Pediatric low-grade gliomas represent the most common childhood brain tumor class. While often curable, some tumors fail to respond and even successful treatments can have life-long side effects. Many clinical trials are underway for pediatric low-grade gliomas. However, these trials are expensive and challenging to organize due to the heterogeneity of patients and subtypes. Advances in sequencing technologies are helping to mitigate this by revealing the molecular landscapes of mutations in pediatric low-grade glioma. Functionalizing these mutations in the form of preclinical models is the next step in both understanding the disease mechanisms as well as for testing therapeutics. However, such models are often more difficult to generate due to their less proliferative nature, and the heterogeneity of tumor microenvironments, cell(s)-of-origin, and genetic alterations. In this review, we discuss the molecular and genetic alterations and the various preclinical models generated for the different types of pediatric low-grade gliomas. We examined the different preclinical models for pediatric low-grade gliomas, summarizing the scientific advances made to the field and therapeutic implications. We also discuss the advantages and limitations of the various models. This review highlights the importance of preclinical models for pediatric low-grade gliomas while noting the challenges and future directions of these models to improve therapeutic outcomes of pediatric low-grade gliomas.

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

Purpose of review

The modeling of biological processes in vitro provides an important tool to better understand mechanisms of development and disease, allowing for the rapid testing of therapeutics. However, a critical constraint in traditional monolayer culture systems is the absence of the multicellularity, spatial organization, and overall microenvironment present in vivo. This limitation has resulted in numerous therapeutics showing efficacy in vitro, but failing in patient trials. In this review, we discuss several organoid and "organ-on-a-chip" systems with particular regard to the modeling of neurological diseases and gastrointestinal disorders.

Recent findings

Recently, the in vitro generation of multicellular organ-like structures, coined organoids, has allowed the modeling of human development, tissue architecture, and disease with human-specific pathophysiology. Additionally, microfluidic "organ-on-a-chip" technologies add another level of physiological mimicry by allowing biological mediums to be shuttled through 3D cultures.

Summary

Organoids and organ-chips are rapidly evolving in vitro platforms which hold great promise for the modeling of development and disease.

This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES elements. Additionally, we describe how to generate a neural stem cell culture from Gt(ROSA)26Sort^{m4(ACTB-tdTomato, EGFP)Luo/}J mice, and validate the MADR plasmids in vitro and in vivo by neonatal mouse brain electroporation. This protocol can be generalized to analyze any transgenic element using MADR technology. For complete details on the use and execution of this protocol, please refer to Kim et al. (2019).

Mosaic analysis by dual recombinase-mediated cassette exchange (MADR) is a technology that allows stable and locus-specific integration of transgenic elements into recipient cells carrying loxP and FRT sites. Nevertheless, most cell lines lack these recombination-specific sites. This protocol describes a method to introduce the minimum requirements into cells, leading to the generation of de novo primary MADR recipient cells or MADR "Proxy" cells. These cell lines allow the combinatorial use of a wide range of transgenic elements through MADR. For complete details on the use and execution of this protocol, please refer to Kim et al. (2019).

Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system allows for stable piggyBac transposition-mediated integration into cells, identification of cells that have been transfected using a fluorescent nuclear reporter, and robust transgene activation or suppression upon the addition of doxycycline (dox) to the cell culture or the diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. More recently, we have developed a transgenic system as an alternative to piggyBac called mosaic analysis by dual recombinase-mediated cassette exchange (MADR), as well as additional in vitro transfection techniques and in vivo dox chow applications. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning of respective genetic element of interest (GOI) into response plasmid Basic Protocol 2: In vitro nucleofection of iPSC-derived human/mouse neural progenitor cells and subsequent derivation of stable inducible cell lines Alternate Protocol: In vitro electroporation of iPSC-derived human/mouse neural progenitor cells Support Protocol: Recovery stage after in vitro transfection Basic Protocol 3: Adding doxycycline to cells to induce/reverse GOI Basic Protocol 4: Assessing gene expression in vitro by non-invasive bioluminescence imaging of luciferase activity.

RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins in vivo via proximity-based biotinylation. RPL applied to U1 identified proteins involved in both U1 canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5'-3' proximity and unexplored subcellular localizations of poly(A)⁺ RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA-protein interactions important for health and disease.

Neurosurgery as one of the most technologically demanding medical fields rapidly adapts the newest developments from multiple scientific disciplines for treating brain tumors. Despite half a century of clinical trials, survival for brain primary tumors such as glioblastoma (GBM), the most common primary brain cancer, or rare ones including primary central nervous system lymphoma (PCNSL), is dismal. Cancer therapy and research have currently shifted toward targeted approaches, and personalized therapies. The orchestration of novel and effective blood-brain barrier (BBB) drug delivery approaches, targeting of cancer cells and regulating tumor microenvironment including the immune system are the key themes of this review. As the global pandemic due to SARS-CoV-2 virus continues, neurosurgery and neuro-oncology must wrestle with the issues related to treatment-related immune dysfunction. The selection of chemotherapeutic treatments, even rare cases of hypersensitivity reactions (HSRs) that occur among immunocompromised people, and number of vaccinations they have to get are emerging as a new chapter for modern Nano neurosurgery.

Research models in cancer have greatly evolved in the last decade, with the advent of several new methods both in vitro and in vivo. While in vivo models remain the gold standard for preclinical studies, these methods present a series of disadvantages such as a high cost and long periods of time to produce results compared with in vitro models. We have previously developed a method named Mosaic Analysis by Dual Recombinase-mediated cassette exchange (MADR) that generates autochthonous gliomas in immunocompetent mice through the transgenesis of personalized driver mutations, which highly mimic the spatial and temporal tumor development of their human counterparts. Due to the control of single-copy expression of transgenes, it allows for comparing the visualization of tumor cells and non-tumor cells. Here we describe a method to generate murine-derived glioma organoids (MGOs) and cell line cultures from these murine models by physical and enzymatic methods for in vitro downstream applications. Tumor cells can be readily distinguished from non-tumor cell populations, in both organoids and monolayer cell cultures, and isolated due to the use of personalized fluorescent reporter transgenes. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D murine-derived glioma organoids Basic Protocol 2: Generation of 2D glioma monolayer cell lines.

AbstractBackgroundRecent findings have indicated the presence of a progenitor domain at the marginal zone/layer 1 of the cerebral cortex, and it has been suggested that these progenitors have neurogenic and gliogenic potential. However, their contribution to the histogenesis of the cortex remains poorly understood due to difficulties associated with genetically manipulating these unique cells in a population-specific manner.ResultsWe have adapted the electroporation technique to target pial surface cells for rapid genetic manipulation at postnatal day 2. In vivo data show that most of these cells proliferate and progressively differentiate into both neuronal and glial subtypes. Furthermore, these cells localize to the superficial layers of the optic tectum and cerebral cortex prior to migration away from the surface.ConclusionsWe provide a foundation upon which future studies can begin to elucidate the molecular controls governing neural progenitor fate, migration, differentiation, and contribution to cortical and tectal histogenesis. Furthermore, specific genetic targeting of such neural progenitor populations will likely be of future clinical interest.