Diffuse Glioma Heterogeneity and Its Therapeutic Implications.

Diffuse gliomas represent a heterogeneous group of universally lethal brain tumors characterized by minimally effective genotype-targeted therapies. Recent advances have revealed that a remarkable level of genetic, epigenetic, and environmental heterogeneity exists within each individual glioma. Together, these interconnected layers of intratumoral heterogeneity result in extreme phenotypic heterogeneity at the cellular level, providing for multiple mechanisms of therapeutic resistance and forming a highly adaptable and resilient disease. In this review, we discuss how glioma intratumoral heterogeneity and malignant cellular state plasticity drive resistance to existing therapies and look to a future in which these challenges may be overcome. SIGNIFICANCE: Glioma intratumoral heterogeneity and malignant cell state plasticity represent formidable hurdles to the development of novel targeted therapies. However, the convergence of genotypically diverse glioma cells into a limited set of epigenetically encoded transcriptional cell states may present an opportunity for a novel therapeutic strategy we call "State Selective Lethality." In this approach, cellular states (as opposed to genetic perturbations/mutations) are the subject of therapeutic targeting, and plasticity-mediated resistance is minimized through the design of cell state "trapping agents."

WNT3A-loaded exosomes enable cartilage repair.

Cartilage defects repair poorly. Recent genetic studies suggest that WNT3a may contribute to cartilage regeneration, however the dense, avascular cartilage extracellular matrix limits its penetration and signalling to chondrocytes. Extracellular vesicles actively penetrate intact cartilage. This study investigates the effect of delivering WNT3a into large cartilage defects in vivo using exosomes as a delivery vehicle. Exosomes were purified by ultracentrifugation from conditioned medium of either L-cells overexpressing WNT3a or control un-transduced L-cells, and characterized by electron microscopy, nanoparticle tracking analysis and marker profiling. WNT3a loaded on exosomes was quantified by western blotting and functionally characterized in vitro using the SUPER8TOPFlash reporter assay and other established readouts including proliferation and proteoglycan content. In vivo pathway activation was assessed using TCF/Lef:H2B-GFP reporter mice. Wnt3a loaded exosomes were injected into the knees of mice, in which large osteochondral defects were surgically generated. The degree of repair was histologically scored after 8 weeks. WNT3a was successfully loaded on exosomes and resulted in activation of WNT signalling in vitro. In vivo, recombinant WNT3a failed to activate WNT signalling in cartilage, whereas a single administration of WNT3a loaded exosomes activated canonical WNT signalling for at least one week, and eight weeks later, improved the repair of osteochondral defects. WNT3a assembled on exosomes, is efficiently delivered into cartilage and contributes to the healing of osteochondral defects.

Activating mutations in BRAF disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mice and humans.

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.

Tumor Microenvironment Is Critical for the Maintenance of Cellular States Found in Primary Glioblastomas.

Glioblastoma (GBM), an incurable tumor, remains difficult to model and more importantly to treat due to its genetic/epigenetic heterogeneity and plasticity across cellular states. The ability of current tumor models to recapitulate the cellular states found in primary tumors remains unexplored. To address this issue, we compared single-cell RNA sequencing of tumor cells from 5 patients across four patient-specific glioblastoma stem cell (GSC)-derived model types, including glioma spheres, tumor organoids, glioblastoma cerebral organoids (GLICO), and patient-derived xenografts. We find that GSCs within the GLICO model are enriched for a neural progenitor-like cell subpopulation and recapitulate the cellular states and their plasticity found in the corresponding primary parental tumors. These data demonstrate how the contribution of a neuroanatomically accurate human microenvironment is critical and sufficient for recapitulating the cellular states found in human primary GBMs, a principle that may likely apply to other tumor models. SIGNIFICANCE: It has been unclear how well different patient-derived GBM models are able to recreate the full heterogeneity of primary tumors. Here, we provide a complete transcriptomic characterization of the major model types. We show that the microenvironment is crucial for recapitulating GSC cellular states, highlighting the importance of tumor-host cell interactions.See related commentary by Luo and Weiss, p. 907.This article is highlighted in the In This Issue feature, p. 890.

Stem Cells, Self-Renewal, and Lineage Commitment in the Endocrine System.

The endocrine system coordinates a wide array of body functions mainly through secretion of hormones and their actions on target tissues. Over the last decades, a collective effort between developmental biologists, geneticists, and stem cell biologists has generated a wealth of knowledge related to the contribution of stem/progenitor cells to both organogenesis and self-renewal of endocrine organs. This review provides an up-to-date and comprehensive overview of the role of tissue stem cells in the development and self-renewal of endocrine organs. Pathways governing crucial steps in both development and stemness maintenance, and that are known to be frequently altered in a wide array of endocrine disorders, including cancer, are also described. Crucially, this plethora of information is being channeled into the development of potential new cell-based treatment modalities for endocrine-related illnesses, some of which have made it through clinical trials.

High-Resolution Patterned Cellular Constructs by Droplet-Based 3D Printing.

Bioprinting is an emerging technique for the fabrication of living tissues that allows cells to be arranged in predetermined three-dimensional (3D) architectures. However, to date, there are limited examples of bioprinted constructs containing multiple cell types patterned at high-resolution. Here we present a low-cost process that employs 3D printing of aqueous droplets containing mammalian cells to produce robust, patterned constructs in oil, which were reproducibly transferred to culture medium. Human embryonic kidney (HEK) cells and ovine mesenchymal stem cells (oMSCs) were printed at tissue-relevant densities (107 cells mL-1) and a high droplet resolution of 1 nL. High-resolution 3D geometries were printed with features of ≤200 µm; these included an arborised cell junction, a diagonal-plane junction and an osteochondral interface. The printed cells showed high viability (90% on average) and HEK cells within the printed structures were shown to proliferate under culture conditions. Significantly, a five-week tissue engineering study demonstrated that printed oMSCs could be differentiated down the chondrogenic lineage to generate cartilage-like structures containing type II collagen.