Development of a human in vitro blood-brain tumor barrier model of diffuse intrinsic pontine glioma to better understand the chemoresistance.

BACKGROUND: Pediatric diffuse intrinsic pontine glioma (DIPG) represents one of the most devastating and lethal brain tumors in children with a median survival of 12 months. The high mortality rate can be explained by the ineligibility of patients to surgical resection due to the diffuse growth pattern and midline localization of the tumor. While the therapeutic strategies are unfortunately palliative, the blood-brain barrier (BBB) is suspected to be responsible for the treatment inefficiency. Located at the brain capillary endothelial cells (ECs), the BBB has specific properties to tightly control and restrict the access of molecules to the brain parenchyma including chemotherapeutic compounds. However, these BBB specific properties can be modified in a pathological environment, thus modulating brain exposure to therapeutic drugs. Hence, this study aimed at developing a syngeneic human blood-brain tumor barrier model to understand how the presence of DIPG impacts the structure and function of brain capillary ECs. METHODS: A human syngeneic in vitro BBB model consisting of a triple culture of human (ECs) (differentiated from CD34+-stem cells), pericytes and astrocytes was developed. Once validated in terms of BBB phenotype, this model was adapted to develop a blood-brain tumor barrier (BBTB) model specific to pediatric DIPG by replacing the astrocytes by DIPG-007, -013 and -014 cells. The physical and metabolic properties of the BBTB ECs were analyzed and compared to the BBB ECs. The permeability of both models to chemotherapeutic compounds was evaluated. RESULTS: In line with clinical observation, the integrity of the BBTB ECs remained intact until 7 days of incubation. Both transcriptional expression and activity of efflux transporters were not strongly modified by the presence of DIPG. The permeability of ECs to the chemotherapeutic drugs temozolomide and panobinostat was not affected by the DIPG environment. CONCLUSIONS: This original human BBTB model allows a better understanding of the influence of DIPG on the BBTB ECs phenotype. Our data reveal that the chemoresistance described for DIPG does not come from the development of a "super BBB". These results, validated by the absence of modification of drug transport through the BBTB ECs, point out the importance of understanding the implication of the different protagonists in the pathology to have a chance to significantly improve treatment efficiency.

Polymer "ruthenium-cyclopentadienyl" conjugates - New emerging anti-cancer drugs.

In this work, we aimed to understand the biological activity and the mechanism of action of three polymer-'ruthenium-cyclopentadienyl' conjugates (RuPMC) and a low molecular weight parental compound (Ru1) in cancer cells. Several biological assays were performed in ovarian (A2780) and breast (MCF7, MDA-MB-231) human cancer derived cell lines as well as in A2780cis, a cisplatin resistant cancer cell line. Our results show that all compounds have high activity towards cancer cells with low IC50 values in the micromolar range. We observed that all Ru-PMC compounds are mainly found inside the cells, in contrast with the parental low molecular weight compound Ru1 that was mainly found at the membrane. All compounds induced mitochondrial alterations. PMC3 and Ru1 caused F-actin cytoskeleton morphology changes and reduced the clonogenic ability of the cells. The conjugate PMC3 induced apoptosis at low concentrations comparing to cisplatin and could overcame the platinum resistance of A2780cis cancer cells. A proteomic analysis showed that these compounds induce alterations in several cellular proteins which are related to the phenotypic disorders induced by them. Our results suggest that PMC3 is foreseen as a lead candidate to future studies and acting through a different mechanism of action than cisplatin. Here we established the potential of these Ru compounds as new metallodrugs for cancer chemotherapy.

Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms.

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.

An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies.

Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.

Comparative proteomic analysis of blood eosinophils reveals redox signaling modifications in patients with FIP1L1-PDGFRA-associated chronic eosinophilic leukemia.

The FIP1L1-PDGFRA (F/P) fusion gene, which was identified as a recurrent molecular finding in hypereosinophilic syndrome (HES), lead to a constitutively increased tyrosine kinase activity of the fusion protein. Despite data obtained in animals or cell lines models, the mechanisms underlying the predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. To define more precisely intrinsic molecular events associated with F/P gene, we performed a proteomic analysis comparing F/P+ eosinophils (F/P-Eos) and eosinophils from healthy donors (C-Eos). Using 2D-DIGE and mass spectrometry techniques, we identified 41 proteins significantly overexpressed between F/P-Eos and C-Eos. Among them, 17.8% belonged to the oxidoreductase family. We further observed a down-expression of peroxiredoxin-2 (PRX-2) and an overexpression of src-homology-2 domain containing tyrosine phosphatase (SHP-1), enzymes regulating PDGFR downstream pathways, and especially intracellular reactive oxygen species (ROS) production. This profile, confirmed in immunoblot analysis, appears specific to F/P-Eos compared to controls and patients with idiopathic HES. In this clonal disorder possibly involving a pluripotent hematopoietic stem cell, we postulate that the well documented relationships between PDGFRA downstream signals and intracellular ROS levels might influence the phenotype of this leukemia.