Dataset Information

Characterization of the secretome of glial cells exposed to glioblastoma secreted factors

ABSTRACT: Glioblastoma (GBM), the most malignant primary brain tumor, is characterized by widespread heterogeneity, leading to poor and unpredictable clinical outcomes. Recent studies have provided evidences that the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. Here we investigate how GBM cells modulate glial cells, and how this modulation can influence back on the malignant phenotype of GBM cells. Primary mouse glial cultures were established and cultured in serum-free media (unprimed) or exposed to secretome derived from GL261 GBM cells (primed). Conditioned media (CM) from each glial culture were collected and a proteomic analysis was conducted. Glial cells CM (unprimed and primed) were also used in GL261 GBM cells to evaluate its impact in critical hallmarks of GBM cells, including viability, migration, and activation of tumor-related intracellular signaling pathways. 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, consistent with a pattern of reactive astrogliosis. At the functional levels, CM derived from unprimed glial cells favored an increase in cell migration capacity, while CM from primed glial cells was more efficient in promoting GBM 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. Together, our results demonstrate that glial cells can have a different impact on the progression of GBM tumors, suggesting 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. 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.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Primary Cell

SUBMITTER: Sandra Anjo

LAB HEAD: Bruno Manadas

PROVIDER: PXD006007 | Pride | 2017-10-09

REPOSITORIES: Pride

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

Oliveira Ana Isabel AI Anjo Sandra I SI Vieira de Castro Joana J Serra Sofia C SC Salgado António J AJ Manadas Bruno B Costa Bruno M BM

Cell communication and signaling : CCS 20171002 1

<h4>Background</h4>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.<h4>Me ...[more]

PMID: 28969644

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Project description:Glial cells are central players in the pathogenesis of neurodevelopmental disorders like Fetal Alcohol Spectrum Disorder (FASD). Ethanol can modulate glial differentiation and astrocyte function imposing dramatic changes to the developing brain. Since ethanol can affect a vast number of intracellular targets and signaling pathways and many effects of early alcohol exposure on cell physiology can persist into adulthood, we tested the hypothesis that ethanol exposure in ferrets during a period equivalent to the last months of human gestation leads to a long-lasting change in astrocyte secretome in vitro. Animals were treated every other day with ethanol (5g/kg) or saline between post-natal day (P)10-30. At P31, astrocyte cultures were made and cells were submitted to stable isotope labeling by amino acids (SILAC). 24h-conditioned media of cells obtained from ethanol- or saline-treated animals (ET-CM or SAL-CM) were collected and analyzed by quantitative mass spectrometry in tandem with liquid chromatography. Here we show that 65 out of 280 quantifiable proteins displayed significant differences comparing ET-CM to SAL-CM. Among 59 proteins found reduced in ET-CM we found components of the extracellular matrix like Laminin subunits α2, α4, β1, β2 and γ1 and the proteoglycans Biglycan, Heparin Sulfate Proteoglycan 2 and Lumican. Proteins with trophic function like Insulin-Like Growth Factor Binding Protein 4, Pigment Epithelium-Derived Factor and Clusterin as well as proteins involved on modulation of proteolysis like TIMP-1 and PAI-1 were also found reduced. On the other hand, pro-synaptogeneic proteins like Thrombospondin-1, Hevin as well as the modulator of extracelular matrix expression, Angiotensinogen, were found increased in ET-CM. The analysis of interactome maps through Ingenuity Pathway Analysis demonstrated that the Amyloid beta A4 protein precursor (APP), which was found reduced in ET-CM, was previously shown to interact with ten other proteins that exhibited significant changes in the ET-CM, rendering it a suitable target for a more accurate analysis about the effects of ethanol on its biology. Taken together our results strongly suggest that early exposure to teratogens such alcohol may lead to an enduring change in astrocyte secretome, affecting mainly the expression of trophic and matricellular proteins, which in turn may lead to permanent alterations in brain function.

Project description:Glioblastoma (GBM) is the most prevalent primary malignant brain tumor in adults and remains almost invariably lethal due to its aggressive and invasive behavior. Despite many clinical trials have been put forward, the standard treatment that stands for more than a decade consists of surgical resection, followed by temozolomide (TMZ) chemotherapy and radiation treatment. However, this strategy has improved the average survival time to 14.6 months when compared to 12,1 months only with surgery and radiation1 . Therefore, due to the low prognosis, different efforts have been employed in the search for new drugs and therapeutic protocols for this malignancy. A class of drugs that has emerged as a promising candidate for GBM is Histone Deacetylase inhibitors (iHDAC). Indeed, pre-clinical studies have demonstrated the efficiency of different iHDACs as anti-tumor agents, especially when associated with other therapies, including chemotherapy and radiation2. In fact, the use of iHDACs reduced the expression of genes involved in DNA repair, formation of mitotic spindles and homologous chromosome segregation, leading to glioma apoptosis in vitro 3. In addition, it has been reported that small iHDACs molecules, such as Trichostatin (TSA), disrupts cellular signaling networks relevant to tumorigenesis, leading to an increased interest in testing iHDACs in clinical trials against different types of tumors 4 . In agreement, a high activity of HDAC has also been associated with tumor progression, corroborating the pharmacological inhibition of HDACs as an interesting anti-cancer molecular target. Previous works from our group are in line with this reasoning showing that iHDACs negatively regulate the migratory capacity of GBM in vitro, suggesting that these inhibitors may regulate the molecular signature of GBM secretome, making the tumor's microenvironment repulsive for cell migration5. In fact, the communication between tumor cells and their microenvironment may occur directly or indirectly through a variety of secreted molecules, such as growth factors, cytokines, chemokines and microRNAs6. One relevant secreted molecule described by our group is Nodal, an important morphogen involved in several cellular processes related to tumorigenesis. In this previous study, our group brings an original approach on the mechanisms that dynamically control intra and extracellular nodal availability during GBM tumorigenesis7. Thus, the heterotypic secretome that derives from the tumor cells, may be an important source of key regulators of the tumorigenic process6 . To better understand whether iHDACs can also affect the repertoire of secreted molecules, and therefore directly modulate the surrounding micro-environment displaying deleterious effects on tumor cells, we investigated the secretome of iHDAC treated GBM cells in vitro using a high throughput methodology of protein identification and quantification couple to label free mass spectrometry. Using this strategy we were able to successfully identify secreted molecules upon HDAC activity knockdown. Interestingly, we identified proteins exclusive to the iHDAC treated secretome as well as proteins found in both control and treated groups. These proteins were classified using the Matrisome classification system corroborating that the molecular signature of tumor secreted proteins, that belong to the ECM, was disrupted. In vitro experiments using 2D and 3D cell culture systems coupled to decellularized ECM analysis by confocal microscopy confirmed that HDAC activity knockdown gave rise to a disrupted pattern of ECM protein staining. Taken together our results first describe in the literature the relevance of epigenetic remodeling HDAC dependent for the molecular signature of GBM cells shedding light on putative molecular targets to approach the malignancy.

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