Cell morphology best predicts tumorigenicity and metastasis in vivo across multiple TNBC cell lines of different metastatic potential.

BACKGROUND: Metastasis is the leading cause of death in breast cancer patients. For metastasis to occur, tumor cells must invade locally, intravasate, and colonize distant tissues and organs, all steps that require tumor cell migration. The majority of studies on invasion and metastasis rely on human breast cancer cell lines. While it is known that these cells have different properties and abilities for growth and metastasis, the in vitro morphological, proliferative, migratory, and invasive behavior of these cell lines and their correlation to in vivo behavior is poorly understood. Thus, we sought to classify each cell line as poorly or highly metastatic by characterizing tumor growth and metastasis in a murine model of six commonly used human triple-negative breast cancer xenografts, as well as determine which in vitro assays commonly used to study cell motility best predict in vivo metastasis. METHODS: We evaluated the liver and lung metastasis of human TNBC cell lines MDA-MB-231, MDA-MB-468, BT549, Hs578T, BT20, and SUM159 in immunocompromised mice. We characterized each cell line's cell morphology, proliferation, and motility in 2D and 3D to determine the variation in these parameters between cell lines. RESULTS: We identified MDA-MB-231, MDA-MB-468, and BT549 cells as highly tumorigenic and metastatic, Hs578T as poorly tumorigenic and metastatic, BT20 as intermediate tumorigenic with poor metastasis to the lungs but highly metastatic to the livers, and SUM159 as intermediate tumorigenic but poorly metastatic to the lungs and livers. We showed that metrics that characterize cell morphology are the most predictive of tumor growth and metastatic potential to the lungs and liver. Further, we found that no single in vitro motility assay in 2D or 3D significantly correlated with metastasis in vivo. CONCLUSIONS: Our results provide an important resource for the TNBC research community, identifying the metastatic potential of 6 commonly used cell lines. Our findings also support the use of cell morphological analysis to investigate the metastatic potential and emphasize the need for multiple in vitro motility metrics using multiple cell lines to represent the heterogeneity of metastasis in vivo.

Neural dependency in wound healing and regeneration.

In response to injury, humans and many other mammals form a fibrous scar that lacks the structure and function of the original tissue, whereas other vertebrate species can spontaneously regenerate damaged tissues and structures. Peripheral nerves have been identified as essential mediators of wound healing and regeneration in both mammalian and nonmammalian systems, interacting with the milieu of cells and biochemical signals present in the post-injury microenvironment. This review examines the diverse functions of peripheral nerves in tissue repair and regeneration, specifically during the processes of wound healing, blastema formation, and organ repair. We compare available evidence in mammalian and nonmammalian models, identifying critical nerve-mediated mechanisms for regeneration and providing future perspectives toward integrating these mechanisms into a therapeutic framework to promote regeneration.

Biophysical control of plasticity and patterning in regeneration and cancer.

Cells and tissues display a remarkable range of plasticity and tissue-patterning activities that are emergent of complex signaling dynamics within their microenvironments. These properties, which when operating normally guide embryogenesis and regeneration, become highly disordered in diseases such as cancer. While morphogens and other molecular factors help determine the shapes of tissues and their patterned cellular organization, the parallel contributions of biophysical control mechanisms must be considered to accurately predict and model important processes such as growth, maturation, injury, repair, and senescence. We now know that mechanical, optical, electric, and electromagnetic signals are integral to cellular plasticity and tissue patterning. Because biophysical modalities underly interactions between cells and their extracellular matrices, including cell cycle, metabolism, migration, and differentiation, their applications as tuning dials for regenerative and anti-cancer therapies are being rapidly exploited. Despite this, the importance of cellular communication through biophysical signaling remains disproportionately underrepresented in the literature. Here, we provide a review of biophysical signaling modalities and known mechanisms that initiate, modulate, or inhibit plasticity and tissue patterning in models of regeneration and cancer. We also discuss current approaches in biomedical engineering that harness biophysical control mechanisms to model, characterize, diagnose, and treat disease states.

Sensory nerves enhance triple-negative breast cancer invasion and metastasis via the axon guidance molecule PlexinB3.

In breast cancer, nerve presence has been correlated with more invasive disease and worse prognosis, yet the mechanisms by which different types of peripheral nerves drive tumor progression remain poorly understood. In this study, we identified sensory nerves as more abundant in human triple-negative breast cancer (TNBC) tumors. Co-injection of sensory neurons isolated from the dorsal root ganglia (DRG) of adult female mice with human TNBC cells in immunocompromised mice increased the number of lung metastases. Direct in vitro co-culture of human TNBC cells with the dorsal root ganglia (DRG) of adult female mice revealed that TNBC cells adhere to sensory neuron fibers leading to an increase in migration speed. Species-specific RNA sequencing revealed that co-culture of TNBC cells with sensory nerves upregulates the expression of genes associated with cell migration and adhesion in cancer cells. We demonstrated that lack of the semaphorin receptor PlexinB3 in cancer cells attenuate their adhesion to and migration on sensory nerves. Together, our results identify a mechanism by which nerves contribute to breast cancer migration and metastasis by inducing a shift in TNBC cell gene expression and support the rationale for disrupting neuron-cancer cell interactions to target metastasis.

Potassium channel-driven bioelectric signalling regulates metastasis in triple-negative breast cancer.

BACKGROUND: There is a critical need to better understand the mechanisms that drive local cell invasion and metastasis to develop new therapeutics targeting metastatic disease. Bioelectricity is an important mediator of cellular processes and changes in the resting membrane potential (RMP) are associated with increased cancer cell invasion. However, whether the RMP can be used to target invading cancer cells is unknown. METHODS: We employed both genetic and pharmacological manipulation of potassium channel activity and characterized the effects on breast cancer cell migration and invasion in vitro, and metastasis in an animal model of breast cancer. FINDINGS: Our data demonstrate that altering the RMP of triple-negative breast cancer (TNBC) cells by manipulating potassium channel expression increases in vitro invasion, in vivo tumour growth and metastasis, and is accompanied by changes in gene expression associated with cell adhesion. INTERPRETATION: We describe a novel mechanism for RMP-mediated cell migration involving cadherin-11 and the MAPK pathway. Importantly, we identify a new strategy to target metastatic TNBC in vivo by repurposing an FDA-approved potassium channel blocker. Our results demonstrate that bioelectricity regulates cancer cell invasion and metastasis which could lead to a new class of therapeutics for patients with metastatic disease. FUNDING: This work was supported by the National Institutes of Health (R00-CA207866 to M.J.O.), Tufts University (Start-up funds from the School of Engineering to M.J.O., Tufts Collaborates Award to M.J.O. and M.L.), Allen Discovery centre program (Paul G. Allen Frontiers Group (12,171) to M.L.), and Breast Cancer Alliance Young Investigator Grant to M.J.O, Laidlaw Scholar funding to D.S. M.L. also gratefully acknowledges support of the Barton Family Foundation.

Beyond Neurons: Long Distance Communication in Development and Cancer.

Cellular communication is important in all aspects of tissue and organism functioning, from the level of single cells, two discreet populations, and distant tissues of the body. Long distance communication networks integrate individual cells into tissues to maintain a complex organism during development, but when communication between cells goes awry, disease states such as cancer emerge. Herein we discuss the growing body of evidence suggesting that communication methods known to be employed by neurons, also exist in other cell types. We identify three major areas of long-distance communication: bioelectric signaling, tunneling nanotubes (TNTs), and macrophage modulation of networks, and draw comparisons about how these systems operate in the context of development and cancer. Bioelectric signaling occurs between cells through exchange of ions and tissue-level electric fields, leading to changes in biochemical gradients and molecular signaling pathways to control normal development and tumor growth and invasion in cancer. TNTs transport key morphogens and other cargo long distances, mediating electrical coupling, tissue patterning, and malignancy of cancer cells. Lastly macrophages maintain long distance signaling networks through trafficking of vesicles during development, providing communication relays and priming favorable microenvironments for cancer metastasis. By drawing comparisons between non-neural long distance signaling in the context of development and cancer we aim to encourage crosstalk between the two fields to cultivate new hypotheses and potential therapeutic strategies.

Biomaterial-based cell delivery strategies to promote liver regeneration.

Chronic liver disease and cirrhosis is a widespread and untreatable condition that leads to lifelong impairment and eventual death. The scarcity of liver transplantation options requires the development of new strategies to attenuate disease progression and reestablish liver function by promoting regeneration. Biomaterials are becoming an increasingly promising option to both culture and deliver cells to support in vivo viability and long-term function. There is a wide variety of both natural and synthetic biomaterials that are becoming established as delivery vehicles with their own unique advantages and disadvantages for liver regeneration. We review the latest developments in cell transplantation strategies to promote liver regeneration, with a focus on the use of both natural and synthetic biomaterials for cell culture and delivery. We conclude that future work will need to refine the use of these biomaterials and combine them with novel strategies that recapitulate liver organization and function in order to translate this strategy to clinical use.

Injectable hydrogel enables local and sustained co-delivery to the brain: Two clinically approved biomolecules, cyclosporine and erythropoietin, accelerate functional recovery in rat model of stroke.

Therapeutic delivery to the brain is limited by the blood-brain barrier and is exacerbated by off-target effects associated with systemic delivery, thereby precluding many potential therapies from even being tested. Given the systemic side effects of cyclosporine and erythropoietin, systemic administration would be precluded in the context of stroke, leaving only the possibility of local delivery. We wondered if direct delivery to the brain would allow new reparative therapeutics, such as these, to be identified for stroke. Using a rodent model of stroke, we employed an injectable drug delivery hydrogel strategy to circumvent the blood-brain barrier and thereby achieved, for the first time, local and sustained co-release to the brain of cyclosporine and erythropoietin. Both drugs diffused to the sub-cortical neural stem and progenitor cell (NSPC) niche and were present in the brain for at least 32 days post-stroke. Each drug had a different outcome on brain tissue: cyclosporine increased plasticity in the striatum while erythropoietin stimulated endogenous NSPCs. Only their co-delivery, but not either drug alone, accelerated functional recovery and improved tissue repair. This platform opens avenues for hitherto untested therapeutic combinations to promote regeneration and repair after stroke.

Defined extracellular ionic solutions to study and manipulate the cellular resting membrane potential.

All cells possess an electric potential across their plasma membranes and can generate and receive bioelectric signals. The cellular resting membrane potential (RMP) can regulate cell proliferation, differentiation and apoptosis. Current approaches to measure the RMP rely on patch clamping, which is technically challenging, low-throughput and not widely available. It is therefore critical to develop simple strategies to measure, manipulate and characterize the RMP. Here, we present a simple methodology to study the RMP of non-excitable cells and characterize the contribution of individual ions to the RMP using a voltage-sensitive dye. We define protocols using extracellular solutions in which permeable ions (Na+, Cl- and K+) are substituted with non-permeable ions [N-Methyl-D-glucamine (NMDG), gluconate, choline, SO42-]. The resulting RMP modifications were assessed with both patch clamp and a voltage sensitive dye. Using an epithelial and cancer cell line, we demonstrate that the proposed ionic solutions can selectively modify the RMP and help determine the relative contribution of ionic species in setting the RMP. The proposed method is simple and reproducible and will make the study of bioelectricity more readily available to the cell biology community.This article has an associated First Person interview with the first author of the paper.

Local delivery of stabilized chondroitinase ABC degrades chondroitin sulfate proteoglycans in stroke-injured rat brains.

Central nervous system (CNS) injuries, such as stroke and spinal cord injuries, result in the formation of a proteoglycan-rich glial scar, which acts as a barrier to axonal regrowth and limits the regenerative capacity of the CNS. Chondroitinase ABC (ChABC) is a potent bacterial enzyme that degrades the chondroitin sulfate proteoglycan (CSPG) component of the glial scar and promotes tissue recovery; however, its use is significantly limited by its inherent instability at physiological temperatures. Here, we demonstrate that ChABC can be stabilized using site-directed mutagenesis and covalent modification with poly(ethylene glycol) chains (i.e. PEGylation). Rosetta protein structure modeling was used to screen >20,000 single point mutations, and four potentially stabilizing mutations were tested in vitro. One of the mutations, N1000G (asparagine ➔ glycine at residue 1000), significantly improved the long-term activity of the protein, doubling its functional half-life. PEGylation of this ChABC mutant inhibited unfolding and aggregation and resulted in prolonged bioactivity with a 10-fold increase in activity compared to the unmodified protein after two days. Local, affinity-controlled release of the modified protein (PEG-N1000G-ChABC) was achieved by expressing it as a fusion protein with Src homology 3 (SH3) and delivering the protein from a methylcellulose hydrogel modified with SH3 binding peptides. This affinity-based release strategy provided sustained PEG-N1000G-ChABC-SH3 release over several days in vitro. Direct implantation of the hydrogel delivery vehicle containing stabilized PEG-N1000G-ChABC-SH3 onto the rat brain cortex in a sub-acute model of stroke resulted in significantly reduced CSPG levels in the penumbra of 49% at 14 and 40% at 28 days post-injury compared to animals treated with the vehicle alone.

Local Delivery of Brain-Derived Neurotrophic Factor Enables Behavioral Recovery and Tissue Repair in Stroke-Injured Rats.

IMPACT STATEMENT: We developed a biocomposite that can be mixed with brain-derived neurotrophic factor (BDNF) and dispensed onto the surface of the brain to provide sustained, local release of the protein using a procedure that avoids additional damage to neural tissue. The composite is simple to fabricate, and provides sustained release without nanoparticle encapsulation of BDNF, preserving material and protein bioactivity. We demonstrate that when delivered epicortically to a rat model of stroke, this composite allows BDNF to diffuse into the brain, resulting in enhanced behavioral recovery and synaptic plasticity in the contralesional hemisphere.

Bioelectric Control of Metastasis in Solid Tumors.

As the leading cause of death in cancer, there is an urgent need to develop treatments to target the dissemination of primary tumor cells to secondary organs, known as metastasis. Bioelectric signaling has emerged in the last century as an important controller of cell growth, and with the development of current molecular tools we are now beginning to identify its role in driving cell migration and metastasis in a variety of cancer types. This review summarizes the currently available research for bioelectric signaling in solid tumor metastasis. We review the steps of metastasis and discuss how these can be controlled by bioelectric cues at the level of a cell, a population of cells, and the tissue. The role of ion channel, pump, and exchanger activity and ion flux is discussed, along with the importance of the membrane potential and the relationship between ion flux and membrane potential. We also provide an overview of the evidence for control of metastasis by external electric fields (EFs) and draw from examples in embryogenesis and regeneration to discuss the implications for endogenous EFs. By increasing our understanding of the dynamic properties of bioelectric signaling, we can develop new strategies that target metastasis to be translated into the clinic.

Initial cell maturity changes following transplantation in a hyaluronan-based hydrogel and impacts therapeutic success in the stroke-injured rodent brain.

Ischemic stroke results in a loss of neurons for which there are no available clinical strategies to stimulate regeneration. While preclinical studies have demonstrated that functional recovery can be obtained by transplanting an exogenous source of neural progenitors into the brain, it remains unknown at which stage of neuronal maturity cells will provide the most benefit. We investigated the role of neuronal maturity on cell survival, differentiation, and long-term sensorimotor recovery in stroke-injured rats using a population of human cortically-specified neuroepithelial progenitor cells (cNEPs) delivered in a biocompatible, bioresorbable hyaluronan/methylcellulose hydrogel. We demonstrate that transplantation of immature cNEPs result in the greatest tissue and functional repair, relative to transplantation of more mature neurons. The transplantation process itself resulted in the least cell death and phenotypic changes in the immature cNEPs, and the greatest acute cell death in the mature cells. The latter negatively impacted host tissue and negated any potential positive effects associated with cell maturity and the hydrogel vehicle, which itself showed some functional and tissue benefit. Moreover, we show that more mature cell populations are drastically altered during the transplantation process itself. The phenotype of the cells before and after transplantation had an enormous impact on their survival and the consequent tissue and behavioral response, emphasizing the importance of characterizing injected cells in transplantation studies more broadly.

Combined delivery of chondroitinase ABC and human induced pluripotent stem cell-derived neuroepithelial cells promote tissue repair in an animal model of spinal cord injury.

The lack of tissue regeneration after traumatic spinal cord injury in animal models is largely attributed to the local inhibitory microenvironment. To overcome this inhibitory environment while promoting tissue regeneration, we investigated the combined delivery of chondroitinase ABC (chABC) with human induced pluripotent stem cell-derived neuroepithelial stem cells (NESCs). ChABC was delivered to the injured spinal cord at the site of injury by affinity release from a crosslinked methylcellulose (MC) hydrogel by injection into the intrathecal space. NESCs were distributed in a hydrogel comprised of hyaluronan and MC and injected into the spinal cord tissue both rostral and caudal to the site of injury. Cell transplantation led to reduced cavity formation, but did not improve motor function. While few surviving cells were found 2 weeks post injury, the majority of live cells were neurons, with only few astrocytes, oligodendrocytes, and progenitor cells. At 9 weeks post injury, there were more progenitor cells and a more even distribution of cell types compared to those at 2 weeks post injury, suggesting preferential survival and differentiation. Interestingly, animals that received cells and chABC had more neurons than animals that received cells alone, suggesting that chABC influenced the injury environment such that neuronal differentiation or survival was favoured.

In Vitro Maturation of Human iPSC-Derived Neuroepithelial Cells Influences Transplant Survival in the Stroke-Injured Rat Brain.

Stem cell transplantation is a promising strategy for brain tissue regeneration; yet, despite some success, cell survival following transplantation remains low. In this study, we demonstrate that cell viability is enhanced by control over maturation of neuronal precursor cells, which are delivered in an injectable blend of hyaluronan and methylcellulose. We selected three subpopulations of human neuronal precursor cells derived from a cortically specified neuroepithelial stem cell (cNESC) population based on differences in expression of multipotent and neuron-specific proteins: early-, mid-, and late-differentiated neurons. These cells were transplanted into an endothelin-1 stroke-injured rat brain and their survival and fate were investigated 1 week later. Significantly, more cells were found in the brain after transplanting early- or mid- differentiated cNESCs compared to the late-differentiated population. The mid-differentiated population also had significantly more ß-III tubulin-positive cells than either the early- or late-differentiated populations. These results suggest that maturity has a significant impact on cell survival following transplantation and cells with an intermediate maturity differentiate to neurons.

Blood vessel formation during tail regeneration in the leopard gecko (Eublepharis macularius): The blastema is not avascular.

Unique among amniotes, many lizards are able to self-detach (autotomize) their tail and then regenerate a replacement. Tail regeneration involves the formation of a blastema, an accumulation of proliferating cells at the site of autotomy. Over time, cells of the blastema give rise to most of the tissues in the replacement tail. In non-amniotes capable of regenerating (such as urodeles and some teleost fish), the blastema is reported to be essentially avascular until tissue differentiation takes place. For tail regenerating lizards less is known. Here, we investigate neovascularization during tail regeneration in the leopard gecko (Eublepharis macularius). We demonstrate that the gecko tail blastema is not an avascular structure. Beginning with the onset of regenerative outgrowth, structurally mature (mural cell supported) blood vessels are found within the blastema. Although the pattern of blood vessel distribution in the regenerate tail differs from that of the original, a hierarchical network is established, with vessels of varying luminal diameters and wall thicknesses. Using immunostaining, we determine that blastema outgrowth and tissue differentiation is characterized by a dynamic interplay between the pro-angiogenic protein vascular endothelial growth factor (VEGF) and the anti-angiogenic protein thrombospondin-1 (TSP-1). VEGF-expression is initially widespread, but diminishes as tissues differentiate. In contrast, TSP-1 expression is initially restricted but becomes more abundant as VEGF-expression wanes. We predict that variation in the neovascular response observed between different regeneration-competent species likely relates to the volume of the blastema. J. Morphol. 278:380-389, 2017. © 2017 Wiley Periodicals, Inc.

The anatomy and histology of caudal autotomy and regeneration in lizards.

Abstract Caudal autotomy-the ability to self-detach the tail-is a dramatic adaptation common to many structural-grade lizards. For most species, tail loss is followed by the equally dramatic phenomenon of tail regeneration. Here we review the anatomy and histology of caudal autotomy and regeneration in lizards, drawing heavily from research published over the past 2 decades. The autotomous tail is characterized by various structural adaptations, which act to minimize blood loss and trauma to adjacent tissues. The early phase of wound healing involves a leukocytic response but limited inflammation. Reepithelialization via a specialized wound epithelium is not only critical for scar-free healing but also necessary for subsequent tissue patterning and regenerative outgrowth. Regeneration begins with the formation of the blastema, a mass of proliferating mesenchymal-like cells. As the blastema expands, it is invaded by blood vessels and the spinal cord. Whereas the replacement tail outwardly resembles the original appendage, it differs in several notable respects, including the tissue composition and organization of the skeleton, muscular system, and spinal cord. Increasingly, the lizard tail is being recognized among biomedical scientists as an important model for the study of wound healing and multitissue restoration.

An osteological and histological investigation of cranial joints in geckos.

Cranial kinesis is a widespread feature of gekkotan lizards. Previous studies of kinesis in lizards often described the relevant, mobile joints as synovial, thus characterized by the presence of a synovial cavity lined with articular cartilage. To date however, detailed investigations of cranial joint histology are lacking. We examined eight cranial joints (quadrate-articular, quadrate-pterygoid, quadrate-otooccipital, quadrate-squamosal, epipterygoid-prootic, epipterygoid-pterygoid, basisphenoid-pterygoid, and frontal-parietal) in five gekkotan species (Oedura lesueuerii, Eublepharis macularius, Hemitheconyx caudicinctus, Tarentola annularis, and Chondrodactylous bibronii) using microcomputed tomography and serial histology. Particular focus was given to the relationship between the bony and soft-tissue components of the joint. Our results demonstrate that only three of these joints are synovial: the quadrate-articular, epipterygoid-pterygoid, and basisphenoid-pterygoid joints. The frontal-parietal and quadrate-pterygoid joints are syndesmosis (fibrous), the epipterygoid-prootic and quadrate-otooccipital joints are synchondroses (cartilaginous without a synovial cavity) and the quadrate-squamosal joint was not present. Based on previous descriptions, we determine that the structure of some cranial joints is variable among lizard taxa. We caution that osteology does not necessarily predict cranial joint histology. Although the functional implications of these findings remain to be explored we note that the development of synovial joints appears to be associated with a neural crest origin for the elements involved.