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

Abstract

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

Zika Virus Infection Promotes Local Inflammation, Cell Adhesion Molecule Upregulation, and Leukocyte Recruitment at the Blood-Brain Barrier

“…Coculture was allowed for 6 days, and Pe was tested via the LY protocol. The following day, human primary astrocytes were plated on poly-L-lysine-coated 12-well plates, and filters were transferred according to published protocol (35). In this set up, there is no contact between the different cell types.…”

“…Finally, we performed ZIKV infection of a triple-culture BBB model, where pericytes are cultured at the bottom of the transwell filter, coculture with hBLECs is allowed for 7 days prior to infection (MOI, 1), and human primary astrocytes are added to the wells (35) (Fig. S5e).…”

Are In Vitro Human Blood–Brain–Tumor-Barriers Suitable Replacements for In Vivo Models of Brain Permeability for Novel Therapeutics?

“…The inclusion of glioma cells (e.g., U87) increased BBTB permeability 2-3-fold compared to hCMEC/D3 monocultures [ 28 ] ( Figure 5 ). Similarly, CD34 + -ECs cultured with pericyte-glioma (DIPG-007, DIPG-013 and DIPG-014) BBTB were more permeable than EC–pericyte–astrocyte BBBs [ 33 ].…”

“…Using immortalized hBMEC/ci18 EC, HASTR/ci35 astrocyte, HBPC pericyte tri-cultures, Ito and colleagues determined a poor BBB permeability threshold of 18 × 10 −6 cm/s [ 19 ], comparable to the CNS permeability threshold of 15 × 10 −6 cm/s derived by Mantle and colleagues using hPSC-derived EC monocultures and CNS-permeable Alzheimer’s drugs [ 22 ] ( Figure 6 ). Using a BBB and BBTB model, Deligne and colleagues demonstrated comparable permeability coefficients for CD34 + -EC and astrocyte versus CD34+-EC and DIPG co-cultures, with CNS permeable TMZ more permeable than CNS impermeable panobinostat in all cultures, except for DIPG-007 [ 33 ] ( Figure 6 ). This reflects the heterogeneity of patient tumors and derived primary cell lines.…”

Tumor‐associated macrophages as treatment targets in glioma

“…Due to accumulated progression and massive angiogenesis of gliomas, the microenvironment eventually becomes hypoxic [43]. The properties of tumor cells, such as tumor progression and invasion, can be influenced by hypoxia [55] or even pushed into a more malignant type [56]. For example, under hypoxic conditions, lactic acid is prone to accumulate in the TME, leading to a low pH environment.…”