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.

The ST6GALNAC5 gene that encodes an α2,6-sialyltransferase involved in the biosynthesis of α-series gangliosides, was previously identified as one of the genes that mediate breast cancer metastasis to the brain. We have shown that the expression of ST6GALNAC5 in MDA-MB-231 breast cancer cells resulted in the expression of G_D1α ganglioside at the cell surface. By using a human blood-brain barrier in vitro model recently developed, consisting in CD34⁺ derived endothelial cells co-cultivated with pericytes, we show that ST6GALNAC5 expression decreased the interactions between the breast cancer cells and the human blood-brain barrier.

Around 7–17% of metastatic breast cancer patients will develop brain metastases, associated with a poor prognosis. To reach the brain parenchyma, cancer cells need to cross the highly restrictive endothelium of the Blood-Brain Barrier (BBB). As treatments for brain metastases are mostly inefficient, preventing cancer cells to reach the brain could provide a relevant and important strategy. For that purpose an in vitro approach is required to identify cellular and molecular interaction mechanisms between breast cancer cells and BBB endothelium, notably at the early steps of the interaction. However, while numerous studies are performed with in vitro models, the heterogeneity and the quality of BBB models used is a limitation to the extrapolation of the obtained results to in vivo context, showing that the choice of a model that fulfills the biological BBB characteristics is essential. Therefore, we compared pre-established and currently used in vitro models from different origins (bovine, mice, human) in order to define the most appropriate tool to study interactions between breast cancer cells and the BBB. On each model, the BBB properties and the adhesion capacities of breast cancer cell lines were evaluated. As endothelial cells represent the physical restriction site of the BBB, all the models consisted of endothelial cells from animal or human origins. Among these models, only the in vitro BBB model derived from human stem cells both displayed BBB properties and allowed measurement of meaningful different interaction capacities of the cancer cell lines. Importantly, the measured adhesion and transmigration were found to be in accordance with the cancer cell lines molecular subtypes. In addition, at a molecular level, the inhibition of ganglioside biosynthesis highlights the potential role of glycosylation in breast cancer cells adhesion capacities.

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

Background: Diffuse midline glioma (DMG) is a rare and aggressive pediatric brain tumor, with a median survival of less than 12 months. Due to its location in the pons, surgical resection is impossible, leaving radiation therapy as the only palliative treatment option. Unfortunately, radiation therapy yields minimal improvement in survival. Thus, characterization of the vascular component of the DMG microenvironment at the cellular and molecular levels following radiotherapy to improve therapeutic strategies. Methods: A human syngeneic blood-brain tumor barrier (BBTB) in vitro model, comprising endothelial cells, pericytes and DMG cells was submitted to a single dose of radiation (2 Gγ to 6 Gγ) and was characterized for its physical and metabolic properties over a period of 7 days post-exposure. The results were then compared to the effects of the same irradiation protocol on a physiologic blood-brain barrier (BBB) model. Results: Following irradiation, the endothelial permeability of the BBB ECs and the BBTB ECs was preserved for up to 7 days but associated with Claudin-5 heterogeneous distribution at the ECs borders and decrease of expression after irradiation. Nevertheless, irradiation was found to potentiate the effect of TNFα on the physical integrity of the BBB, which was less important for the BBTB. The metabolic properties of the BBB and BBTB were modulated by radiation at the transcriptional level. Interestingly, different regulations were observed in endothelial cells and pericytes. Notably, pericytes have demonstrated compensatory effects. Immunoblots confirmed the decrease of BCRP, MRP4 and MFSD2A in BBTB endothelial cells after irradiation. Despite significant reduced efficiency, P-gp/BCRP efflux pump activity remains functional in endothelial cells and pericytes following irradiation. Conclusions: Irradiation sensitizes the BBB, but to a lesser extent the BBTB, to the effects of pro-inflammatory cytokines. The observed decrease in P-gp/BCRP activity, as well as the involvement of MFSD2A, MRP4 and Claudin-5 regulation, warrant further investigations.