Microenvironment-Driven Dynamic Chromatin Changes in Glioblastoma Recapitulate Early Neural Development at Single-Cell Resolution.

The tumor microenvironment is necessary for recapitulating the intratumoral heterogeneity and cell state plasticity found in human primary glioblastoma (GBM). Conventional models do not accurately recapitulate the spectrum of GBM cellular states, hindering elucidation of the underlying transcriptional regulation of these states. Using our glioblastoma cerebral organoid model, we profiled the chromatin accessibility of 28,040 single cells in five patient-derived glioma stem cell lines. Integration of paired epigenomes and transcriptomes within the context of tumor-normal host cell interactions was used to probe the gene-regulatory networks underlying individual GBM cellular states in a way not readily possible in other in vitro models. These analyses identified the epigenetic underpinnings of GBM cellular states and characterized dynamic chromatin changes reminiscent of early neural development that underlie GBM cell state transitions. Despite large differences between tumors, a shared cellular compartment made up of neural progenitor-like cells and outer radial glia-like cells was observed. Together, these results shed light on the transcriptional regulation program in GBM and offer novel therapeutic targets across a broad range of genetically heterogenous GBMs. SIGNIFICANCE: Single-cell analyses elucidate the chromatin landscape and transcriptional regulation of glioblastoma cellular states and identify a radial glia-like population, providing potential targets to disrupt cell states and improve therapeutic efficacy.

A review of protocols for brain organoids and applications for disease modeling.

Recent breakthroughs in human stem cell technologies have enabled the generation of 3D brain organoid platforms for modeling human neurodevelopment and disease. Here, we review advances in brain organoid development, approaches for generating whole-brain or cerebral organoids and region-specific brain organoids, and their applications in disease modeling. We present a comprehensive overview of various brain organoid generation protocols, including culture steps, media, timelines, and technical considerations associated with each protocol, and highlight the advantages and disadvantages of each protocol. We also discuss the current limitations as well as increasing sophistication of brain organoid technology, and future directions for the field. These insights provide a valuable assessment of multiple commonly used brain organoid models and main considerations for investigators who are considering implementing brain organoid technologies in their laboratories.

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.

Modeling Patient-Derived Glioblastoma with Cerebral Organoids.

The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a "normal" human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor microtubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening.

Genome-wide meta-analysis and replication studies in multiple ethnicities identify novel adolescent idiopathic scoliosis susceptibility loci.

Adolescent idiopathic scoliosis (AIS) is the most common musculoskeletal disorder of childhood development. The genetic architecture of AIS is complex, and the great majority of risk factors are undiscovered. To identify new AIS susceptibility loci, we conducted the first genome-wide meta-analysis of AIS genome-wide association studies, including 7956 cases and 88 459 controls from 3 ancestral groups. Three novel loci that surpassed genome-wide significance were uncovered in intragenic regions of the CDH13 (P-value_rs4513093 = 1.7E-15), ABO (P-value_ rs687621 = 7.3E-10) and SOX6 (P-value_rs1455114 = 2.98E-08) genes. Restricting the analysis to females improved the associations at multiple loci, most notably with variants within CDH13 despite the reduction in sample size. Genome-wide gene-functional enrichment analysis identified significant perturbation of pathways involving cartilage and connective tissue development. Expression of both SOX6 and CDH13 was detected in cartilage chondrocytes and chromatin immunoprecipitation sequencing experiments in that tissue revealed multiple HeK27ac-positive peaks overlapping associated loci. Our results further define the genetic architecture of AIS and highlight the importance of vertebral cartilage development in its pathogenesis.

Short interfering RNA induced generation and translation of stable 5' mRNA cleavage intermediates.

Sequence-specific degradation of homologous mRNA is the main mechanism by which short-interfering RNAs (siRNAs) suppress gene expression. Generally, it is assumed that the mRNA fragments resulting from Ago2 cleavage are rapidly degraded, thus making the transcript translation-incompetent. However, the molecular mechanisms involved in the post-cleavage mRNA decay are not completely understood and the fate of cleavage intermediates has been poorly studied. Using specific siRNAs and short-hairpin RNAs (shRNAs) we show that the 5' and 3' mRNA cleavage fragments of human papilloma virus type 16 (HPV-16) E6/7 mRNA, over-expressed in cervical malignancies, are unevenly degraded. Intriguingly, the 5' mRNA fragment was more abundant and displayed a greater stability than the corresponding 3' mRNA fragment in RNAi-treated cells. Further analysis revealed that the 5' mRNA fragment was polysome-associated, indicating its active translation, and this was further confirmed by using tagged E7 protein to show that C-terminally truncated proteins were produced in treated cells. Overall, our findings provide new insight into the degradation of siRNA-targeted transcripts and show that RNAi can alter protein expression in cells as a result of preferential stabilization and translation of the 5' cleavage fragment. These results challenge the current model of siRNA-mediated RNAi and provide a significant step forward towards understanding non-canonical pathways of siRNA gene silencing.

2'-OMe-phosphorodithioate-modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity.

Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2'-O-Methyl (2'-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2'-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM domain containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumours following MePS2-modified siRNA treatment, leading to a synergistic anti-tumour effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types.

CD62L as a therapeutic target in chronic lymphocytic leukemia.

PURPOSE: Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the disease remains incurable with standard therapies and relapse is inevitable. A growing body of evidence indicates that alterations in the adhesion properties of neoplastic cells play a pivotal role in the development and progression of CLL. EXPERIMENTAL DESIGN: The expression of 71 cell surface molecules was examined on CLL peripheral blood mononuclear cells (PBMCs) over 3 weeks in culture. The most highly upregulated marker, CD62L, was examined further for expression on CD5(+)/CD19(+) CLL cells in vitro and in lymph node and bone marrow biopsies. The prosurvival role of CD62L was examined using a functional blocking antibody and therapeutic potential evaluated by comparison with current chemotherapy agents. RESULTS: Blocking CD62L resulted in apoptosis of CLL cells but not PBMCs from healthy donors suggesting a novel role for CD62L in CLL cell survival. The beneficial effect of coculturing CLL cells with bone marrow stromal cells or endothelial cells does not protect CLL cells from anti-CD62L-related toxicity. Moreover, combining fludarabine or mafosfamide with the anti-CD62L in vitro produced an additive effect both with and without stromal cells. CONCLUSION: This is the first reported data showing that blocking the activation and homing marker, CD62L, regulates CLL cell survival in vitro. These data also suggest that therapeutic antibodies against CD62L may provide additional clinical benefit to patients with CLL receiving current standard chemotherapy protocols.

Efficient Biodistribution and Gene Silencing in the Lung epithelium via Intravenous Liposomal Delivery of siRNA.

RNA interference (RNAi) may provide a therapeutic solution to many pulmonary epithelium diseases. However, the main barrier to the clinical use of RNAi remains the lack of efficient delivery vectors. Research has mainly concentrated on the intranasal route of delivery of short interfering RNA (siRNA) effector molecules for the treatment of respiratory diseases. However, this may be complicated in a diseased state due to the increased fluid production and tissue remodeling. Therefore, we investigated our hydration of a freeze-dried matrix (HFDM) formulated liposomes for systemic delivery to the lung epithelium. Here, we show that 45 ± 2% of epithelial murine lung cells receive siRNA delivery upon intravenous (IV) liposomal administration. Furthermore, we demonstrate that liposomal siRNA delivery resulted in targeted gene and protein knockdown throughout the lung, including lung epithelium. Taken together, this is the first description of lung epithelial delivery via cationic liposomes, and provides a proof of concept for the use of IV liposomal RNAi delivery to specifically knockdown targeted genes in the respiratory system. This approach may provide an attractive alternate therapeutic delivery strategy for the treatment of lung epithelium diseases.Molecular Therapy - Nucleic Acids (2013) 2, e96; doi:10.1038/mtna.2013.22; published online 4 June 2013.

Predicting polymorphic EST-SSRs in silico.

The public availability of large quantities of gene sequence data provides a valuable resource of the mining of Simple Sequence Repeat (SSR) molecular genetic markers for genetic analysis. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the characterization of barley EST-SSRs and the identification of putative polymorphic SSRs from EST data. Polymorphic SSRs are distinguished from monomorphic SSRs by the representation of varying motif lengths within an alignment of sequence reads. Two measures of confidence are calculated, redundancy of a polymorphism and co-segregation with accessions. The utility of this method is demonstrated through the discovery of 597 candidate polymorphic SSRs, from a total of 452 642 consensus expressed sequences. PCR amplification primers were designed for the identified SSRs. Ten primer pairs were validated for polymorphism in barley and for transferability across species. Analysis of the polymorphisms in relation to SSR motif, length, position and annotation is discussed.

RNA interference for the treatment of papillomavirus disease.

Human Papillomavirus (HPV)-induced diseases are a significant burden on our healthcare system and current therapies are not curative. Vaccination provides significant prophylactic protection but effective therapeutic treatments will still be required. RNA interference (RNAi) has great promise in providing highly specific therapies for all HPV diseases yet this promise has not been realised. Here we review the research into RNAi therapy for HPV in vitro and in vivo and examine the various targets and outcomes. We discuss the idea of using RNAi with current treatments and address delivery of RNAi, the major issue holding back clinical adoption. Finally, we present our view of a potential path to the clinic.