Lipophilic Fluorescent Dye Research Articles

Confounding factors in vesicle uptake studies using fluorescent lipophilic membrane dyes

ABSTRACTSmall extracellular vesicles (sEVs) such as exosomes are nanocarriers of proteins, RNAs and DNAs. Isolation of pure sEV populations remains challenging, with reports of protein and lipoprotein contaminants in the isolates. Cellular uptake – a cornerstone for understanding exosome and sEV function – is frequently examined using lipophilic dyes such as PKH67 or CellMask to label the vesicles. In this study, we investigated whether contaminants can confound the outcomes from sEV and exosomes uptake experiments. sEVs were isolated from blood plasma of fasted or non-fasted rats as well as from serum-supplemented or serum-free conditioned cell culture medium using size-exclusion chromatography (SEC). Eluent fractions were characterized using nanoparticle tracking, protein and triglyceride assays and immunoassays. SEC fractions were labelled with different lipophilic dyes and cellular uptake was quantified using endothelial cells or primary cardiomyocytes. We report co-isolation of sEVs with apolipoprotein B-containing lipoproteins. Cellular dye transfer did not correspond to sEV content of the SEC fractions, but was severely affected by lipoprotein and protein content. Overnight fasting of rats decreased lipoprotein content and also decreased dye transfer, while late, sEV-poor/protein-rich fractions demonstrated even greater dye transfer. The potential for dye transfer to occur in the complete absence of sEVs was clearly shown by experiments using staining of sEV-depleted serum or pure protein sample. In conclusion, proteins and lipoproteins can make a substantial contribution to transfer of lipophilic dyes to recipient cells. Considering the likelihood of contamination of sEV and exosome isolates, lipophilic dye staining experiments should be carefully controlled, and conclusions interpreted with caution.

Elucidation of rubber biosynthesis and accumulation in the rubber producing shrub, guayule (Parthenium argentatum Gray).

Guayule biosynthesizes and accumulates rubber particles predominantly in epithelial cells in the parenchyma tissue, and this biosynthesis and accumulation is accompanied by remodeling of the roles of epithelial cells. The mechanism underlying the biosynthesis and accumulation of large quantities of rubber particles and resin in the parenchyma tissue of the stem bark of guayule (Parthenium argentatum Gray) remained unanswered up to now. Here, we focused on rubber particle biosynthesis and accumulation in guayule and performed histochemical analyses using a lipophilic fluorescent dye specific for lipids and spectral confocal laser scanning microscopy. Unmixing images were constructed based on specific spectra of cis-polyisoprene and resin and showed that guayule accumulates a large amount of resin in the resin canals in parenchyma tissue and in pith. Interestingly, the fluorescence signals of rubber were predominantly detected in a specific single layer of epithelial cells around the resin canals. These epithelial cells accumulated large rubber particles and essentially no resin. Immunoblotting and immunostaining of guayule homologue of small rubber particle proteins (GHS), which contributes to the biosynthesis of rubber in guayule, showed that GHS is one of several small rubber particle proteins and is localized around rubber particles in epithelial cells. De novo sequencing of the rubber particle proteins showed the presence of all known organelle proteins, suggesting that epithelial cells biosynthesize rubber particles, followed by remodeling of the cells for the accumulation of rubber particles with subsequent decomposition of the organelles. These results indicate that epithelial cells around resin canals are bifunctional cells dedicated to the biosynthesis and accumulation of rubber particles.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye.

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

UbasM: An effective balanced optical clearing method for intact biomedical imaging

Optical clearing methods can facilitate deep optical imaging in biological tissue by reducing light scattering and this has enabled accurate three-dimensional signal visualization and quantification of complex biological structures. Unfortunately, existing optical clearing approaches present a compromise between maximizing clearing capability, the preservation of fluorescent protein emission and membrane integrity and the speed of sample processing – with the latter typically requiring weeks for cm scale tissue samples. To address this challenge, we present a new, convenient, aqueous optical clearing agent, termed UbasM: Urea-Based Amino-Sugar Mixture, that rapidly renders fixed tissue samples highly transparent and reliably preserves emission from fluorescent proteins and lipophilic dyes in membrane integrity preserved tissues. UbasM is simple, inexpensive, reproducible and compatible with all labeling methods that we have encountered. It can enable convenient, volumetric imaging of tissue up to the scale of whole adult mouse organs and should be useful for a wide range of light microscopy and tomography techniques applied to biomedical research, especially the study on organism-level systems biology at multiple levels.

Abstract 4775: PTHrP regulate CSC and promote EMT in the PyMT model of breast cancer progression

Abstract PTH-related peptide (PTHrP) was first discovered in cancer patients as the primary cause of humoral hypercalcemia of malignancy (HHM). We previously showed that PTHrP ablation in the MMTV-PyMT murine model of breast cancer progression can dramatically prolongs tumor latency, slows tumor growth & metastases suggesting that PTHrP may regulate cancer stem cells (CSCs). CSCs & epithelial-mesenchymal transition (EMT) have been recognized as driving forces in tumor initiation & progression, & have been characterized previously using ALDH & CD49/EpCAM markers. Here, we aim to determine the role of PTHrP in CSCs & EMT regulation. For this purpose, we analyzed tumor cells derived from Pthrpflox/flox;Cre- (Control) & Pthrpflox/flox;Cre+ (KO) tumors. Cells were incubated with ALDH reagent followed by CD49f/EpCAM antibody labelling & subjected to FACS analysis. Using CD49f/EpCAM labelling, cells separated into two distinct populations, luminal progenitor cells (CD49f+EpCAM+) & EMT cells (CD49f+EpCAM-). In control tumors, ~85% of cells were characterized as luminal progenitor and ~15% as EMT. In contrast, in KO tumors, ~97% of cells were characterized as luminal progenitor whereas only ~2% were EMT. Using ALDH staining alone, ~8% & ~10% of the cells from the control & KO tumor were ALDH+, respectively. Furthermore, the majority of the ALDH+ cells were predominately contained within the luminal progenitor population in both the control & KO tumors cells. Next, we confirmed the dividing capacity of the two cell populations using a lipophilic fluorescent dye, PKH26. As expected, luminal progenitor cells exhibited a fast-dividing phenotype, whereas EMT cells displayed a slow dividing phenotype. However, using mammosphere assay, we did not see any significant difference in mammosphere forming efficiency between control & KO tumor. In summary, PTHrP ablation significantly diminishes the EMT population in KO compared to the control tumor. In conclusion, PTHrP may shift the tumor cell phenotype towards EMT, which suggests a mechanism underlying PTHrP - driven metastases. Citation Format: Rui Zhang, Jiarong Li, Andrew C. Karaplis, Richard Kremer. PTHrP regulate CSC and promote EMT in the PyMT model of breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4775. doi:10.1158/1538-7445.AM2017-4775

Abstract 5134: Novel trifunctional immunoliposome delivery system to minimize resistance and side effects for anticancer therapy

Abstract Background: Breast cancer (BC) is the second leading cause of cancer death in women worldwide. About 25% of BCs have overexpression of the HER2 receptor. Although HER2 targeted therapies have shown considerable improvement in HER2-positive BC patients’ outcome, treatment resistance remains a clinical challenge. Here, we sought to develop and evaluate a novel tri-functional immune-liposome (TFIL) that overcomes HER2 treatment resistance and associated cardiotoxicity by dually targeting HER2 on BC cells and CD3 receptors on cytotoxic T-lymphocytes (CTLs). Material and methods: Anti-HER2 (Trastuzumab) and anti-CD3 (OKT-3) antibodies, were conjugated to lipid nanoparticles by the micelle-transfer method, and the resulting formulation was purified by dextran gradient ultra-centrifugation. Targeted lipid nanoparticles were formulated with a fluorescent lipophilic dye, DiD, for studying receptor binding and internalization. Studies were conducted with HER2-positive BT474 cells and CD3-positive Jurkat cells using flow cytometry analyses. Doxorubicin (DXR) was encapsulated in the nanoparticles by the remote-loading technique for cell-kill experiments. In vitro cell-kill studies were conducted by co-culturing BT474 as the target cells, and peripheral blood mononuclear cells as the effector cells, at varying ratios. Results: Purified formulations were successfully characterized for conjugation by determining the protein to lipid ratio. Flow cytometry analyses demonstrated successful cell binding and/or internalization of the TFIL with both the HER2 and CD3-positive cell lines. Moreover, these dual-targeted nanoparticles were able to retarget T cells to kill HER2 positive BC cells, and showed improved efficacy compared to non-targeted and plain HER2-targeted formulations in vitro. Conclusion: A novel drug delivery system, TFIL, that targets HER2 receptors on tumor cells, CD3 on CTLs, and is able to slowly release DXR was successfully developed and evaluated in vitro on HER2 overexpressing BC cells. Our findings show great promise at overcoming resistance and associated cardiotoxicity to present HER2 targeted BC therapies, and may translate into improved anti-tumor activity clinically compared to other treatment options. Citation Format: Tanaya R. Vaidya, Sihem Ait-Oudhia. Novel trifunctional immunoliposome delivery system to minimize resistance and side effects for anticancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5134. doi:10.1158/1538-7445.AM2017-5134

Evaluation of persistence and distribution of intra-dermally administered PKH26 labelled goat bone marrow derived mesenchymal stem cells in cutaneous wound healing model.

The current study was designed to study the persistence and distribution of caprine bone marrow derived mesenchymal stem cells (cBM-MSCs) when administered intra-dermally in experimentally induced cutaneous wounds in rabbits. MSC's from goat bone marrow were isolated and their differentiation potential towards adipogenic and osteogenic lineages were assayed in vitro. The isolated cells were phenotypically analysed using flow cytometry for the expression of MSC specific matrix receptors (CD73, CD105 and Stro-1) and absence of hematopoietic lineage markers. Further, these in vitro expanded MSCs were stained with PKH26 lipophilic cell membrane red fluorescent dye and prepared for transplantation into cutaneous wounds created on rabbits. Five, 2cm linear full thickness skin incisions were created on either side of dorsal midline of New Zealand white rabbits (n=4). Four wounds in each animal were implanted intra-dermally with PKH26 labelled cBM-MSCs suspended in 500µl of Phosphate Buffer Saline (PBS). Fifth wound was injected with PBS alone and treated as negative control. The skin samples were collected from respective wounds on 3, 7, 10 and 14days after the wound creation, and cryosections of 6µM were made from it. Fluorescent microscopy of these cryosections showed that the PKH26 labelled transplanted cells and their daughter cells demonstrated a diffuse pattern of distribution initially and were later concentrated towards the wound edges and finally appeared to be engrafted with the newly developed skin tissues. The labelled cells were found retained in the wound bed throughout the period of 14days of experimental study with a gradual decline in their intensity of red fluorescence probably due to the dye dilution as a result of multiple cell division. The retention of transplanted MSCs within the wound bed even after the complete wound healing suggests that in addition to their paracrine actions as already been reported, they may have direct involvement in various stages of intricate wound healing process which needs to be explored further.

Optimization of liposome delivery using low intensity ultrasound and microbubbles

Liposomes are versatile drug delivery microvehicles to carry small molecule therapeutics and have a steadily growing global market share. Liposomal formulations can stabilize therapeutic compounds, promote cellular uptake, and improve biodistribution. The controlled delivery of liposomes can be further enhanced by ultrasound and microbubbles. Here we study the effect of ultrasound-mediated liposome delivery with microbubbles. By incorporating the fluorescent lipophilic dye, DiO, we investigated the effect of different liposome loading techniques and microbubble conjugation on liposome delivery in vitro. We found that active-loading liposomes demonstrated a better ultrasound-mediated delivery efficacy when conjugated with microbubbles than when used as a free drug. We also investigated the acoustic parameters: pressure (70 to 2000 kPa), duty cycle (10 to 50%), and PRF (2 to 1000 Hz); at a fixed 1 MHz center frequency for optimal liposome delivery with low cell detachment. Our results indicate that the liposome-carried microbubbles could be a promising new drug delivery vehicle to incorporate into the current chemoradiation therapy regime against cancer.

Imaging the accumulated intracellular microalgal lipids as a response to temperature stress.

Over the last few decades, many scientists considered microalgae as promising actors for future biofuels because of the high lipid productivity inside their cells. Moreover, much attention has been paid to algal lipids as they can be used in biodiesel production. In this study, we optimized the different suitable conditions such as incubation time, incubation temperature, Dimethylesulfoxide and Nile red concentrations ofthe lipophilic fluorescence dye Nile red as an excellent and fast vital stain to detect and quantify intracellular lipids. This was achieved using the green alga Nannochloropsis salina. In addition, investigating the accumulation of lipid vesicles inside different isolated microalgal species as a response to temperature stress. Furthermore, the confocal laser scanning microscopy (LS510) for imaging and measuring the size and volume of the accumulated lipid vesicles was used.

The toll-like receptor 2 agonist Pam3CSK4 is neuroprotective after spinal cord injury

Microglia/macrophage activation and recruitment following spinal cord injury (SCI) is associated with both detrimental and reparative functions. Stimulation of the innate immune receptor Toll-like receptor-2 (TLR2) has shown to be beneficial following SCI, and it increases axonal regeneration following optic nerve crush. However, the mechanism(s) remain unclear. As microglia express high levels of TLR2, we hypothesized that modulating the microglial response to injury using a specific TLR2 agonist, Pam3CSK4, would prevent secondary-mediated white matter degeneration following SCI. To test this hypothesis, we documented acute changes in microglia, axons, and oligodendroglia over time using two-photon excitation and an ex vivo laser-induced SCI (LiSCI) model. We utilized double transgenic mice that express GFP in either microglia or oligodendroglia, and YFP in axons, and we applied the lipophilic fluorescent dye (Nile Red) to visualize myelin. We found that treatment with Pam3CSK4 initiated one hour after injury induced a significant increase in the extent and timing of the microglial response to injury compared to vehicle controls. This enhanced response was observed 2 to 4h following SCI and was most prominent in areas closer to the ablation site. In addition, Pam3CSK4 treatment significantly reduced axonal dieback rostral and caudal to the ablation at 6h post-SCI. This protective effect of Pam3CSK4 was also mirrored when assessing secondary bystander axonal damage (i.e., axons spared by the primary injury that then succumb to secondary degeneration), and when assessing the survival of oligodendroglia. Following these imaging experiments, custom microarray analysis of the ex vivo spinal cord preparations revealed that Pam3CSK4-treatment induced an alternative (mixed M1:M2) microglial activation profile. In summary, our data suggest that by providing a second “sterile” activation signal to microglia through TLR2/TLR1 signaling, the microglial response to injury can be modulated in situ and is highly neuroprotective.

Light/magnetic hyperthermia triggered drug released from multi-functional thermo-sensitive magnetoliposomes for precise cancer synergetic theranostics

Precise delivery of antineoplastic drugs to specific tumor region has drawn much attention in recent years. Herein, a light/magnetic hyperthermia triggered drug delivery with multiple functionality is designed based on methotrexate (MTX) modified thermo-sensitive magnetoliposomes (MTX-MagTSLs). In this system, MTX and oleic acid modified magnetic nanoparticles (MNPs) can be applied in biological and magnetic targeting. Meanwhile, lipophilic fluorescent dye Cy5.5 and MNPs are encapsulated into the bilayer of liposomes, which can not only achieve dual-imaging effect to verify the MTX-MagTSLs accumulation in tumor region, but also provide an appropriate laser irradiation region to release Doxorubicin (Dox) under alternating magnetic field (AMF). Both in vitro and in vivo results revealed that MTX-MagTSLs possessed an excellent targeting ability towards HeLa cells and HeLa tumor-bearing mice. Furthermore, the heating effect of MTX-MagTSLs was amplified 4.2-fold upon combination with AMF and local precise near-infrared laser irradiation (808nm) (DUAL-mode) to rapidly reach the phase change temperature (Tm) of MTX-MagTSLs in 5min compared with either AMF or laser stimulation alone, resulting in a significantly enhanced release of Dox at tumor region and precise cancer synergetic theranostics.

Labeling Mesenchymal Stromal Cells with PKH26 or VybrantDil Significantly Diminishes their Migration, but does not affect their Viability, Attachment, Proliferation and Differentiation Capacities

Fluorescent dyes such as PKH26 and VybrantDil facilitate rapid and simple labeling of cells for later detection in various assays. But covering the cell surface with lipophilic fluorescent dyes may impair cellular functions. We therefore investigated the effects of PKH26 and VybrantDil on viability, proliferation, differentiation, attachment and migration of human mesenchymal stromal cells (MSCs) in vitro. To this end, MSCs were harvested from bone marrow of 12 individuals, expanded employing methods compliant to good manufacturing practice, and stained with PKH26 or VybrantDil. MSCs without label served as controls. The intensity of fluorescent staining as function of label and incubation time was investigated. Viability and proliferation were enumerated by cell counting. The osteogenic and adipogenic differentiations of MSCs were explored by cytochemical staining and transcript analyses, the cell migration and attachment by specific in vitro assays. We report that labeling of human MSCs with PKH26 yielded brighter signals facilitating prolonged detection compared to VybrantDil. No significant effects of PKH26 and VybrantDil were recorded when the viability, proliferation, attachment, osteogenic and adipogenic differentiation of human MSCs were investigated. But loading cells with PKH26 or VybrantDil significantly diminished the migration of the MSCs in vitro. We conclude that analyses depending on cellular migration may be biased when the cells are loaded with these lipophilic dyes.

In vivo imaging and quantification of regional adiposity in zebrafish.

Adipose tissues (ATs) are lipid-rich structures that supply and sequester energy-dense lipid in response to the energy status of an organism. As such, ATs provide an organism energetic insurance during periods of adverse physiological burden. ATs are deposited in diverse anatomical locations, and excessive accumulation of particular regional ATs modulates disease risk. Therefore, a model system that facilitates the visualization and quantification of regional adiposity holds significant biomedical promise. The zebrafish (Danio rerio) has emerged as a new model system for AT researchin which the entire complement of regional ATs can be imaged and quantified in live individuals. Here we present detailed methods for labeling adipocytes in live zebrafish using fluorescent lipophilic dyes, and for identifying and quantifying regional zebrafish ATs.

Validated flow cytometry allows rapid quantitative assessment of immune responses in amphibians

Assessments of amphibian immune state have been commonly made through indirect methods like phytohemagglutinin (PHA) injections, or by direct methods like cell counts from blood smears. Here we validate a simple method to assess immune responses in amphibians by means of flow cytometry with a fluorescent lipophilic dye (3,3′ Dipentyloxacarbocyanine), which removes the need for specific antibodies. We experimentally altered the immunological state ofPelobates cultripestadpoles by exposing some to exogenous corticosterone. We then determined the immune state of each tadpole through both blood smears and flow cytometry. We found that both techniques showed similar patterns of the proportion of white blood cells. Once validated, flow cytometry also allowed quantitation of changes in absolute number of white cells. We discuss the suitability of both techniques attending to the accuracy of each technique, body size requirements, or the tractability in field studies.

A classification system for zebrafish adipose tissues

ABSTRACTThe zebrafish model system offers significant utility for in vivo imaging of adipose tissue (AT) dynamics and for screening to identify chemical and genetic modifiers of adiposity. In particular, AT can be quantified accurately in live zebrafish using fluorescent lipophilic dyes. Although this methodology offers considerable promise, the comprehensive identification and classification of zebrafish ATs has not been performed. Here, we use fluorescent lipophilic dyes and in vivo imaging systematically to identify, classify and quantify the zebrafish AT pool. We identify 34 regionally distinct zebrafish ATs, including five visceral ATs and 22 subcutaneous ATs. For each of these ATs, we describe detailed morphological characteristics to aid their identification in future studies. Furthermore, we quantify the areas for each AT and construct regression models to allow prediction of expected AT size and variation across a range of developmental stages. Finally, we demonstrate the utility of this resource for identifying effects of strain variation and high-fat diet on AT growth. Altogether, this resource provides foundational information on the identity, dynamics and expected quantities of zebrafish ATs for use as a reference for future studies.

Semi‐fluorinated alkanes for topical delivery of Cyclosporine

PurposeCyclosporine A (CsA) is an anti‐inflammatory agent that has been frequently used to treat ocular inflammatory conditions, such as dry eye syndrome. However, the poor water solubility of CsA makes it difficult to formulate into an acceptable ocular dosage form. Semi‐fluorinated alkanes (SFAs) are a novel class of inert, non‐toxic and amphiphilic liquids that form clear solutions with CsA and have been suggested as efficient carriers for topical administration of CsA. The aim of this study was to assess the corneal bioavailability of CsA from SFAs compared to currently marketed formulations.MethodsAn ex vivo porcine eye model was used to study the penetration of a) Restasis® (0.05% CsA ophthalmic emulsion), b) Ikervis® (0.1% CsA ophthalmic emulsion), and c) 0.05% or 0.1% CsA in SFAs. The amount of drug penetrated per gram of cornea between 0.5 and 4 h after application was assayed by HPLC and statistically compared using a two‐way ANOVA. Drug distribution in different layers of the cornea was also visualized by substituting CsA with a lipophilic fluorescent dye and viewing corneal sections under a fluorescent microscope.ResultsSignificant improvement in corneal penetration of CsA could be observed for 0.05% CsA in SFAs (C1 h = 5.844 ± 2.408 ng/g) over Restasis (C1 h = 761 ± 221 ng/g), with the area under curve (AUC) being more than 8‐folds greater. The AUC of 0.1% CsA in SFAs (C1 h = 12.556 ± 4.017 ng/g) was 3.6 folds greater than Ikervis (C1 h = 2.900 ± 341 ng/g). Microscopic examinations revealed that the dye incorporated into SFAs tended to accumulate mainly in the corneal epithelium.ConclusionsOverall, this study showed that SFAs can significantly improve the corneal absorption of lipophilic drugs, such as CsA, and could therefore be a promising platform for drug delivery to the eye.

Deletion of angiotensin II type 2 receptor accelerates adipogenesis in murine mesenchymal stem cells via Wnt10b/beta-catenin signaling

Recent evidence suggests that the renin-angiotensin system (RAS) has a vital role in adipocyte biology and the pathophysiology of metabolic syndrome. Obesity is the main culprit of metabolic syndrome; and mesenchymal stem cells (MSCs) have been forwarded as a major source of adipocyte generation. Previously, we reported that MSCs have a local RAS and that pharmacological blockade of angiotensin II type 2 receptor (AT2R) promotes adipogenesis in human MSCs. However, the definitive roles of AT2R and how AT2R functions in adipogenesis remains unknown. To this end, we employed AT2R-null murine MSCs to characterize how AT2R affects the differentiation of MSCs into adipocytes. Murine MSCs were isolated from AT2R-null mice and wild-type littermates, grown to confluency, and then differentiated into adipocytes. Adipogenesis was quantitated by assessing the lipid droplet accumulation. Using the lipophilic fluorescent dye, the AT2R-null cells showed significantly increased total fluorescence (261.6±49.6% vs littermate) on day 7. Oil red O staining followed by extraction of the absorbed dye and measurement of the absorbance on day 14 also exhibited significantly increased lipid droplet accumulation in the AT2R-null cells (202.7±14.1% vs littermate). We also examined the expression of adipogenic marker genes by quantitative RT-PCR. The AT2R-null group exhibited significantly increased expression of PPAR-gamma, fatty acid synthase, and adiponectin (vs littermate). We further examined the role of Wnt10b/beta-catenin signaling, which reportedly has an important inhibitory role in adipogenesis. The AT2R-null group exhibited significantly decreased Wnt10b expression accompanied by decreased beta-catenin (vs littermate). Our results thus revealed that the AT2R inhibits adipogenic differentiation in murine MSCs. Moreover, this inhibitory effect is associated with Wnt10b/beta-catenin signaling. These results provide important insights into the pathophysiology of obesity and obesity-related consequences such as metabolic syndrome, hinting at possible future therapies.

Abstract 871: Nanoliposomal targeting of ephrin receptor A2 (EphA2): Preclinical in vitro and in vivo rationale

Abstract Ephrins receptors are cell to cell adhesion molecules that mediate signaling and are implicated in neuronal repulsion, cell migration and angiogenesis. Ephrin receptor A2 (EphA2) is part of the ephrin family and was shown to be overexpressed in several solid tumors including ovarian, gastric and lung cancer and is associated with poor prognosis. MM-310 is a novel EphA2 targeted docetaxel nanoliposome. Similar to other nanoparticles, MM-310 leverages organ specificity through enhanced permeability and retention, but can also leverage cellular specificity through its EphA2 targeting arm. Binding of targeted liposomes to cells in vitro was assessed using flow cytometry in a large panel cell lines. 3D spheroids were used to assess targeting as well as liposome penetration. In order to test the effect of targeting on liposome microdistribution in vivo, primary and metastatic tumor bearing animals were injected with a mixture of EphA2-targeted liposome (EphA2-Ls) and non-targeted liposome (NT-Ls) labeled with two different lipophilic fluorescent dyes. Tissues were assessed using fluorescent microscopy. In order to evaluate the contribution of EphA2-targeting to efficacy, four gastric xenograft models were treated with either MM-310 or a non-targeted version of the drug NT-310, and compared to free docetaxel. In cell suspension models, we observed a high level of specificity for EphA2-Ls, with more than one hundred-fold increase in liposome cell association. 3D spheroid assays showed that EphA2-Ls binds and penetrates EphA2+ spheroids, while non-targeted liposomes show minimal penetration. Tissue microdistribution analysis in triple negative breast and esophageal tumor models following injection of the EphA2-Ls/NT-Ls mixtures showed a target mediated shift in the microdistribution of liposomes. EphA2-Ls penetrated deeper within the lesions while the NT-Ls deposited at high levels in areas close to the microvasculature. The target mediated shift in microdistribution was also observed in lung metastasis model, with a pattern of distribution that potentially matches disseminated tumor cells. In the same animals, targeting did not affect microdistribution in normal organs such as liver, spleen and skin. Four models of gastric and esophageal cancers were used to test the potential link between cell targeting and tumor growth control. While NT-310 and MM-310 were both able to control tumor growth leading to regression in most models, in three out of four models there was a statistically significant difference between MM-310 and NT-310 at 25 mpk. Biomarker analysis is underway to evaluate the key parameters necessary to mediate targeting. In conclusion, the data suggests clear evidence of targeting in 2D cell suspension, 3D spheroids, and in primary as well as metastatic tumor models in vivo. In vivo efficacy data showed evidence of the contribution of EphA2 targeting to tumor growth control and regression in several gastric cancer models. Citation Format: Walid S. Kamoun, Lia Luus, Christine Pien, Tad Kornaga, Shinji Oyama, Zhaohua Richard Huang, Suresh Tipparaju, Dmitri B. Kirpotin, James D. Marks, Alexander Koshkaryev, Melissa Geddie, Lihui Xu, Alexey Lugovosky, Daryl C. Drummond. Nanoliposomal targeting of ephrin receptor A2 (EphA2): Preclinical in vitro and in vivo rationale. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 871.

Endothelial Progenitor Cells Combined with Cytosine Deaminase-Endostatin for Suppression of Liver Carcinoma.

Transplantation of gene transfected endothelial progenitor cells (EPCs) provides a novel method for treatment of human tumors. To study treatment of hepatocellular carcinoma using cytosine deaminase (CD)- and endostatin (ES)-transfected endothelial progenitor cells (EPCs), mouse bone marrow-derived EPCs were cultured and transfected with Lenti6.3-CD-EGFP and Lenti6.3-ES-Monomer-DsRed labeled with superparamagnetic iron oxide (SPIO) nanoparticles. DiD (lipophilic fluorescent dye)-labeled EPCs were injected into normal mice and mice with liver carcinoma. The EPCs loaded with CD-ES were infused into the mice through caudal veins and tumor volumes were measured. The tumor volumes in the EPC + SPIO + CD/5-Fc + ES group were found to be smaller as a result and grew more slowly than those from the EPC + SPIO + LV (lentivirus, empty vector control) group. Survival times were also measured after infusion of the cells into the mice. The median survival time was found to be longer in the EPC + SPIO + CD/5-Fc + ES group than in the others. In conclusion, the EPCs transfected with CD-ES suppressed the liver carcinoma cells in vitro, migrated primarily to the carcinoma, inhibited tumor growth, and also extended the median survival time for the mice with liver carcinoma.

The carbocyanine dye DiD labels in vitro and in vivo neural stem cells of the subventricular zone as well as myelinated structures following in vivo injection in the lateral ventricle.

Carbocyanines are fluorescent lipophilic cationic dyes used since the early 1980s as neuronal tracers. Several applications of these compounds have been developed thanks to their low cell toxicity, lateral diffusion within the cellular membranes, and good photostability. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine 4-chlorobenzenesulfonate (DiD) is an interesting component of this family because, in addition to the classic carbocyanine properties, it has a longer wavelength compared with its analogues. That makes DiD an excellent carbocyanine for labeling cells and tissues with significant intrinsic fluorescence. Drug encapsulation, drug delivery, and cellular transplantation are also fields using DiD-based systems where having detailed knowledge about its behavior as a single entity is important. Recently, promising studies concerned neural stem cells from the subventricular zone of the lateral ventricle in the brain (their natural niche) and their potential therapeutic use. Here, we show that DiD is able to label these stem cells in vitro and present basilar information concerning its pharmacokinetics, concentrations, and microscope protocols. Moreover, when DiD is injected in vivo in the cerebrospinal fluid present in the lateral ventricle of rat, it also labels stem cells as well as myelinated structures of the caudoputamen. This analysis provides a database to consult when planning experiments concerning DiD and neural stem cells from the subventricular zone.