Temporal and regional expression changes and co-staining patterns of metabolic and stemness-related markers during glioblastoma progression.

Glioblastomas (GBMs) are characterized by high heterogeneity, involving diverse cell types, including those with stem-like features contributing to GBM's malignancy. Moreover, metabolic alterations promote growth and therapeutic resistance of GBM. Depending on the metabolic state, antimetabolic treatments could be an effective strategy. Against this background, we investigated temporal and regional expression changes and co-staining patterns of selected metabolic markers [pyruvate kinase muscle isozyme 1/2 (PKM1/2), glucose transporter 1 (GLUT1), monocarboxylate transporter 1/4 (MCT1/4)] in a rodent model and patient-derived samples of GBM. To understand the cellular sources of marker expression, we also examined the connection of metabolic markers to markers related to stemness [Nestin, Krüppel-like factor 4 (KLF4)] in a regional and temporal context. Rat tumour biopsies revealed a temporally increasing expression of GLUT1, higher expression of MCT1/4, Nestin and KLF4, and lower expression of PKM1 compared to the contralateral hemisphere. Patient-derived tumours showed a higher expression of PKM2 and Nestin in the tumour centre vs. edge. Whereas rare co-staining of GLUT1/Nestin was found in tumour biopsies, PKM1/2 and MCT1/4 showed a more distinct co-staining with Nestin in rats and humans. KLF4 was mainly co-stained with GLUT1, MCT1 and PKM1/2 in rat and human tumours. All metabolic markers yielded individual co-staining patterns among themselves. Co-staining mainly occurred later in tumour progression and was more pronounced in tumour centres. Also, positive correlations were found amongst markers that showed co-staining. Our results highlight a link between metabolic alterations and stemness in GBM progression, with complex distinctions depending on studied markers, time points and regions.

Incidence of microvascular dysfunction is increased in hyperlipidemic mice, reducing cerebral blood flow and impairing remote memory.

Introduction: The development of cognitive dysfunction is not necessarily associated with diet-induced obesity. We hypothesized that cognitive dysfunction might require additional vascular damage, for example, in atherosclerotic mice. Methods: We induced atherosclerosis in male C57BL/6N mice by injecting AAV-PCSK9DY (2x1011 VG) and feeding them a cholesterol-rich Western diet. After 3 months, mice were examined for cognition using Barnes maze procedure and for cerebral blood flow. Cerebral vascular morphology was examined by immunehistology. Results: In AAV-PCSK9DY-treated mice, plaque burden, plasma cholesterol, and triglycerides are elevated. RNAseq analyses followed by KEGG annotation show increased expression of genes linked to inflammatory processes in the aortas of these mice. In AAV-PCSK9DY-treated mice learning was delayed and long-term memory impaired. Blood flow was reduced in the cingulate cortex (-17%), caudate putamen (-15%), and hippocampus (-10%). Immunohistological studies also show an increased incidence of string vessels and pericytes (CD31/Col IV staining) in the hippocampus accompanied by patchy blood-brain barrier leaks (IgG staining) and increased macrophage infiltrations (CD68 staining). Discussion: We conclude that the hyperlipidemic PCSK9DY mouse model can serve as an appropriate approach to induce microvascular dysfunction that leads to reduced blood flow in the hippocampus, which could explain the cognitive dysfunction in these mice.

Epithelial and Mesenchymal-like Pancreatic Cancer Cells Exhibit Different Stem Cell Phenotypes Associated with Different Metastatic Propensities.

Pancreatic ductal adenocarcinoma (PDAC) is mostly diagnosed at advanced or even metastasized stages, limiting the prognoses of patients. Metastasis requires high tumor cell plasticity, implying phenotypic switching in response to changing environments. Here, epithelial-mesenchymal transition (EMT), being associated with an increase in cancer stem cell (CSC) properties, and its reversion are important. Since it is poorly understood whether different CSC phenotypes exist along the EMT axis and how these impact malignancy-associated properties, we aimed to characterize CSC populations of epithelial and mesenchymal-like PDAC cells. Single-cell cloning revealed CSC (Holoclone) and non-CSC (Paraclone) clones from the PDAC cell lines Panc1 and Panc89. The Panc1 Holoclone cells showed a mesenchymal-like phenotype, dominated by a high expression of the stemness marker Nestin, while the Panc89 Holoclone cells exhibited a SOX2-dominated epithelial phenotype. The Panc89 Holoclone cells showed enhanced cell growth and a self-renewal capacity but slow cluster-like invasion. Contrarily, the Panc1 Holoclone cells showed slower cell growth and self-renewal ability but were highly invasive. Moreover, cell variants differentially responded to chemotherapy. In vivo, the Panc1 and Panc89 cell variants significantly differed regarding the number and size of metastases, as well as organ manifestation, leading to different survival outcomes. Overall, these data support the existence of different CSC phenotypes along the EMT axis in PDAC, manifesting different metastatic propensities.

Maternal microbiota communicates with the fetus through microbiota-derived extracellular vesicles.

BACKGROUND: Reports regarding the presence of bacteria in the fetal environment remain limited and controversial. Recently, extracellular vesicles secreted by the human gut microbiota have emerged as a novel mechanism for host-microbiota interaction. We aimed to investigate the presence of bacterial extracellular vesicles in the fetal environment during healthy pregnancies and determine whether extracellular vesicles derived from the gut microbiota can cross biological barriers to reach the fetus. RESULTS: Bacterial extracellular vesicles were detectable in the amniotic fluid of healthy pregnant women, exhibiting similarities to extracellular vesicles found in the maternal gut microbiota. In pregnant mice, extracellular vesicles derived from human maternal gut microbiota were found to reach the intra-amniotic space. CONCLUSIONS: Our findings reveal maternal microbiota-derived extracellular vesicles as an interaction mechanism between the maternal microbiota and fetus, potentially playing a pivotal role in priming the prenatal immune system for gut colonization after birth. Video Abstract.

Multiscale morphological analysis of bone microarchitecture around Mg-10Gd implants.

The utilization of biodegradable magnesium (Mg)-based implants for restoration of bone function following trauma represents a transformative approach in orthopaedic application. One such alloy, magnesium-10 weight percent gadolinium (Mg-10Gd), has been specifically developed to address the rapid degradation of Mg while enhancing its mechanical properties to promote bone healing. Previous studies have demonstrated that Mg-10Gd exhibits favorable osseointegration; however, it exhibits distinct ultrastructural adaptation in comparison to conventional implants like titanium (Ti). A crucial aspect that remains unexplored is the impact of Mg-10Gd degradation on the bone microarchitecture. To address this, we employed hierarchical three-dimensional imaging using synchrotron radiation in conjunction with image-based finite element modelling. By using the methods outlined, the vascular porosity, lacunar porosity and the lacunar-canaliculi network (LCN) morphology of bone around Mg-10Gd in comparison to Ti in a rat model from 4 weeks to 20 weeks post-implantation was investigated. Our investigation revealed that within our observation period, the degradation of Mg-10Gd implants was associated with significantly lower (p < 0.05) lacunar density in the surrounding bone, compared to Ti. Remarkably, the LCN morphology and the fluid flow analysis did not significantly differ for both implant types. In summary, a more pronounced lower lacunae distribution rather than their morphological changes was detected in the surrounding bone upon the degradation of Mg-10Gd implants. This implies potential disparities in bone remodelling rates when compared to Ti implants. Our findings shed light on the intricate relationship between Mg-10Gd degradation and bone microarchitecture, contributing to a deeper understanding of the implications for successful osseointegration.

Establishment of a Rodent Glioblastoma Partial Resection Model for Chemotherapy by Local Drug Carriers-Sharing Experience.

Local drug delivery systems (LDDS) represent a promising therapy strategy concerning the most common and malignant primary brain tumor glioblastoma (GBM). Nevertheless, to date, only a few systems have been clinically applied, and their success is very limited. Still, numerous new LDDS approaches are currently being developed. Here, (partial resection) GBM animal models play a key role, as such models are needed to evaluate the therapy prior to any human application. However, such models are complex to establish, and only a few reports detail the process. Here, we report our results of establishing a partial resection glioma model in rats suitable for evaluating LDDS. C6-bearing Wistar rats and U87MG-spheroids- and patient-derived glioma stem-like cells-bearing athymic rats underwent tumor resection followed by the implantation of an exemplary LDDS. Inoculation, tumor growth, residual tumor tissue, and GBM recurrence were reliably imaged using high-resolution Magnetic Resonance Imaging. The release from an exemplary LDDS was verified in vitro and in vivo using Fluorescence Molecular Tomography. The presented GBM partial resection model appears to be well suited to determine the efficiency of LDDS. By sharing our expertise, we intend to provide a powerful tool for the future testing of these very promising systems, paving their way into clinical application.

Exploring the Usability of α-MSH-SM-Liposome as an Imaging Agent to Study Biodegradable Bone Implants In Vivo.

Novel biodegradable metal alloys are increasingly used as implant materials. The implantation can be accompanied by an inflammatory response to a foreign object. For studying inflammation in the implantation area, non-invasive imaging methods are needed. In vivo imaging for the implanted area and its surroundings will provide beneficiary information to understand implant-related inflammation and help to monitor it. Therefore, inflammation-sensitive fluorescent liposomes in rats were tested in the presence of an implant to evaluate their usability in studying inflammation. The sphingomyelin-containing liposomes carrying alpha-melanocyte-stimulating hormone (α-MSH)-peptide were tested in a rat bone implant model. The liposome interaction with implant material (Mg-10Gd) was analyzed with Mg-based implant material (Mg-10Gd) in vitro. The liposome uptake process was studied in the bone-marrow-derived macrophages in vitro. Finally, this liposomal tracer was tested in vivo. It was found that α-MSH coupled sphingomyelin-containing liposomes and the Mg-10Gd implant did not have any disturbing influence on each other. The clearance of liposomes was observed in the presence of an inert and biodegradable implant. The degradable Mg-10Gd was used as an alloy example; however, the presented imaging system offers a new possible use of α-MSH-SM-liposomes as tools for investigating implant responses.

Performance and reproducibility of 13C and 15N hyperpolarization using a cryogen-free DNP polarizer.

The setup, operational procedures and performance of a cryogen-free device for producing hyperpolarized contrast agents using dissolution dynamic nuclear polarization (dDNP) in a preclinical imaging center is described. The polarization was optimized using the solid-state, DNP-enhanced NMR signal to calibrate the sample position, microwave and NMR frequency and power and flip angle. The polarization of a standard formulation to yield ~ 4 mL, 60 mM 1-13C-pyruvic acid in an aqueous solution was quantified in five experiments to P(13C) = (38 ± 6) % (19 ± 1) s after dissolution. The mono-exponential time constant of the build-up of the solid-state polarization was quantified to (1032 ± 22) s. We achieved a duty cycle of 1.5 h that includes sample loading, monitoring the polarization build-up, dissolution and preparation for the next run. After injection of the contrast agent in vivo, pyruvate, pyruvate hydrate, lactate, and alanine were observed, by measuring metabolite maps. Based on this work sequence, hyperpolarized 15N urea was obtained (P(15N) = (5.6 ± 0.8) % (30 ± 3) s after dissolution).

Dynamic in vivo monitoring of fracture healing process in response to magnesium implant with multimodal imaging: pilot longitudinal study in a rat external fixation model.

Rodent models are commonly used in pre-clinical research of magnesium (Mg)-based and other types of biomaterials for fracture treatment. Most studies selected unstable fixation methods, and there is a lack of multimodal longitudinal in vivo monitoring of bone healing. The purpose of this study is to develop a rat femoral fracture model stabilized by external fixation with intra-medullary Mg implant, and to investigate the dynamic bone union process with several imaging techniques offering complementing insights into the process. Pure Mg pins were prepared, followed by an in vitro degradation test. Male Sprague-Dawley rats in the experimental group underwent femoral osteotomy stabilized by external fixators with intra-medullary implantation of Mg pins, and the control group underwent external fixation without intra-medullary implants. Post-operative radiograph, micro-CT and B-mode ultrasonography were acquired directly after surgery, and re-examined at week 4, 8 and 12. Bone tissue volume, in vivo implant degradation, histological staining and MRI images were analyzed using ex vivo samples. Both groups achieved fracture union at week 12, and the dynamic healing process was illustrated by in vivo radiograph, micro-CT and ultrasonography. Bilateral whole femur ex vivo analysis further demonstrated increased ratio of bone tissue volume in the surgical femur with Mg implants, and in vivo degradation of Mg pins was slower than in vitro results. Titanium screws rather than intra-medullary Mg pins were the source of artifact in MRI. This pilot study showed the rat fracture model with external fixation and intra-medullary Mg implantation to be an effective method for dynamic in vivo monitoring of the bone healing process. Future application of the animal model may facilitate pre-clinical translational research of biodegradable orthopaedic implant materials for fracture treatment.

Utilizing Sphingomyelinase Sensitizing Liposomes in Imaging Intestinal Inflammation in Dextran Sulfate Sodium-Induced Murine Colitis.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the gastrointestinal tract, resulting in severe symptoms. At the moment, the goal of medical treatments is to reduce inflammation. IBD is treated with systemic anti-inflammatory compounds, but they have serious side effects. The treatment that is most efficient and causes the fewest side effects would be the delivery of the drugs on the disease site. This study aimed to investigate the suitability of sphingomyelin (SM) containing liposomes to specifically target areas of inflammation in dextran sulfate sodium-induced murine colitis. Sphingomyelin is a substrate to the sphingomyelinase enzyme, which is only present outside cells in cell stress, like inflammation. When sphingomyelin consisting of liposomes is predisposed to the enzyme, it causes the weakening of the membrane structure. We demonstrated that SM-liposomes are efficiently taken up in intestinal macrophages, indicating their delivery potential. Furthermore, our studies showed that sphingomyelinase activity and release are increased in a dextran sulfate sodium-induced IBD mouse model. The enzyme appearance in IBD disease was also traced in intestine samples of the dextran sulfate sodium-treated mice and human tissue samples. The results from the IBD diseased animals, treated with fluorescently labeled SM-liposomes, demonstrated that the liposomes were taken up preferentially in the inflamed colon. This uptake efficiency correlated with sphingomyelinase activity.

Multimodal Targeted Nanoparticle-Based Delivery System for Pancreatic Tumor Imaging in Cellular and Animal Models.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), which ranks forth on the cancer-related death statistics still is both a diagnostic and a therapeutic challenge. Adenocarcinoma of the exocrine human pancreas originates in most instances from malignant transformation of ductal epithelial cells, alternatively by Acinar-Ductal Metaplasia (ADM). RA-96 antibody targets to a mucin M1, according to the more recent nomenclature MUC5AC, an extracellular matrix component excreted by PDAC cells. In this study, we tested the usability of multimodal nanoparticle carrying covalently coupled RA-96 Fab fragments for pancreatic tumor imaging. METHODS: In order to make and evaluate a novel, better targeting, theranostic nanoparticle, iron nanoparticles and the optical dye indocyanin green (ICG) were encapsulated into the cationic sphingomyelin (SM) consisting liposomes. RA-96 Fab fragment was conjugated to the liposomal surface of the nanoparticle to increase tumor homing ability. ICG and iron nanoparticle-encapsulated liposomes were studied in vitro with cells and (i) their visibility in magnetic resonance imaging (MRI), (ii) optical, (iii) Magnetic particle spectroscopy (MPS) and (iv) photoacoustic settings was tested in vitro and also in in vivo models. The targeting ability and MRI and photoacoustic visibility of the RA-96-nanoparticles were first tested in vitro cell models where cell binding and internalization were studied. In in vivo experiments liposomal nanoparticles were injected into the tail vain using an orthotopic pancreatic tumor xenograft model and subcutaneous pancreatic cancer cell xenografts bearing mice to determine in vivo targeting abilities of RA-96-conjugated liposomes Results: Multimodal liposomes could be detected by MRI, MPS and by photoacoustic imaging in addition to optical imaging showing a wide range of imaging utility. The fluorescent imaging of ICG in pancreatic tumor cells Panc89 and Capan-2 revealed an increased association of ICG-encapsulated liposomes carrying RA-96 Fab fragments in vitro compared to the control liposomes without covalently linked RA-96. Fluorescent molecular tomography (FMT) studies showed increased accumulation of the RA96-targeted nanoparticles in the tumor area compared to non-targeted controls in vivo. Similar accumulation in the tumor sites could be seen with liposomal ferric particles in MRI. Fluorescent tumor signal was confirmed by using an intraoperative fluorescent imaging system, which showed fluorescent labeling of pancreatic tumors. CONCLUSION: These results suggest that RA-96-targeted liposomes encapsulating ICG and iron nanoparticles can be used to image pancreatic tumors with a variety of optical and magnetic imaging techniques. Additionally, they might be a suitable drug delivery tool to improve treatment of PDAC patients.

Telmisartan prevents high-fat diet-induced neurovascular impairments and reduces anxiety-like behavior.

Angiotensin II receptor blockers (telmisartan) prevent rodents from diet-induced obesity and improve their metabolic status. Hyperglycemia and obesity are associated with reduced cerebral blood flow and neurovascular uncoupling which may lead to behavioral deficits. We wanted to know whether a treatment with telmisartan prevents these changes in obesity.We put young mice on high-fat diet and simultaneously treated them with telmisartan. At the end of treatment, we performed laser speckle imaging and magnetic resonance imaging to assess the effect on neurovascular coupling and cerebral blood flow. Different behavioral tests were used to investigate cognitive function.Mice developed diet-induced obesity and after 16, not 8 weeks of high-fat diet, however, the response to whisker pad stimulation was about 30% lower in obese compared to lean mice. Simultaneous telmisartan treatment increased the response again by 10% compared to obese mice. Moreover, telmisartan treatment normalized high-fat diet-induced reduction of cerebral blood flow and prevented a diet-induced anxiety-like behavior. In addition to that, telmisartan affects cellular senescence and string vessel formation in obesity.We conclude, that telmisartan protects against neurovascular unit impairments in a diet-induced obesity setting and may play a role in preventing obesity related cognitive deficits in Alzheimer's disease.

Tissue responses after implantation of biodegradable Mg alloys evaluated by multimodality 3D micro-bioimaging in vivo.

The local response of tissue triggered by implantation of degradable magnesium-based implant materials was investigated in vivo in a murine model. Pins (5.0 mm length by 0.5 mm diameter) made of Mg, Mg-10Gd, and Ti were implanted in the leg muscle tissue of C57Bl/6N mice (n = 6). Implantation was generally well tolerated as documented by only a mild short term increase in a multidimensional scoring index. Lack of difference between the groups indicated that the response was systemic and surgery related rather than material dependent. Longitudinal in vivo monitoring utilizing micro-computed tomography over 42 days demonstrated the highest and most heterogeneous degradation for Mg-10Gd. Elemental imaging of the explants by micro X-ray fluorescence spectrometry showed a dense calcium-phosphate-containing degradation layer. In order to monitor resulting surgery induced and/or implant material associated local cell stress, sphingomyelin based liposomes containing indocyanine green were administered. An initial increase in fluorescent signals (3-7 days after implantation) indicating cell stress at the site of the implantation was measured by in vivo fluorescent molecular tomography. The signal decreased until the 42nd day for all materials. These findings demonstrate that Mg based implants are well tolerated causing only mild and short term adverse reactions.

Lipid-Iron Nanoparticle with a Cell Stress Release Mechanism Combined with a Local Alternating Magnetic Field Enables Site-Activated Drug Release.

Most available cancer chemotherapies are based on systemically administered small organic molecules, and only a tiny fraction of the drug reaches the disease site. The approach causes significant side effects and limits the outcome of the therapy. Targeted drug delivery provides an alternative to improve the situation. However, due to the poor release characteristics of the delivery systems, limitations remain. This report presents a new approach to address the challenges using two fundamentally different mechanisms to trigger the release from the liposomal carrier. We use an endogenous disease marker, an enzyme, combined with an externally applied magnetic field, to open the delivery system at the correct time only in the disease site. This site-activated release system is a novel two-switch nanomachine that can be regulated by a cell stress-induced enzyme at the cellular level and be remotely controlled using an applied magnetic field. We tested the concept using sphingomyelin-containing liposomes encapsulated with indocyanine green, fluorescent marker, or the anticancer drug cisplatin. We engineered the liposomes by adding paramagnetic beads to act as a receiver of outside magnetic energy. The developed multifunctional liposomes were characterized in vitro in leakage studies and cell internalization studies. The release system was further studied in vivo in imaging and therapy trials using a squamous cell carcinoma tumor in the mouse as a disease model. In vitro studies showed an increased release of loaded material when stress-related enzyme and magnetic field was applied to the carrier liposomes. The theranostic liposomes were found in tumors, and the improved therapeutic effect was shown in the survival studies.

Comparison of photoacoustic and fluorescence tomography for the in vivo imaging of ICG-labelled liposomes in the medullary cavity in mice.

Few reports quantitatively compare the performance of photoacoustic tomography (PAT) versus fluorescence molecular tomography (FMT) in vivo. We compared both modalities for the detection of signals from injected ICG liposomes in the tibial medullary space of 10 BALB/c mice in vivo and ex vivo. Signals significantly correlated between modalities (R²â€¯= 0.69) and within each modality in vivo versus ex vivo (PAT: R²â€¯= 0.70, FMT: R²â€¯= 0.76). Phantom studies showed that signals at 4 mm depth are detected down to 3.3 ng ICG by PAT and 33 ng by FMT, with a nominal spatial resolution below 0.5 mm in PAT and limited to 1 mm in FMT. Our study demonstrates comparable in vivo sensitivity, but superior ex vivo sensitivity and in vivo resolution for our ICG liposomes of the VevoLAZR versus the FMT2500. PAT provides a useful new tool for the high-resolution imaging of bone marrow signals, for example for monitoring drug delivery.

Alpha-MSH Targeted Liposomal Nanoparticle for Imaging in Inflammatory Bowel Disease (IBD).

BACKGROUND: The purpose of our study was to find a novel targeted imaging and drug delivery vehicle for inflammatory bowel disease (IBD). IBD is a common and troublesome disease that still lacks effective therapy and imaging options. As an attempt to improve the disease treatment, we tested αMSH for the targeting of nanoliposomes to IBD sites. αMSH, an endogenous tridecapeptide, binds to the melanocortin-1 receptor (MC1-R) and has anti-inflammatory and immunomodulating effects. MC1-R is found on macrophages, neutrophils and the renal tubule system. We formulated and tested a liposomal nanoparticle involving αMSH in order to achieve a specific targeting to the inflamed intestines. METHODS: NDP-αMSH peptide conjugated to Alexa Fluor™ 680 was linked to the liposomal membrane via NSuccinyl PE and additionally loaded into the lumen of the liposomes. Liposomes without the αMSH-conjugate and free NDP-αMSH were used as a control. The liposomes were also loaded with ICG to track them. The liposomes were tested in DSS treated mice, which had received DSS via drinking water order to develop a model IBD. Inflammation severity was assessed by the Disease Activity Index (DAI) score and ex vivo histological CD68 staining of samples taken from different parts of the intestine. The liposome targeting was analyzed by analyzing the ICG and ALEXA 680 fluorescence in the intestine compared to the biodistribution. RESULTS: NPD-αMSH was successfully labeled with Alexa and retained its biological activity. Liposomes were identified in expected regions in the inflamed bowel regions and in the kidneys, where MC1-R is abundant. In vivo liposome targeting correlated with the macrophage concentration at the site of the inflammation supporting the active targeting of the liposomes through αMSH. The liposomal αMSH was well tolerated by animals. CONCLUSION: This study opens up the possibility to further develop an αMSH targeted theranostic delivery to different clinically relevant applications in IBD inflammation but also opens possibilities for use in other inflammations like lung inflammation in Covid 19.

Using Alendronic Acid Coupled Fluorescently Labelled SM Liposomes as a Vehicle for Bone Targeting.

BACKGROUND: We recently developed a liposomal nanoparticle system that can be used for drug delivery and simultaneously be monitored by optical or photoacoustic imaging devices. Here we tested the efficacy of alendronate as a homing molecule in SM-liposomes for bone targeting. METHODS: Alendronate was immobilized covalently on the liposomal surface and the fluorescent dye indocyanine green was used as a payload in the liposomes. The indocyanine green delivery was analyzed by 3D optical tomography, optical fluorescence scanner, photoacoustic imaging, and by ex-vivo biodistribution studies. RESULTS: The results show that the alendronate, coupled to the liposomal surface, increases sphingomyelin containing liposome targeting up to several-folds. CONCLUSION: The alendronate targeted liposomes open possibilities for an application in active bone targeting.

Dasatinib prevents skeletal metastasis of osteotropic MDA-MB-231 cells in a xenograft mouse model.

PURPOSE: Bone metastasis in breast cancer has been linked to activity of c-Src kinase, one of the extensively explored tyrosine kinases in cell biology. The impact of TNF-related apoptosis inducing ligand (TRAIL) and TRAIL receptors has just recently been integrated into this conception. METHODS: An osteotropic clone of MDA-MB-231 cells simulated a model for bone metastasis of triple-negative breast cancer (TNBC). The effects of Dasatinib, a clinically established inhibitor of Src kinases family and Abl were evaluated in vitro and in vivo. In vivo effects of Dasatinib treatment on the occurrence of skeletal metastases were tested in a xenograft mouse model after intra-cardiac injection of osteotropic MDA-MB-231-cells. Ex vivo analyses of the bone sections confirmed intraosseous growth of metastases and allowed determination of osteoclastic activity. RESULTS: Treatment of osteotropic MDA-MB-231 cells with Dasatinib inhibited proliferation rates in vitro. A shift in TRAIL-receptor expression towards an induction of oncogenic TRAIL-R2 was observed. In vivo, 15 of 30 mice received an intra-peritoneal treatment with Dasatinib. These mice showed significantly less skeletal metastases in bioluminescence scans. Moreover, a pronounced increase in bone volume was observed in the treatment group, as detected by µ-Computed Tomography. Dasatinib treatment also led to a greater increase in bone density in tibiae without metastatic affection, which was accompanied by reduced recruitment of osteoclasts. CONCLUSION: Our observations support the concept of utilizing Dasatinib in targeting early-stage bone metastatic TNBC and sustaining bone health.

Utilizing ICG Spectroscopical Properties for Real-Time Nanoparticle Release Quantification In vitro and In vivo in Imaging Setups.

BACKGROUND: Nanoparticle imaging and tracking the release of the loaded material from the nanoparticle system have attracted significant attention in recent years. If the release of the loaded molecules could be monitored reliably in vivo, it would speed up the development of drug delivery systems remarkably. METHODS: Here, we test a system that uses indocyanine green (ICG) as a fluorescent agent for studying release kinetics in vitro and in vivo from the lipid iron nanoparticle delivery system. The ICG spectral properties like its concentration dependence, sensitivity and the fluctuation of the absorption and emission wavelengths can be utilized for gathering information about the change of the ICG surrounding. RESULTS: We have found that the absorption, fluorescence, and photoacoustic spectra of ICG in lipid iron nanoparticles differ from the spectra of ICG in pure water and plasma. We followed the ICG containing liposomal nanoparticle uptake into squamous carcinoma cells (SCC) by fluorescence microscopy and the in vivo uptake into SCC tumors in an orthotopic xenograft nude mouse model under a surgical microscope. CONCLUSION: Absorption and emission properties of ICG in the different solvent environment, like in plasma and human serum albumin, differ from those in aqueous solution. Photoacoustic spectral imaging confirmed a peak shift towards longer wavelengths and an intensity increase of ICG when bound to the lipids. The SCC cells showed that the ICG containing liposomes bind to the cell surface but are not internalized in the SCC-9 cells after 60 minutes of incubation. We also showed here that ICG containing liposomal nanoparticles can be traced under a surgical camera in vivo in orthotopic SCC xenografts in mice.

Universal membrane-labeling combined with expression of Katushka far-red fluorescent protein enables non-invasive dynamic and longitudinal quantitative 3D dual-color fluorescent imaging of multiple bacterial strains in mouse intestine.

BACKGROUND: The human gastrointestinal (GI) tract microbiota has been a subject of intense research throughout the 3rd Millennium. Now that a general picture about microbiota composition in health and disease is emerging, questions about factors determining development of microbiotas with specific community structures will be addressed. To this end, usage of murine models for colonization studies remains crucial. Optical in vivo imaging of either bioluminescent or fluorescent bacteria is the basis for non-invasive detection of intestinal colonization of bacteria. Although recent advances in in vivo fluorescence imaging have overcome many limitations encountered in bioluminescent imaging of intestinal bacteria, such as requirement for live cells, high signal attenuation and 2D imaging, the method is still restricted to bacteria for which molecular cloning tools are available. RESULTS: Here, we present usage of a lipophilic fluorescent dye together with Katushka far-red fluorescent protein to establish a dual-color in vivo imaging system to monitor GI transit of different bacterial strains, suitable also for strains resistant to genetic labeling. Using this system, we were able to distinguish two different E. coli strains simultaneously and show their unique transit patterns. Combined with fluorescence molecular tomography, these distinct strains could be spatially and temporally resolved and quantified in 3D. CONCLUSIONS: Developed novel method for labeling microbes and identify their passage both temporally and spatially in vivo makes now possible to monitor all culturable bacterial strains, also those that are resistant to conventional genetic labeling.