Guidance Cues Research Articles

Gellan-Xanthan Hydrogel Conduits with Intraluminal Electrospun Nanofibers as Physical, Chemical and Therapeutic Cues for Peripheral Nerve Repair.

Optimal levels of functional recovery in peripheral nerve injuries remain elusive due to the architectural complexity of the neuronal environment. Commercial nerve repair conduits lack essential guidance cues for the regenerating axons. In this study, the regenerative potential of a biosimulated nerve repair system providing three types of regenerative cues was evaluated in a 10 mm sciatic nerve-gap model over 4 weeks. A thermo-ionically crosslinked gellan-xanthan hydrogel conduit loaded with electrospun PHBV-magnesium oleate-N-acetyl-cysteine (PHBV-MgOl-NAC) nanofibers was assessed for mechanical properties, nerve growth factor (NGF) release kinetics and PC12 viability. In vivo functional recovery was based on walking track analysis, gastrocnemius muscle mass and histological analysis. As an intraluminal filler, PHBV-MgOl-NAC nanofibers improved matrix resilience, deformation and fracture of the hydrogel conduit. NGF release was sustained over 4 weeks, governed by Fickian diffusion and Case-II relaxational release for the hollow conduit and the nanofiber-loaded conduit, respectively. The intraluminal fibers supported PC12 proliferation by 49% compared to the control, preserved up to 43% muscle mass and gradually improved functional recovery. The combined elements of physical guidance (nanofibrous scaffolding), chemical cues (N-acetyl-cysteine and magnesium oleate) and therapeutic cues (NGF and diclofenac sodium) offers a promising strategy for the regeneration of severed peripheral nerves.

Mechanisms of oligodendrocyte progenitor developmental migration.

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), develop from oligodendrocyte progenitor cells (OPCs) that must first migrate extensively throughout the developing brain and spinal cord. Specified at particular times from discrete regions in the developing CNS, OPCs are one of the most migratory of cell types and disperse rapidly. A variety of factors act on OPCs to trigger intracellular changes that regulate their migration. We will discuss factors that act as long-range guidance cues, those that act to regulate cellular motility, and those that are critical in determining the final positioning of OPCs. In addition, recent evidence has identified the vasculature as the physical substrate used by OPCs for their migration. Several new findings relating to this oligodendroglial-vascular signaling axis reveal new insight on the relationship between OPCs and blood vessels in the developing and adult brain.

G-protein coupled receptor Smo positively regulates proliferation and migration of adult neural stem cells in vitro

To investigate the role of G-protein coupled receptor Smoothened (Smo) in regulating proliferation and migration of adult neural stem cells (ANSCs) and explore the underlying mechanism. Cultured ANSCs were treated with purmorphamine (PM, an agonist of Smo) or cyclopamine (CPM, an inhibitor of Smo), and the changes in cell proliferation migration abilities were assessed using cell counting kit-8 (CCK8) assay and wound healing assay, respectively. The mRNA expressions of membrane receptor Patched 1 (Ptch1), Smo, glioma-associated oncogene homolog 1 (Gli1), axon guidance cue slit1 (Slit1) and brain-derived neurotrophic factor (BDNF) in the treated cells were detected using real-time quantitative PCR (RT-PCR). PM significantly promoted the proliferation (P < 0.01) and migration of ANSCs (P < 0.01), and up-regulated the mRNA expressions of Ptch1, Smo, Gli1, Slit1 and BDNF. Treatment with CPM significantly inhibited the proliferation and migration of ANSCs. Modulating Smo activity can positively regulate the proliferation and migration of ANSCs possibly by regulating the expressions of BDNF and Slit1.

Evaluating the effectiveness of crosswalk tactile paving on street-crossing behavior: A field trial study for people with visual impairment

People with visual impairment cannot easily obtain external information through vision and thus they face more challenges when traveling than sighted people. The travel environment for people with visual impairment includes pedestrian safety concerns, especially when crossing roads at intersections. Tactile paving can be used as a guidance cue for street-crossing purposes but its use is not yet widespread globally (except at some test sites in Japan). This study investigated the effectiveness of tactile paving on crosswalks based on a field trial conducted in China. A drone and three-axis accelerometer were used to collect participants’ behavioral data. Several quantitative indices for street-crossing behavior, including trajectory, maximum distance of the directional deviation, crossing speed, crossing time, and gait regularity and symmetry, were investigated to measure the participants’ street-crossing performance. Before-after comparative analysis of the quantitative index results was conducted to compare the participants’ use of crosswalks with and without tactile paving. The results reveal that tactile paving helps people with visual impairment to maintain a straight crossing path, avoid directional deviation, reduce their crossing time, and improve their gait regularity and symmetry. Study participants reported positive impressions of the effectiveness of crosswalk tactile paving based on one-to-one interviews conducted after the crosswalk tests. The results also indicate that crosswalk tactile paving is more effective for people with blindness than for those with low vision. This study’s findings could serve as a theoretical basis for the deployment of various barrier-free traffic facilities (especially street-crossing facilities) for people with visual impairment.

Translational control in neurovascular brain development.

The human brain carries out complex tasks and higher functions and is crucial for organismal survival, as it senses both intrinsic and extrinsic environments. Proper brain development relies on the orchestrated development of different precursor cells, which will give rise to the plethora of mature brain cell-types. Within this process, neuronal cells develop closely to and in coordination with vascular cells (endothelial cells (ECs), pericytes) in a bilateral communication process that relies on neuronal activity, attractive or repulsive guidance cues for both cell types and on tight-regulation of gene expression. Translational control is a master regulator of the gene-expression pathway and in particular for neuronal and ECs, it can be localized in developmentally relevant (axon growth cone, endothelial tip cell) and mature compartments (synapses, axons). Herein, we will review mechanisms of translational control relevant to brain development in neurons and ECs in health and disease.

Collagen fibers provide guidance cues for capillary regrowth during regenerative angiogenesis in zebrafish

Although well investigated, the importance of collagen fibers in supporting angiogenesis is not well understood. In this study, we demonstrate that extracellular collagen fibers provide guidance cues for endothelial cell migration during regenerative angiogenesis in the caudal zebrafish fin. Inhibition of collagen cross-linking by β-Aminopropionitrile results in a 70% shorter regeneration area with 50% reduced vessel growth and disintegrated collagen fibers. The disrupted collagen scaffold impedes endothelial cell migration and induces formation of abnormal angioma-like blood vessels. Treatment of the Fli//colRN zebrafish line with the prodrug Nifurpirinol, which selectively damages the active collagen-producing 1α2 cells, reduced the regeneration area and vascular growth by 50% with wider, but less inter-connected, capillary segments. The regenerated area contained larger vessels partially covered by endothelial cells embedded in atypical extracellular matrix containing cell debris and apoptotic bodies, macrophages and granulocytes. Similar experiments performed in early embryonic zebrafish suggested that collagens are important also during embryonic angiogenesis. In vitro assays revealed that collagen I allows for the most efficient endothelial cell migration, followed by collagen IV relative to the complete absence of exogenous matrix support. Our data demonstrates severe vascular defects and restricted fin regeneration when collagens are impaired. Collagen I therefore, provides support and guidance for endothelial cell migration while collagen IV is responsible for proper lumen formation and vascular integrity.

Electrotaxis-on-Chip to Quantify Neutrophil Migration Towards Electrochemical Gradients.

Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including wound healing and immune response to injuries to epithelial barriers (e.g. lung pneumocytes). Immune cells are known to migrate towards both chemical (chemotaxis), physical (mechanotaxis) and electric stimuli (electrotaxis). Electrotaxis is the guided migration of cells along electric fields, and has previously been reported in T-cells and cancer cells. However, there remains a need for engineering tools with high spatial and temporal resolution to quantify EF guided migration. Here we report the development of an electrotaxis-on-chip (ETOC) platform that enables the quantification of dHL-60 cell, a model neutrophil-like cell line, migration toward both electrical and chemoattractant gradients. Neutrophils are the most abundant white blood cells and set the stage for the magnitude of the immune response. Therefore, developing engineering tools to direct neutrophil migration patterns has applications in both infectious disease and inflammatory disorders. The ETOC developed in this study has embedded electrodes and four migration zones connected to a central cell-loading chamber with migration channels [10 µm X 10 µm]. This device enables both parallel and competing chemoattractant and electric fields. We use our novel ETOC platform to investigate dHL-60 cell migration in three biologically relevant conditions: 1) in a DC electric field; 2) parallel chemical gradient and electric fields; and 3) perpendicular chemical gradient and electric field. In this study we used differentiated leukemia cancer cells (dHL60 cells), an accepted model for human peripheral blood neutrophils. We first quantified effects of electric field intensities (0.4V/cm-1V/cm) on dHL-60 cell electrotaxis. Our results show optimal migration at 0.6 V/cm. In the second scenario, we tested whether it was possible to increase dHL-60 cell migration to a bacterial signal [N-formylated peptides (fMLP)] by adding a parallel electric field. Our results show that there was significant increase (6-fold increase) in dHL60 migration toward fMLP and cathode of DC electric field (0.6V/cm, n=4, p-value<0.005) vs. fMLP alone. Finally, we evaluated whether we could decrease or re-direct dHL-60 cell migration away from an inflammatory signal [leukotriene B4 (LTB4)]. The perpendicular electric field significantly decreased migration (2.9-fold decrease) of dHL60s toward LTB4 vs. LTB4 alone. Our microfluidic device enabled us to quantify single-cell electrotaxis velocity (7.9 µm/min ± 3.6). The magnitude and direction of the electric field can be more precisely and quickly changed than most other guidance cues such as chemical cues in clinical investigation. A better understanding of EF guided cell migration will enable the development of new EF-based treatments to precisely direct immune cell migration for wound care, infection, and other inflammatory disorders.

Locomotor recovery after spinal cord injury: intimate dependence between axonal regeneration and re-connection.

Locomotor recovery after spinal cord injury: intimate dependence between axonal regeneration and re-connection.

Unraveling Axon Guidance during Axotomy and Regeneration.

During neuronal development and regeneration axons extend a cytoskeletal-rich structure known as the growth cone, which detects and integrates signals to reach its final destination. The guidance cues “signals” bind their receptors, activating signaling cascades that result in the regulation of the growth cone cytoskeleton, defining growth cone advance, pausing, turning, or collapse. Even though much is known about guidance cues and their isolated mechanisms during nervous system development, there is still a gap in the understanding of the crosstalk between them, and about what happens after nervous system injuries. After neuronal injuries in mammals, only axons in the peripheral nervous system are able to regenerate, while the ones from the central nervous system fail to do so. Therefore, untangling the guidance cues mechanisms, as well as their behavior and characterization after axotomy and regeneration, are of special interest for understanding and treating neuronal injuries. In this review, we present findings on growth cone guidance and canonical guidance cues mechanisms, followed by a description and comparison of growth cone pathfinding mechanisms after axotomy, in regenerative and non-regenerative animal models.

Dual role for CXCL12 signaling in semilunar valve development.

SummaryCxcl12-null embryos have dysplastic, misaligned, and hyperplastic semilunar valves (SLVs). In this study, we show that CXCL12 signaling via its receptor CXCR4 fulfills distinct roles at different stages of SLV development, acting initially as a guidance cue to pattern cellular distribution within the valve primordia during the endocardial-to-mesenchymal transition (endoMT) phase and later regulating mesenchymal cell proliferation during SLV remodeling. Transient, anteriorly localized puncta of internalized CXCR4 are observed in cells undergoing endoMT. In vitro, CXCR4+ cell orientation in response to CXCL12 requires phosphatidylinositol 3-kinase (PI3K) signaling and is inhibited by suppression of endocytosis. This dynamic intracellular localization of CXCR4 during SLV development is related to CXCL12 availability, potentially enabling activation of divergent downstream signaling pathways at key developmental stages. Importantly, Cxcr7-/- mutants display evidence of excessive CXCL12 signaling, indicating a likely role for atypical chemokine receptor CXCR7 in regulating ligand bioavailability and thus CXCR4 signaling output during SLV morphogenesis.

Molecular Mechanisms Underlying Motor Axon Guidance in Drosophila.

Decoding the molecular mechanisms underlying axon guidance is key to precise understanding of how complex neural circuits form during neural development. Although substantial progress has been made over the last three decades in identifying numerous axon guidance molecules and their functional roles, little is known about how these guidance molecules collaborate to steer growth cones to their correct targets. Recent studies in Drosophila point to the importance of the combinatorial action of guidance molecules, and further show that selective fasciculation and defasciculation at specific choice points serve as a fundamental strategy for motor axon guidance. Here, I discuss how attractive and repulsive guidance cues cooperate to ensure the recognition of specific choice points that are inextricably linked to selective fasciculation and defasciculation, and correct pathfinding decision-making.

Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems.Key points• Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1• Biolayer interferometry allows target protein quantitation in expression media• High production yields in the bioreactor do not impair Netrin-1 proteoglycan qualityGraphical abstract

Interaction of micropatterned topographical and biochemical cues to direct neurite growth from spiral ganglion neurons

Functional outcomes with neural prosthetic devices, such as cochlear implants, are limited in part due to physical separation between the stimulating elements and the neurons they stimulate. One strategy to close this gap aims to precisely guide neurite regeneration to position the neurites in closer proximity to electrode arrays. Here, we explore the ability of micropatterned biochemical and topographic guidance cues, singly and in combination, to direct the growth of spiral ganglion neuron (SGN) neurites, the neurons targeted by cochlear implants. Photopolymerization of methacrylate monomers was used to form unidirectional topographical features of ridges and grooves in addition to multidirectional patterns with 90o angle turns. Microcontact printing was also used to create similar uni- and multi-directional patterns of peptides on polymer surfaces. Biochemical cues included peptides that facilitate (laminin, LN) or repel (EphA4-Fc) neurite growth. On flat surfaces, SGN neurites preferentially grew on LN-coated stripes and avoided EphA4-Fc-coated stripes. LN or EphA4-Fc was selectively adsorbed onto the ridges or grooves to test the neurite response to a combination of topographical and biochemical cues. Coating the ridges with EphA4-Fc and grooves with LN lead to enhanced SGN alignment to topographical patterns. Conversely, EphA4-Fc coating on the grooves or LN coating on the ridges tended to disrupt alignment to topographical patterns. SGN neurites respond to combinations of topographical and biochemical cues and surface patterning that leverages both cues enhance guided neurite growth.

Rapid Generation of Neurospheres from Hippocampal Neurons Using Extracellular-Matrix-Mimetic Scaffolds

3D models of brain organoids represent an innovative and promising tool in neuroscience studies. However, the process of neurosphere formation in vitro remains complicated and is not always very effective. This is largely due to the lack of growth factors, guidance cues, and scaffold structures commonly found in tissues. Here we present a new, simple, and efficient method for generating neurospheres using scaffolds composed of electrospun nylon fibers with a diameter of 40-180 nm, which makes them similar to the brain extracellular matrix (ECM) components. Several main advantages of the proposed method should be highlighted. The method is fast, and the biomaterial consumption is low. Also, the resulting neurospheres are attached to the scaffold nanofibers. This not only provides the experimental convenience but also suggests that the resulting organoid models can potentially demonstrate fundamentally new properties, being closer to the nervous tissue in vivo. We demonstrate the influence of the fibrous scaffold structure on the formation, morphology, and composition of neurospheres and confirm adequate functional activity of the cellular components of these spheroids. The proposed approach can be further used for drug screening, modeling of neurodevelopmental, neurodegenerative disorders, and, potentially, therapeutic tissue engineering.

Stereocontrolled Synthesis of Lyso‐phosphatidyl β‐D‐Glucoside

AbstractLyso‐phosphatidyl β‐D‐glucoside (lysoPtdGlc) is during embryonic development a neurite growth cone guidance cue associated with G‐protein coupled receptor 55, and has been found to be involved in a variety of physiological and pathological events, including inflammatory pain, and cancer. Hence, the ability to prepare large quantities of lysoPtdGlc is an important objective. Herein, we report the convergent chemical synthesis of lysoPtdGlc and its glyceryl epimer (S‐lysoPtdGlc). High stereocontrol during β‐glycosyl H‐phosphonate construction was achieved using a microfluidic approach, and common but undesirable regioisomerization due to acyl migration on the glyceryl subunit was successfully suppressed during deprotection. Purification of the final product was optimized to obtain sufficiently large amounts of lysoPtdGlc and S‐lysoPtdGlc for biological assaying purposes.

Assessing the combination of magnetic field stimulation, iron oxide nanoparticles, and aligned electrospun fibers for promoting neurite outgrowth from dorsal root ganglia in vitro

Magnetic fiber composites combining superparamagnetic iron oxide nanoparticles (SPIONs) and electrospun fibers have shown promise in tissue engineering fields. Controlled grafting of SPIONs to the fibers post-electrospinning generates biocompatible magnetic composites without altering desired fiber morphology. Here, for the first time, we assess the potential of SPION-grafted scaffolds combined with magnetic fields to promote neurite outgrowth by providing contact guidance from the aligned fibers and mechanical stimulation from the SPIONs in the magnetic field. Neurite outgrowth from primary rat dorsal root ganglia (DRG) was assessed from explants cultured on aligned control and SPION-grafted electrospun fibers as well as on non-grafted fibers with SPIONs dispersed in the culture media. To determine the optimal magnetic field stimulation to promote neurite outgrowth, we generated a static, alternating, and linearly moving magnet and simulated the magnetic flux density at different areas of the scaffold over time. The alternating magnetic field increased neurite length by 40% on control fibers compared to a static magnetic field. Additionally, stimulation with an alternating magnetic field resulted in a 30% increase in neurite length and 62% increase in neurite area on SPION-grafted fibers compared to DRG cultured on PLLA fibers with untethered SPIONs added to the culture media. These findings demonstrate that SPION-grafted fiber composites in combination with magnetic fields are more beneficial for stimulating neurite outgrowth on electrospun fibers than dispersed SPIONs. Statement of significanceAligned electrospun fibers improve axonal regeneration by acting as a passive guidance cue but do not actively interact with cells, while magnetic nanoparticles can be remotely manipulated to interact with neurons and elicit neurite outgrowth. Here, for the first time, we examine the combination of magnetic fields, magnetic nanoparticles, and aligned electrospun fibers to enhance neurite outgrowth. We show an alternating magnetic field alone increases neurite outgrowth on aligned electrospun fibers. However, combining the alternating field with magnetic nanoparticle-grafted fibers does not affect neurite outgrowth compared to control fibers but improves outgrowth compared to freely dispersed magnetic nanoparticles. This study provides the groundwork for utilizing magnetic electrospun fibers and magnetic fields as a method for promoting axonal growth.

Coordination of endothelial cell positioning and fate specification by the epicardium

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.

Anisometric Microstructures to Determine Minimal Critical Physical Cues Required for Neurite Alignment.

In nerve regeneration, scaffolds play an important role in providing an artificial extracellular matrix with architectural, mechanical, and biochemical cues to bridge the site of injury. Directed nerve growth is a crucial aspect of nerve repair, often introduced by engineered scaffolds imparting linear tracks. The influence of physical cues, determined by well-defined architectures, has been mainly studied for implantable scaffolds and is usually limited to continuous guiding features. In this report, the potential of short anisometric microelements in inducing aligned neurite extension, their dimensions, and the role of vertical and horizontal distances between them, is investigated. This provides crucial information to create efficient injectable 3D materials with discontinuous, in situ magnetically oriented microstructures, like the Anisogel. By designing and fabricating periodic, anisometric, discreet guidance cues in a high-throughput 2D in vitro platform using two-photon lithography techniques, the authors are able to decipher the minimal guidance cues required for directed nerve growth along the major axis of the microelements. These features determine whether axons grow unidirectionally or cross paths via the open spaces between the elements, which is vital for the design of injectable Anisogels for enhanced nerve repair.

Magnetoreception: An exciting field of challenges, hopes, wishes and beyond

The ability of animals to sense the geomagnetic field and perceive guidance cues for orientation and navigation has been a subject of lively debate since the beginning. A wealth of behavioral evidences has been accumulated and several sophisticated theoretical models including electromagnetic induction, ferrimagnetism, radical pairs model based on cryptochrome and biocompass model based on MagR and MagR/Cry protein complex have been proposed. However, the mechanism of magnetoreception remains largely unknown up to date, and thus exciting. How do migratory organisms find their way has been listed by Science as one of 125 most compelling scientific questions that cannot currently be answered, but are on the horizon. Here in this historical review, we firstly focused on examining the magnetoreception research throughout the last two centuries emerged in the literatures. We then reviewed the four major hypotheses behind this scientific mystery, and highlighted pros and cons of each hypothesis. We also discussed that if it was possible that a universal mechanism can be applied to explain all questions in animal magnetoreception, from the perspective of biodiversity and evolution. One of the heated controversies in magnetoreception is that can human navigate by sensing the geomagnetic field? We explored the research history of human magnetoreception in this review. The question can be traced back to 1873 when Charles Darwin wrote a letter to Nature , arguing that animals including human have the “instinct” to navigate. Then after almost 100 years, Robin Baker claimed to find a human compass sense of magnetic field in “Manchester Experiments” in Science in 1980, but the replication efforts all failed for more than 30 years by other groups. This controversy topic was then re-visited in 2019 and two groups published results suggesting many humans are able to unconsciously detect changes in Earth-strength magnetic fields, which raised more questions in this field. Putting the research history into perspective, we then discussed the unique features of biological magnetoreception, the approaches currently used in this field, the challenges we were facing and the possible solutions. We listed the contradictions in four current major models to explain the mechanism of biological magnetic sensing, and why the controversies could happen. We proposed that the pattern to perceive magnetic field should be regarded as “massive event”, since all molecules/receptors would respond to the changes of external magnetic field spontaneously, which was fundamentally different than the “single event” pattern when a chemical ligand binding to its receptor. We argue that a better understanding of the differences between magnetoreception and other sensory systems would help us to resolve this scientific mystery in the future.

Abstract LB199: Trafficking of Semaphorin4D is regulated by endoplasmic reticulum chaperone GRP78

Abstract Localization of endoplasmic reticulum (ER) chaperones to the cell surface is an adaptive mechanism for cells to expand the functionality of these proteins in response to stress by activating signaling pathways distinct from their ER functions, thereby impacting cell survival and signaling. In cancer, cell-surface ER chaperones have been reported to regulate oncogenic signaling pathways, proliferation and cell adhesion through interactions with other cell surface proteins. The emerging role of peripheral cell surface GRP78 (csGRP78), traditionally regarded as an ER luminal chaperone, as a multifunctional co-receptor for many different types of cell surface proteins presents novel opportunities for therapeutic interventions. One such cell surface protein is Semaphorin4D (Sema4D). Sema4D was originally discovered as a cell surface cleavable ligand and neuronal axon guidance cue that mobilizes the actin cytoskeleton to facilitate neuronal growth cone homing during development. In cancer, cell surface Sema4D expression is pro-metastatic and pro-angiogenic. Sema4D has been shown to be highly expressed in the breast cancer and epidemiological studies have demonstrated that Sema4D expression in breast cancer patients confers a poor prognosis and high probability of metastatic organotropism. Our recent advances have preliminarily identified a critical role for GRP78 in facilitating Semaphorin4D (Sema4D) trafficking. We show that Sema4D forms a direct protein-protein interaction with GRP78 and that GRP78 may facilitate membrane trafficking of Sema4D to the cell surface, where it exerts pro-angiogenic and metastatic effects. Furthermore, csGRP78 may interact with and facilitate many of the functions of Sema4D. Understanding the conditions by which GRP78 contributes to surface expression of Sema4D and the downstream effects may lead to novel therapeutics that abrogate the effects of Sema4D in cancer and to the elucidation of novel mechanisms by which Sema4D trafficking in cancer is regulated by GRP78. Citation Format: Anthony Carlos, Yuan-Li Tsai, Yan Ma, Amy Lee. Trafficking of Semaphorin4D is regulated by endoplasmic reticulum chaperone GRP78 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB199.