Breast cancer brain metastases : in vitro study of mechanisms responsible for the crossing of the Blood-Brain Barrier

Dans le monde, une femme sur neuf sera atteinte d’un cancer du sein et environ un tiers développeront alors des métastases, principalement au niveau des poumons, des os et du cerveau. Dans ce dernier cas, le pronostic de survie des patientes est extrêmement faible, et ce, malgré le développement constant de nouvelles molécules anticancéreuses. En effet, la majorité de ces composés sont inefficaces du fait de leur incapacité à franchir la barrière hémato-encéphalique (BHE) pour aller cibler les métastases cérébrales. Ainsi, bloquer le passage des cellules tumorales vers l’endothélium cérébral peut être envisagé comme l’une des stratégies thérapeutiques de réduction de la formation des métastases cérébrales. Néanmoins, cette approche implique l’identification des mécanismes moléculaires mis en jeu lors des interactions entre les cellules tumorales et les cellules endothéliales de la BHE. C’est dans ce contexte que s’inscrivent ces travaux de doctorat en démontrant que la pertinence des résultats obtenus à partir d’études in vitro est largement dépendante de la qualité du modèle de BHE utilisé. L’utilisation de différents modèles in vitro montre en effet que seul le modèle généré à partir de cellules souches humaines fournit des résultats conformes aux données cliniques quant aux capacités d’interactions des cellules tumorales mammaires avec la BHE en fonction de leur agressivité relative. Ce modèle permet ainsi l’étude spécifique des mécanismes responsables des interactions entre les cellules tumorales et les cellules de la BHE lors de la formation des métastases cérébrales chez l’homme.Worldwide, one in nine women will suffer from breast cancer and about a third of them will develop distant metastases, mainly in the lung, the bones and the brain. Brain metastases are associated with poor prognosis and reduced overall survival because most of the available anticancer agents proved to be ineffective because of their failure to cross the Blood-Brain Barrier (BBB) to target brain metastases. Hence, prevent cancer cells to cross the BBB can be considered as one of therapeutic strategies to reduce brain metastases development. However, this includes the prior identification of still unknown molecular mechanisms involved during interactions between cancer cells and BBB endothelial cells. It is in this context that this thesis work has been drawn up. It shows that the relevance of results obtained from in vitro studies heavily depends on the quality of the chosen BBB model. The use of several in vitro models demonstrates that only the one generated from human stem cells provides results in accordance with clinical data. This model also enables study of specific mechanisms responsible for interactions between cancer cells and BBB endothelial cells during brain metastases formation

Adapting coculture in vitro models of the blood–brain barrier for use in cancer research: maintaining an appropriate endothelial monolayer for the assessment of transendothelial migration

International audienceAlthough brain metastases are the most common brain tumors in adults, there are few treatment options in this setting. To colonize the brain, circulating tumor cells must cross the blood-brain barrier (BBB), which is situated within specialized, restrictive microvascular endothelium. Understanding how cancer cells manage to transmigrate through the BBB might enable this process to be prevented. In vitro models are dedicated tools for characterizing the cellular and molecular mechanisms that underlie transendothelial migration process, as long as they accurately mimic the brain endothelium's in vivo characteristics. The objective of the present study was to adapt an existing in vitro model of the human BBB for use in studying cancer cell transmigration. The model is based on the coculture of endothelial cells (ECs, derived from cord blood hematopoietic stem cells) and brain pericytes. To allow the migration of cancer cells into the lower compartment, our model had to be transposed onto inserts with a larger pore size. However, we encountered a problem when culturing ECs on large (3-μm)-pore inserts: the cells crossed the membrane and formed a non-physiological second layer on the lower face of the insert. Using 3-μm-pore inserts (in a 12-well plate format), we report here on a method that enables the maintenance of a single monolayer of ECs on the insert's upper face only. Under these chosen conditions, the ECs exhibited typical BBB properties found in the original model (including restricted paracellular permeability and the expression of continuous tight junctions). This modified in vitro model of the human BBB enabled us to investigate the migratory potential of the MDA-MB-231 cell line (derived from highly metastatic human breast cancer cells). Last, the results obtained were compared with the rate of transmigration through endothelia with no BBB features

Accumulation of GD1α Ganglioside in MDA-MB-231 Breast Cancer Cells Expressing ST6GalNAc V

International audienceα-Series gangliosides define a particular sub-class of glycosphingolipids containing sialic acid α2,6-linked to GalNAc residue that was isolated as a minor compound from the brain. The sialyltransferase ST6GalNAc V was cloned from mouse brain and showed α2,6-sialyltransferase activity almost exclusively for GM1b, to form GD1α and is considered as the main enzyme involved in the biosynthesis of α-series gangliosides. Recently, ST6GALNAC5 was identified as one of the genes over-expressed in breast cancer cell populations selected for their ability to produce brain metastasis. However, the capacity of human breast cancer cells to produce α-series gangliosides has never been clearly demonstrated. Here, we show by stable transfection and MS-MS analysis of total glycosphingolipids that ST6GALNAC5 expressing MDA-MB-231 breast cancer cells accumulate GD1α ganglioside (IV3Neu5Ac1, III6Neu5Ac1Gg4-Cer)

Selection of a Relevant In Vitro Blood-Brain Barrier Model to Investigate Pro-Metastatic Features of Human Breast Cancer Cell Lines.

International audienceAround 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

ST6GALNAC5 Expression Decreases the Interactions between Breast Cancer Cells and the Human Blood-Brain Barrier.

International audienceThe 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 GD1α 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

BBB properties and cancer cells adhesion assay on HUVECs <i>in vitro</i> model.

<p>(A) Integrity measurement. The HUVECs model presents a discontinuous staining of tight junction proteins ZO-1 (left panel) and Claudin-5 (right panel) associated with a high Pe^LY of 2.35 ± 0.19 x 10⁻³ cm/min. Interruptions of the staining are indicated by white arrows. Nuclei are stained with Hoechst, bar = 50 μm. (B) Quantification of cancer cells adhesion. The number of adherent MDA-MB-231 was set up to 100% and equal to 1748. HUVECs: Human Umbilical vein endothelial cells; Pe^LY: Lucifer Yellow Endothelial Permeability; ZO-1: Zonula-Occludens-1. The results are mean of triplicate and representative of three independent experiments. N.S.: Non significant.</p

BBB properties and cancer cells adhesion assay on animal <i>in vitro</i> BBB models.

<p>BBB Integrity measurement. The bovine (A) and the murine (C) models present a continuous staining of tight junction proteins ZO-1 (left panel) and Claudin-5 (right panel) associated with a Pe^LY of respectively 0.39 ± 0.07 x 10⁻³ cm/min and 0.36 ± 0.19 x 10⁻³ cm/min. Nuclei are stained with Hoechst (blue), bar = 50 μm. Quantification of cancer cells adhesion. The number of adherent MDA-MB-231 was set up to 100% and equal to 286 for the bovine model (B) and 442 the murine model (D); Pe^LY: Lucifer Yellow Endothelial Permeability; ZO-1: Zonula-Occludens-1, NS: Non Significant. The results are mean of triplicate and representative of two independent experiments. ***p<0.001.</p

Comparative results obtained for BBB characteristics and cancer cells adhesion.

<p>Comparative results obtained for BBB characteristics and cancer cells adhesion.</p

Adhesion analysis of breast cancer cell lines after glycosphingolipid biosynthesis inhibition.

<p>(A) Visualization of glycosphingolipid synthesis inhibition. In control condition, MDA-MB-231 expressed the glycosphingolipid G_M1 (green) at the cell surface (left panel). After 10 μM PPMP treatment during 5 days, G_M1 expression at the cell surface was significantly reduced (right panel). Nuclei are stained with Hoechst, bar = 50 μm. (B) Quantification of MDA-MB-231 cells adhesion after PPMP treatment. The number of adherent non-treated MDA-MB-231 was set up to 100%. (C) Quantification of MCF-7 cells adhesion after PPMP treatment. The number of adherent non-treated MCF-7 was set up to 100%. The results are mean of triplicate and representative of three independent experiments. ***p<0.001.</p