Red Fluorescent Cells Research Articles

Rapid discovery of death ligands with one-bead-two-compound combinatorial library methods.

The one-bead-one-compound (OBOC) technology enables one to generate thousands to millions of chemical molecules on resin beads (90 μm diameter) such that each bead displays 10(13) copies of the same chemical entity. Whole-cell binding assays have been developed to screen OBOC combinatorial libraries for ligands that bind to specific cell surface receptors. While very powerful, this screening method does not address the downstream cell signaling properties of the binding ligand. We have modified the OBOC technology by introducing a fixed known cell adhesion ligand to the outer layer of each bead. This one-bead-two-compound (OB2C) library configuration allows the bound cells to interact with the random immobilized chemical molecules on each bead. The bound cells can then be probed for specific cellular responses such as apoptosis and activation or inhibition of a specific cell signaling pathway. To validate this concept, an OB2C combinatorial library was created such that a random hexapeptide plus a high affinity lymphoma targeting ligand LLP2A were displayed on each bead. This LLP2A-X(6) OB2C library was then screened with human T-cell leukemia cells (Molt-4) for cell death responses. After 5 days of incubation, propidium iodide was added to the bead library to stain dead cells. Beads coated by red fluorescent cells were isolated for sequence analysis. Two ligands identified by this method, when added to the lymphoid cancer cells, were able to induce cell death.

Transplantation of Neonatal Gut Neural Crest Progenitors Reconstructs Ganglionic Function in Benzalkonium Chloride-Treated Homogenic Rat Colon

To value the possibility and the future feasibility of the use of autograft cells transplantation in disorders of the enteric neural system, we postulate that isolated neonatal nongenetically modified neural crest progenitors could survive and differentiate into neurons and glia in homogenic denervated rats and, therefore, restore partial intestinal function after transplantation. Neural crest progenitors were isolated from neonatal rats. After passages, the cells were labeled with CM-DiI. The labeled cells were then delivered into the muscular distal denervated colon of rats whose neural plexuses were eliminated using benzalkonium chloride. The treated colons of recipients were harvested at 1, 4, and 8 wk, and identified by immunofluorescent staining. The physiologic and functional improvements on treated colons were well examined after transplantation 8 wk. Progenitors could generate neurospheres and differentiate into neurons and glia in vitro. After transplantation, red fluorescent cells were observed in the injected tissue for up to 8 wk, and they differentiated into neurons and glia in the host colon. Functional examinations indicated that symptoms and intestinal dysfunction of the denervated model were reversed. We provide herein further evidence that autologous cell transplantation is a feasible therapy for enteric nervous system disorders.

The Effects of Platelet-Rich Plasma on Bone Marrow Stromal Cell Transplants for Tendon Healing In Vitro

In this study, we investigated the effect of platelet-rich plasma (PRP) and bone marrow-derived stromal cell (BMSC)-seeded interposition in an in vitro canine tendon repair model. Bone marrow, peripheral blood, and tendons were harvested from mixed-breed dogs. Bone marrow-derived stromal cells were cultured and passaged from adherent cells of bone marrow suspension. Platelet-rich plasma was purified from peripheral blood using a commercial kit. A total of 192 flexor digitorum profundus tendons were used for the study. Tendons repaired with a simple suture were used as a control group. In treatment groups, a collagen gel patch was interposed at the tendon repair site before suture. There were 3 treatment groups, according to the type of collagen patch: a patch with PRP, a patch with BMSC, and a patch with PRP and BMSC. The repaired tendons were evaluated by biomechanical testing and by histological survey after 2 and 4 weeks in tissue culture. To evaluate viability, cells were labeled with PKH26 red fluorescent cell linker (Sigma, St. Louis, MO) and surveyed under confocal microscopy after culture. The maximum breaking strength and stiffness of the healing tendons with the BMSC-seeded PRP patch were significantly higher than those of the healing tendons without a patch or with a cell-seeded patch (p < .02). Viable BMSCs were present at both 2 and 4 weeks. Platelet-rich plasma enhanced the effect of BMSC-seeded collagen gel interposition in this in vitro model. Based on these results, we now plan to investigate this effect in vivo.

Recognition of yeast by murine macrophages requires mannan but not glucan.

The first barrier against infection by Candida albicans involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in C. albicans; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology. Candida glabrata and Saccharomyces cerevisiae are taken up by J774 macrophage cells more rapidly than C. albicans, and C. albicans yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake.

Human Serum Mannose-binding Lectin Senses Wall Teichoic Acid Glycopolymer of Staphylococcus aureus, Which Is Restricted in Infancy

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.

Cytochrome-P450 2B1 gene silencing attenuates puromycin aminonucleoside-induced cytotoxicity in glomerular epithelial cells

Previously, we demonstrated that cytochrome P450 2B1 (CYP2B1) can generate reactive oxygen species in puromycin aminonucleoside (PAN)-induced nephrotic syndrome, an animal model of minimal-change disease in humans. In this study we found that overexpression of CYP2B1 in rat glomerular epithelial cells in vitro significantly increased PAN-induced reactive oxygen species generation, cytotoxicity, cell death, and collapse of the actin cytoskeleton. All of these pathological changes were markedly attenuated by siRNA-induced CYP2B1 silencing. The cellular CYP2B1 protein content was significantly decreased whereas its mRNA level was markedly increased, suggesting regulation by protein degradation rather than transcriptional inhibition in the PAN-treated glomerular epithelial cells. This degradation of CYP2B1 was accompanied by the induction of heme oxygenase-1, an important indicator of heme-induced oxidative stress. In PAN-treated CYP2B1-silenced glomerular epithelial cells the induction of heme oxygenase-1 and caspase-3 activity were significantly decreased. Further, cleavage of the stress-induced pro-apoptotic endoplasmic reticulum-specific pro-caspase-12 was prevented in the silenced cells. Our results support a pivotal role of CYP2B1 for reactive oxygen species production in the endoplasmic reticulum in PAN-induced cytotoxicity.

Abstract 4814: Tunneling nanotubes facilitate intercellular communication and trafficking between mesothelioma cells

Abstract Introduction: Mesothelioma is an aggressive malignancy which spreads via local invasion. We have observed fine, long, non-adherent cable-like structures connecting mesothelioma cells in vitro which are consistent with tunneling nanotubes, an entity recently described in malignant as well as neuronal and immune cells. We hypothesized that tunneling nanotubes facilitate intercellular communication between distant and neighboring cells via transfer of intracellular components. Materials and Methods: Biphasic and sarcomatoid mesothelioma cells, as well as primary mesothelioma and lung cancer cells, were grown using medium of variable concentrations of serum, glucose, and cytokines at low or normal pH. Fluorescent lipophilic dyes were used to stain cytoplasmic membranes and mitochondria. Red and green fluorescent protein-expressing cells were examined following mixing of the two populations. Brightfield and fluorescent microscopy were performed, as was time-lapse imaging to determine time frames for transport via the nanotubes. To determine composition, nanotubes and cells were fixed and immunofluorescent staining was performed. Electron microscopy was also performed to elucidate nanotube structure and cell membrane insertions. Results: Nanotubes connected cells in close and distant proximity. The proportion of cells growing one or more nanotubes was highest in medium with low pH and/or low serum, high glucose, and cytokines. We have reproduced this finding in primary tumor cells from resected mesothelioma cells following surgery, as well as mesothelioma and lung cancer cells from pleural effusions. Brightfield and fluorescent microscopy revealed transfer of material (including mitochondria and proteins) between cells following nanotube formation, confirming the tubes are tunneling. Cells positive for both GFP and RFP and connected by nanotubes were observed, indicating protein transfer was bidirectional. Fluorescent immunostaining elucidated the presence of actin, vimentin, Zo-1, and fascin in nanotubes, supporting actin as a key component. Electron microscopy demonstrated the presence of early nanotubes and insertion of mature nanotubes into receptive cells. Conclusions: Tunneling nanotubes facilitate intercellular communication and trafficking between cells from cell lines and primary tumors. For the first time, we demonstrate that specific medium conditions can reproducibly promote increased growth of nanotubes to accelerate cell-to-cell communication via these structures, as well the ability to fix these sensitive structures for immunostaining. We also demonstrate that proteins and mitochondria are among cell components transported bidirectionally via nanotubes. Tunneling nanotubes provide a significant alternative for cell-to-cell communication between invasive cancer cells, as well as a plausible mechanism for tumoral resistance to therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4814.

Impact of third-party bone marrow mesenchymal stem cells on allogenic skin transplantation

To investigate the effect of the third-party bone marrow-derived mesenchymal stem cells (BMSCs) on the allogeneic skin transplantation. 40 female C57BL/6 mice and 50 male BALB/C mice were respectively used as donors and recipients of skin transplantation. 50 BALB/C mice were divided randomly into 5 groups: Blank control group, Cyclophosphamide group BMSCs group, Cyclophosphamide + BMSCs group and CM-DiI staining group, with 10 mice in each group. Before skin transplantation, high-dose abdominal injection of Cyclophosphamide (200 mg/kg, 2 d) was performed in recipient mice. On the transplantation day, a bonus of 1 x 10(5) BMSCs of the SD rat (SD-BMSCs) were injected through the tail vein. The observation of skin grafts, mixed lymphocyte culture (MLC), HE staining, the observation of CM-DiI-labeled SD-BMSCs and FACS were used. The skin graft survival time was significantly prolonged in the Cyclophosphamide + BMSCs group, as compared with the blank control group, the Cyclophosphamide group, the BMSCs group respectively. When BMSC and lymphocyte mixed at the ratio of 1:1 and 1:10, rat BMSCs inhibited T lymphocyte proliferation. More angiogenesis and less lymphocyte infiltration were found in the experimental group than them in other groups. Red fluorescent cells were found in CM-DiI staining group under long-term observation. The SD-BMSCs can he detected by flow cytometry in the cell group and the Cyclophosphamide + BMSCs group. BMSCs can survive in the heterogeneous recipient body; the third-party BMSCs transplantation can prolong skin graft survival time; BMSCs can inhibit T lymphocyte activation and proliferation.

Isolation, Culture and Biological Characteristics of Primordial Germ Cells from Beijing Fatty Chicken

The aim of this study was to explore the isolation and culture process of Beijing fatty chicken primordial germ cells (PGCs) and investigate their biological characteristics. The PGCs isolated from the genital ridges of Beijing fatty chicken (Gallus domesticus) embryos after 5.5 days of incubation were co-cultured with mice embryonic fibroblasts (MEF). The results showed that the PGCs of the Beijing fatty chicken were positive for periodic acid Schiff (PAS) and alkaline phosphatase (AKP) staining. These cells could proliferate for a prolonged time in vitro and maintain diploid karyotype. Immunocytochemical staining showed that they expressed SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81, and real-time PCR showed that they expressed Cvh, CDH and Dazl. They could form simple embryoid bodies and differentiate into osteoblasts in vitro. In addition, after transfected with pEGFP-N3, pEYFP-N1 and pDsRed-N1 vectors by liposomal transfection, enhanced green, yellow and red fluorescent protein-positive cells could be visualized using a laser confocal microscope. The above results suggested that PGCs from the Beijing fatty chicken not only had strong self-renewal ability, but also had the potential to differentiate towards mesoblast cells. These cells are suitable for genetic manipulation as nuclear donors.

&lt;I&gt;In Vitro&lt;/I&gt; Biocompatibility of Ni-Free Ti-Based Shape Memory Alloys for Biomedical Applications

This study was undertaken to evaluate the in vitro biocompatibility of newly developed Ni-free Ti-based shape memory alloys (SMAs) in comparison to that of commercial pure titanium (cpTi). This study compared Ti-24 mol%Nb-3 mol%Al (Ti-Nb-Al) and Ti-7 mol%Cr-3 mol%Sn (Ti-Cr-Sn) to cpTi from a cell-compatibility perspective. In all, 63 samples (21 samples for each group) were prepared, which were machined into 10-mm-diameter, 0.15-mm-thick, mirror-polished disks. Their surface morphology was evaluated using scanning electron microscopy (SEM). The chemical composition of the sample surface was determined using an energy dispersive X-ray analyzer (EDX). Sample surface roughness was measured using a non-contact 3D profiler. After sample surface observations, the cell proliferation and viability of African green monkey kidney fibroblast cell line COS7 in direct contact with these new alloys were evaluated by DNA quantification, by live cell imaging using CelLuminate Red fluorescent cell stain as a new method, and by cytoskeletal observations by immunofluorescent actin labelling. Cell proliferation was examined after 1, 3, and 5 days of culture. Results were the following. (i) Each sample showed high purity and a very smooth surface, showing no morphological differences among groups. (ii) The COS7 cells took in sufficient CelLuminate Red to visualize the cells using epifluorescent microscopy, and (iii) cell proliferation with Ti-Cr-Sn was lower than with either cpTi or Ti-Nb-Al. These results suggest that Ti-Nb-Al alloy showed biocompatibility as high as that of cpTi and that it is more suitable for biomedical applications.

Progressive epithelial to mesenchymal transitions in ARCaP E prostate cancer cells during xenograft tumor formation and metastasis.

The mechanism of epithelial to mesenchymal transition (EMT) could be adopted by tumor cells for migration and invasion. We have reported that ARCaP(E) human prostate cancer cells undergo EMT-like changes during xenograft growth in athymic mice. In this report, we assessed the extent of EMT by tracking changes in cloned ARCaP(E) cells expressing red fluorescence protein during successive orthotopic prostate tumor formation. Cancer cells with stromal-like morphology were isolated and examined for EMT-like changes. EMT-like morphologic and expression changes were detected after one round of in vivo tumor formation. Importantly, when recovered tumor cells were used in second round xenograft tumor formation, a large fraction of ARCaP(E) cells showed drastic EMT-like changes, with markedly enlarged cell size and divergent cell shapes similar to those of mesenchymal stromal cells. The morphologic change was accompanied by increased growth and metastasis, as tumor incidence increased while red fluorescent tumor cells could be detected from circulating blood, bone marrow, peritoneal ascites, and lung of the tumor-bearing mice. Recovered clones from these samples had lost epithelial markers but many showed activated stromal marker vimentin expression. The EMT appeared permanent since the newly acquired morphology was sustained after continuous passages. Results from this study demonstrate that through interaction with the host tumor microenvironment, cancer cells acquire cellular plasticity. During xenograft tumor formation and metastasis, a single clone of cancer cells could yield a heterogeneous population, with a substantial number of tumor cells adopting mesenchymal stroma-like phenotypes.

Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos

BackgroundThe use of genetically-encoded fluorescent proteins has revolutionized the fields of cell and developmental biology and in doing so redefined our understanding of the dynamic morphogenetic processes that shape the embryo. With the advent of more accessible and sophisticated imaging technologies as well as an abundance of fluorescent proteins with different spectral characteristics, the dynamic processes taking place in situ in living cells and tissues can now be probed. Photomodulatable fluorescent proteins are one of the emerging classes of genetically-encoded fluorescent proteins.ResultsWe have compared PA-GFP, PS-CFP2, Kaede and KikGR four readily available and commonly used photomodulatable fluorescent proteins for use in ES cells and mice. Our results suggest that the green-to-red photoconvertible fluorescent protein, Kikume Green-Red (KikGR), is most suitable for cell labeling and lineage studies in ES cells and mice because it is developmentally neutral, bright and undergoes rapid and complete photoconversion. We have generated transgenic ES cell lines and strains of mice exhibiting robust widespread expression of KikGR. By efficient photoconversion of KikGR we labeled subpopulations of ES cells in culture, and groups of cells within ex utero cultured mouse embryos. Red fluorescent photoconverted cells and their progeny could be followed for extended periods of time.ConclusionTransgenic ES cells and mice exhibiting widespread readily detectable expression of KikGR are indistinguishable from their wild type counterparts and are amenable to efficient photoconversion. They represent novel tools for non-invasive selective labeling specific cell populations and live imaging cell dynamics and cell fate. Genetically-encoded photomodulatable proteins such as KikGR represent emergent attractive alternatives to commonly used vital dyes, tissue grafts and genetic methods for investigating dynamic behaviors of individual cells, collective cell dynamics and fate mapping applications.

The effects of bone marrow stromal cell transplants on tendon healing in vitro

The purpose of this study was to investigate the effect of bone marrow stromal cells (BMSCs) on tendon healing in a canine ex vivo model. Bone marrow was harvested and BMSCs were isolated and cultured according to established protocols. Cells were seeded into 0.5 mg/ml collagen gels and cultured for 24 h to allow gel contraction, and then implanted between the lacerated ends of repaired flexor digitorum profundus tendons. Tendons repaired with a gel patch alone and without a gel patch served as control groups. After 2 and 4 weeks in culture, the repaired tendons were evaluated for breaking strength and stiffness. Cell viability was assessed by labeling the cells with PKH26 red fluorescent cell linker. The maximal strength and stiffness of repaired tendons with the BMSC-seeded patch were significantly higher than the repaired tendons without a patch or with a patch without cells, at both 2 and 4 weeks ( p < 0.05). Viable BMSC were present between the cut tendon ends at both 2 and 4 weeks. We conclude that BMSC-seeded gel patch transplantation has the potential to enhance flexor tendon healing, and we plan to investigate this effect in vivo.

In Vivo Tracking of Dual‐Labeled Mesenchymal Stem Cells Homing Into the Injured Common Carotid Artery

The aim of this study is to conduct in vivo, noninvasive magnetic resonance imaging of labeled rat bone mesenchymal stem cells (BMSCs) as they home into the site of injured common carotid artery following allograft transplantation. Our study was approved by the Institutional Committee on Animal Research. Purified rat BMSCs were dual labeled with superparamagnetic iron oxide (SPIO) particle and fluorescent DiI dye, and subsequently transplanted into recipient rats injured in the left common carotid arteries. Immediately before and 3 hr, 3, 7 and 12 days after transplantation, the labeled cells were monitored in vivo using a 7T micromagnetic resonance imaging (7T micro-MRI) scanner. The signal-to-noise ratios (SNRs) at the injured sites were corroborated with histological examination using Prussian blue staining and fluorescent imaging. Rat BMSCs were labeled with SPIO and DiI at 100% efficiency. When compared with the baseline level before transplantation, the SNR decreased significantly on Days 3 and 7 after injection in the experimental group (Dunnet t test, P < 0.05), whereas insignificant differences were observed after 3 hr and 12 days (Dunnet t test, P > 0.05). In the control group, no significant differences in SNR were found among different time points (ANOVA, P > 0.05). Histological analyses illustrated that red fluorescence and Prussian blue-positive cells were mainly distributed around the lesion areas of injured common carotid arteries. Rat BMSCs can be efficiently labeled with SPIO and DiI, and the directional homing of labeled cells to the site of injured common carotid arteries after intravascular transplantation could be tracked in vivo with 7T micro-MRI.

Reprogramming after Chromosome Transfer into Mouse Blastomeres

It is well known that oocytes can reprogram differentiated cells, allowing animal cloning by nuclear transfer. We have recently shown that fertilized zygotes retain reprogramming activities, suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells. When chromosomes from one of the two blastomeres were replaced with the chromosomes of an embryonic or CD4(+) T lymphocyte donor cell, we observed nuclear reprogramming and efficient contribution of the manipulated cell to the developing blastocyst. Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic "cloned" animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines.

Flow cytometric observation of picophytoplankton community structure in the cascade reservoirs along the Wujiang River, SW China

Picophytoplankton community structure has been seasonally investigated in the cascade reservoirs along the Wujiang River from April 2006 to January 2007. Besides picoeukaryotes, two groups of picocyanobacteria have also been detected by flow cytometry. One is a phycoerythrin-rich picocyanobacteria (PE-rich Pcy), the other is a red-fluorescing cells with lacking orange fluorescence and could be a phycocyanin-rich picocyanobacteria (PC-rich Pcy). The average abundances of PC-rich Pcy, PE-rich Pcy and picoeukaryotes were 103, 104 and 102 cells mL-1, respectively. PE-rich Pcy was the dominant population but showed a reduction with eutrophication, and therefore the community structure of picophytoplankton transformed from dominant PE-rich Pcy to dominant PE-rich Pcy and PC-rich Pcy, which suggested they are excellent indicators for the change of trophic state. Picophytoplankton community structure also presented a seasonal variation, indicating the different response of each picophytoplankton group to water temperature.

Can autologous myoblasts be used as a potential bulking agent?

To investigate the behaviour of donor myoblasts at the vesico-ureteric junction (VUJ) and to evaluate their potential as an autologous bulking agent, as myoblast transplantation has been shown to regenerate damaged or degenerated tissue, and it was postulated that they could be used to treat vesico-ureteric reflux. Muscle biopsies were obtained from the lower limb muscles of 10 pigs. The quality of the cells was evaluated by electrophysiological and immunohistochemical tests. The cell membranes of myoblasts were labelled with PKH26, a fluorescent dye. Six weeks after taking of the muscle biopsies all pigs underwent cell transplantation; 30 x 10(6) cells suspended in transplantation medium (in 1-mL syringes) were injected at the VUJ, into the proximal urethra and the rhabdosphincter. At the VUJ volumes of 1 mL were injected, whereas in the urethra and rhabdosphincter small cell depots (0.1 mL) were injected. All the pigs were killed 8 weeks later, and the myoblasts and newly formed myofibres were identified using fluorescence microscopy, with a histological evaluation and investigation of potential local inflammatory reaction. Two to three intact layers of autologous myoblasts were found in the outer aspects of the large cell depots in the VUJ. Immunohistochemistry further showed that the myoblasts were only viable at these outermost borders of the large bulking areas, whereas necrosis with red fluorescent cell detritus was visible in the remaining central aspects of the large bulk of cells. By contrast, cells survived and formed myotubes in the wall of the proximal urethra and the rhabdosphincter where the small cell depots had been injected. In small depots, transplanted autologous myoblasts can survive and differentiate into myofibres, while in a large bulk of cells the vast majority of cells become necrotic. The present results show that myoblasts cannot be used for augmentation of large volumes of tissue or as a bulking agent.

Color-coded imaging of splenocyte-pancreatic cancer cell interactions in the tumor microenvironment

In spite of advances in surgical and medical care pancreatic cancer remains a leading cause of cancer-related death in the United States. An understanding of cancer cell interactions with host cells is critical to our ability to develop effective antitumor therapeutics for pancreatic cancer. We report here a color-coded model system for imaging cancer cell interactions with host immune cells within the native pancreas. A human pancreatic cancer cell line engineered to express green fluorescent protein (GFP) in the nucleus and red fluorescent protein (DsRed2) in the cytoplasm was orthotopically implanted into the pancreas of a nude mouse. After 10-14 days red or green fluorescent splenocytes from immune-competent donors were delivered systemically to the pancreatic cancer-bearing nude mice. Animals were imaged after splenocyte delivery using high-resolution intravital imaging systems. At 1 day after iv injection red or green fluorescent spleen cells were found distributed in lung, liver, spleen and pancreas. By 4 days after cell delivery, however, the immune cells could be clearly imaged surrounding the tumor cells within the pancreas as well as collecting within lymphatic tissues such as lymph nodes and spleen. With the high-resolution intravital imaging afforded by the Olympus IV100 and OV100 systems the interactions of the dual-colored cancer cells and the red fluorescent spleen cells could be clearly imaged in this orthotopic pancreatic cancer model. This color-coded in vivo imaging technology offers a novel approach to imaging the interactions of cancer and immune cells in the tumor microenvironment (TME).

Stem Cell–Derived, Tissue-Engineered Pulmonary Artery Augmentation Patches In Vivo

Reconstruction of the right ventricular outflow tract is a frequently encountered component of many congenital cardiac repairs. We sought to tissue engineer pulmonary artery augmentation patches from retrovirally labeled endothelial progenitor and mesenchymal stem cells and determine the persistence of the seeded cells in vivo. Autologous ovine endothelial progenitor and mesenchymal stem cells were labeled with a retroviral vector encoding green and red fluorescent proteins, coseeded onto biopolymers, and cultured for 5 days. The tissue-engineered patches were implanted into the main pulmonary artery with 1, 2, 4, and 6 week in vivo maturation (n = 8). In vivo evaluation included ultrasonography and angiography, with preimplant and explanted specimens evaluated using histologic examination and immunofluorescence. Echocardiography at each time demonstrated laminar pulmonary artery flow without a pressure gradient across the replaced segment. Pulmonary angiography did not exhibit stenosis or aneurysmal change. Gross appearance of all explanted patches showed progressive tissue formation with increased length of time in vivo. Retrovirally labeled cellular persistence was 96%, 82%, 85%, and 66% at 1, 2, 4, and 6 weeks after implantation, respectively. Early in the in vivo remodeling period, the number of green fluorescent protein-positive endothelial progenitor cells was 1.6 fold greater than the red fluorescent protein-positive mesenchymal stem cells. As in vivo remodeling continued, red fluorescent protein-expressing mesenchymal stem cells were expressed 1.2 to 1.7 times that of the green fluorescent protein-positive endothelial progenitor cells. The data demonstrate the successful creation of an anatomically functional, autologous tissue-engineered pulmonary artery using coseeded progenitor cell sources. Labeled implanted stem cells persisted in the engineered construct, suggesting that in vitro seeding is necessary to engineer tissue. This study demonstrates an effective method to track multiple cell types after implantation.

Filamentous Actin Regulates Insulin Exocytosis through Direct Interaction with Syntaxin 4

Glucose-induced insulin exocytosis is coupled to associations between F-actin and SNARE proteins, although the nature and function of these interactions remains unknown. Toward this end we show here that both Syntaxin 1A and Syntaxin 4 associated with F-actin in MIN6 cells and that each interaction was rapidly and transiently diminished by stimulation of cells with d-glucose. Of the two isoforms, only Syntaxin 4 was capable of interacting directly with F-actin in an in vitro sedimentation assay, conferred by the N-terminal 39-112 residues of Syntaxin 4. The 39-112 fragment was capable of selective competitive inhibitory action, disrupting endogenous F-actin-Syntaxin 4 binding in MIN6 cells. Disruption of F-actin-Syntaxin 4 binding correlated with enhanced glucose-stimulated insulin secretion, mediated by increased granule accumulation at the plasma membrane and increased Syntaxin 4 accessibility under basal conditions. However, no increase in basal level Syntaxin 4-VAMP2 association occurred with either latrunculin treatment or expression of the 39-112 fragment. Taken together, these data disclose a new underlying mechanism by which F-actin negatively regulates exocytosis via binding and blocking Syntaxin 4 accessibility, but they also reveal the existence of additional signals and/or steps required to trigger the subsequent docking and fusion steps of exocytosis.