Crosstalk between glial and glioblastoma cells triggers the "go-or-grow" phenotype of tumor cells.

BACKGROUND: Glioblastoma (GBM), the most malignant primary brain tumor, leads to poor and unpredictable clinical outcomes. Recent studies showed the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. In this context, we investigated how GBM cells modulate resident glial cells, particularly their paracrine activity, and how this modulation can influence back on the malignant phenotype of GBM cells. METHODS: Conditioned media (CM) of primary mouse glial cultures unexposed (unprimed) or exposed (primed) to the secretome of GL261 GBM cells were analyzed by proteomic analysis. Additionally, these CM were used in GBM cells to evaluate their impact in glioma cell viability, migration capacity and activation of tumor-related intracellular pathways. RESULTS: The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells. At the functional levels, CM derived from unprimed glial cells favored an increase in GBM cell migration capacity, while CM from primed glial cells promoted cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation. CONCLUSIONS: Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the "go-or-grow" phenotypic switch of GBM cells.

The secretome of macrophages has a differential impact on spinal cord injury recovery according to the polarization protocol.

Introduction: The inflammatory response after spinal cord injury (SCI) is an important contributor to secondary damage. Infiltrating macrophages can acquire a spectrum of activation states, however, the microenvironment at the SCI site favors macrophage polarization into a pro-inflammatory phenotype, which is one of the reasons why macrophage transplantation has failed. Methods: In this study, we investigated the therapeutic potential of the macrophage secretome for SCI recovery. We investigated the effect of the secretome in vitro using peripheral and CNS-derived neurons and human neural stem cells. Moreover, we perform a pre-clinical trial using a SCI compression mice model and analyzed the recovery of motor, sensory and autonomic functions. Instead of transplanting the cells, we injected the paracrine factors and extracellular vesicles that they secrete, avoiding the loss of the phenotype of the transplanted cells due to local environmental cues. Results: We demonstrated that different macrophage phenotypes have a distinct effect on neuronal growth and survival, namely, the alternative activation with IL-10 and TGF-ß1 (M(IL-10+TGF-ß1)) promotes significant axonal regeneration. We also observed that systemic injection of soluble factors and extracellular vesicles derived from M(IL-10+TGF-ß1) macrophages promotes significant functional recovery after compressive SCI and leads to higher survival of spinal cord neurons. Additionally, the M(IL-10+TGF-ß1) secretome supported the recovery of bladder function and decreased microglial activation, astrogliosis and fibrotic scar in the spinal cord. Proteomic analysis of the M(IL-10+TGF-ß1)-derived secretome identified clusters of proteins involved in axon extension, dendritic spine maintenance, cell polarity establishment, and regulation of astrocytic activation. Discussion: Overall, our results demonstrated that macrophages-derived soluble factors and extracellular vesicles might be a promising therapy for SCI with possible clinical applications.

Treating Parkinson's Disease with Human Bone Marrow Mesenchymal Stem Cell Secretome: A Translational Investigation Using Human Brain Organoids and Different Routes of In Vivo Administration.

Parkinson's disease (PD) is the most common movement disorder, characterized by the progressive loss of dopaminergic neurons from the nigrostriatal system. Currently, there is no treatment that retards disease progression or reverses damage prior to the time of clinical diagnosis. Mesenchymal stem cells (MSCs) are one of the most extensively studied cell sources for regenerative medicine applications, particularly due to the release of soluble factors and vesicles, known as secretome. The main goal of this work was to address the therapeutic potential of the secretome collected from bone-marrow-derived MSCs (BM-MSCs) using different models of the disease. Firstly, we took advantage of an optimized human midbrain-specific organoid system to model PD in vitro using a neurotoxin-induced model through 6-hydroxydopamine (6-OHDA) exposure. In vivo, we evaluated the effects of BM-MSC secretome comparing two different routes of secretome administration: intracerebral injections (a two-site single administration) against multiple systemic administration. The secretome of BM-MSCs was able to protect from dopaminergic neuronal loss, these effects being more evident in vivo. The BM-MSC secretome led to motor function recovery and dopaminergic loss protection; however, multiple systemic administrations resulted in larger therapeutic effects, making this result extremely relevant for potential future clinical applications.

Immunomodulatory and regenerative effects of the full and fractioned adipose tissue derived stem cells secretome in spinal cord injury.

Adipose tissue derived stem cells (ASCs) are recognized to secret a myriad of molecules (secretome) know to modulate inflammatory response, promote axonal growth as well vascular remodeling and cellular survival. In previous works we have reported the benefit effects of ASCs transplanted to the injury site in a rat model of spinal cord injury (SCI). Emerging evidence have shown that the therapeutic actions of these cells are a consequence of their intense paracrine activity mediated by their secretome, which includes soluble bioactive molecules and vesicles. In this study, we intended to dissect the vesicular and protein individual function, comparing with whole secretome therapeutic effect. Therefore, we identified a beneficial effect of the whole secretome on neurite growth compared with protein or vesicular fraction alone and characterized their impact on microglia in vitro. Moreover, in a compression SCI mice model, from the motor tests performed, a statistical difference was found on beam balance test revealing differences in motor recovery between the use of the whole the secretome or their protein fraction. Finally, two different delivery methods, local or peripheral (IV), of ASC secretome were tested in vivo. Results indicate that when injected intravenously the secretome of ASCs has a beneficial effect on motor recovery of spinal cord injury animals compared with a single local injection and respective controls. Overall, our results showed that the whole secretome performed better than the fractions individually, raising ASC secretome mode of action as a synergy of proteic and vesicular fraction on SCI context. Also, when intravenously delivered, ASC secretome can promote SCI animal's motor recovery highlighting their therapeutic potential.

Astrocyte signaling impacts the effects of human bone marrow mesenchymal stem cells secretome application into the hippocampus: A proliferation and morphometrical analysis on astrocytic cell populations.

The central nervous system (CNS) has a limited auto-regeneration capacity, which makes it challenging for the development of new therapies. Previous studies from our lab have demonstrated the applicability of human bone marrow mesenchymal stem cells (hBM-MSCs) secretome as a possible therapeutic tool for CNS. Astrocytes, glial cells present in all brain regions, are important players in brain function through their vast influence in extracellular homeostasis, neuro-vascular regulation, synaptic modulation and neurogenesis. Thus, in the present work, we aimed to evaluate the specific impact of MSCs secretome on hippocampal proliferation and astrocyte morphology, in both WT and dnSNARE mice, a transgenic model that presents impaired astrocytic exocytosis and consequently impaired astrocytic function. Results demonstrated increased levels of proliferation for WT when treated with secretome. Additionally, it was possible to observe that dnSNARE animals injected with hBM-MSCs secretome disclosed increased levels of proliferating GFAP stained cells at the SGZ. Morphometrical evaluation found increased process hypertrophy and branching of dnSNARE astrocytes when treated with secretome. These results are closely related with the trophic factors present in the secretome, namely FGF-2, BDNF, GDNF, IGF-1, VEGF, CADH2, PEDF and miR-16. Moreover, the impaired exocytosis of astrocytes may also have implications for the response to the proliferative stimulus, given the established autocrine signaling through this mechanism.

Influence of passage number on the impact of the secretome of adipose tissue stem cells on neural survival, neurodifferentiation and axonal growth.

Mesenchymal stem cells (MSCs), and within them adipose tissue derived stem cells (ASCs), have been shown to have therapeutic effects on central nervous system (CNS) cell populations. Such effects have been mostly attributed to soluble factors, as well as vesicles, present in their secretome. Yet, little is known about the impact that MSC passaging might have in the secretion therapeutic profile. Our aim was to show how human ASCs (hASCs) passage number influences the effect of their secretome in neuronal survival, differentiation and axonal growth. For this purpose, post-natal rat hippocampal primary cultures, human neural progenitor cell (hNPCs) cultures and dorsal root ganglia (DRGs) explants were incubated with secretome, collected as conditioned media (CM), obtained from hASCs in P3, P6, P9 and P12. Results showed no differences when comparing percentages of MAP-2 positive cells (a mature neuronal marker) in neuronal cultures or hNPCs, after incubation with hASCs secretome from different passages. The same was observed regarding DRG neurite outgrowth. In order to characterize the secretomes obtained from different passages, a proteomic analysis was performed, revealing that its composition did not vary significantly with passage number P3 to P12. Results allowed us to identify several key proteins, such as pigment epithelium derived factor (PEDF), DJ-1, interleucin-6 (IL-6) and galectin, all of which have already proven to play neuroprotective and neurodifferentiating roles. Proteins that promote neurite outgrowth were also found present, such as semaphorin 7A and glypican-1. We conclude that cellular passaging does not influence significantly hASCs's secretome properties especially their ability to support post-natal neuronal survival, induce neurodifferentiation and promote axonal growth.

Old and new challenges in Parkinson's disease therapeutics.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic neurons and/or loss od neuronal projections, in several dopaminergic networks. Current treatments for idiopathic PD rely mainly on the use of pharmacologic agents to improve motor symptomatology of PD patients. Nevertheless, so far PD remains an incurable disease. Therefore, it is of utmost importance to establish new therapeutic strategies for PD treatment. Over the last 20 years, several molecular, gene and cell/stem-cell therapeutic approaches have been developed with the aim of counteracting or retarding PD progression. The scope of this review is to provide an overview of PD related therapies and major breakthroughs achieved within this field. In order to do so, this review will start by focusing on PD characterization and current treatment options covering thereafter molecular, gene and cell/stem cell-based therapies that are currently being studied in animal models of PD or have recently been tested in clinical trials. Among stem cell-based therapies, those using MSCs as possible disease modifying agents for PD therapy and, specifically, the MSCs secretome contribution to meet the clinical challenge of counteracting or retarding PD progression, will be more deeply explored.

Mesenchymal stem cells secretome-induced axonal outgrowth is mediated by BDNF.

Mesenchymal stem cells (MSCs) have been used for cell-based therapies in regenerative medicine, with increasing importance in central and peripheral nervous system repair. However, MSCs grafting present disadvantages, such as, a high number of cells required for transplantation and low survival rate when transplanted into the central nervous system (CNS). In line with this, MSCs secretome which present on its composition a wide range of molecules (neurotrophins, cytokines) and microvesicles, can be a solution to surpass these problems. However, the effect of MSCs secretome in axonal elongation is poorly understood. In this study, we demonstrate that application of MSCs secretome to both rat cortical and hippocampal neurons induces an increase in axonal length. In addition, we show that this growth effect is axonal intrinsic with no contribution from the cell body. To further understand which are the molecules required for secretome-induced axonal outgrowth effect, we depleted brain-derived neurotrophic factor (BDNF) from the secretome. Our results show that in the absence of BDNF, secretome-induced axonal elongation effect is lost and that axons present a reduced axonal growth rate. Altogether, our results demonstrate that MSCs secretome is able to promote axonal outgrowth in CNS neurons and this effect is mediated by BDNF.

Modulation of the Mesenchymal Stem Cell Secretome Using Computer-Controlled Bioreactors: Impact on Neuronal Cell Proliferation, Survival and Differentiation.

In recent years it has been shown that the therapeutic benefits of human mesenchymal stem/stromal cells (hMSCs) in the Central Nervous System (CNS) are mainly attributed to their secretome. The implementation of computer-controlled suspension bioreactors has shown to be a viable route for the expansion of these cells to large numbers. As hMSCs actively respond to their culture environment, there is the hypothesis that one can modulate its secretome through their use. Herein, we present data indicating that the use of computer-controlled suspension bioreactors enhanced the neuroregulatory profile of hMSCs secretome. Indeed, higher levels of in vitro neuronal differentiation and NOTCH1 expression in human neural progenitor cells (hNPCs) were observed when these cells were incubated with the secretome of dynamically cultured hMSCs. A similar trend was also observed in the hippocampal dentate gyrus (DG) of rat brains where, upon injection, an enhanced neuronal and astrocytic survival and differentiation, was observed. Proteomic analysis also revealed that the dynamic culturing of hMSCs increased the secretion of several neuroregulatory molecules and miRNAs present in hMSCs secretome. In summary, the appropriate use of dynamic culture conditions can represent an important asset for the development of future neuro-regenerative strategies involving the use of hMSCs secretome.

Unveiling the Differences of Secretome of Human Bone Marrow Mesenchymal Stem Cells, Adipose Tissue-Derived Stem Cells, and Human Umbilical Cord Perivascular Cells: A Proteomic Analysis.

The use of human mesenchymal stem cells (hMSCs) has emerged as a possible therapeutic strategy for CNS-related conditions. Research in the last decade strongly suggests that MSC-mediated benefits are closely related with their secretome. Studies published in recent years have shown that the secretome of hMSCs isolated from different tissue sources may present significant variation. With this in mind, the present work performed a comparative proteomic-based analysis through mass spectrometry on the secretome of hMSCs derived from bone marrow (BMSCs), adipose tissue (ASCs), and human umbilical cord perivascular cells (HUCPVCs). The results revealed that BMSCs, ASCs, and HUCPVCs differed in their secretion of neurotrophic, neurogenic, axon guidance, axon growth, and neurodifferentiative proteins, as well as proteins with neuroprotective actions against oxidative stress, apoptosis, and excitotoxicity, which have been shown to be involved in several CNS disorder/injury processes. Although important changes were observed within the secretome of the cell populations that were analyzed, all cell populations shared the capability of secreting important neuroregulatory molecules. The difference in their secretion pattern may indicate that their secretome is specific to a condition of the CNS. Nevertheless, the confirmation that the secretome of MSCs isolated from different tissue sources is rich in neuroregulatory molecules represents an important asset not only for the development of future neuroregenerative strategies but also for their use as a therapeutic option for human clinical trials.

Failure of Y-27632 to improve the culture of adult human adipose-derived stem cells.

Y-27632 is a well-known inhibitor of the Rho-associated coiled kinase (ROCK) and has been shown to significantly improve the culture of a variety of multipotent stem cell types. However, the effects of Y-27632 on the expansion of adult human adipose-derived stem cell (hADSC) cultures remain to be established. Here, we aimed to characterize the effects of Y-27632 on the culture of hADSCs. Adult hADSCs were isolated from subjects submitted to elective plastic surgery procedures and cultivated in vitro under optimized conditions. Our results show that the continuous supplementation of hADSC cultures with Y-27632 led to decreased numbers of cells and decreased global metabolic viability of hADSC cultures when compared with control conditions. This effect appeared to be dependent on the continuous presence of the drug and was shown to be concentration-dependent and significant for 10 µM and 20 µM of Y-27632. Moreover, the Y-27632-induced decrease in hADSC numbers was not linked to a block in global cell proliferation, as cell numbers consistently increased from the moment of plating until passaging. In addition, Y-27632 was not able to increase the number of hADSCs present in culture 24 hours after passaging. Taken together, our results suggest that, in contrast to other stem cell types, Y-27632 supplementation is not a suitable strategy to enhance hADSC culture expansion.

Combinatorial Activity of Flavonoids with Antibiotics Against Drug-Resistant Staphylococcus aureus.

The use of resistance-modifying agents is a potential strategy that is used to prolong the effective life of antibiotics in the face of increasing antibiotic resistance. Since certain flavonoids are potent bacterial efflux pump inhibitors, we assessed morin, rutin, quercetin, hesperidin, and (+)-catechin for their combined activity with the antibiotics ciprofloxacin, tetracycline, erythromycin, oxacillin, and ampicillin against drug-resistant strains of Staphylococcus aureus, including methicillin-resistant S. aureus. Four established methods were used to determine the combined efficacy of each combination: microdilution checkerboard assays, time-kill determinations, the Etest, and dual disc-diffusion methods. The cytotoxicity of the flavonoids was additionally evaluated in a mouse fibroblast cell line. Quercetin and its isomer morin decreased by 3- to 16-fold the minimal inhibitory concentration of ciprofloxacin, tetracycline, and erythromycin against some S. aureus strains. Rutin, hesperidin, and (+)-catechin did not promote any potentiation of antibiotics. Despite the potential cytotoxicity of these phytochemicals at a high concentration (fibroblast IC50 of 41.8 and 67.5 mg/L, respectively), quercetin is commonly used as a supplement for several therapeutic purposes. All the methods, with exception of the time-kill assay, presented a high degree of congruence without any apparent strain specificity.

Evaluation of the best method to assess antibiotic potentiation by phytochemicals against Staphylococcus aureus.

The increasing occurrence of bacterial resistance to antibiotics has now reached a critical level. Finding antibiotic coadjuvants capable to inhibit the bacterial resistance mechanisms would be a valuable mid-term solution, until new classes of antibiotics are discovered. Selected plant alkaloids were combined with 5 antibiotics against 10 Staphylococcus aureus strains, including strains expressing distinct efflux pumps and methicillin-resistant S. aureus strains. The efficacy of each combination was assessed using the microdilution checkerboard, time-kill, Etest, and disc diffusion methods. The cytotoxicity of the alkaloids was evaluated in a mouse fibroblast cell line. Potentiation was obtained in 6% of all 190 combinations, especially with the combination of: ciprofloxacin with reserpine (RES), pyrrolidine (PYR), and quinine (QUIN); tetracycline with RES; and erythromycin with PYR. The highest cytotoxicity values were found for QUIN (half maximal inhibitory concentration [IC50] = 25 ± 2.2 mg/L) and theophylline (IC50 = 100 ± 4.7 mg/L).