Search Results (45)

Background: Preclinical cell tracking is enhanced with a multimodal imaging approach. Bioluminescence imaging (BLI) is a highly sensitive optical modality that relies on engineering cells to constitutively express a luciferase gene. Magnetic particle imaging (MPI) is a newer imaging modality that directly detects superparamagnetic iron oxide (SPIO) particles used to label cells. Here, we compare BLI and MPI for imaging cells in vitro and in vivo. Methods: Mouse 4T1 breast carcinoma cells were transduced to express firefly luciferase, labeled with SPIO (ProMag), and imaged as cell samples after subcutaneous injection into mice. Results: For cell samples, the BLI and MPI signals were strongly correlated with cell number. Both modalities presented limitations for imaging cells in vivo. For BLI, weak signal penetration, signal attenuation, and scattering prevented the detection of cells for mice with hair and for cells far from the tissue surface. For MPI, background signals obscured the detection of low cell numbers due to the limited dynamic range, and cell numbers could not be accurately quantified from in vivo images. Conclusions: It is important to understand the shortcomings of these imaging modalities to develop strategies to improve cellular detection sensitivity. Full article

Luciferase (luc) bioluminescence (BL) is the most used light-emitting protein that has been engineered to be expressed in multiple cancer cell lines, allowing for the detection of tumor nodules in vivo as it can penetrate most tissues. The goal of this study was to develop an oncolytic adenovirus (OAd)-resistant human triple-negative breast cancer (TNBC) that could express luciferase. Thus, when combining an OAd with chemotherapies or targeted therapies, we would be able to monitor the ability of these compounds to enhance OAd antitumor efficacy using BL in real time. The TNBC cell line HCC1937 was stably transfected with the plasmid pGL4.50[luc2/CMV/Hygro] (HCC1937/luc2). Once established, HCC1937/luc2 was orthotopically implanted in the 4th mammary gland fat pad of NSG (non-obese diabetic severe combined immunodeficiency disease gamma) female mice. Bioluminescence imaging (BLI) revealed that the HCC1937/luc2 cell line developed orthotopic breast tumor and lung metastasis over time. However, the integration of luc plasmid modified the HCC1937 phenotype, making HCC1937/luc2 more sensitive to OAdmCherry compared to the parental cell line and blunting the interferon (IFN) antiviral response. Testing two additional luc cell lines revealed that this was not a universal response; however, proper controls would need to be evaluated, as the integration of luciferase could affect the cells’ response to different treatments. Full article

Goat may represent a valid large animal model for human pathogens and new vaccines testing. Appropriate vaccine administration is a critical component of a successful immunization program. The wrong route of administration may reduce the efficacy of the vaccine, whereas the proper administration strategy can enhance it. Viral vectors have been employed successfully for goat and sheep immunization; however, no data concerning the vaginal route are available. A viral vector’s ability to transduce the site of inoculation is of primary interest. In this study, a fast and reliable ex vivo assay for testing the transduction capability of an Ad5-based vector when intravaginally administered was developed. An Ad5 vector delivering an expression cassette with a bicistronic reporter gene, Ad5-CMV-turboGFP-IRES-Luc2, was constructed. We demonstrated Ad5-CMV-turboGFP-IRES-Luc2’s ability to transduce caprine vaginal mucosa by ex vivo bioluminescent imaging (BLI) employing a simple CCD camera apparatus for chemiluminescence western immunoblotting. These data, though simple, provide valuable insights into developing a vaginal immunization strategy using a viral vector-based vaccine to protect against pathogens causing genital diseases. Full article

Bioluminescence imaging (BLI) is an indispensable technique for visualizing the dynamics of diverse biological processes in mammalian animal models, including cancer, viral infections, and immune responses. However, a critical scientific challenge remains: non-invasively visualizing homeostatic and disease mechanisms in freely moving animals to understand the molecular basis of exercises, social behavior, and other phenomena. Classical BLI relies on prolonged camera exposure to accumulate the limited number of photons that traveled from deep tissues in anesthetized or constrained animals. Recent advancements in synthetic bioluminescence reactions, utilizing artificial luciferin–luciferase pairs, have considerably increased the number of detectable photons from deep tissues, facilitating high-speed BLI to capture moving objects. In this review, I provide an overview of emerging synthetic bioluminescence reactions that enable the non-invasive imaging of freely moving animals. This approach holds the potential to uncover unique physiological processes that are inaccessible with current methodologies. Full article

Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO^® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design. Full article

The advancement of stem cell therapy has offered transformative therapeutic outcomes for a wide array of diseases over the past decades. Consequently, stem cell tracking has become significant in revealing the mechanisms of action and ensuring safe and effective treatments. Fluorescence stands out as a promising choice for stem cell tracking due to its myriad advantages, including high resolution, real-time monitoring, and multi-fluorescence detection. Furthermore, combining fluorescence with other tracking modalities—such as bioluminescence imaging (BLI), positron emission tomography (PET), photoacoustic (PA), computed tomography (CT), and magnetic resonance (MR)—can address the limitations of single fluorescence detection. This review initially introduces stem cell tracking using fluorescence imaging, detailing various labeling strategies such as green fluorescence protein (GFP) tagging, fluorescence dye labeling, and nanoparticle uptake. Subsequently, we present several combinations of strategies for efficient and precise detection. Full article

Bioluminescence imaging (BLI) is a widely used technique in preclinical scientific research, particularly in the development of mRNA-based medications and non-invasive tumor diagnostics. It has become an essential tool in current science. However, the current state of bioluminescence imaging is primarily qualitative, making it challenging to obtain quantitative measurements and to draw accurate conclusions. This fact is caused by the unique properties of optical photons and tissue interactions. In this paper, we propose an experimental setup and Geant4-simulations to gain a better understanding of the optical properties and processes involved in bioluminescence imaging. Our goal is to advance the field towards more quantitative measurements. We will discuss the details of our experimental setup, the data we collected, the outcomes of the Geant4-simulations, and additional information on the underlying physical processes. Full article

Virulent Aeromonas hydrophila (vAh) strains that cause motile Aeromonas septicemia (MAS) in farmed channel catfish (Ictalurus punctatus) have been an important problem for more than a decade. However, the routes of infection of vAh in catfish are not well understood. Therefore, it is critical to study the pathogenicity of vAh in catfish. To this goal, a new bioluminescence expression plasmid (pAKgfplux3) with the chloramphenicol acetyltransferase (cat) gene was constructed and mobilized into vAh strain ML09-119, yielding bioluminescent vAh (BvAh). After determining optimal chloramphenicol concentration, plasmid stability, bacteria number–bioluminescence relationship, and growth kinetics, the catfish were challenged with BvAh, and bioluminescent imaging (BLI) was conducted. Results showed that 5 to 10 µg/mL chloramphenicol was suitable for stable bioluminescence expression in vAh, with some growth reduction. In the absence of chloramphenicol, vAh could not maintain pAKgfplux3 stably, with the half-life being 16 h. Intraperitoneal injection, immersion, and modified immersion (adipose fin clipping) challenges of catfish with BvAh and BLI showed that MAS progressed faster in the injection group, followed by the modified immersion and immersion groups. BvAh was detected around the anterior mouth, barbels, fin bases, fin epithelia, injured skin areas, and gills after experimental challenges. BLI revealed that skin breaks and gills are potential attachment and entry portals for vAh. Once vAh breaches the skin or epithelial surfaces, it can cause a systemic infection rapidly, spreading to all internal organs. To our best knowledge, this is the first study that reports the development of a bioluminescent vAh and provides visual evidence for catfish–vAh interactions. Findings are expected to provide a better understanding of vAh pathogenicity in catfish. Full article

Bladder cancer (BCa) is the sixth most prevalent cancer in men and seventeenth most prevalent cancer in women worldwide. Current treatment paradigms have limited therapeutic impact, suggesting an urgent need for the investigation of novel therapies. To best emulate the progression of human BCa, a pre-clinical intravesical murine model is required in conjunction with existing non-invasive imaging modalities to detect and evaluate cancer progression. Non-invasive imaging modalities reduce the number of required experimental models while allowing for longitudinal studies of novel therapies to investigate long-term efficacy. In this review, we discuss the individual and multi-modal use of non-invasive imaging modalities; bioluminescence imaging (BLI), micro-ultrasound imaging (MUI), magnetic resonance imaging (MRI), and positron emission tomography (PET) in BCa evaluation. We also provide an update on the potential and the future directions of imaging modalities in relation to intravesical murine models of BCa. Full article

Activated hepatic stellate cells (HSCs) play a detrimental role in liver fibrosis progression. Natural killer (NK) cells are known to selectively recognize abnormal or transformed cells via their receptor activation and induce target cell apoptosis and, therefore, can be used as a potential therapeutic strategy for liver cirrhosis. In this study, we examined the therapeutic effects of NK cells in the carbon tetrachloride (CCl₄)-induced liver cirrhosis mouse model. NK cells were isolated from the mouse spleen and expanded in the cytokine-stimulated culture medium. Natural killer group 2, member D (NKG2D)-positive NK cells were significantly increased after a week of expansion in culture. The intravenous injection of NK cells significantly alleviated liver cirrhosis by reducing collagen deposition, HSC marker activation, and macrophage infiltration. For in vivo imaging, NK cells were isolated from codon-optimized luciferase-expressing transgenic mice. Luciferase-expressing NK cells were expanded, activated and administrated to the mouse model to track them. Bioluminescence images showed increased accumulation of the intravenously inoculated NK cells in the cirrhotic liver of the recipient mouse. In addition, we conducted QuantSeq 3′ mRNA sequencing-based transcriptomic analysis. From the transcriptomic analysis, 33 downregulated genes in the extracellular matrix (ECM) and 41 downregulated genes involved in the inflammatory response were observed in the NK cell-treated cirrhotic liver tissues from the 1532 differentially expressed genes (DEGs). This result indicated that the repetitive administration of NK cells alleviated the pathology of liver fibrosis in the CCl₄-induced liver cirrhosis mouse model via anti-fibrotic and anti-inflammatory mechanisms. Taken together, our research demonstrated that NK cells could have therapeutic effects in a CCl₄-induced liver cirrhosis mouse model. In particular, it was elucidated that extracellular matrix genes and inflammatory response genes, which were mainly affected after NK cell treatment, could be potential targets. Full article

Orthotopic glioblastoma xenografts are paramount for evaluating the effect of innovative anti-cancer treatments. In longitudinal studies, tumor growth (or regression) of glioblastoma can only be monitored by noninvasive imaging. For this purpose, bioluminescence imaging (BLI) has gained popularity because of its low cost and easy access. In the context of the development of new nanomedicines for treating glioblastoma, we were using luciferase-expressing GL261 cell lines. Incidentally, using BLI in a specific GL261 glioblastoma model with cells expressing both luciferase and the green fluorescent protein (GL261-luc-GFP), we observed an apparent spontaneous regression. By contrast, the magnetic resonance imaging (MRI) analysis revealed that the tumors were actually growing over time. For other models (GL261 expressing only luciferase and U87 expressing both luciferase and GFP), data from BLI and MRI correlated well. We found that the divergence in results coming from different imaging modalities was not due to the tumor localization nor the penetration depth of light but was rather linked to the instability in luciferase expression in the viral construct used for the GL261-luc-GFP model. In conclusion, the use of multi-modality imaging prevents possible errors in tumor growth evaluation, and checking the stability of luciferase expression is mandatory when using BLI as the sole imaging modality. Full article

Magnetic particle imaging (MPI) provides hotspot tracking and direct quantification of superparamagnetic iron oxide nanoparticle (SPIO)-labelled cells. Bioluminescence imaging (BLI) with the luciferase reporter gene Akaluc can provide complementary information on cell viability. Thus, we explored combining these technologies to provide a more holistic view of cancer cell fate in mice. Akaluc-expressing 4T1Br5 cells were labelled with the SPIO Synomag-D and injected into the mammary fat pads (MFP) of four nude mice. BLI was performed on days 0, 6 and 13, and MPI was performed on days 1, 8 and 14. Ex vivo histology and fluorescence microscopy of MFP and a potential metastatic site was conducted. The BLI signal in the MFP increased significantly from day 0 to day 13 (p < 0.05), mirroring tumor growth. The MPI signal significantly decreased from day 1 to day 14 (p < 0.05) due to SPIO dilution in proliferating cells. Both modalities detected secondary metastases; however, they were visualized in different anatomical regions. Akaluc BLI complemented MPI cell tracking, allowing for longitudinal measures of cell viability and sensitive detection of distant metastases at different locations. We predict this multimodal imaging approach will help to evaluate novel therapeutics and give a better understanding of metastatic mechanisms. Full article

Sanyin formula (SYF) is used as a complementary treatment for triple-negative breast cancer (TNBC). The purpose of this study was to identify the potential functional components and clarify the underlying molecular mechanisms of SYF in TNBC. High-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) was used to identify the main components of SYF extracts. Network pharmacology and bioinformatic analyses were carried out to identify potential candidate targets of SYF in TNBC. Cell proliferation was determined with a Celigo imaging cytometer. Wound-healing and Transwell assays were adopted to evaluate cell migration. A Transwell cell-invasion assay was performed with Matrigel-coated membranes. In vivo bioluminescence imaging (BLI) and pathological analyses illustrated the effect of SYF on cancer cell metastasis in tumour-bearing mice. The inhibitory mechanism of SYF was investigated via quantitative PCR (qPCR) and Western blotting. We found that 3,4-dihydroxyphenyllactic acid, kaempferol, p-coumaric acid, and vanillic acid may be the active components of SYF. Molecular docking confirmed that kaempferol, p-coumaric acid, vanillic acid, and 3,4-dihydroxyphenyllactic acid bound stably to proteins such as AKR1C3, MMPs, and STAT3. SYF extract suppressed TNBC cell proliferation, migration, invasion, and metastasis by inhibiting JAK/STAT3 signalling and then regulating downstream genes, such as MMP-2/MMP-9. SYF regulates the expression of genes involved in cell proliferation, migration, and invasion by regulating the JAK/STAT3 signalling pathway and finally inhibits tumour cell metastasis in TNBC. The present study clarifies the mechanism by which SYF inhibits TNBC metastasis and lays an experimental foundation for the continued clinical development of SYF targeting the JAK/STAT3 pathway. Full article

Prepubertal boys with cancer may suffer from reduced fertility and maturity following gonadotoxic chemoradiotherapy. Thus, a viable method of immature testicular tissue (ITT) preservation is required in this cohort. In this study, we used poly-L-lactic acid electrospun scaffolds with two levels of fineness to support the development of ITT transplanted from transgenic donors to wild-type recipient mice. The purpose of this study was to evaluate the potential of ITT transplantation and spermatogenesis after using the two scaffolds, employing bioluminescence imaging for evaluation. The results suggest that ITT from 4-week-old mice possessed the most potential in spermatogenesis on the 70th day, together with the fine electrospun scaffolds. Moreover, bioluminescent imaging intensity was observed in recipient mice for up to 107 days, approximately six times more than the coarse electrospun scaffold and the control group. This occurs since the fine scaffold is more akin to the microenvironment of native testicular tissue as it reduces stiffness resulting from micronization and body fluid infiltration. The thermal analysis also exhibited recrystallization during the biodegradation process, which can lead to a more stable microenvironment. Overall, these findings present the prospect of fertility preservation in prepubertal males and could serve as a framework for future applications. Full article

The vascular disrupting activity of a promising tubulin-binding agent (OXi6196) was demonstrated in mice in MDA-MB-231 human breast tumor xenografts growing orthotopically in mammary fat pad and syngeneic RENCA kidney tumors growing orthotopically in the kidney. To enhance water solubility, OXi6196, was derivatized as its corresponding phosphate prodrug salt OXi6197, facilitating effective delivery. OXi6197 is stable in water, but rapidly releases OXi6196 in the presence of alkaline phosphatase. At low nanomolar concentrations OXi6196 caused G2/M cell cycle arrest and apoptosis in MDA-MB-231 breast cancer cells and monolayers of rapidly growing HUVECs underwent concentration-dependent changes in their morphology. Loss of the microtubule structure and increased bundling of filamentous actin into stress fibers followed by cell collapse, rounding and blebbing was observed. OXi6196 (100 nM) disrupted capillary-like endothelial networks pre-established with HUVECs on Matrigel^®. When prodrug OXi6197 was administered to mice bearing orthotopic MDA-MB-231-luc tumors, dynamic bioluminescence imaging (BLI) revealed dose-dependent vascular shutdown with >80% signal loss within 2 h at doses ≥30 mg/kg and >90% shutdown after 6 h for doses ≥35 mg/kg, which remained depressed by at least 70% after 24 h. Twice weekly treatment with prodrug OXi6197 (20 mg/kg) caused a significant tumor growth delay, but no overall survival benefit. Similar efficacy was observed for the first time in orthotopic RENCA-luc tumors, which showed massive hemorrhage and necrosis after 24 h. Twice weekly dosing with prodrug OXi6197 (35 mg/kg) caused tumor growth delay in most orthotopic RENCA tumors. Immunohistochemistry revealed extensive necrosis, though with surviving peripheral tissues. These results demonstrate effective vascular disruption at doses comparable to the most effective vascular-disrupting agents (VDAs) suggesting opportunities for further development. Full article