Crossing Barriers Cancer Model for Studying Monocyte Migration across Endothelial Barriers.

Monocytes originate in the bone marrow and make up 2-10% of all white blood cells, circulating in the blood to damaged tissue and disease sites, where they differentiate into macrophages or dendritic cells. In solid tumors such as glioblastoma, monocytes recruited from the blood to the tumor site differentiate into tumor-associated macrophages (TAMs), which play a key role in tumor progression and metastasis. The endothelial vessel wall plays a significant role in the migration, activation, and polarization of these monocytes.

A genome-wide in vivo CRISPR activation screen identifies BACE1 as a therapeutic vulnerability of lung cancer brain metastasis.

Brain metastasis occurs in up to 40% of patients with non-small cell lung cancer (NSCLC). Considerable genomic heterogeneity exists between the primary lung tumor and respective brain metastasis; however, the identity of the genes capable of driving brain metastasis is incompletely understood. Here, we carried out an in vivo genome-wide CRISPR activation screen to identify molecular drivers of brain metastasis from an orthotopic xenograft model derived from a patient with NSCLC.

Growth Dynamics of Brain Tumor Through the Lens of MT-Weighted MRI.

Magnetic resonance imaging (MRI) is a well-established medical imaging technique that is used to visualize internal structures of the body without the use of ionizing radiation. Instead, MRI uses radio waves and a strong magnetic field to construct detailed cross-sectional images of various parts of the body, including the brain and spine. Magnetization transfer MRI is a well-established medical imaging technique based on the underlying physics of nuclear magnetic dipole-dipole interaction between water and macromolecules in a relaxation mechanism.

Bioluminescent Imaging of Patient-Derived Brain Tumors in Live Mice.

Patient-derived tumor xenograft (PDX) models are crucial for understanding brain tumor progression and for pre-clinical evaluation of therapeutic interventions. Luciferase-based bioluminescence imaging offers a non-invasive approach to visualizing and quantifying tumor development across multiple time points. This chapter presents a methodology for employing the IVIS® (in vivo imaging) system to analyze tumor dynamics in luciferase expressing PDX brain tumor models.

An In Vivo Model of Recurrent Glioblastoma.

Glioblastoma (GBM) is a highly aggressive and prevalent adult brain tumor. Despite an intensive standard of care consisting of surgical resection followed by radiation therapy and chemotherapy with temozolomide, patients invariably succumb to the recurrent tumor and face an overall survival of less than 15 months. Recurrent GBM often differs greatly from the primary counterparts and show greater resistance to genotoxic therapies.

Using GlioVis to Explore, Analyze, and Visualize Brain Tumor Multi-Omics Datasets.

With the prevalence of large multi-omics datasets in recent years, it has become more important than ever to integrate and functionalize these large quantities of information to better answer research questions in cancer research. GlioVis is a web-based tool allowing users to access and analyze proteomic, transcriptomic, genomic, and other clinical data from primary brain cancer datasets. GlioVis provides an excellent way for researchers and scientists to explore genes of interest and analyze their own transcriptomic data in a user-friendly portal, however, collaboration with bioinformaticians and biostatisticians is recommended to validate and confirm these preliminary analyses.

Cleavage Under Targets & Release Using Nuclease (CUT&RUN) Protocols for the Study of Genetic and Epigenetic Regulation in Cancer.

Cancer cell malignancy is regulated by a variety of mechanisms, collectively termed the hallmarks of cancer. At the core of these regulative processes are genetic and epigenetic perturbations that play an essential role in neoplasmic formation. Advances in molecular techniques have enabled the in-depth research of the epigenome by means of epigenetic profiling, a process that has been dominated by the chromatin immunoprecipitation (ChIP) method.

Unmasking Stemness and Immune Cell Markers in Brain Tumors with Immunohistochemistry.

Immunohistochemistry (IHC) is a useful method to detect and visualize targeted proteins in tissues, with a high degree of specificity and clarity. Through the strategic use of chromogens and counterstains, it enables a contrast-rich and accurate evaluation of specific molecules, such as surface markers. Here, we outline a comprehensive IHC followed with counterstaining protocol for the positive selection of stem-like cells and immune cells in brain tumor tissue.

CRISPR-Cas9-Guided Genetic Manipulation of Patient-Derived Brain Tumor Cells.

CRISPR-Cas9 technology has revolutionized scientific research and has provided scientists with the ability to change DNA bases specifically and precisely at predetermined sites. The CRISPR-Cas9 knockout (KO) and activation (a) platforms developed by Hart et al. and Sanson et al.

Chimeric Antigen Receptor T Cell Production for Brain Cancers.

Chimeric Antigen Receptor (CAR) T cells are T cells that have been engineered to specifically recognize antigens of interest on target cells, may that be in cancer or other pathologies. To date, CAR T cell therapy has solely been approved for use in liquid cancers, with many groups and trial working on CAR T cells for solid tumors. Herein, we describe the outline CAR T cell production using human peripheral blood mononuclear cells (PBMCs) and third-generation packaging vectors for use in in vitro and in vivo experimental assays.

Efficient Migration Assay for Brain Tumor Stem Cells.

A defining characteristic of brain tumor stem cells (BTSCs) is their ability to migrate and invade the brain parenchyma. This invasive behavior is particularly critical in understanding how BTSCs originating from primary tumors contribute to the formation of secondary brain tumor lesions. In this chapter, we present a streamlined and unbiased method to assess BTSC migration capacity.

Proximity Ligation Assay.

The Proximity Ligation Assay (PLA) is a powerful method for visualizing protein-protein interactions with high specificity and sensitivity. By tethering DNA probes to antibodies directed against two target proteins, the PLA enables the detection of protein complexes within a 40-nm range. Upon colocalization of the target proteins, the DNA probes can anneal a connector oligonucleotide, initiating rolling circle amplification and generating a fluorescent signal detectable by microscopy.

O-Methylguanine-DNA Methyltransferase (MGMT) Promoter Methylation Analysis in Glioblastoma Patients.

The alkylating agent temozolomide is used to treat patients who have glioblastoma. However, treatment is only effective when O6-methylguanine-DNA methyltransferase is silenced in the cancer cells. The best way to assess whether or not a patient will be responsive to treatment is to determine if the promoter region of the O6-methylguanine-DNA methyltransferase gene is methylated.

Synergy Assay for Screening Small Molecule Combinations in Brain Cancer Stem Cells.

Brain tumors are among the most heterogeneous tumors characterized. This heterogeneity drives resistance to single-drug therapies, making combination treatment a more promising approach to improve treatment outcomes. To optimize drug combinations and guide treatment development in this new era of combinatorial therapy, methods for quantifying synergistic, additive or antagonistic interactions between treatments are needed.

Isolation and Culture of Human Neural Stem Cells.

A major hurdle in brain tumor therapy development is a lack of adequate controls to ensure therapeutic safety in human patients. Neural stem cells (NSCs) are healthy brain cells that are commonly used by neurobiologists and neuroscientists as controls for therapy development. Consequently, it is important that these cells are properly isolated and cultured to maintain cell integrity to efficiently uncover potential toxicities associated with developing therapies for downstream applications, including omics studies.

Isolation and Propagation of Brain Tumor Stem Cells.

Shortly after the discovery of cancer stem cells in acute myeloid leukemia, cancer stem cells in several cancer types were discovered, including brain cancer. Brain tumor stem cells (BTSCs), initially characterized by the expression of CD133, are a population of cells that can proliferate and form spheres in vitro and form tumors in vivo. Due to the belief that they are treatment-resistant and seed-recurrent diseases, they are an ideal preclinical model that can be used to study brain cancer.

Brain Tumor Stem Cells: New Perspectives.

Brain cancer remains to be one of the most formidable challenges in oncology, due to its complexity, heterogeneity, and aggressive nature. Despite advances in treatment, many patients continue to face poor prognoses, underscoring the dire need for ongoing research and innovation to address this clinical enigma. Brain tumor stem cells (BTSCs) have shown to play a significant factor in the transformation and progression of brain tumors.

Targeted Radionuclide Therapy Using a Lutetium-177 Labeled Human Anti-CD133 Antibody.

Purpose: Targeted radionuclide therapy against cancer stem cell-specific markers, such as CD133, constitutes a promising strategy to eliminate resilient cancer stem cells for improved outcomes in refractory tumors. Here, we report the synthesis and evaluation of [Lu]Lu-DOTA-RW03, a CD133-targeted radioimmunotherapy.

Procedures: A fully human, anti-CD133 antibody (RW03) was conjugated with DOTA-NHS and radiolabeled with lutetium-177 to yield [Lu]Lu-DOTA-RW03.

DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload.

We describe a series of DAP12-associated receptors that can be used to achieve multi-targeting within a small genetic payload. Empirical evaluation of scaffold/binder combinations is required to define the optimal synthetic receptor configuration. When two DAP12-associated synthetic receptors were expressed in T cells from a single vector, the surface levels of individual receptors was reduced when compared to T cells engineered with vectors that express a single receptor.

Functional profiling of murine glioma models highlights targetable immune evasion phenotypes.

Cancer-intrinsic immune evasion mechanisms and pleiotropy are a barrier to cancer immunotherapy. This is apparent in certain highly fatal cancers, including high-grade gliomas and glioblastomas (GBM). In this study, we evaluated two murine syngeneic glioma models (GL261 and CT2A) as preclinical models for human GBM using functional genetic screens, single-cell transcriptomics and machine learning approaches.

De novo GTP synthesis is a metabolic vulnerability for the interception of brain metastases.

Patients with brain metastases (BM) face a 90% mortality rate within one year of diagnosis and the current standard of care is palliative. Targeting BM-initiating cells (BMICs) is a feasible strategy to treat BM, but druggable targets are limited. Here, we apply Connectivity Map analysis to lung-, breast-, and melanoma-pre-metastatic BMIC gene expression signatures and identify inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo GTP synthesis pathway, as a target for BM.

Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells.

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. In this study, we investigated functional drivers of post-treatment recurrent GBM through integrative genomic analyses, genome-wide genetic perturbation screens in patient-derived GBM models and independent lines of validation. Specific genetic dependencies were found consistent across recurrent tumor models, accompanied by increased mutational burden and differential transcript and protein expression compared to its primary GBM predecessor.

Automatic Vehicle Fueling System using PLC Controlled Robotic Arm - A Simulation Design.

The objective of this research is to simulate an automatic fuelling system using a PLC LogixPro simulation. The system includes the "FASS" concept, which is Fast, Accurate, Safe and Simple, to allow car users to have an efficient fuel filling system. The design concept consists of three processes - identification of the vehicle, payment, and filling with the fuel.

Alkylating agents are possible inducers of glioblastoma and other brain tumors.

Epidemiological evidence of an association between exposure to chemical carcinogens and an increased risk for development of glioblastoma (GBM) is limited to weak statistical associations in cohorts of firefighters, farmers, residents exposed to air pollution, and soldiers exposed to toxic chemicals (e.g., military burn pits, oil-well fire smoke).