Lateral Tail Vein Injection Research Articles

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice.

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the affected cells located in different sites. Intravenous injection is a widely used systemic delivery route in preclinical studies that assess treatments intended for body-wide administration. In adult mice, it involves the intravenous administration of the therapeutic agent into the mouse's lateral tail veins. When mastered, tail-vein injections are safe and fast, and only require simple and commonly available tools. However, tail-vein injections are technically challenging and require extensive training and continuous practice to ensure the accurate delivery of the intended dose. Here we describe a detailed, optimized, lateral tail-vein injection protocol that we have developed based on our experience and on recommendations that had been previously reported by other groups. Other than the mouse restrainers and insulin syringes, this protocol requires only reagents and equipment that are readily available in most labs. We found that following this protocol results in consistently successful intravenous delivery of adeno-associated virus (AAV) into the tail veins of unsedated 7-9 week-old mice. Additionally, we describe the optimized protocols for the histological detection of fluorescent reporter proteins and vector genome per diploid genome (vg/dg) quantification used to assess AAV transduction and biodistribution. The goal of this protocol is to aid experimenters in easily performing tail-vein injections successfully and consistently, which can reduce the practice time needed to master the technique.

Trichinella spiralis-Secreted Products Promote Collagen Capsule Formation through TGF-β1/Smad3 Pathway.

Trichinella spiralis (T. spiralis) muscle larvae colonize in the host's skeletal muscle cells, which are surrounded by collagen capsules. The mechanism underlying muscle stage larva-induced collagen capsule formation remains unknown. To clarify the mechanism, a T. spiralis muscular-infected mouse model was established by a single lateral tail vein injection with 20,000 T. spiralis newborn larvae (NBL). The infected mice were treated with or without SB525334 (TGF-β1 receptor type I inhibitor). Diaphragms were obtained post-infection, and the expression levels of the TGF-β1/Smad3 pathway-related genes and collagen genes (type IV and VI) were observed during the process of collagen capsule formation. The changes in myoblasts under stimulation of the excretory-secretory (ES) products of NBL with or without SB525334 were further investigated. Results showed that the expression levels of type IV collagen gene, type VI collagen gene, Tgfb1, and Smad3 were significantly increased in infected mice muscle cells. The expression levels of all the above genes were enhanced by the products of NBL in myoblast cells. These changes were reversed by co-treatment with SB525334 in vivo and in vitro. In conclusion, the TGF-β1/Smad3 pathway can be activated by T. spiralis infection in muscle cells. The activated TGF-β1/Smad3 pathway can stimulate the secretion of collagens by myocytes and plays a promoting role in the process of collagen capsule formation. The research has the limitation that the protein identification of the products of NBL has yet to be performed. Therefore, the specific components in the T. spiralis ES products that induce collagen synthesis should be further investigated.

Heme Oxygenase-1 (HMX1) Loss of Function Increases the In-Host Fitness of the Saccharomyces 'boulardii' Probiotic Yeast in a Mouse Fungemia Model.

The use of yeast-containing probiotics is on the rise; however, these products occasionally cause fungal infections and possibly even fungemia among susceptible probiotic-treated patients. The incidence of such cases is probably underestimated, which is why it is important to delve deeper into the pathomechanism and the adaptive features of S. ‘boulardii’. Here in this study, the potential role of the gene heme oxygenase-1 (HMX1) in probiotic yeast bloodstream-derived infections was studied by generating marker-free HMX1 deletion mutants with CRISPR/Cas9 technology from both commercial and clinical S. ‘boulardii’ isolates. The six commercial and clinical yeasts used here represented closely related but different genetic backgrounds as revealed by comparative genomic analysis. We compared the wild-type isolates against deletion mutants for their tolerance of iron starvation, hemolytic activity, as well as kidney burden in immunosuppressed BALB/c mice after lateral tail vein injection. Our results reveal that the lack of HMX1 in S. ‘boulardii’ significantly (p < 0.0001) increases the kidney burden of the mice in most genetic backgrounds, while at the same time causes decreased growth in iron-deprived media in vitro. These findings indicate that even a single-gene loss-of-function mutation can, surprisingly, cause elevated fitness in the host during an opportunistic systemic infection. Our findings indicate that the safety assessment of S. ‘boulardii’ strains should not only take strain-to-strain variation into account, but also avoid extrapolating in vitro results to in vivo virulence factor determination.

Assembly and operation of an easy-to-make portable device for facilitating mouse lateral tail-vein injection

Drug delivery by lateral tail-vein injection in mice is widely used in preclinical research, but the technique is laborious to perform because the tail vein is hardly visible and too small to be cannulated. Misinjections of test components can lead to defective or even false experiment results. We present a simple but useful injection-assistant device to visualize the tail vein of mice. The device consists of a light-emitting diode (LED) circuit and a finger component. The finger component consists of an open-looped ring to slide on the finger, a slot to accommodate the mouse's tail and a lamp cage in which to set the LED lamp. Once the mouse's tail has been illuminated, the tail vein can be clearly seen as a dark line along the bright background of the tail, which facilitates venipuncture and improves the success rate of tail-vein injection. If the protocol provided has been followed correctly, a robust tail-vein injection-assistant device can be set up in 3 h with low-cost components.

Erratum: Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells.

An erratum was issued for:Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells. The author list was updated. The author list was updated from: Katie A. Thies1,Sue E. Knoblaugh2, and Steven T. Sizemore1 1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center 2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University to: Katie A. Thies1, Sarah Steck1, Sue E. Knoblaugh2, and Steven T. Sizemore1 1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center 2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University.

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous Administration is the Best Route of Mesenchymal Stem Cells Migration in Improving Liver Function Enzyme of Acute Liver Failure

Background: Mesenchymal stem cells (MSCs) migrate and transmigrate to acute liver failure (ALF) area due to vascular endothelial growth factor (VEGF) stimulation as an attractant molecule then actively giving the paracrine signaling and or differentiating into primary hepatocytes, however the best route of MSCs transplanted to liver injury area remains unclear. &amp;nbsp; Aim: In this study we compare intravenous (IV) and intraperitoneal (IP) route of MSCs administration by analyzing serum glutamic pyruvic transaminase (SGPT), serum glutamic-oxaloacetic transaminase (SGOT) and bilirubin level as improvement markers of liver function and VEGF as attractant-proliferation molecule on days 2 and 5. &amp;nbsp; Materials and methods: Eighteen male Sprague-Dawley rats weighting 200 g were used in this study. They were divided in three study groups: vehicle control, IP and IV groups. The IV group was treated by MSCs at dose 1&amp;times;106 by lateral tail vein injection and IP group received 1&amp;times;106 MSCs via IP injection. The level of SGPT, SGOT and bilirubin were measured by an automatic analyzer, the VEGF level using enzyme-linked immunosorbent assay (ELISA), while the CD73 expression was evaluated using immunohistochemistry. &amp;nbsp; Results: This study showed that IV injection of MSCs was more efficient for increasing liver function than IP treatment group that confirmed by the observed significant decrease in SGPT, SGOT and bilirubin level on days 2 and 5 (p&lt;0.001). This effect was most likely mediated by the significant increase of VEGF level (p&lt;0.05) on days 2 and 5. &amp;nbsp; Conclusion: Our result conclude that an IV administration of MSCs was more efficacious than the IP administration for liver injury &amp;nbsp;regeneration.&amp;nbsp;

Xenogeneic transplantation of human WJ-MSCs rescues mice from acute radiation syndrome via Nrf-2-dependent regeneration of damaged tissues.

There is an unmet medical need for radiation countermeasures that can be deployed for treatment of exposed individuals during ionizing radiation (IR) accidents or terrorism. Wharton's jelly mesenchymal stem cells (WJ-MSCs) from human umbilical cord have been shown to avoid allorecognition and induce a tissue-regenerating microenvironment, which makes them an attractive candidate for mitigating IR injury. We found that WJ-MSCs protected mice from a lethal dose of IR even when transplanted up to 24hours after irradiation, and a combination of WJ-MSCs and antibiotic (tetracycline) could further expand the window of protection offered by WJ-MSCs. This combinatorial approach mitigated IR-induced damage to the hematopoietic and gastrointestinal system. WJ-MSCs increased the serum concentration of the cytoprotective cytokines granulocyte colony-stimulating factor (G-CSF) and IL-6 in mice. Knockdown of G-CSF and IL-6 in WJ-MSCs before injection to lethally irradiated mice or transplantation of WJ-MSCs to lethally irradiated Nrf-2 knockout mice significantly nullified the therapeutic protective efficacy. Hence, WJ-MSCs could be a potential cell-based therapy for individuals accidentally exposed to radiation.

Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa

Targeted transcriptional modulation in the central nervous system (CNS) can be achieved by adeno-associated virus (AAV) delivery of CRISPR activation (CRISPRa) and interference (CRISPRi) transgenes. To enable AAV packaging, we constructed minimal CRISPRa and CRISPRi transgenes by fusing catalytically inactive Staphylococcus aureus Cas9 (dSaCas9) to the transcriptional activator (VP64 and VP160) and repressor (KRAB and SID4X) domains along with truncated regulatory elements. We then evaluated the performance of these constructs in two reporter assays (bioluminescent and fluorescent), five endogenous genes (Camk2a, Mycn, Nrf2, Keap1, and PDGFRA), and two cell lines (neuro-2a [N2a] and U87) by targeting the promoter and/or enhancer regions. To enable systemic delivery of AAVs to the CNS, we have also generated an AAV1-PHP.B by inserting a 7-mer PHP.B peptide on AAV1 capsid. We showed that AAV1-PHP.B can efficiently cross the blood-brain barrier (BBB) and be taken up by the brain tissue upon lateral tail vein injection in mice. Importantly, a single-dose intravenous administration of AAV1-PHP.B expressing CRISPRa was shown to achieve targeted transgene activation in the mouse brain. This proof-of-concept study will contribute to the development of a non-invasive, specific and potent AAV-CRISPR system for correcting transcriptional misregulation in broad brain areas and multiple neuroanatomical structures.

Pharmacodynamics of a Long-Acting Echinocandin, CD101, in a Neutropenic Invasive-Candidiasis Murine Model Using an Extended-Interval Dosing Design.

Echinocandins are important in the prevention and treatment of invasive candidiasis but limited by current dosing regimens that include daily intravenous administration. The novel echinocandin CD101 has a prolonged half-life of approximately 130 h in humans, making it possible to design once-weekly dosing strategies. The present study examined the pharmacodynamic activity of CD101 using the neutropenic invasive candidiasis mouse model against select Candida albicans (n = 4), C. glabrata (n = 3), and C. parapsilosis (n = 3) strains. The CD101 MIC ranged from 0.03 to 1 mg/liter. Plasma pharmacokinetic measurements were performed using uninfected mice after intraperitoneal administration of 1, 4, 16, and 64 mg/kg. The elimination half-life was prolonged at 28 to 41 h. Neutropenic mice were infected with each strain by lateral tail vein injection, treated with a single dose of CD101, and monitored for 7 days, at which time the organism burden was enumerated from the kidneys. Dose-dependent activity was observed for each organism. The pharmacokinetic/pharmacodynamic (PK/PD) index of the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC index) correlated well with efficacy (R2, 0.74 to 0.93). The median stasis 24-h free-drug AUC/MIC targets were as follows: for C. albicans, 2.92; for C. glabrata, 0.07; and for C. parapsilosis, 2.61. The PK/PD targets for 1-log10 kill endpoint were 2- to 4-fold higher. Interestingly, the aforementioned PK/PD targets of CD101 were numerically lower for all three species than those of other echinocandins. In summary, CD101 is a promising, novel echinocandin with advantageous pharmacokinetic properties and potent in vivo pharmacodynamic activity.

PH triggered re-assembly of nanosphere to nanofiber: The role of peptide conformational change for enhanced cancer therapy

pH-triggered conformational change and subsequent re-assembly of nanostructures provide a new strategy in nanomedicine for controlled drug release and enhanced therapy. Here, we reported the development of a novel pH-responsive nano-assembly as a drug carrier from peptide amphiphile (PA) consisting of mimicking peptide and stearic acid moieties. The mimicking peptide is a basic 17-amino acid peptide derived from antennapedia homeodomain, and undergoes a conformational transition of the secondary structure from β-sheet at pH7.4 to α-helix at pH5.0. Such transition therefore leads to simultaneous evolution of the self-assembled structure of PA from nanosphere to nanofiber, promotes assemblies retention and then release drugs in the cytoplasm of tumor cell. In vitro studies showed that the doxorubicin (Dox)-loaded PA nanoparticle (PA@Dox) could be uptaken efficiently by the cell due to the membrane penetrating capability of the mimicking peptide and subsequently the released Dox further induce apoptosis of murine colon carcinoma CT26 (MCCC) cell. In a mouse xenograft model of MCCC, administration of PA@Dox via lateral tail vein injection could remarkably retard the tumor growth. The overall results suggested that the PA-based nanocarriers adopting the novel strategy of pH-triggered secondary structural change could enhance therapeutic efficacy and be used as a promising platform for potential development of new generation of drug carriers for cancer therapy.

Abstract B26: Role of exosomal annexin a2 in angiogenesis and breast cancer metastasis

Abstract Background: Early detection of cancer using circulating biomarkers is a realistic possibility with the discovery of exosomes. Cells under both physiological and pathological conditions secrete a wide array of membranous vesicles containing specific protein and RNA signatures. Exosomes, which are 40-100 nm in size, comprise a major portion of these vesicles. Exosomes play important roles in promoting tumor progression and metastasis, but the mechanisms by which they act are not yet understood. Annexin A2 (AnxA2) is a 36 kDa calcium dependent phospholipid-binding protein up-regulated in many cancer types and promotes tumorigenesis and angiogenesis by acting as a receptor for angiostatin and tissue plasminogen activator (t-PA) leading to plasmin mediated angiogenesis and metastasis. Recently, it was found that AnxA2 is one of the most commonly expressed proteins in exosomes, but its function has never been characterized. We propose to explore the correlation and functionality of exosome associated AnxA2 (exo-AnxA2) in breast cancer. Methods: We used two breast cancer models: MCF10 progression model (MCF10A non to correlate exo-AnxA2 with breast cancer progression, and MDA-MB-231/ its organ specific metastatic variants (BoM-1833 bone, MDA-MB-231-831 brain and MDA-MB-231-4175 lungs). Exosome characterization was performed via Western blotting (WB), particle size analyzer, Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). In vitro and in vivo angiogenesis assays were performed to explore the role of exo-AnxA2 in angiogenesis. AnxA2 targeting peptide (LCKLSL) was used to inhibit exo-AnxA2. For metastasis studies, experimentally induced (intracardiac) and spontaneous (mammary fat pad) metastasis models were used. Animals were primed for one month (lateral tail vein injection) with PBS or MDA-MB-231/variants derived exosomes having WT AnxA2 (exo-WTAnxA2) or sh-AnxA2 (exo-shAnxA2). After one month of priming, the animals were challenged with luciferase positive MDA-MB-231/variants and the differences in the extent of metastasis were studied. We performed clinical analysis AnxA2 levels in 50 breast cancer serum samples and 50 age matched normal serum samples by analyzing total serum AnxA2 levels and serum exosomal AnxA2 levels via ELISA. We also correlated the AnxA2 levels with types of cancer (ER, PR, HER2 or Triple negative). Further, we correlated the exosomal AnxA2 levels with stage (early versus late stage) and metastatic sites (no metastasis, lung, brain and/or bone) of patients. Result: AFM and WB characterization of exo-AnxA2 along the progression model revealed that AnxA2 is highly expressed in cancer exosomes vs. non-malignant and pre-malignant exosomes. Both in vitro and in vivo angiogenesis studies with LCKLSL showed exo-AnxA2 to be a potent inducer of angiogenesis (∼ 45% and ∼42.4% decrease with LCKLSL respectively). Furthermore, both the metastasis models showed a significant decrease in the extent of organ specific metastasis (lungs ∼43%, brain ∼ 30% and bone ∼41%) in animals primed with exo-shAnxA2 vs. exo-WTAnxA2. Histological analyses of the organs showed increased micro and macro metastasis in the brain, lungs and bones in exo-WTAnxA2 primed animals when compared to exo-shAnxA2. Clinical analysis of the serum samples revealed that although total serum AnxA2 is increased in breast cancer serum samples vs. normal, the correlation was much better between high exo-AnxA2 levels and metastatic breast cancer. Conclusion: Exo-AnxA2 plays a major role in promoting angiogenesis and breast cancer metastasis both in vitro and in vivo. Further, exo-AnxA2 is over expressed in breast cancer serum samples vs. control. Thus, exo-AnxA2 can be a potential diagnostic and prognostic marker in breast cancer patients to detect and monitor metastasis. All the authors accept the responsibility for the statements made in the abstract. (Supported by NIMHHD grant under Award Number P20MD006882) Citation Format: Sayantan Maji, Ann Marilyn Leitch, Irina Akopova, Phung Nguyen, Jamboor K. Vishwanatha. Role of exosomal annexin a2 in angiogenesis and breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B26.

A critical evaluation of drug delivery from ligand modified nanoparticles: Confounding small molecule distribution and efficacy in the central nervous system

In this work, we sought to test how surface modification of poly(lactic-co-glycolic acid) (PLGA) nanoparticles with peptide ligand alters the brain specific delivery of encapsulated molecules. For biodistribution studies, nanoparticles modified with rabies virus glycoprotein (RVG29) were loaded with small molecule drug surrogates and administered to healthy mice by lateral tail vein injection. Mice were perfused 2h after injection and major anatomical regions of the CNS were dissected (striatum, midbrain, cerebellum, hippocampus, cortex, olfactory bulb, brainstem, and cervical, thoracic, lumbar and sacral spinal cord). For functional studies, surface modified nanoparticles were loaded with the chemotherapeutic camptothecin (CPT) and administered to mice bearing intracranial GL261-Luc2 gliomas. Outcome measures included tumor growth, as measured by bioluminescent imaging, and median survival time. We observed that small molecule delivery from PLGA nanoparticles varied by as much as 150% for different tissue regions within the CNS. These differences were directly correlated to regional differences in cerebral blood volume. Although the presence of RVG29 enhanced apparent brain delivery for multiple small molecule payloads, we observed minimal evidence for targeting to muscle or spinal cord, which are the known sites for rabies virus entry into the CNS, and enhancements in brain delivery were not prolonged due to an apparent aqueous instability of the RVG29 ligand. Furthermore, we have identified concerning differences in apparent delivery kinetics as measured by different payloads: nanoparticle encapsulated DiR was observed to accumulate in the brain, whereas encapsulated Nile red was rapidly cleared. Although systemically administered CPT loaded nanoparticles slowed the growth of orthotopic brain tumors to prolong survival, the presence of RVG29 did not enhance therapeutic efficacy compared to control nanoparticles. These data are consistent with a model of delivery of hydrophobic small molecules to the brain that does not rely on internalization of polymer nanoparticles in target tissue. We discuss an important risk for discordance between biodistribution, as typically measured by drug surrogate, and therapeutic outcome, as determined by clinically relevant measurement of drug function in a disease model. These results pose critical considerations for the methods used to design and evaluate targeted drug delivery systems in vivo.

Abstract 2267: Defining roles of SIN3 isoforms in breast cancer metastasis

Abstract Metastasis continues to be the most difficult clinical challenge for breast cancer. These patients have unacceptable survival rates that have not changed in the past 20 years. Therefore, more effective therapeutic options are needed. The purpose of this study is to better understand molecular mechanisms of the SIN3 chromatin complexes that have been functionally associated with breast cancer progression to identify novel targeting strategies. Alteration of the composition of SIN3 complexes regulates the metastatic potential of breast cancer cells; and inhibition of SIN3 complexes induces differentiation and inhibits invasion of breast cancer cells. However, differences in the function of SIN3 isoforms (SIN3A and SIN3B) in breast cancer metastasis have not been characterized. To better define the role of SIN3 isoforms in breast cancer metastasis, we generated stable knockdown of SIN3 isoforms individually and in combination using 3 non-overlapping shRNA in two different metastatic breast cancer cell lines (MDA-MB-231 and -435). No significant change was noted in the proliferation rate of the transduced cells in vitro. Stable knockdown of SIN3B caused a significant decrease in invasion through Matrigel (28 ± 4 invaded cells/field compared to 50 ± 8 for control; p = 0.027) using a modified Boyden chamber that was corroborated by the decreased presence of stellate projections and a more epithelial phenotypic growth in 3D. Surprisingly, stable knockdown of SIN3A significantly increased invasion through Matrigel (98 ± 14 invaded cells/field; p = 0.008) and increased the presence of stellate projections in the 3D growth assay. Dual knockdown of SIN3A and SIN3B caused a significant decrease in invasiveness (19 ± 5 invaded cells/field; p = 0.003) and decreased presence of stellate projections similar to the phenotype noted with individual knockdown of SIN3B. These results were confirmed in vivo with an experimental metastasis assay (lateral tail vein injection in athymic nude mice) in which SIN3B knockdown significantly decreased lung metastasis (70% decrease compared to control; p = 0.009) and SIN3A knockdown increased metastasis (163% increase compared to control; p = 0.096). These results demonstrate key functional differences between SIN3 isoforms in regulating the process of metastasis. Our data suggests metastasis suppressive roles of SIN3A and metastasis promoting roles of SIN3B that will be important in discovering novel therapeutic strategies for metastatic breast cancer patients. Citation Format: Monica J. Lewis, Jianzhong Liu, Douglas R. Hurst. Defining roles of SIN3 isoforms in breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2267. doi:10.1158/1538-7445.AM2015-2267

The use of eutectic mixture of lidocaine and prilocaine in mice (Mus musculus) for tail vein injections

ObjectiveTo investigate a topical local anesthesia technique as a means to prevent and/or diminish pain in mice in a laboratory setting associated with tail vein injections performed by personnel in training. Study designProspective, randomized experimental trial. AnimalsThirty six adult female, 23–28 g CD-1 mice from an in-house training colony. They were acclimated to routine training and handling classes. MethodsEutectic mixture of local anesthetics (EMLA) cream (2.5% lidocaine/2.5% prilocaine) or a bland ointment control (n = 18) was applied on the tail prior to intravenous injection. The injections were performed by novices, who had never attempted the procedure, and experienced personnel. All participants were blinded to treatment groups. Three injection attempts were allowed per animal. The mice were observed and scored by blinded evaluators for behavioral and physiological changes, including respiratory rate, vocalization, tail flick, and escape behaviors, during and after the injection. ResultsThis study demonstrates that aversive behaviors induced by lateral tail vein injection were not changed by the preemptive application of EMLA cream. The aversive behaviors associated with lateral tail vein injection were significantly affected by the number of injection attempts and the individual's experience level. Conclusions and clinical relevanceTopical EMLA cream did not reduce signs of aversive reaction to tail vein injection and thus we did not find support for its use in mouse training programs for tail vein injections.

Abstract 2812: mTOR kinase mediates dissemination and colonization of breast cancer metastasis

Abstract Background: Early events of metastatic dissemination arise from the ability of tumor cells to acquire a number of biological changes which allow for their survival in distal microenvironments. Among the early events that initiate disseminated tumor populations include epithelial-to-mesenchymal transition (EMT) and increased potential for invasion that will ultimately lead to colonization of distal sites. The PI3K/mTOR pathway has been linked to clinic-pathalogical features of invasion and metastasis of multiple tumor types. Results: We sought to investigate using both in vitro and in vivo models of metastasis a panel of PI3K/mTOR selective inhibitors, in which INK128, a selective mTOR kinase inhibitor in clinical development was included. Using live-cell time lapse imaging experiments to monitor cell migration and invasion we found that INK128 significantly inhibited motility and velocity of metastatic breast cancer models in wound assays and decreased the ability of tumor cells to migrate through membranes and matrigel. The ability of INK128 to selectively inhibit tumor cell migration in vitro prompted us to assess whether INK128 can prevent tumor dissemination in vivo. In metastatic breast cancer xenograft models we monitored the growth of tumors implanted in mammary fat pads (MFP) and metastatic dissemination by serial noninvasive whole body bioluminescent imaging. We first assessed and characterized the degree of spontaneous metastasis to the lung, liver, auxillary lymph nodes, chest cavity of mice and subsequently the effect of mTOR kinase inihibition on primary tumor growth on the MFP versus overt metastatic dissemination upon daily, oral treatment with INK128. We show that INK128 blocked drastically metastatic spread in a dose-dependent manner without significantly affecting the growth of primary tumors. Interestingly, we found that INK128 did not affect the ability of breast cancer cells to enter into the lungs following an alternative experimental lung metastasis model by lateral tail vein injection but INK128 greatly diminished the capacity of breast cancer cells to subsequently colonize and expand. Conclusion: Our results position mTOR on a metastasis-sustaining axis that engages downstream pathways to support the biological machinery of migration, invasion, and fitness of initiating breast cancer cells during the establishment of thoracic metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2812. doi:1538-7445.AM2012-2812

Susceptibility to gold nanoparticle-induced hepatotoxicity is enhanced in a mouse model of nonalcoholic steatohepatitis

Although the safety of gold nanoparticle (AuNP) use is of growing concern, most toxicity studies of AuNPs had focused on their chemical characteristics, including their physical dimensions, surface chemistry, and shape. The present study examined the susceptibility of rodents with healthy or damaged livers to AuNP-induced hepatotoxicity. To induce a model of liver injury, mice were fed a methionine- and choline-deficient (MCD) diet for 4 weeks. Sizes and biodistribution of 15-nm PEGylated AuNPs were analyzed by transmission electron microscopy. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were estimated with an automatic chemical analyzer, and liver sections were subjected to pathological examination. Activities of antioxidant enzymes were determined by biochemical assay. Lateral tail vein injection of MCD diet-fed mice with 5mgkg−1 AuNPs significantly elevated the serum ALT and AST levels compared to MCD diet-fed mice injected with mPEG (methylpolyethylene glycol). Similarly, severe hepatic cell damage, acute inflammation, and increased apoptosis and reactive oxygen species (ROS) production were observed in the livers of AuNP-injected mice on the MCD diet; these liver injuries were attenuated in mice fed a normal chow diet. The results suggest that AuNPs display toxicity in a stressed liver environment by stimulating the inflammatory response and accelerating stress-induced apoptosis. These conclusions may point to the importance of considering health conditions, including liver damage, in medical applications of AuNPs.

Reprogrammed Murine Fibroblasts Differentiated into Hematopoietic Progenitors Are Able to Successfully Engraft and Repopulate the Bone Marrow

Cellular therapy using embryonic stem cells has always been an area of great interest due to the pluripotent characteristics of stem cells. In 2006, Takahashi and Yamanaka (Cell 126, 663–676) demonstrated that somatic cells can be reprogrammed into a stem cell-like state, termed induced pluripotent stem (iPS) cells, by ectopic expression of Oct4, Sox2, Klf4 and c Myc. A later report (Nakagawa et al. Nat. Biotechnol. 26:101–106, 2008) showed that iPS cells can be produced in the absence of the c Myc oncogene. We have used this latter strategy to successfully reprogram somatic cells derived from C57BL/6 mouse tail fibroblast to iPS cells. Retrovirus infected fibroblasts exhibited stem cell-like morphology by 14 days post infection. These iPS cells were then infected with a retrovirus that expressed HOXB4. Recombinant leukemia inhibitor factor (LIF) supplement was removed from media at this time and the cells allowed to differentiate into embryoid bodies. These cells were screened for specific differentiation stem cell markers, such as Oct4, Nanog, Sall4 and SSEA-1. iPS cells were converted into embryonic bodies and then infected with retroviruses expressing HOXB4. Embryoid bodies stably expressing HOXB4 were induced to hematopoietic differentiation by treatment of thrombopoietin (TPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), interferon gamma (IFNg) and fms-like tyrosine kinase (FLT3 ligand). Evaluation of iPS-derived hematopoietic cells on smears show strikingly similarity in morphology to the W4 mouse embryonic stem (ES) cells differentiated into hematopoietic cells as a control. Flow cytometry analysis of iPS-derived hematopoietic cells after 1 week exposure to cytokines revealed 7% B220+ cells (B cells), 11% Ter119+ cells (erythroid), and 13% Gr-1+ cells (granulocytes) similar to W4 ES cells. The iPS-derived hematopoietic cells were transplanted into irradiated immunodeficient mice via lateral tail vein injection. Transplantation of these iPS-derived hematopoietic progenitors tagged with GFP into irradiated SCID mice revealed that the hematopoietic progenitors were able to home to the bone marrow after 1 week of transplantation. Importantly, after 1 month, GFP+ engrafted cells remained in the bone marrow suggesting a long-term engraftment. This long term engraftment of the iPS-derived hematopoietic cells to the bone marrow constitutes an important step toward potential therapy of numerous patient-specific blood based diseases.

Comparison of the lateral tail vein and the retro-orbital venous sinus as routes of intravenous drug delivery in a transgenic mouse model

In mice, intravenous injections are commonly administered in the lateral tail vein. This technique is sometimes difficult to carry out and may cause stress to mice. Though injection through the retro-orbital venous sinus can provide certain advantages over lateral tail vein injection, this method is poorly defined and infrequently used. To compare the efficacy of these two routes of drug delivery, the authors injected MAFIA transgenic mice with the depletion agent AP20187, which selectively induces apoptosis in macrophages. Each mouse received five consecutive daily injections through either the lateral tail vein or the retro-orbital venous sinus. The authors then compared macrophage depletion in different tissues (lung, spleen, bone marrow and peritoneal exudate cells). Both routes of injection were similarly effective. A separate experiment using BALB/c mice indicated that retro-orbital venous sinus injection was the less stressful of the two methods.

Murine Mesenchymal Stem Cells Fail To Suppress Acute Graft Versus Host Disease.

Mesenchymal stem cells (MSCs) are multipotent cells of potential clinical interest given their capacity for in vitro expansion and intriguing immunologic properties. Studies using human and murine MSCs demonstrated their ability to suppress stimulated T cells in vitro. Consequently, much interest has been generated in the ability of MSCs to prevent graft versus host disease (GVHD). In fact, a limited number of case reports suggest a therapeutic role of human MSCs in the treatment of GVHD. Although encouraging, no systematic study has been performed to assess the ability of MSCs to suppress GVHD in vivo. In the current study we utilize a purified population of adult bone marrow derived murine MSCs previously shown to be immunosuppressive in vitro to evaluate the therapeutic potential in vivo of MSCs in an established model of murine GVHD.Methods: 8–12 week old C57Bl/6xBalb/c F1 mice were given 750cGy irradiation in a Cs135 gamma irradiator. 16–20 hours after irradiation, the mice received one of four groups of donor cells via lateral tail vein injection: 1) 10e6 C57Bl/6 (B6) bone marrow cells (BM) (n=5), 2) 10e6 B6 BM cells + 30e6 B6 spleen cells (n=12) (GVHD inoculum), 3) 10e6 B6 BM cells + 30e6 B6 spleen cells + 1e6 B6 MSCs (n=4) or 4) 10e6 B6 BM cells + 30e6 B6 spleen cells + 1.5e5 B6 MSCs (n=7). Mice were weighed and assessed for physical signs of GVHD such as ruffled fur, desquamation, diarrhea and hunching prior to receiving irradiation and on a weekly basis following irradiation and injection of the cellular inoculum.Results: In accordance with previous studies, the injection of 30e6 parental (B6) spleen cells combined with 10e6 (B6) parental BM cells into an F1 (B6xBalb/c) recipient following 750cGy irradiation resulted in a reliable model of GVHD. All mice receiving this inoculum demonstrated physical signs of GVHD including hunching and ruffled fur by three weeks post injection with the progression to desquamation and diarrhea by 5 weeks post injection. Similar to mice receiving the GVHD inoculum, mice receiving 30e6 B6 spleen cells + 10e6 B6 BM cells + either 1e6 B6 MSCs or 1.5e5 B6 MSCs demonstrated physical signs of GVHD by 3 weeks post injection. Control mice receiving only 10e6 B6 BM cells after 750cGy irradiation remained healthy and did not demonstrate any signs of GVHD. As demonstrated in figure 1, coinjection of either 1e6 B6 MSCs or 1.5e5 B6 MSCs with 30e6 B6 spleen cells + 10e6 B6 BM cells did not result in any significant change in weight loss compared to those mice receiving the GVHD inoculum. Similarly, the survival of mice receiving the GVHD inoculum was not improved by the coinjection of either 1e6 B6 MSCs or 1.5e5 B6 MSCs (25% vs 0% vs 28.57% at 6 weeks post injection).Conclusion: Previous studies have supported an in vitro immunosuppressive function of MSCs and a limited number of human studies have highlighted the potential ability of human MSCs to suppress GVHD. Despite these previous findings the current study demonstrates that the intravenous injection of MHC matched murine MSCs at the time of GVHD induction in an established murine model of GVHD does not affect the onset or severity of GVHD as measured by physical exam, weight loss and survival. [Display omitted]