Marie-Pierre Junier

Glioblastoma (GBM) remains one of the most aggressive human cancers, driven by cellular plasticity and dynamic interactions with the neurovascular niche. Yet, further refinement of existing preclinical models is needed to improve the reproduction of the human brain–vascular interface and facilitate the study of tumor-host crosstalk and invasion. To address this limitation, we developed a human induced pluripotent stem cell (hiPSC)-derived cortico-endothelial (CO + EO) assembloid model by fusing cortical and endothelial organoids. These assembloids spontaneously form branched vascular networks enriched in tight junction proteins (CLDN5, OCLN, ZO-1), thereby recapitulating key BBB-like features and providing a physiologically relevant human platform to investigate GBM–neurovascular interactions. Using this system, we uncovered a previously unrecognized CELF2-dependent glioma stem cell (GSC) tropism. CELF2-expressing GSCs preferentially infiltrate neural regions, where they induce neuronal apoptosis and disrupt endothelial tight junction structure, supporting an aggressive phenotype. In contrast, CELF2-deficient GSCs lose neurotropism, acquire mesenchymal features, and are redirected toward vascular compartments. This endothelial affinity depends on the scaffold protein AHNAK, strongly expressed at the plasma membrane of CELF2-deficient cells and enriched at tumor-endothelial interfaces. AHNAK knockdown partially abolishes endothelial infiltration, demonstrating its role in GBM vascular tropism. Moreover, analysis of patient GBM specimens confirmed that CELF2-positive tumor cells are enriched in poorly vascularized, mitotically active regions and are largely excluded from vessel-rich zones, closely resembling the behavior observed in assembloids. Transcriptomic profiling further revealed that CELF2 promotes a neuronal progenitor-like program while repressing mesenchymal and vascular-associated gene signatures, thereby shaping tumor identity, invasive behavior, and tissue preference. Collectively, this study establishes CO + EO assembloids as an original human model of GBM plasticity at the neurovascular interface. We identify CELF2 as a master regulator of GSC tropism and AHNAK as a mediator of vascular affinity, revealing a molecular axis that governs GBM invasion and providing potential avenues for future therapeutic intervention.

An acousto-fluidic bioreactor allowing the culture of patient-derived gliobastoma tumoroids in acoustic levitation and model immune cell infiltration using the transverse acoustic radiation force in the levitation plane.

Background: Glioblastomas (GBMs) are the most aggressive and common neoplasms that affect glial cells, presenting rapid growth, invasion, and resistance to treatments. Studies have demonstrated the potentially inhibitory effect of flavonoids on glioblastoma cells’ stemness and viability. However, further research is needed to explore sensitivity and the mechanism of action in chemoresistant cells. Methods: In this study, we characterized the impact of apigenin treatment on the viability and differentiation of human GBM cells in vitro and its effects on tumorigenesis and regulation of the inflammatory response in vivo. Results: The flavonoid apigenin reduced the viability of U-251 cells, patient-derived cells TG-1 and OB-1 stem cells in a dose-dependent manner, associated with the induction of acidic vesicle organelles formation and apoptosis. Treatment with apigenin also inhibited migration and induced neural differentiation in the remaining viable cells, characterized by a decrease in the expression of the precursor marker nestin and an increase in the expression of astrocyte and neuron markers, GFAP and β-III tubulin, respectively. The xenotransplantation of apigenin-pretreated U251 cells into rat brains did not lead to tumor formation, unlike untreated cells. The surrounding area of transplanted untreated U251 cells exhibited reactive microglia and astrocytes, along with increased VEGF expression, which was absent in implant sites of apigenin-pretreated GBM cells. Moreover, in this implant area, we observed a significant decrease in the expression of mRNA for inflammatory factors IL-1β, TNF, and NOS2, and the downregulation of IL-10 and IL-4. Conclusions: These results demonstrate that apigenin inhibits the growth of tumoral cells, affecting the viability of tumor stem cells and impairing tumorigenicity, while altering the regulatory profile of immunomodulatory proteins. Therefore, this flavonoid can be considered for further studies to determine its use as an adjuvant to the treatment of human GBMs.

We describe an acoustofluidic device that allows scaffold-free structuration and culture of multi-cellular tumoroids composed of patient-derived glioblastoma cells only or in combination with non-cancerous cell. A PDMS chip of controlled height was created to allow acoustic levitation of cells using a 2 MHz transducer held on top of the chip. Cells are introduced into the chip through a dedicated inlet upstream of the resonant cavity. The specific design of the cavity together with the acoustic field allow the formation of tumoroids of cells in a precise and controlled manner within the levitation chamber. The acoustic and fluidic environment of the device was determined through experiments confronted with numerical simulations. The control of the flow within the chip was optimized to allow long-term culture of tumoroids and injection of cell culture media without disturbing the tumoroids in levitation. The tumoroids can be also structured, with sequential injections of the different cell types. Using microglia, we show that the acoustofluidic device allows the formation and culture in acoustic levitation of tumoroids mixing cancer cells with other cells populating the tumor as well as immune cell infiltration within the tumoroids. These results demonstrate the suitability of acoustofluidic levitation as an original 3D culture method adapted to the exploration of cancer growth at multiple levels.

Deciphering shared features between patients through unsupervised analyses of tumor single-cell transcriptomes is hindered by the predominant clustering of malignant cells based on the patient's tumor of origin. In contrast, cancer-associated non-malignant cells within tumors cluster according to their somatic cell type (e.g. macrophage, fibroblast, astrocyte), regardless of the individual patient. We investigated the origin of this contrasting clustering behavior using computational analyses and novel data sampling techniques across 160 tumors representing 9 distinct cancer types. This led to the unexpected disclosing of tumor-specific malignant cell types. We demonstrate that tumor-driven malignant cell clustering is independent from technical or computational biases, nor is it reducible to defined gene sets with tumor-specific expression across all examined cancers. On the opposite, we unveil redundant information dispersed across the transcriptome that encodes a unique identity shared by the malignant cells within each tumor. Additionally, we found a similar genomic encoding for the identity of normal cell types. Finally, we demonstrate that malignant cell identities are maintained across space and over time, as are the identities of normal somatic cells types. These findings suggest the establishment of a distinct type of malignant cells within each tumor, robustly and diffusively encoded across the entire transcribed genome.

Glioblastomas (GBs) are incurable brain tumors. The persistence of aggressive stem-like tumor cells after cytotoxic treatments compromises therapeutic efficacy, leading to GBM recurrence. Forcing the GBM cells to irreversibly abandon their aggressive stem-like phenotype may offer an alternative to conventional cytotoxic treatments. Here, we show that the RNA binding protein CELF2 is strongly expressed in mitotic and OLIG2-positive GBM cells, while it is downregulated in differentiated and non-mitotic cells by miR-199a-3p, exemplifying GBM intra-tumor heterogeneity. Using patient-derived cells and human GBM samples, we demonstrate that CELF2 plays a key role in maintaining the proliferative/OLIG2 cell phenotype with clonal and tumorigenic properties. Indeed, we show that CELF2 deficiency in patient-derived GSCs drastically reduced tumor growth in the brains of nude mice. We further show that CELF2 promotes TRIM28 and G9a expression, which drive a H3K9me3 epigenetic profile responsible for the silencing of the SOX3 gene. Thus, CELF2, which is positively correlated with OLIG2 and Ki67 expression in human GBM samples, is inversely correlated with SOX3 and miR-199a-3p. Accordingly, the invalidation of SOX3 in CELF2-deficient patient-derived cells rescued proliferation and OLIG2 expression. Finally, patients expressing SOX3 above the median level of expression tend to have a longer life expectancy. CELF2 is therefore a crucial target for the malignant potential of GBM and warrants attention when developing novel anticancer strategies.

Cell motility is critical for tumor malignancy. Metabolism being an obligatory step in shaping cell behavior, we looked for metabolic weaknesses shared by motile cells across the diverse genetic contexts of patients’ glioblastoma. Computational analyses of single-cell transcriptomes from thirty patients’ tumors isolated cells with high motile potential and highlighted their metabolic specificities. These cells were characterized by enhanced mitochondrial load and oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). Functional assays with patients’ tumor-derived cells and -tissue organoids, and genetic and pharmacological manipulations confirmed that the cells depend on enhanced ROS production and MPST activity for their motility. MPST action involved protection of protein cysteine residues from damaging hyperoxidation. Its knockdown translated in reduced tumor burden, and a robust increase in mice survival. Starting from cell-by-cell analyses of the patients’ tumors, our work unravels metabolic dependencies of cell malignancy maintained across heterogeneous genomic landscapes.