Embryonic stem cell secreted factors decrease invasiveness of triple-negative breast cancer cells through regulome modulation.

Stem cell microenvironments decrease the invasiveness of cancer cells, and elucidating the mechanisms associated with disease regression could further the development of targeted therapies for aggressive cancer subtypes. To this end, we applied an emerging technology, TRanscriptional Activity CEll aRray (TRACER), to investigate the reprogramming of triple-negative breast cancer (TNBC) cells in conditions that promoted a less aggressive phenotype. The repressive environment was established through exposure to mouse embryonic stem cell conditioned media (mESC CM). Assessment of carcinogenic phenotypes indicated that mESC CM exposure decreased proliferation, invasion, migration, and stemness in TNBC cells. Protein expression analysis revealed that mESC CM exposure increased expression of the epithelial protein E-cadherin and decreased the mesenchymal protein MMP9. Gene expression analysis showed that mESC CM decreased epithelial to mesenchymal transition (EMT) markers fibronectin, vimentin, and Snail. Over a period of 6 d, TRACER quantified changes in activity of 11 transcription factors (TFs) associated with oncogenic progression. The EMT profile was decreased in association with the activity of 7 TFs (Smad3, NF-κΒ, MEF2, GATA, Hif1, Sp1, and RXR). Further examination of Smad3 and GATA expression and phosphorylation revealed that mESC CM exposure decreased noncanonical Smad3 phosphorylation and Smad3-mediated gene expression, increased GATA3 expression and phosphorylation, and resulted in a synergistic decrease in migration of GATA3 overexpressing MDA-MB-231 cells. Collectively, the application of TRACER to examine TF activity associated with the transition of cancer cells to a less aggressive phenotype, as directed by mESC CM, identified novel mechanistic events linking the embryonic microenvironment to both favorable changes and cellular plasticity in TNBC cell phenotypes.

Multifunctional, multichannel bridges that deliver neurotrophin encoding lentivirus for regeneration following spinal cord injury.

Therapeutic strategies following spinal cord injury must address the multiple barriers that limit regeneration. Multiple channel bridges have been developed that stabilize the injury following implantation and provide physical guidance for regenerating axons. These bridges have now been employed as a vehicle for localized delivery of lentivirus. Implantation of lentivirus loaded multiple channel bridges produced transgene expression that persisted for at least 4 weeks. Expression was maximal at the implant at the earliest time point, and decreased with increasing time of implantation, as well as rostral and caudal to the bridge. Immunohistochemical staining indicated transduction of macrophages, Schwann cells, fibroblasts, and astrocytes within the bridge and adjacent tissue. Subsequently, the delivery of lentivirus encoding the neurotrophic factors NT-3 or BDNF significantly increased the extent of axonal growth into the bridge relative to empty scaffolds. In addition to promoting axon growth, the induced expression of neurotrophic factors led to myelination of axons within the channels of the bridge, where the number of myelinated axons was significantly enhanced relative to control. Combining gene delivery with biomaterials to provide physical guidance and create a permissive environment can provide a platform to enhance axonal growth and promote regeneration.

Hydrogels to modulate lentivirus delivery in vivo from microporous tissue engineering scaffolds.

Numerous strategies to induce tissue regeneration employ scaffolds to create space and present biological cues that promote development. In this report, microporous scaffolds that provide structural support were filled with hydrogels to regulate cell adhesion and migration and were investigated as delivery vehicles for gene therapy vectors in vivo. Porous scaffolds were filled with either lentivirus-entrapped collagen or fibrin hydrogels, both of which support cell adhesion yet have varied rates for degradation and cell infiltration. Empty scaffolds and alginate hydrogels were employed as controls, with the latter not supporting cell infiltration. Hydrogel-filled scaffolds retained the lentivirus more effectively than empty scaffolds, and transgene expression was observed for all scaffold conditions. Empty and fibrin-filled scaffolds had maximal transgene expression in vivo, followed by collagen and alginate, with similar levels. Transduced macrophages and dendritic cells were initially present at the scaffold boundary and adjacent tissue and within the scaffold at later time points for all but the alginate condition. At days 3 and 7, expression was also imaged throughout the spleen and thymus, which may result from cell migration from the implant. These studies demonstrate that hydrogels can modulate gene delivery from scaffolds used in cell transplantation and regenerative medicine.

Patterned transgene expression in multiple-channel bridges after spinal cord injury.

Patterning of gene delivery on sub-millimeter length scales within tissue engineering scaffolds is fundamental to recreating the complex architectures of tissues. Surface-mediated delivery of lipoplexes mixed with fibronectin was investigated to pattern vectors within 250 microm channels in poly(lactide-co-glycolide) (PLG) bridges. Initial studies performed in vitro on PLG surfaces indicated that a DNA density of 0.07 microg mm(-2) inside each channel with a weight ratio of DNA to fibronectin of 1:20 maximized the number of transfected cells and the levels of transgene expression. Patterned vectors encoding for nerve growth factor (NGF) resulted in localized neurite extension within the channel. Translation to three-dimensional multiple-channel bridges enabled patterned transfection of different vectors throughout the channels for DNA:fibronectin ratios of 1:4 and multiple DNA depositions, with a large increase of neural cell bodies and neurite extension for delivery of DNA encoding for NGF. In vivo, the immobilization of non-viral vectors within the channels resulted in localized transfection within the pore structure of the bridge immediately around the channels of the bridge containing DNA. This surface immobilization strategy enables patterned gene delivery in vitro and in vivo on length scales of hundreds of microns and may find utility in strategies aimed at regenerating tissues with complex architectures.

The contribution of plasmid design and release to in vivo gene expression following delivery from cationic polymer modified scaffolds.

Tissue engineering scaffolds capable of gene delivery can provide a structure that supports tissue formation while also inducing the expression of inductive factors. Sustained release strategies are hypothesized to maintain elevated plasmid concentrations locally that can enhance gene transfer. In this report, we investigate the relationship between plasmid release kinetics and the extent and duration of transgene expression. Scaffolds were fabricated from polymer microspheres modified with cationic polymers (polyethylenimine, poly(L-lysine), poly(allylamine hydrochloride), polydiallyldimethylammonium) or polydopamine (PD), with PD enhancing incorporation and slowing release. In vivo implantation of scaffolds into the peritoneal fat pad had no significant changes in the level and duration of transgene expression between PD and unmodified scaffolds. Control studies with plasmid dried onto scaffolds, which exhibited a rapid release, and scaffolds with extended leaching to reduce initial quantities released had similar levels and duration of expression. Changing the plasmid design, from a cytomegalovirus (CMV) to an ubiquitin C (UbC) promoter substantially altered the duration of expression. These studies suggest that the initial dose released and vector design affect the extent and duration of transgene expression, which may be sustained over several weeks, potentially leading to numerous applications in cell transplantation and regenerative medicine.

Isolated swine heart ventricle perfusion model for implant assisted-magnetic drug targeting.

An isolated swine heart ventricle perfusion model was developed and used under physiologically relevant conditions to study implant assisted-magnetic drug targeting (IA-MDT). A stent coil was fabricated from a ferromagnetic SS 430 wire and used to capture 100-nm diameter magnetite particles that mimicked magnetic drug carrier particles (MDCPs). Four key cases were studied: (1) no stent and no magnet (control), (2) no magnet but with a stent, (3) no stent but with a magnet (traditional MDT), and (4) with a stent and a magnet (IA-MDT). When applied, the magnetic field was fixed at 0.125T. The performance of the system was based on the capture efficiency (CE) of the magnetite nanoparticles. The experiments done in the absence of the magnetic field showed minimal retention of any nanoparticles whether the stent was present or not. The experiments done in the presence of the magnetic field showed a statistically significant increase in the retention of the nanoparticles, with a marked difference between the traditional and IA-MDT cases. Compared to the control case, in one case there was nearly an 11-fold increase in CE for the IA-MDT case compared to only a threefold increase in CE for the traditional MDT case. This enhanced performance by the IA-MDT case was typical of all the experiments. Histology images of the cross-section of the coronary artery revealed that the nanoparticles were captured mainly in the vicinity of the stent. Overall, the IA-MDT results from this work with actual tissue were very encouraging and similar to those obtained from other non-tissue and theoretical studies; but, they did point to the need for further studies of IA-MDT.