The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis.

The tumor microenvironment in brain metastases is characterized by high myeloid cell content associated with immune suppressive and cancer-permissive functions. Moreover, brain metastases induce the recruitment of lymphocytes. Despite their presence, T-cell-directed therapies fail to elicit effective anti-tumor immune responses. Here, we seek to evaluate the applicability of radio-immunotherapy to modulate tumor immunity and overcome inhibitory effects that diminish anti-cancer activity. Radiotherapy-induced immune modulation resulted in an increase in cytotoxic T-cell numbers and prevented the induction of lymphocyte-mediated immune suppression. Radio-immunotherapy led to significantly improved tumor control with prolonged median survival in experimental breast-to-brain metastasis. However, long-term efficacy was not observed. Recurrent brain metastases showed accumulation of blood-borne PD-L1+ myeloid cells after radio-immunotherapy indicating the establishment of an immune suppressive environment to counteract re-activated T-cell responses. This finding was further supported by transcriptional analyses indicating a crucial role for monocyte-derived macrophages in mediating immune suppression and regulating T-cell function. Therefore, selective targeting of immune suppressive functions of myeloid cells is expected to be critical for improved therapeutic efficacy of radio-immunotherapy in brain metastases.

Compensatory CSF2-driven macrophage activation promotes adaptive resistance to CSF1R inhibition in breast-to-brain metastasis.

Tumor microenvironment-targeted therapies are emerging as promising treatment options for different cancer types. Tumor-associated macrophages and microglia (TAMs) represent an abundant nonmalignant cell type in brain metastases and have been proposed to modulate metastatic colonization and outgrowth. Here we demonstrate that targeting TAMs at distinct stages of the metastatic cascade using an inhibitor of colony-stimulating factor 1 receptor (CSF1R), BLZ945, in murine breast-to-brain metastasis models leads to antitumor responses in prevention and intervention preclinical trials. However, in established brain metastases, compensatory CSF2Rb-STAT5-mediated pro-inflammatory TAM activation blunted the ultimate efficacy of CSF1R inhibition by inducing neuroinflammation gene signatures in association with wound repair responses that fostered tumor recurrence. Consequently, blockade of CSF1R combined with inhibition of STAT5 signaling via AC4-130 led to sustained tumor control, a normalization of microglial activation states and amelioration of neuronal damage.

Aging-regulated anti-apoptotic long non-coding RNA Sarrah augments recovery from acute myocardial infarction.

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.

Microenvironmental Regulation of Tumor Progression and Therapeutic Response in Brain Metastasis.

Cellular and non-cellular components of the tumor microenvironment (TME) are emerging as key regulators of primary tumor progression, organ-specific metastasis, and therapeutic response. In the era of TME-targeted- and immunotherapies, cancer-associated inflammation has gained increasing attention. In this regard, the brain represents a unique and highly specialized organ. It has long been regarded as an immunological sanctuary site where the presence of the blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCB) restricts the entry of immune cells from the periphery. Consequently, tumor cells that metastasize to the brain were thought to be shielded from systemic immune surveillance and destruction. However, the detailed characterization of the immune landscape within border-associated areas of the central nervous system (CNS), such as the meninges and the choroid plexus, as well as the discovery of lymphatics and channels that connect the CNS with the periphery, have recently challenged the dogma of the immune privileged status of the brain. Moreover, the presence of brain metastases (BrM) disrupts the integrity of the BBB and BCB. Indeed, BrM induce the recruitment of different immune cells from the myeloid and lymphoid lineage to the CNS. Blood-borne immune cells together with brain-resident cell-types, such as astrocytes, microglia, and neurons, form a highly complex and dynamic TME that affects tumor cell survival and modulates the mode of immune responses that are elicited by brain metastatic tumor cells. In this review, we will summarize recent findings on heterotypic interactions within the brain metastatic TME and highlight specific functions of brain-resident and recruited cells at different rate-limiting steps of the metastatic cascade. Based on the insight from recent studies, we will discuss new opportunities and challenges for TME-targeted and immunotherapies for BrM.

Controlling the dose-dependent, synergistic and temporal effects of NGF and GDNF by encapsulation in PLGA microparticles for use in nerve guidance conduits for the repair of large peripheral nerve defects.

Neurotrophic factor delivery via biodegradable nerve guidance conduits may serve as a promising treatment for the repair of large peripheral nerve defects. However, a platform for controlled delivery is required because of their short in vivo half-life and their potential to impede axonal regeneration when used in supraphysiological doses. In this study, we investigated the dose-dependent, synergistic and temporal effects of NGF and GDNF on neurite outgrowth, adult dorsal root ganglia axonal outgrowth, Schwann cell migration and cytokine production in vitro. Using the optimal dose and combination of NGF and GDNF, we developed a PLGA microparticle-based delivery platform to control their delivery. The dose-dependent effects of both NGF and GDNF individually were found to be non-linear with a saturation point. However, the synergistic effect between NGF and GDNF was found to outweigh their dose-dependent effects in terms of enhancing Schwann cell migration and axonal outgrowth while allowing a 100-fold reduction in dose. Moreover, a temporal profile that mimics the physiological flux of NGF and GDNF in response to injury, compared to one that resembles an early burst release delivery profile, was found to enhance their bioactivity. The optimized NGF- and GDNF-loaded microparticles were then incorporated into a guidance conduit, and their capacity to enhance nerve regeneration across a 15 mm sciatic nerve defect in rats was demonstrated. Enhanced nerve regeneration was seen in comparison to non-treated defects and very encouragingly, to a similar level compared to the clinical gold standard of autograft. Taken together, we suggest that this delivery platform might have significant potential in the field of peripheral nerve repair; allowing spatial and temporal control over the delivery of potent neurotrophic factors to enhance the regenerative capacity of biomaterials-based nerve guidance conduits.

Long-term neurite orientation on astrocyte monolayers aligned by microtopography.

After spinal cord injury neuronal connections are not easily re-established. Success has been hampered by the lack of orientation of neurites inside scar tissue and a lack of neurites crossing out of the site of injury. Oriented scaffolds in biodegradable polymers could be an excellent way to support both the orientation of neurites within the injury site as well as aiding their crossing out of the lesion. To establish the validity of using grooved micro-topography in polycaprolactone in combination with glia we have studied the long-term (3 weeks) orientation of neuronal cells on monolayers of astrocytes on the top of grooved topographies of various dimensions. We find that neurites are significantly aligned by groove/ridge type topographies which are "buried" under a monolayer of astrocytes for up to 3 weeks. This alignment is significantly lower than that of neurites growing directly on the topography, but these neurons do not survive on the poly-l-lysine coated polymer for more than a week. The alignment of neurites on the astrocyte layer to the underlying topography decreases over time, and with groove width. Topographies with 12.5 or 25 microm lateral dimension appear optimal for the long-term alignment and can support myelination. We have shown for the first time that micro-topography can act through an overlaid astrocyte layer and results in aligned neurites in long-term culture and that these can be myelinated by endogenous oligodendrocytes.

Olfactory Derived Stem Cells Delivered in a Biphasic Conduit Promote Peripheral Nerve Repair In Vivo.

Peripheral nerve injury presents significant therapeutic challenges for recovery of motor and sensory function in patients. Different clinical approaches exist but to date there has been no consensus on the most effective method of treatment. Here, we investigate a novel approach to peripheral nerve repair using olfactory derived stem (ONS) cells delivered in a biphasic collagen and laminin functionalized hyaluronic acid based nerve guidance conduit (NGC). Nerve regeneration was studied across a 10-mm sciatic nerve gap in Sprague Dawley rats. The effect of ONS cell loading of NGCs with or without nerve growth factor (NGF) supplementation on nerve repair was compared to a cell-free NGC across a variety of clinical, functional, electrophysiological, and morphologic parameters. Animals implanted with ONS cell loaded NGCs demonstrated improved clinical and electrophysiological outcomes compared to cell free NGC controls. The nerves regenerated across ONS cell loaded NGCs contained significantly more axons than cell-free NGCs. A return of the nocioceptive withdrawal reflex in ONS cell treated animals indicated an advanced repair stage at a relatively early time point of 8 weeks post implantation. The addition of NGF further improved the outcomes of the repair indicating the potential beneficial effect of a combined stem cell/growth factor treatment strategy delivered on NGCs. Stem Cells Translational Medicine 2017;6:1894-1904.

A Physicochemically Optimized and Neuroconductive Biphasic Nerve Guidance Conduit for Peripheral Nerve Repair.

Clinically available hollow nerve guidance conduits (NGCs) have had limited success in treating large peripheral nerve injuries. This study aims to develop a biphasic NGC combining a physicochemically optimized collagen outer conduit to bridge the transected nerve, and a neuroconductive hyaluronic acid-based luminal filler to support regeneration. The outer conduit is mechanically optimized by manipulating crosslinking and collagen density, allowing the engineering of a high wall permeability to mitigate the risk of neuroma formation, while also maintaining physiologically relevant stiffness and enzymatic degradation tuned to coincide with regeneration rates. Freeze-drying is used to seamlessly integrate the luminal filler into the conduit, creating a longitudinally aligned pore microarchitecture. The luminal stiffness is modulated to support Schwann cells, with laminin incorporation further enhancing bioactivity by improving cell attachment and metabolic activity. Additionally, this biphasic NGC is shown to support neurogenesis and gliogenesis of neural progenitor cells and axonal outgrowth from dorsal root ganglia. These findings highlight the paradigm that a successful NGC requires the concerted optimization of both a mechanical support phase capable of bridging a nerve defect and a neuroconductive phase with an architecture capable of supporting both Schwann cells and neurons in order to achieve functional regenerative outcome.

Roofed grooves: rapid layer engineering of perfusion channels in collagen tissue models.

Surface patterning (micro-moulding) of dense, biomimetic collagen is a simple tool to produce complex tissues using layer-by-layer assembly. The aim here was to channelise three-dimensional constructs for improved perfusion. Firstly, collagen fibril accumulation was measured by comparative image analysis to understand the mechanisms of structure formation in plastically compressed collagen during µ-moulding. This showed that shape (circular or rectangular) and dimensions of the template affected collagen distribution around moulded grooves and consequently their stability. In the second part, this was used for effective fabrication of multi-layered plastically compressed collagen constructs with internal channels by roofing the grooves with a second layer. Using rectangular templates of 25/50/100 µm widths and 75 µm depth, grooves were µ-moulded into the fluid-leaving surface of collagen layers with predictable width/depth fidelities. These grooves were then roofed by addition of a second plastically compressed collagen layer on top to produce µ-channels. Resulting µ-channels retained their dimensions and were stable over time in culture with fibroblasts and could be cell seeded with a lining layer by simple transfer of epithelial cells. The results of this study provide a valuable platform for rapid fabrication of complex collagen-based tissues in particular for provision of perfusing microchannels through the bulk material for improved core nutrient supply.

Engineering a microvascular capillary bed in a tissue-like collagen construct.

Previous studies have shown that plastic compression (PC) of collagen gels allows a rapid and controlled fabrication of matrix- and cell-rich constructs in vitro that closely mimic the structure and characteristics of tissues in vivo. Microvascular endothelial cells, the major cell type making up the blood vessels in the body, were added to the PC collagen to determine whether cells attach, survive, grow, and express endothelial cell characteristics when seeded alone or in coculture with other cells. Endothelial cells seeded on the PC collagen containing human foreskin fibroblasts (HFF) or human osteoblasts (HOS) formed vessel-like structures over 3 weeks in culture without the addition of exogenous growth factors in the medium. In contrast, on the PC scaffolds without HFF or HOS, human dermal microvascular endothelial cells (HDMEC) exhibited a typical cobblestone morphology for 21 days under the same conditions. We propose that the coculture of primary endothelial cells with PC collagen constructs, containing a stromal cell population, is a valuable technique for in vitro modeling of proangiogenic responses toward such biomimetic constructs in vivo. A major observation in the cocultures was the absence of gel contraction, even after 3 weeks of fibroblast culture. This collagen form could, for example, be of great value in tissue engineering of the skin, as contractures are both aesthetically and functionally disabling.

Development of conical soluble phosphate glass fibers for directional tissue growth.

One of the challenges of tissue engineering is the regulation of vascularization and innervations of the implant by the host. Here, we propose that using soluble phosphate glass (SPG) fibers, incorporated in dense collagen constructs will allow us to control the rate and direction of tissue ingrowth. The idea here was to generate channels with tailored direction using conical phosphate glass fibers. The changing surface area-to-mass ratio of conical fibers will make them to dissolve faster from their narrow ends opening up channels in that direction ahead of any ingrowing cells. In this study, we show that SPG fibers can be manipulated to produce conical shape fibers using graded dissolution. Our result shows that 40 µm fibers of composition ratio 0.5 (P(2)O(5)):0.25 (CaO):0.25 (Na(2)O) and dissolution time of 8-10 h have a mean reduction in fiber diameter of 8.85 ± 2.8 µm over 19.5 mm fiber length, i.e., a mean rate of 0.5 µm/mm (n=20) change. These conically shaped fibers can also be manipulated and potentially used to promote uniaxial cell-tissue ingrowth for improved innervations and vascularization of tissue engineered constructs.

Alignment hierarchies: engineering architecture from the nanometre to the micrometre scale.

Natural tissues are built of metabolites, soluble proteins and solid extracellular matrix components (largely fibrils) together with cells. These are configured in highly organized hierarchies of structure across length scales from nanometre to millimetre, with alignments that are dominated by anisotropies in their fibrillar matrix. If we are to successfully engineer tissues, these hierarchies need to be mimicked with an understanding of the interaction between them. In particular, the movement of different elements of the tissue (e.g. molecules, cells and bulk fluids) is controlled by matrix structures at distinct scales. We present three novel systems to introduce alignment of collagen fibrils, cells and growth factor gradients within a three-dimensional collagen scaffold using fluid flow, embossing and layering of construct. Importantly, these can be seen as different parts of the same hierarchy of three-dimensional structure, as they are all formed into dense collagen gels. Fluid flow aligns collagen fibrils at the nanoscale, embossed topographical features provide alignment cues at the microscale and introducing layered configuration to three-dimensional collagen scaffolds provides microscale- and mesoscale-aligned pathways for protein factor delivery as well as barriers to confine protein diffusion to specific spatial directions. These seemingly separate methods can be employed to increase complexity of simple extracellular matrix scaffolds, providing insight into new approaches to directly fabricate complex physical and chemical cues at different hierarchical scales, similar to those in natural tissues.