3D Mice Research Articles

Generation of G51D and 3D mice reveals decreased α-synuclein tetramer-monomer ratios promote Parkinson’s disease phenotypes

Mutations in the α-Synuclein (αS) gene promote αS monomer aggregation that causes neurodegeneration in familial Parkinson’s disease (fPD). However, most mouse models expressing single-mutant αS transgenes develop neuronal aggregates very slowly, and few have dopaminergic cell loss, both key characteristics of PD. To accelerate neurotoxic aggregation, we previously generated fPD αS E46K mutant mice with rationally designed triple mutations based on the α-helical repeat motif structure of αS (fPD E46K→3 K). The 3 K variant increased αS membrane association and decreased the physiological tetramer:monomer ratio, causing lipid- and vesicle-rich inclusions and robust tremor-predominant, L-DOPA responsive PD-like phenotypes. Here, we applied an analogous approach to the G51D fPD mutation and its rational amplification (G51D → 3D) to generate mutant mice. In contrast to 3 K mice, G51D and 3D mice accumulate monomers almost exclusively in the cytosol while also showing decreased αS tetramer:monomer ratios. Both 1D and 3D mutant mice gradually accumulate insoluble, higher-molecular weight αS oligomers. Round αS neuronal deposits at 12 mos immunolabel for ubiquitin and pSer129 αS, with limited proteinase K resistance. Both 1D and 3D mice undergo loss of striatal TH+ fibers and midbrain dopaminergic neurons by 12 mos and a bradykinesia responsive to L-DOPA. The 3D αS mice have decreased tetramer:monomer equilibria and recapitulate major features of PD. These fPD G51D and 3D mutant mice should be useful models to study neuronal αS-toxicity associated with bradykinetic motor phenotypes.

Realization of NIR-II 3D whole-body contour and tumor blood vessels imaging in small animals using rotational stereo vision technique.

Optical imaging in the second near-infrared (NIR-II, 1000 to 1700nm) region is capable of deep tumor vascular imaging due to low light scattering and low autofluorescence. Non-invasive real-time NIR-II fluorescence imaging is instrumental in monitoring tumor status. Our aim is to develop an NIR-II fluorescence rotational stereo imaging system for 360-deg three-dimensional (3D) imaging of whole-body blood vessels, tumor vessels, and 3D contour of mice. Our study combined an NIR-II camera with a 360-deg rotational stereovision technique for tumor vascular imaging and 3D surface contour for mice. Moreover, self-made NIR-II fluorescent polymer dots were applied in high-contrast NIR-II vascular imaging, along with a 3D blood vessel enhancement algorithm for acquiring high-resolution 3D blood vessel images. The system was validated with a custom-made 3D printing phantom and in vivo experiments of 4T1 tumor-bearing mice. The results showed that the NIR-II 3D 360-deg tumor blood vessels and mice contour could be reconstructed with 0.15mm spatial resolution, 0.3mm depth resolution, and 5mm imaging depth in an ex vivo experiment. The pioneering development of an NIR-II 3D 360-deg rotational stereo imaging system was first applied in small animal tumor blood vessel imaging and 3D surface contour imaging, demonstrating its capability of reconstructing tumor blood vessels and mice contour. Therefore, the 3D imaging system can be instrumental in monitoring tumor therapy effects.

Influences of repeated exposure to low levels of TiO2 nanoparticles on Kruppel-like factors in 3D Caco-2 spheroids and mouse intestines.

Kruppel-like factors (KLFs) are a set of transcription factors (TFs) involved in the regulation of many basic biological processes, and recent studies suggested that nanoparticles (NPs) were capable to change KLFs in different models even at non-cytotoxic concentrations. In this study, we repeatedly exposed 3D Caco-2 spheroids and mice to TiO2 NPs, one of the most frequently used metal oxide NPs, and investigated the changes of KLF-signaling pathways based on RNA-sequencing. Although the internalization of TiO2 NPs did not induce cytotoxicity in vitro, repeated exposure (three times within 7 days) to 15.7 ng/ml TiO2 NPs increased KLF4 but decreased KLF6. Consistently, KLF4/KLF6-regulated gene ontology terms were altered, including those involved in the regulation of gene expression. We further verified that repeated exposure to 15.7 ng/ml TiO2 NPs increased the expression of KLF4 and proto-oncogene, bHLH transcription factor (MYC), but decreased the expression of KLF6 and activating transcription factor 3 (ATF3). But with the increase of NP concentrations, the expression of these genes was decreased. In mice following intragastrical exposure to 4.39 and 43.9 mg/kg TiO2 NPs (once a day for 5 continuous days), we observed increased expression of klf4, klf6, myc, and atf3, along with morphological changes of intestines. We concluded that repeated exposure to low levels of TiO2 NPs altered KLF-signaling pathways in intestinal cells both in vitro and in vivo.

INTRAPULMONARY TREATMENT WITH A NOVEL TLR4 AGONIST CONFERS PROTECTION AGAINST KLEBSIELLA PNEUMONIA.

Objectives: Nosocomial pneumonia is a common complication in critically ill patients. The goal of this study was to examine the efficacy of the Toll-like receptor 4 agonist 3-deacyl phosphorylated hexacyl disaccharide (3D PHAD), in a clinically relevant murine model of pneumonia, and assess the cellular mechanisms that mediate the protective response. Design: Mice received intrapulmonary 3D PHAD (20 μg) or vehicle for 2 consecutive days before challenge with intrapulmonary Klebsiella pneumoniae (2.3 × 10 3 colony-forming units). Mice were followed for 14-day survival, pulmonary K. pneumoniae burden, lung leukocyte profile, leukocyte phagocytic capacity, and cytokine production. Pneumonia severity and leukocyte recruitment were further assessed by histological evaluation. Setting: Research laboratory. Subjects: Wild-type, male C57BL/6 J mice. Interventions: Intrapulmonary treatment with 20 μg 3D PHAD for 2 consecutive days. Measurements and main results: Intrapulmonary treatment with 3D PHAD decreased lung K. pneumoniae colony-forming units and pneumonia severity with an associated improvement in survival compared with mice treated with vehicle. The numbers of neutrophils, monocytes, and macrophages in the lungs of 3D PHAD-treated mice were higher than those in vehicle-treated mice before infection but were not significantly different from vehicle-treated mice at 48 h after K. pneumoniae challenge. Lung innate leukocytes from 3D PHAD-treated mice had increased phagocytic capacity. Treatment with 3D PHAD alone increased cytokines in the lungs but decreased cytokines in plasma during K. pneumoniae pneumonia as compared with control. Conclusions: Intrapulmonary treatment with 3D PHAD augments innate immunity in the lung and facilitates resistance to K. pneumoniae pneumonia.

TO901317 activation of LXR-dependent pathways mitigate amyloid-beta peptide-induced neurotoxicity in 3D human neural stem cell culture scaffolds and AD mice

Alzheimer's disease (AD) is the major cause of neurodegeneration worldwide and is characterized by the accumulation of amyloid beta (Aβ) in the brain, which is associated with neuronal loss and cognitive impairment. Liver X receptor (LXR), a critical nuclear receptor, and major regulator in lipid metabolism and inflammation, is suggested to play a protective role against the mitochondrial dysfunction noted in AD. In our study, our established 3D gelatin scaffold model and a well characterized in vivo (APP/PS1) murine model of AD were used to directly investigate the molecular, biochemical and behavioral effects of neuronal stem cell exposure to Aβ to improve understanding of the in vivo etiology of AD. Herein, human neural stem cells (hNSCs) in our 3D model were exposed to Aβ, and had significantly decreased cell viability, which correlated with decreased mRNA and protein expression of LXR, Bcl-2, CREB, PGC1α, NRF-1, and Tfam, and increased caspase 3 and 9 activities. Cotreatment with a synthetic agonist of LXR (TO901317) significantly abrogated these Aβ-mediated effects in hNSCs. Moreover, TO901317 cotreatment both significantly rescues hNSCs from Aβ-mediated decreases in ATP levels and mitochondrial mass, and significantly restores Aβ-induced fragmented mitochondria to almost normal morphology. TO901317 cotreatment also decreases tau aggregates in Aβ-treated hNSCs. Importantly, TO901317 treatment significantly alleviates the impairment of memory, decreases Aβ aggregates and increases proteasome activity in APP/PS1 mice; whereas, these effects were blocked by cotreatment with an LXR antagonist (GSK2033). Together, these novel results improve our mechanistic understanding of the central role of LXR in Aβ-mediated hNSC dysfunction. We also provide preclinical data unveiling the protective effects of using an LXR-dependent agonist, TO901317, to block the toxicity observed in Aβ-exposed hNSCs, which may guide future treatment strategies to slow or prevent neurodegeneration in some AD patients.

Zn-based physiometacomposite nanoparticles: distribution, tolerance, imaging, andantiviral and anticancer activity.

The aim of this study was toinvestigate the distribution, tolerance, and anticancerandantiviral activity of Zn-based physiometacomposites (PMCs).Manganese, iron, nickel and cobalt-doped ZnO, ZnS or ZnSe were synthesized. Cell uptake, distribution into 3D culture and mice, andbiochemical and chemotherapeutic activity were studied by fluorescence/bioluminescence, confocal microscopy, flow cytometry, viability, antitumor and virus titer assays.Luminescence and inductively coupled plasma mass spectrometry analysis showed that nanoparticle distribution was liver >spleen >kidney >lung >brain, without tissue or blood pathology. Photophysical characterization as ex vivo tissue probes and LL37 peptide, antisense oligomeror aptamer delivery targeting RAS/Ras binding domain (RBD) was investigated. Treatment at25μg/ml for48 h showed ≥98-99% cell viability, 3D organoid uptake, 3-log inhibition of β-Galactosidase and porcine reproductive respiratory virus infection. Data support the preclinical development of PMCs for imaging and delivery targeting cancer and infectious disease.

Co-infections as Modulators of Disease Outcome: Minor Players or Major Players?

Human host and pathogen interaction is dynamic in nature and often modulated by co-pathogens with a functional role in delineating the physiological outcome of infection. Co-infection may present either as a pre-existing pathogen which is accentuated by the introduction of a new pathogen or may appear in the form of new infection acquired secondarily due to a compromised immune system. Using diverse examples of co-infecting pathogens such as Human Immunodeficiency Virus, Mycobacterium tuberculosis and Hepatitis C Virus, we have highlighted the role of co-infections in modulating disease severity and clinical outcome. This interaction happens at multiple hierarchies, which are inclusive of stress and immunological responses and together modulate the disease severity. Already published literature provides much evidence in favor of the occurrence of co-infections during SARS-CoV-2 infection, which eventually impacts the Coronavirus disease-19 outcome. The availability of biological models like 3D organoids, mice, cell lines and mathematical models provide us with an opportunity to understand the role and mechanism of specific co-infections. Exploration of multi-omics-based interactions across co-infecting pathogens may provide deeper insights into their role in disease modulation.

Mechanistic Similarities between 3D Human Bronchial Epithelium and Mice Lung, Exposed to Copper Oxide Nanoparticles, Support Non-Animal Methods for Hazard Assessment.

The diversity and increasing prevalence of products derived from engineered nanomaterials (ENM), warrants implementation of non-animal approaches to health hazard assessment for ethical and practical reasons. Although non-animal approaches are becoming increasingly popular, there are almost no studies of side-by-side comparisons with traditional in vivo assays. Here, transcriptomics is used to investigate mechanistic similarities between healthy/asthmatic models of 3D air-liquid interface (ALI) cultures of donor-derived human bronchial epithelia cells, and mouse lung tissue, following exposure to copper oxide ENM. Only 19% of mouse lung genes with human orthologues are not expressed in the human 3D ALI model. Despite differences in taxonomy and cellular complexity between the systems, a core subset of matching genes cluster mouse and human samples strictly based on ENM dose (exposure severity). Overlapping gene orthologue pairs are highly enriched for innate immune functions, suggesting an important and maybe underestimated role of epithelial cells. In conclusion, 3D ALI models based on epithelial cells, are primed to bridge the gap between traditional 2D in vitro assays and animal models of airway exposure, and transcriptomics appears to be a unifying dose metric that links in vivo and in vitro test systems.

A Single Heat Exposure Improves Heat Tolerance in a Mouse Exertional Heat Stroke Model

Exertional heat stroke (EHS) is an ongoing medical issue for military personnel. Currently there is poor insight into the etiology of organ damage sustained during EHS and what factors may increase susceptibility. Recent evidence suggests that prior heat injury can increase the likelihood of subsequent EHS. Individuals that have experienced prior EHS report being less tolerant to heat and are flagged in the military for extra surveillance when conducting training exercises. The goal of this study was to determine the impact of prior EHS on heat tolerance and running performance during a subsequent EHS event. Using C57BL/6J male mice, we hypothesized that prior EHS would decrease thermoregulatory performance in the heat, as measured by total heating time to collapse (min), total distance run (m), maximal speed achieved (m/min), and thermal load (°C·min) compared to those without prior EHS. Using a forced running EHS protocol (environmental temperature [Tenv]=37.5 ± 0.2°C), mice were allowed to recover for 24 hours (EHS 24h; n=31), 3 days (EHS 3D; n=22) or 7 days (EHS 7D; n=21) before being subjected to a secondary EHS. To our surprise, mice exposed to two EHS events exhibited a significant increase in time to collapse, distance ran, and thermal load compared to the single EHS group. Mice in the EHS 24h group performed best in three of four performance measures; heating time to collapse (344.55±8.89 min), distance ran (1908.21±92.30 m) and max speed (8.71±0.23m/min) during their second EHS. EHS 24h mice also displayed the highest maximum core temperature (42.50 ± 0.3°C, Tc Max) prior to loss of consciousness, which was significantly higher than the other groups (p<0.02). EHS 3D mice had the highest thermal load (1035±30.8°C·min) compared with the EHS 7D group only (930.75±24.65°C·min, p<0.01). Overall the EHS 24h mice had the best performance and showed the highest increase in performance from their initial EHS, suggesting an increase in heat tolerance. The EHS 3D and EHS 7D group performance measures showed a decay over time, regressing towards initial heat performance levels. Author views not official US Army or DoD policy.

Impact of Multiple Exertional Heat Strokes on Thermoregulation and Performance

Heat stress can result in beneficial acclimation and/or harmful injury depending on the severity of exposure. The impact of multiple heat exposures on thermoregulatory and physical performance has not been well‐studied, but is an important consideration for warfighters, athletes, and others who routinely encounter hot environments. The aim of this study was to examine the effects of multiple exertional heat strokes (EHS) on thermoregulatory and performance variables. C57BL/6J male mice were implanted with an intra‐abdominal radiotelemetry device to measure core temperature (Tc; ±0.1°C). Four exercise training sessions were conducted at thermoneutral conditions (25°C) prior to heat exposure to familiarize mice with the forced running protocol. For the EHS protocol, mice rested inside a running wheel for approximately 35 minutes until the environmental chamber reached 37.5°C and then began running at 2.5m/min, accelerating 0.3m/min every 10 minutes until reaching their max speed when Tc reached 41.0°C; mice were removed from the heat when they collapsed. After recovering for either one day (1D), three days (3D), or seven days (7D), mice repeated the EHS protocol. Regardless of the number of recovery days, mice spent more time in the heat prior to collapse during the second heat exposure compared to the first (1D: 335±9 minutes vs 275±8 minutes, p<0.001; 3D: 331±9 minutes vs 275±7 minutes, p<0.001; 7D: 320±9 minutes vs 274±7 minutes, p<0.001). Maximum Tc (Tcmax) was higher during the second heat exposure after both 1D and 3D recovery compared to the first heat exposure (1D: 42.50±0.03°C vs 42.15±0.04°C, p<0.001; 3D: 42.40±0.04°C vs 42.13±0.04°C, p<0.001; 7D: 42.17±0.03°C vs 42.12±0.03°C, not significant) and during the second heat exposure Tcmax was higher in 1D compared to 3D (p<0.05) and 7D (p<0.001) and higher in 3D than 7D (p<0.001). Hypothermia depth, a measure of injury severity, was less pronounced after the second heat exposure compared to the first heat (1D: 34.57±0.13°C vs 31.31±0.34°C, p < 0.001; 3D: 33.33±0.19°C vs 31.86±0.22°C, p < 0.001; 7D: 33.59±0.30°C vs 31.77±0.46°C, p < 0.001). Hypothermia depth after the second heat was also less severe in 1D mice compared to 3D mice (p < 0.05) and trended less severe compared to 7D mice (p = 0.07). Taken together, these results demonstrate that a single exertional heat exposure can induce acclimation‐like measures that enhance performance and may protect against injury during subsequent heat exposures. Author views are not official US Army or DoD policy.

Uridine inhibits the stemness of intestinal stem cells in 3D intestinal organoids and mice.

The activity of intestinal stem cells (ISCs) is foremost in maintaining homeostasis and repair of intestines. As a pivotal substrate of RNA and DNA biosynthesis, uridine plays essential roles in nutritional and disease monitoring. Whether uridine influences ISC activity remains undefined. To answer this question, 3-dimensional (3D) mouse intestinal organoids and living mice were used as a model. It was found that uridine causes a significant decrease in the number of crypts per intestinal organoid. Uridine also significantly decreases mRNA expression and protein levels with markers of ISCs in intestinal organoids in a dose-dependent manner, which was instructed via mTOR. In parallel, uridine decreases the expression of marker of ISCs in mouse intestine in vivo. Our findings are the first to demonstrate that uridine is able to govern the functions of ISCs in intestinal organoid and mouse models. Thus, this study may provide a useful reference for developing novel functional food bioactives that maintain intestinal homeostasis.

Performance Comparison of User Interface Devices for Controlling Mining Software in Virtual Reality Environments

Recently, many studies have been conducted to apply virtual reality (VR) technology to the mining industry. To accomplish this, it is necessary to develop user interface devices that can effectively control software. Most VR content in the mining industry requires precise device control for equipment operation or accident response. In this study, we compare the performance of four user interface devices (a 2D mouse, 2D & 3D mice, a VR controller, and a Kinect (Microsoft) sensor and bend-sensing data glove) for controlling mining industry software in a VR environment. The total working time, number of device clicks and click accuracy, were analyzed based on 10 experimenters performing 3D orebody modeling, using each device in the VR environment. Furthermore, we conducted a survey to evaluate the ease of learning, ease of use, immersion and fatigue of each device after the experiment. The results show that the 2D mouse yields a high performance in terms of its working time, click accuracy, ease of learning and ease of use. However, the 2D mouse did not completely leverage the VR environment, owing to low user immersion. The Kinect sensor and bend-sensing data glove could control the software efficiently while maximizing user immersion. Our findings are expected to provide a useful reference for the future development of user interface devices in the mining industry.

A novel approach for computing 3D mice distal femur properties using high-resolution micro-computed tomography scanning

One of the most-scanned joints in preclinical animal models dealing with musculoskeletal pathologies is the mouse knee. While three-dimensional (3D) characterization of bone tissue porosity have previously been performed on cortical bone, it has not yet been comprehensively performed for the subchondral bone (SB) and the calcified cartilage (CC), which compose the subchondral mineralized zone (SMZ). Thus, it remains challenging to assess changes that occur in the SMZ of the mouse knee during pathologies such as osteoarthritis. One of the keys to addressing this challenge is to segment each layer to measure their morphologies, material properties, and porosity. Our study presents a novel approach for computing Tissue Mineral Density, 3D porosity, and the thickness of SB and CC in a mouse distal femur using High-Resolution Micro-Computed Tomography (HR-μCT). We have segmented the Vascular Porosity network, the osteocytes’ lacunae of the SB, and the chondrocytes of the CC by using multi-thresholding and the percentage of chondrocytes porosity. Our results show a low intra- and inter-observer coefficient of variability. Regarding porosity and geometrical properties of both CC and SB, our results are within the range of the literature. Our approach opens new avenues for assessing porosity and vascular changes in the distal femur of preclinical animal models dealing with musculoskeletal pathologies such as osteoarthritis.

Abstract P6-05-08: LINC02273 interacts with hnRNPL and promotes metastasis through directly activating AGR2 in breast cancer

Abstract The prognosis of breast cancer patients with metastasis is still poor even improved by current treatment modalities. Unveiling new biomarkers and molecular mechanisms that underlie metastasis are of vital importance for the treatment of breast cancer. The recent discovery of long noncoding RNAs (lncRNAs) has gained widespread attention. To identify critical lncRNAs that contributed to breast cancer metastasis, we profiled their expression in 5 pairs of primary tumors and lymph nodes metastasis loci by HTA2.0 microarray. LINC02273 is significantly upregulated in metastasis loci and its high expression is associated with poor diseases free survival in a validation set of 254 patients. LINC02273 was mainly located in the cell nucleus . RACE-PCR showed two isoforms and the longest one was the most abundant isoform in breast cancer. Through transwell assay, 3D spheroid invasion assay and mice xenograft metastasis model, we found that LINC02273 promoted breast cancer cell migration, invasion and metastasis. Via mass spectrometry, hnRNPL was found to interact with LINC02273 to enhance its stability, which was further confirmed by Actinomycin D inhibition assay and luciferase reporter assay. Furthermore, ChIRP-seq and ChIP-seq showed that LINC02273 stimulated oncogene AGR2 expression by directly binding to the AGR2 promoter region and increasing H3K27ac modification. Triplex formation assay was performed for verification. We demonstrated that the expression level and oncogenic ability of AGR2 were regulated by hnRNPL through LINC02273. Clinical data and mouse xenograft tumors also revealed the positive correlation of AGR2 and LINC02273. In conclusion, LINC02273, which is stabilized by hnRNPL can promote breast cancer metastasis through upregulation of AGR2 and may serve as a prognostic biomarker for breast cancer. Citation Format: Xiu B, Chi Y, Ji W, Zhang Q, Wu J. LINC02273 interacts with hnRNPL and promotes metastasis through directly activating AGR2 in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-05-08.

Negative Surface Shielded Polymeric Micelles with Colloidal Stability for Intracellular Endosomal/Lysosomal Escape.

The critical process and step in achieving effective antitumor therapies is facilitating endosomal escape, which can enhance the intracellular target delivery of therapeutics. However, the normally adopted approaches tend to result in colloidal instability as a result of the inevitable interactions between the resulting positively charged surfaces of micelles and proteins in vivo. Herein, negatively charged surface shielded polymeric micelles, consisting of polymethylacrylamide derivatives and hydrophilic chitosan ( Mw = 18.8 kDa) linked by 3,3'-dithiodipropionic, are constructed. Until the pH decreases to less than 4.5, the DOX-loaded polymeric micelles (CSO-SS-PDPA/DOX) retain a negative surface charge as a result of the abundant amide groups, which could resist formation of the protein "corona" as visualized by transmission electron microscopy. Robust endosomal escape within tens of minutes due to protonated amine groups and specific redox-responsive drug release is visualized by confocal microscopy. The superior therapeutic efficacy in both 3D tumor spheroids and MCF-7 bearing mice further suggested that the prepared CSO-SS-PDPA/DOX is a promising approach for maintaining colloidal stability while achieving intracellular endosomal/lysosomal escape, which opens new opportunities for drug delivery.

Co-administration of iRGD with peptide HPRP-A1 to improve anticancer activity and membrane penetrability

To improve the specificity and penetration of anticancer peptides against tumors, in this study, we examined the effects of co-administration of the membrane-active peptide HPRP-A1 and the tumor homing/penetrating peptide iRGD. iRGD peptide is widely recognized as an efficient cell membrane penetration peptide targeting to αvβ3 integrins and neuropilin-1 (NRP-1) receptors, which show high expression in many tumor cells. The anticancer activity, cancer specificity and penetration activity in vitro and in vivo of the co-administered peptides were examined on 2D monolayer cells, 3D multi-cellular spheroids (MCS) and xenograft nude mice. Co-administration of iRGD and HPRP-A1 exhibited stronger anticancer activity and tumor specificity against A549 non-small cell lung cancer cells with NRP-1 receptor overexpression compared with HPRP-A1 alone. A549 cells showed uptake of the peptide combination and destruction of the integrity of the cell membrane, as well as adherence to the mitochondrial net, resulting in induction of apoptosis by a caspase-dependent pathway. The iRGD peptide dramatically increased the penetration depth of HPRP-A1 on A549 MCS and anticancer efficacy in an A549 xenograft mouse model. Our results suggest that the co-administration strategy of anticancer and penetrating peptides could be a potential therapeutic approach for cancer treatment in clinical practice.

Anti‐tumor Activity of PCAIs in 3D Cell Culture and Mice Models

PURPOSEOf the four molecular subtypes of breast cancer, TNBC is the most difficult to treat due to its lack of biomarkers and targeted therapies. Metastasis, which is characterized by cancer cell migration and invasion of distal organs, is highly prevalent in TNBC, making the disease harder to manage while lowering survival rates. The PCAIs, developed and modeled around the post‐translational modifications of regulatory proteins such as K‐Ras, were designed to inhibit the interactions of overactive monomeric G‐proteins. These proteins operate downstream of EGFR which is overexpressed in 60% of TNBC cases and are vastly involved in regulating cell survival, motility, apoptosis, and ultimately cancer progression. Previous work in our lab has shown that treatment of various cancer cell lines with PCAIs leads to cell cycle arrest, suppression of cell viability, inhibition of monolayer cell migration and invasion, and induction of caspase 3/7‐mediated apoptotic death. The purpose of this study was to determine the effectiveness of PCAIs at impeding the trademarks of cancer progression in 3D cell culture and in in vivo models.METHODSMDA‐MB‐231 cells were used in 3D cell culture assays to assess the PCAIs' ability to inhibit spheroid formation and invasion while inducing apoptosis. TNBC mice xenografts were used to determine the efficacy of PCAIs in vivo.RESULTSCompounds NSL‐BA‐055 and NSL‐BA‐040 were the most potent of the PCAIs, halting the formation of compact spheroids at 1.0 and 2.0 μM while significantly inhibiting spheroid invasion at 0.2 and 1.0 μM, respectively. Both compounds induced apoptosis and completely obliterated compact TNBC spheroids at 5.0 μM. Five and 10 mg/kg NSL‐BA‐040 inhibited the growth of TNBC xenograft tumors by 60.1 and 71.2%, respectively.CONCLUSIONThe PCAIs' ability to prevent spheroid formation and invasion while inducing spheroid apoptosis suggests their ability to prevent metastasis, tumor formation, growth and potential as effective therapies for TNBC.Support or Funding InformationThe research reported in this publication was supported by the National Cancer Institute (NCI) and National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Grant SC1CA190505 and by the National Institute On Minority Health and Health Disparities (NIMHD) of the NIH under Award Number G12 MD007582 (previous award NIH/NCRR/RCMI G12 RR03020). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

In vitro and in vivo antitumor effects of the VO-chrysin complex on a new three-dimensional osteosarcoma spheroids model and a xenograft tumor in mice.

Osteosarcoma (OS) is the most common primary tumor of bone, occurring predominantly in the second decade of life. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for patients with localized disease. Vanadium is an ultra-trace element that after being absorbed accumulates in bone. Besides, vanadiumcompounds have been studied during recent years to be considered as representative of a new class of non-platinum antitumor agents. Moreover, flavonoids are a wide family of polyphenolic compounds that display many interesting biological effects. Since coordination of ligands to metals can improve the pharmacological properties, we report herein, for the first time, the in vitro and in vivo effects of an oxidovanadium(IV) complex with the flavonoid chrysin on the new 3D human osteosarcoma and xenograft osteosarcoma mice models. The pharmacological results show that VOchrys inhibited the cell viability affecting the shape and volume of the spheroids and VOchrys suppressed MG-63 tumor growth in the nude mice without inducing toxicity and side effects. As a whole, the results presented herein demonstrate that the antitumor action of the complex was very promissory on human osteosarcoma models, whereby suggesting that VOchrys is a potentially good candidate for future use in alternative antitumor treatments.

AB173. TR4 nuclear receptor increases prostate cancer invasion via decreasing the miR-373-3p expression to alter TGFβR2/p-Smad3 signals

ObjectiveTesticular nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily, which may play key roles to influence the metabolic diseases and prostate tumorigenesis. The purpose of our study is to elucidate the mechanisms how TR4 influences the prostate cancer (PCa) metastasis.MethodsWe constructed three different PCa cell lines including C4-2, PC3 and CWR22Rv1 with differential stable expression of TR4. RT-PCR and Western blot analysis were used to validate identified downstream genes. To explore the function of genes, we manipulated cells 2D and 3D invasion assays and mice experiment.Resultswe found TR4 could promote PCa cell invasion using two different cell invasion assays. Mechanism dissection revealed that TR4 might enhance PCa cell invasion via modulation of the microRNA-373-3p (miR-373-3p) expression. An interruption approach using miR-373-3p partially reversed TR4-enhanced PCa cell invasion. Furthermore, we found TR4-miR-373-3p might function through modulation of the TGFβR2/p-Smad3 signals to enhance the PCa cell invasion. The in vivo mouse model using orthotopic xenografted CWR22Rv1 cell line transfected with luciferase-reporter also confirmed in vitro cell line studies showing TR4 enhanced PCa metastasis via modulation of miR-373-3p.ConclusionsOur data suggest that TR4 may represent a key player to influence the PCa metastasis and targeting TR4 miR-373-3p→ TGFβR2/p-Smad3 axis using TR4 antagonist or TR4-siRNA or miR-373-3p may become a new potential therapeutic approach to better suppress PCa metastasis.

Abstract A56: The role of Pentraxin 3 (PTX3) in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a disease with dismal prognosis and is invariably diagnosed late. Current tests used to diagnose or monitor PDAC are invasive and expensive. Tumour-supportive desmoplastic stroma, an important modulator of PDAC, is laid down by pancreatic stellate cells (PSC). All-trans Retinoic Acid (ATRA), a pleiotropic agent modulating multiple signalling pathways, renders activated stellate cells quiescent. Pentraxin 3 (PTX3) gene level expression is downregulated upon rendering stellate cells quiescent. The interaction between PTX3 and TSG-6 has been shown in other disease states to stabilise hyaluranon (HA) present in the extracellular matrix. PDAC extracellular matrix is known to be rich in HA. Methods: Human serum PTX3 levels were measured by ELISA. Twelve pancreatic cell lines, primary and immortalised pancreatic stellate cell lines were screened for PTX3 and TSG-6 expression at protein and mRNA levels. Distribution of PTX3, TSG-6 and HA in organotypic 3D cultures, transgenic mice and patient samples was determined using immunohistochemistry and immunofluorescence. Results: PDAC patients have significantly higher serum PTX3 levels (median 15.32 ng/ml, range 5.42-39.87 ng/ml, n= 43 ) compared to patients with other pancreatic diseases (median=7.09 ng/ml, range 4.45-14.01 ng/ml, n=36, 2 way anova P=0.014) and healthy individuals (median 3.72 ng/ml, range 1.97-8.82 ng/ml, n=22, 2 way anova P=0.001) ROC curve analysis ((AUC=0.946 (95%CI= 0.9-0.99, P<0.0001) demonstrates that serum PTX3 levels greater than 8ng/mL can diagnose pancreatic cancer with a sensitivity of 89.47 (CI =78.48 to 96.04%) and specificity of 72.09 (CI 56.33 to84.67%). This is being validated in an independent cohort. Western blotting and immunofloursecnce demonstrate PTX3 is present maximally in stellate cells and also in some pancreatic cancer cell lines and is secreted in heavily glycosylated form. Rendering PSC quiescent reduced expression and secretion of PTX3. In the 3D organotypic culture model secreted PTX3 stains both cancer cells and PSC. However, in ATRA treated organotypics only the PSC are positive. Immunofluorescent examination reveals HA expression at the cellular membrane of PSC with no expression in cancer cells. In the organotypic model there is strong HA expression in the PSC layer. Immunohistochemistry staining of human PDAC tissues shows PTX3 to be predominantly expressed in the tumour stroma, a small proportion of patients also express PTX3 in the epithelial component of their tumours. Conclusions: PTX3 distinguishes pancreatic cancer from other diseases of the pancreas and can be tested using a simple blood test. Activated PSC produce this marker and its expression is down regulated on rendering these cells quiescent. PTX3 could be used as a marker for monitoring stromal response in stroma-targeted therapy. The interaction of PTX3 with HA in PDAC is the subject of ongoing investigation. Citation Format: Jennifer Watt, Imran Siddique, Thomas Dowe, Tatjana Crnogorac-Jurcevic, Satyajit Bhattacharya, Paola Allavena, Hemant M. Kocher. The role of Pentraxin 3 (PTX3) in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A56.