Mesenchymal stem cells enhance tumorigenic properties of human glioblastoma through independent cell-cell communication mechanisms

Mesenchymal stem cells (MSC) display tumor tropism and have been addressed as vehicles for delivery of anti-cancer agents. As cellular components of the tumor microenvironment, MSC also influence tumor progression. However, the contribution of MSC in brain cancer is not well understood since either oncogenic or tumor suppressor effects have been reported for these cells. Here, MSC were found capable of stimulating human Glioblastoma (GBM) cell proliferation through a paracrine effect mediated by TGFB1. Moreover, when in direct cell-cell contact with GBM cells, MSC elicited an increased proliferative and invasive tumor cell behavior under 3D conditions, as well as accelerated tumor development in nude mice, independently of paracrine TGFB1. A secretome profiling of MSC-GBM co-cultures identified 126 differentially expressed proteins and 10 proteins exclusively detected under direct cell-cell contact conditions. Most of these proteins are exosome cargos and are involved in cell motility and tissue development. These results indicate a dynamic interaction between MSC and GBM cells, favoring aggressive tumor cell traits through alternative and independent mechanisms. Overall, these findings indicate that MSC may exert pro-tumorigenic effects when in close contact with tumor cells, which must be carefully considered when employing MSC in targeted cell therapy protocols against cancer.


proteomics protein digestion
For protein digestion, 50 μg of total protein from CM co-cultures and respective controls was diluted in 50 mM ammonium bicarbonate to a final volume of 60 μL. Then, protein samples were denatured with 0.2% (wt:vol) RapiGest ™ SF Surfactant (Waters) at 80° C for 15 min, reduced with 2.5 μL of 100 mM dithiothreitol at 60° C for 30 min, alkylated with 2.5 μL of 300 mM iodoacetamide at room temperature for 30 min, and enzymatically digested at 37° C overnight with trypsin at a 1:50 (wt/wt) enzyme:protein ratio. After, hydrolyze the RapiGest with 10 µL of 5% trifluoroacetic acid (TFA) and samples were incubated at 37° C for 90 min. Centrifuged samples at 16,000 × g and 6° C for 30 min and 16 μL of the internal standard (yeast alcohol dehydrogenase at 1 pmol/μL) were added.

processing samples and analyzing protein profile by liquid chromatography coupled to tandem mass spectrometry (lC-mS/mS)
Samples were analyzed by nanoACQUITY system (Waters) which contains two binary nanoHPLC pumps. Peptides were desalted and concentrated in a Symmetry C18 column (180 μm × 20 mm, 5 μm; Waters) with 0.1% TFA (15 µL/min for 5 min), and directed to HSS C18 analytical column (75 μm × 150 mm, 1.7 μm; Waters) where they were separated by a gradient elution with a mixture of 2 % DMSO in 0.1% formic acid in water and 5% DMSO in 0.1% formic acid in acetonitrile which proportion was increased from 2 to 45% in 90 minutes at 0.4 µL/min.

protein identification and database analysis
Acquisition of four to nine technical replicates of samples were made. The tandem mass spectrometer Q-Exactive type (Thermo Scientific) was calibrated in the positive mode at the beginning of each analytical sequence. Analytical sequence started after the acquisition of human cell line extract Mino (new mantle cell lymphoma) for quality control of the number of identified proteins [1] the t(11;14. The acquired data were processed by MaxQuant programs [2] specifically designed for high-resolution and quantitative data. Protein identification was performed by Andromeda search tool using the database of the human proteome UniProtKB (Swissprot june 2015). Following criteria were applied to identification: allowed maximum of two incomplete cleavages by trypsin, fixed modification by carbamidometilation of cysteines and variable changes by N-terminal portion acetylation and methionine oxidation. Quantification was based on a modified intensity based absolute quantification labelfree strategy [3] in which P00330 was used as internal normalizer. Cross-validation of protein identification through the Trans-Proteomic Pipeline platform was performed (TPP) using the X! Tandem as search tool and applying the same parameters used in the analysis by Andromeda/MaxQuant.

Statistical analysis
Student t-test was performed and p < 0.05 were considered significant at Perseus software. The proteins identified in MaxQuant platform were considered for analysis only if they could obtain probabilities above 95% in Protein Prophet system module X!Tandem/TPP.

Functional analysis systems biology
Analyzes were performed using the Ingenuity Pathway Analysis software (IPA). The reference database used was the Ingenuity Knowledge Base (Genes) including only direct relations considering molecules and relationships in humans and experimentally observed.