treatment of lung cancers.

Optical Bioluminescence Protocol for Imaging Mice.

The presence, growth, or decline of transfected cell populations expressing the enzyme Luciferase can be followed in live mice using bioluminescence optical imaging techniques. This protocol describes how to verify the imaging equipment, options for injecting the substrate Luciferin into mice, image acquisition considerations, and commonly used data analysis techniques.

Orthotopic hepatic cancer: radiofrequency hyperthermia-enhanced intratumoral herpes simplex virus-thymidine kinase gene therapy.

PurposeTo validate the feasibility of using interventional radiofrequency hyperthermia(RFH) to enhance herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) gene therapy of rat orthotopic hepatic cancer.Material and MethodsRat hepatocellular carcinoma cells (MCA-RH-7777) were transduced with lentivirus/luciferase gene for optical imaging. In-vitro experiments with the luciferase cells and in-vivo experiments on rats with orthotopic hepatic tumors were divided into four treatment groups: (i) HSV-TK/GCV-mediated gene therapy combined with RFH; (ii) gene therapy alone; (iii) RFH alone; and (iv) phosphate buffered saline (PBS). Cell viability was evaluated by MTS assay and confocal microscopy, and HSV-TK gene expression in cells and tumors was quantified by western blotting. Bioluminescent optical imaging and ultrasound imaging were used to monitor and compare the photon signal and tumor size changes among different treatment groups overtime, respectively. The imaging findings were correlated with histology.ResultsFor in-vitro experiments, the combination therapy group (gene therapy + RFH) demonstrated the lowest cell proliferation by MTS assay, compared to the gene therapy alone, RFH alone, and PBS (26.1±3.2% vs 50.4±4.6% vs 82.9±6.3% vs 100%, p<0.01). The combination therapy group also showed fewer survived cells by the confocal microscopy and the lowest bioluminescent signal by the optical imaging. For in-vivo experiments, the combination therapy group demonstrated a significantly decreased signal intensity on the bioluminescent optical imaging (0.57±0.09, 1.06±0.10 vs 3.43±0.27 vs 3.85±0.12, p<0.05) and smallest tumor volume by ultrasound imaging (0.28±0.11 vs 1.28±0.23vs 4.64±0.35 vs 6.37±0.36, p<0.05), compared to the other three groups. Additionally, these imaging findings correlated well with the histological confirmation.ConclusionIt is feasible to use RFH to enhance HSV-TK/GCV gene therapy of hepatic tumors in in-vitro and in-vivo settings, as assessed by molecular imaging. This technical development may provide a novel opportunity for effective treatment of liver malignancies by employing simultaneous integration of radiofrequency technology, interventional oncology, and direct intratumoral gene therapy.

Cationic microbubbles and antibiotic-free miniplasmid for sustained ultrasound–mediated transgene expression in liver

Despite the increasing number of clinical trials in gene therapy, no ideal methods still allow non-viral gene transfer in deep tissues such as the liver. We were interested in ultrasound (US)-mediated gene delivery to provide long term liver expression. For this purpose, new positively charged microbubbles were designed and complexed with pFAR4, a highly efficient small length miniplasmid DNA devoid of antibiotic resistance sequence. Sonoporation parameters, such as insonation time, acoustic pressure and duration of plasmid injection were controlled under ultrasound imaging guidance. The optimization of these various parameters was performed by bioluminescence optical imaging of luciferase reporter gene expression in the liver. Mice were injected with 50μg pFAR4-LUC either alone, or complexed with positively charged microbubbles, or co-injected with neutral MicroMarker™ microbubbles, followed by low ultrasound energy application to the liver. Injection of the pFAR4 encoding luciferase alone led to a transient transgene expression that lasted only for two days. The significant luciferase signal obtained with neutral microbubbles decreased over 2days and reached a plateau with a level around 1 log above the signal obtained with pFAR4 alone. With the newly designed positively charged microbubbles, we obtained a much stronger bioluminescence signal which increased over 2days. The 12-fold difference (p<0.05) between MicroMarker™ and our positively charged microbubbles was maintained over a period of 6months. Noteworthy, the positively charged microbubbles led to an improvement of 180-fold (p<0.001) as regard to free pDNA using unfocused ultrasound performed at clinically tolerated ultrasound amplitude. Transient liver damage was observed when using the cationic microbubble-pFAR4 complexes and the optimized sonoporation parameters. Immunohistochemistry analyses were performed to determine the nature of cells transfected. The pFAR4 miniplasmid complexed with cationic microbubbles allowed to transfect mostly hepatocytes compared to its co-injection with MicroMarker™ which transfected more preferentially endothelial cells.

Multi-modality imaging-monitored creation of rat orthotopic pancreatic head cancer with obstructive jaundice.

PurposeTo investigate the feasibility of using multi-modality imaging to monitor the creation of rat models with orthotopic pancreatic head cancer with obstructive jaundice.Results27 of 52 rats (51.92%) developed pancreatic head cancer. The tumor formation rate was significantly higher in the animal group receiving bioluminescent tumor, compared to the group receiving non-bioluminescent donor tumors [78.1% (25/32 rats) vs 10.0% (2/20 rats), P = 0.0001]. Both ultrasound imaging and MRI clearly characterized the orthotopic tumors. Laboratory biochemistry test for those rats with obstructive jaundice showed elevated levels of bilirubin, aspartate transaminase (AST), alkaline phosphatase (ALT) and gamma-glutamyl transpeptidase (λ-GGT), compared with those rats without jaundice (P < 0.05). Correlative pathology confirmed that all tumors were ductal adenocarcinomas, and located in pancreatic head regions.Materials and MethodsRat pancreatic adenocarcinoma cells (DSL-6A/C1) were first transfected with lentivirus/mCherry-luciferase genes, and then subcutaneously implanted into flanks of donor immunocompetent Lewis rats, to create pancreatic tumor tissues. The tumor tissues from donor rats with either bioluminescence signal or without the signal were then transplanted into the pancreatic heads of 52 recipient Lewis rats. Bioluminescence optical and ultrasound imaging, as well as magnetic resonance imaging (MRI), were performed to follow up the tumor formation and growth in these tumor-transplanted rats. Physical examination and biochemistry test were used to discern the rats with obstructive jaundice. The rats were euthanized for subsequent histologic correlation and confirmation.ConclusionsWe successfully created a new rat model with orthotopic pancreatic head cancer, which can be accurately monitored and visualized by different imaging modalities.

106 ASSESSMENT OF ANTI-BACTERIAL EFFECTS OF PEGYLATED SILVER-COATED CARBON NANOTUBES ON CAUSATIVE BACTERIA OF BOVINE INFERTILITY USING BIOLUMINESCENCE IMAGING SYSTEM

Pathogenic bacteria including Escherichia coli and Salmonella sp. are the major causative agents of endometritis and can cause infertility in livestock animals. Antibiotics are commonly used to terminate bacterial infections, but the development of bacterial antibiotic resistance is often encountered. Nanotechnology associated with silver nanoparticles has been highlighted as an alternative anti-bacterial agent, and pegylated silver-coated single-walled carbon nanotubes have high anti-bacterial effects and are non-toxic to human and murine cells in vitro. Here we verified whether a real-time bioluminescence monitoring system could be an alternative tool to assess anti-bacterial effects of nanotubes in a noninvasive approach. Escherichia coli and Salmonella sp. were transfected with plasmids containing constructs for luciferase enzyme (LuxCDABE) and substrate (luciferin) to create self-illuminating bioluminescent bacteria. Pathogens were grown in LB broth at 37°C, adjusted to 107 cfu mL−1, and placed in 96-well plates for treatments. Pegylated (pSWCNTs-Ag) and non-pegylated (SWCNTs-Ag) nanotubes were prepared and added to culture wells at various concentrations (31.25–125 µg mL−1). The control group corresponded to bacteria without nanotubes (0 µg mL−1). Anti-bacterial effects of nanotubes were determined every 10 min until 1 h, then every 30 min up to 6 h incubation through optical density (600 nm) measurements and bioluminescence imaging (BLI) and quantification using an IVIS system. Optical density and BLI data were compared at each time-point using 2-way ANOVA, with P &lt; 0.05 set for significance. Bioluminescence signals emitted by both bacteria stains appeared within 10 min of incubation. Thereafter, control bacteria showed exponential growth that was detected as early as 25 min post-incubation. Bioluminescence imaging revealed dose-dependent anti-bacterial activities of both pSWCNTs-Ag and SWCNTs-Ag on each E. coli and Salmonella sp. (P &lt; 0.05). Contrary to BLI, the OD values did not always reflect bacteria concentrations, and varied according to nanotube concentrations. No significant differences in anti-bacterial activities were revealed between pSWCNTs-Ag and SWCNTs-Ag based on OD values during 6 h of incubation (P &gt; 0.05); meanwhile, pSWCNTs-Ag nanotubes exhibited stronger anti-bacterial effects than SWCNTs-Ag during the same period using BLI (P &lt; 0.05). In summary, we confirmed previous reports showing dose-dependent eliminations of pathogenic bacteria by silver nanotubes. Pegylated nanotubes exhibited high anti-bacterial activity compared to non-pegylated nanotubes. Bioluminescence imaging system revealed superior resolution to enable precise investigation of anti-bacterial kinetics of silver nanotubes. This feature could be useful for the study of bacterial infections that impair livestock fertility. Work was supported by USDA-ARS Biophotonics Initiative grant #58-6402-3-018.

A transgenic mouse model expressing an ERα folding biosensor reveals the effects of Bisphenol A on estrogen receptor signaling.

Estrogen receptor-α (ERα) plays an important role in normal and abnormal physiology of the human reproductive system by interacting with the endogenous ligand estradiol (E2). However, other ligands, either analogous or dissimilar to E2, also bind to ERα. This may create unintentional activation of ER signaling in reproductive tissues that can lead to cancer development. We developed a transgenic mouse model that constitutively expresses a firefly luciferase (FLuc) split reporter complementation biosensor (NFLuc-ER-LBDG521T-CFLuc) to simultaneously evaluate the dynamics and potency of ligands that bind to ERα. We first validated this model using various ER ligands, including Raloxifene, Diethylstilbestrol, E2, and 4-hydroxytamoxifen, by employing FLuc-based optical bioluminescence imaging of living mice. We then used the model to investigate the carcinogenic property of Bisphenol A (BPA), an environmental estrogen, by long-term exposure at full and half environmental doses. We showed significant carcinogenic effects on female animals while revealing activated downstream ER signaling as measured by bioluminescence imaging. BPA induced tumor-like outgrowths in female transgenic mice, histopathologically confirmed to be neoplastic and epithelial in origin. This transgenic mouse model expressing an ERα folding-biosensor is useful in evaluation of estrogenic ligands and their downstream effects, and in studying environmental estrogen induced carcinogenesis in vivo.

Intratumoral radiofrequency hyperthermia-enhanced direct chemotherapy of pancreatic cancer.

PurposeTo investigate the technical feasibility of using ultrasound-guided intratumoral radiofrequency hyperthermia (RFH) to enhance local chemotherapy of rat orthotopic pancreatic cancers.Materials and MethodsOrthotopic pancreatic cancer masses were established by inoculating luciferase/mCherry labeled-pancreatic cancer cells into the pancreatic tails of Lewis model rats via a laparotomy approach. Twenty-four rats with pancreatic cancer and 24 mice with subcutaneous pancreatic cancer xenografts in four study groups (n = 6/group) received various treatments: i) combination therapy of intratumoral MR imaging-heating-guidewire-mediated RFH (42oC) plus local chemotherapy (gemcitabine); ii) intratumoral chemotherapy alone; iii) RFH alone; and (iv)phosphate-buffered saline (PBS). Transcutaneous ultrasound imaging was used to guide the treatment and subsequently follow changes in tumor sizes. Bioluminescence optical imaging was performed to follow photon signal changes. Sonographic and optical findings were correlated with histology at 14 days.ResultsOptical imaging demonstrated a significantly decreased bioluminescence signal in mice with combination therapy group, compared with the other control groups (0.51±0.18 VS 1.6±0.4 VS 3.18±0.9 VS 3.5±0.96, p < 0.05). Ultrasound imaging showed the smallest tumor volumes of both mice and rat group with the combination therapy, compared with other control groups (0.62±0.16 VS 1.25±0.19 VS 2.28±0.25 VS 2.64±0.26, p < 0.05) and (0.75±0.18 VS 1.31±0.30 VS 1.61±0.28 VS 1.72±0.28, p < 0.05). Both imaging findings were confirmed by histologic correlation.ConclusionIntratumoral RFH can augment the chemotherapeutic effect in an orthotopic pancreatic cancer model.