A Micro-Optic Stalk (µOS) System to Model the Collective Migration of Retinal Neuroblasts.

Contemporary regenerative therapies have introduced stem-like cells to replace damaged neurons in the visual system by recapitulating critical processes of eye development. The collective migration of neural stem cells is fundamental to retinogenesis and has been exceptionally well-studied using the fruit fly model of Drosophila Melanogaster. However, the migratory behavior of its retinal neuroblasts (RNBs) has been surprisingly understudied, despite being critical to retinal development in this invertebrate model. The current project developed a new microfluidic system to examine the collective migration of RNBs extracted from the developing visual system of Drosophila as a model for the collective motile processes of replacement neural stem cells. The system scales with the microstructure of the Drosophila optic stalk, which is a pre-cursor to the optic nerve, to produce signaling fields spatially comparable to in vivo RNB stimuli. Experiments used the micro-optic stalk system, or µOS, to demonstrate the preferred sizing and directional migration of collective, motile RNB groups in response to changes in exogenous concentrations of fibroblast growth factor (FGF), which is a key factor in development. Our data highlight the importance of cell-to-cell contacts in enabling cell cohesion during collective RNB migration and point to the unexplored synergy of invertebrate cell study and microfluidic platforms to advance regenerative strategies.

Collective behaviors of Drosophila-derived retinal progenitors in controlled microenvironments.

Collective behaviors of retinal progenitor cells (RPCs) are critical to the development of neural networks needed for vision. Signaling cues and pathways governing retinal cell fate, migration, and functional organization are remarkably conserved across species, and have been well-studied using Drosophila melanogaster. However, the collective migration of heterogeneous groups of RPCs in response to dynamic signaling fields of development remains incompletely understood. This is in large part because the genetic advances of seminal invertebrate models have been poorly complemented by in vitro cell study of its visual development. Tunable microfluidic assays able to replicate the miniature cellular microenvironments of the developing visual system provide newfound opportunities to probe and expand our knowledge of collective chemotactic responses essential to visual development. Our project used a controlled, microfluidic assay to produce dynamic signaling fields of Fibroblast Growth Factor (FGF) that stimulated the chemotactic migration of primary RPCs extracted from Drosophila. Results illustrated collective RPC chemotaxis dependent on average size of clustered cells, in contrast to the non-directional movement of individually-motile RPCs. Quantitative study of these diverse collective responses will advance our understanding of retina developmental processes, and aid study/treatment of inherited eye disease. Lastly, our unique coupling of defined invertebrate models with tunable microfluidic assays provides advantages for future quantitative and mechanistic study of varied RPC migratory responses.

Invertebrate Retinal Progenitors as Regenerative Models in a Microfluidic System.

Regenerative retinal therapies have introduced progenitor cells to replace dysfunctional or injured neurons and regain visual function. While contemporary cell replacement therapies have delivered retinal progenitor cells (RPCs) within customized biomaterials to promote viability and enable transplantation, outcomes have been severely limited by the misdirected and/or insufficient migration of transplanted cells. RPCs must achieve appropriate spatial and functional positioning in host retina, collectively, to restore vision, whereas movement of clustered cells differs substantially from the single cell migration studied in classical chemotaxis models. Defining how RPCs interact with each other, neighboring cell types and surrounding extracellular matrixes are critical to our understanding of retinogenesis and the development of effective, cell-based approaches to retinal replacement. The current article describes a new bio-engineering approach to investigate the migratory responses of innate collections of RPCs upon extracellular substrates by combining microfluidics with the well-established invertebrate model of Drosophila melanogaster. Experiments utilized microfluidics to investigate how the composition, size, and adhesion of RPC clusters on defined extracellular substrates affected migration to exogenous chemotactic signaling. Results demonstrated that retinal cluster size and composition influenced RPC clustering upon extracellular substrates of concanavalin (Con-A), Laminin (LM), and poly-L-lysine (PLL), and that RPC cluster size greatly altered collective migratory responses to signaling from Fibroblast Growth Factor (FGF), a primary chemotactic agent in Drosophila. These results highlight the significance of examining collective cell-biomaterial interactions on bio-substrates of emerging biomaterials to aid directional migration of transplanted cells. Our approach further introduces the benefits of pairing genetically controlled models with experimentally controlled microenvironments to advance cell replacement therapies.

Targeted Lipid Nanoemulsions Encapsulating Epigenetic Drugs Exhibit Selective Cytotoxicity on CDH1-/FOXM1+ Triple Negative Breast Cancer Cells.

The plasticity of cancer epigenetics makes them plausible candidates for therapeutic intervention. We took advantage of elevated expression of lysophosphatidic acid receptor 1 (LPAR1) in triple negative breast cancer (TNBC) tissues to target decitabine (DAC) and panobinostat (PAN) to breast cancer cells. DAC and PAN were shown to reverse abnormal methylation of DNA and altered chromatin structure, respectively, leading to increased expression of tumor suppressor genes and decreased expression of oncogenes. Although DAC and PAN have therapeutic benefits, they are limited by chemical instability and systemic toxicity. Herein, we present LPAR1-targeted, lipid nanoemulsions (LNEs) encapsulating both DAC and PAN. Our results demonstrated that the cell uptake and in vivo biodistribution of LNEs was dependent on LPAR1 expression in TNBCs. DAC/PAN-LNEs were effective in inhibiting the growth of mesenchymal breast cancer cells by restoring CDH1/E-cadherin and suppressing forkhead box M1 (FOXM1) expression. Epithelial breast cancer cells that inherently express low FOXM1 and high CDH1 were unaffected by DAC/PAN-LNEs. Overall, we successfully designed LPAR1-targeted LNEs that selectively act on CDH1(low)/FOXM1(high) TNBC cell lines.