JoVE Neuroscience is a multidisciplinary section devoted to investigations of the structure, function, physiology, and pathophysiology of the brain and nervous system. Included methodologies range from molecular and cellular level studies to full central and peripheral neural systems. Potential treatment platforms and surgical techniques for neurological diseases and disorders are also presented in this section.

Neuroscience

JoVE Medicine serves as a connection between basic research and clinical applications. Articles may present medical procedures, case studies or clinical trial methodologies. Included studies are often modeled towards predictive, preventative, personalized, and practical approaches to treatment of disease. The goal of research methodologies in this section is to improve overall patient care and prognosis.

Medicine

JoVE Immunology and Infection includes content from a variety of fields working toward a shared initiative of improving global health. Techniques evaluating the biological response to pathogens from the molecular to organismal level are presented as well as therapeutic agents and their efficacy in treating disease.

Immunology and Infection

JoVE Genetics publishes video articles demonstrating experimental approaches to investigate gene function and regulation. This includes methods to study gene expression, mutagenesis, epigenetics, genetic determinants of development, genetic disorders, population genetics and evolution.

Genetics

JoVE Environment is dedicated to research methodologies that address environmental concerns and seek to better understand Earth's ecosystem.  Special consideration is given to experimental methodologies that assess society’s impact on the environment, suggest solutions for protecting Earth’s resources, and develop sustainable fuel sources. Moving towards an environmentally conscious planet is a collaborative effort with contributions from both life and physical scientists.

Environment

JoVE Engineering encompasses a broad range of experimental and instrumental techniques utilized in physics research. Investigations in this area strive to address and answer a broad range of scientific questions, such as device mechanisms and efficiencies, using physical tools. This approach often requires a combination of specialties, and research in this area tends to be interdisciplinary with contributions from mechanical, electrical, and chemical engineers.

Engineering

JoVE Developmental Biology publishes research methodologies in the field of developmental biology. This includes methods that are used to study biological development in vitro and in vivo at the molecular, cellular, tissue, organ and whole organism levels.

Developmental Biology

JoVE Chemistry surveys a broad scope of chemical disciplines. This collection includes fundamental and applied research that investigates molecular interactions, properties, and structure spanning in the following core divisions: analytical, organic, organometallic, inorganic, bioinorganic, and biochemistry. This section also includes the design and preparation of materials and small molecules for advanced or unique applications.

Chemistry

JoVE Cancer Research illustrates experimental approaches in biomedical research and clinical practice aimed at understanding, detecting, treating and preventing cancer. The video articles in this section demonstrate methods to study the cellular and molecular mechanisms of cancer, animal models of cancer research, development of new therapies and diagnostics, tumor imaging, chemical and radiation treatments, surgical procedures and improvements in therapy. These video articles offer researchers and clinical practitioners an efficient way to learn the technologies and experimental methods in cancer research.

Cancer Research

JoVE Biology welcomes all general biology research methodologies. Content in this section canvases all fields of cell, molecular, and organismal biology, ranging from new applications of standard techniques to novel approaches aimed at understanding the functions of life and living organisms. This diverse section includes, but is not limited to, techniques in physical biology, cellular biochemistry, genetics, physiology, systems biology and a combination of eukaryotic and prokaryotic model systems.

Biology

JoVE Bioengineering merges both physical and life sciences to understand and predict biological processes. Applying physical science tools to life science questions allow for the discovery of better technologies to measure, diagnose, and clinically treat disease.

Bioengineering

JoVE Biochemistry publishes experiments that investigate the structure, function, and interactions of biological molecules. The video articles in this section demonstrate methods to study nucleic acids, proteins, carbohydrates and lipids as well as enzyme function, protein folding, molecular interactions and biochemical reactions.

Biochemistry

JoVE Behavior documents observational and experimental techniques that seek to understand human and animal behavior. Work in this field includes analysis of genetic causes and physiological responses to internal or external stimuli as well as the implicated brain regions.

Behavior

Glioblastoma (GB) is a highly aggressive brain cancer with a median survival of under 2 years despite conventional therapy. Research efforts frequently use murine in vivo models to study cancer progression and tumor growth, as these models recapitulate critical aspects of the tumor microenvironment, immune responses, and the appearance of classical GB pathology. Here, we describe a detailed protocol for performing stereotaxic injections to establish GB tumor models in immunocompetent C57BL/6 mice. Stereotaxic surgery enables rapid and precise delivery of GB cells grown as 2D cultures or spheroids into the brain using anatomical landmarks without the need for image guidance. Specifically, GL261 murine cell lines are injected into the striatum, allowing subsequent assessment of behavior and physiology. On day 21 post surgery, animals are euthanized following perfusion and their brains extracted and fixed. Hematoxylin-eosin staining on brain slices enables measurement of tumor area and volume, while immunohistochemistry enables imaging of tumor proliferation markers. This paper provides detailed protocols for a reliable approach to generate in vivo GB models for studying tumor growth and cancer progression. Additionally, this method is adaptable for other GB cell lines, transgenic mice, or patient-derived xenografts broadening its applicability for GB research.

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In Vivo Glioblastoma Tumor Modeling via Stereotaxic Injection in Mice for Tumor Progression Studies

Glioblastoma (GB) is a highly aggressive brain cancer with a median survival of under 2 years despite conventional therapy. Research efforts frequently ...

A compelling body of research has confirmed the critical role of the tumor microenvironment (TME) in the growth and aggressiveness of breast cancer (BC). Cancer-associated fibroblasts (CAF), the predominant cell component of the TME, significantly influences its behavior through complex crosstalk with cancer and other stromal cells in response to tumor-derived activating signals. Murine models are extensively used to investigate the mechanisms underlying CAF's role in BC progression, as they allow for the evaluation of the endogenous TME and the assessment of immune system involvement using syngeneic xenograft studies. Here, we present a set of methods for culturing BC-derived CAFs, manipulating gene expression, and evaluating their pro-tumorigenic functions using both in vitro and in vivo approaches. We also describe protocols for CAF gene expression profiling and transcriptomic analysis, alongside a computational procedure for inferring transcriptional signatures in primary tumors using publicly available bulk RNA-seq data. Additionally, we introduce a user-friendly, web-based tool for analyzing the transcriptomes of stromal and epithelial components from laser capture micro-dissected BC tumors. Ultimately, these methods can provide valuable insights into the molecular mechanisms driving CAF-tumor interactions and hold potential for identifying novel therapeutic targets.

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Cancer-Associated Fibroblasts from Mouse Mammary Tumors as Tools for Molecular and Computational Studies

A compelling body of research has confirmed the critical role of the tumor microenvironment (TME) in the growth and aggressiveness of breast cancer (BC). ...

Colorectal cancer (CRC) is the third most common cancer worldwide. Tumor-infiltrating lymphocytes (TILs) have been identified as an important prognostic marker in CRC. The therapeutic application of TILs has already shown promising results in melanoma and cervical cancer. However, their use in CRC therapy remains in an exploratory phase. A suitable in vitro model to evaluate TIL efficacy is currently unavailable, hindering further advancements in this field. Patient-derived organoid (PDO) models, which closely retain the characteristics of the original tumor tissue and reflect inter-patient heterogeneity, provide an excellent platform for studying the interaction between CRC and TILs. In this study, a method is described to establish a patient-derived CRC organoid model from freshly resected tumor tissue, followed by isolation and expansion of TILs. This system allows co-culture of CRC organoids and TILs, enabling the assessment of TIL-mediated cytotoxicity and immune responses. By analyzing TIL killing efficacy on organoids, the potential outcomes of TIL-based immunotherapy for personalized CRC treatment can be predicted. Moreover, further engineering of TILs may enhance their anti-tumor efficacy, offering a promising strategy for developing more effective cellular therapies. This PDO-TIL co-culture model provides a powerful tool for preclinical evaluation of TIL therapies and personalized treatment strategies in CRC.

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Establishment of a Co-culture System of Patient-Derived Colorectal Tumor Organoids and Tumor-Infiltrating Lymphocytes (TILs)

Colorectal cancer (CRC) is the third most common cancer worldwide. Tumor-infiltrating lymphocytes (TILs) have been identified as an important prognostic ...

As vesicles secreted by cells, exosomes enclose and transfer various nucleic acids, proteins, and lipids, and participate in communication between different cells. Exosomes in the tumor microenvironment play an important role in the biological characteristics of tumor cells and can strongly influence the states of other cells. Therefore, to further study the specific functions of exosomes in the development of tumors, it is undoubtedly necessary to extract and isolate exosomes from tumor tissues. Direct extraction of exosomes from tumor tissue reduces the need for in vitro cell culture, and exosomes from tumor tissue may be more similar to those in the natural state. Therefore, maintaining the natural state of exosomes in tumor tissue is the key to the extraction process. Enzymatic digestion is often combined with ultracentrifugation to extract exosomes from tumor tissues. Here, we provide a novel method to extract exosomes from tumor tissue. We used a serum-free medium to incubate small tumor tissue pieces on a shaking table, repeatedly collected and extracted exosomes naturally released from tumor tissue, and identified the extracted exosome structure, concentration, and characteristics, providing a new method for researchers studying the function of exosomes in tumor development.

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Collection of Naturally Released Exosomes from Tumor Tissue

As vesicles secreted by cells, exosomes enclose and transfer various nucleic acids, proteins, and lipids, and participate in communication between ...

Recent studies demonstrate that small extracellular vesicles (sEVs), which are found in all biofluids, play critical roles in intercellular communication by channeling proteins, DNA, and RNAs. MicroRNAs (miRNAs) that are packaged in sEVs have emerged as critical deliverable regulators in recipient cells. Since sEVs secreted by normal and diseased cells carry different miRNA cargos, recent sEV-miRNA profiling studies suggest that they may help identify novel circulating biomarkers. However, cell/disease-specific sEVs circulating in diverse biofluids, once isolated, provide low miRNA quantities, which are generally difficult to quantify using conventional spectrometric methodologies. Small non-coding RNA Next Generation Sequencing (NGS), which allows for the amplification of cloned miRNA sequences, offers a valuable opportunity to evaluate the miRNA cargos of sEVs. Unfortunately, commercial cDNA library preparation procedures often require RNA inputs well above the unquantifiable amounts available from isolated sEVs.
Thus, considering the robustness and multiplexing capabilities of our existing cDNA library preparation procedure (i.e., initially optimized for the analysis of low-input, highly degraded, formalin-fixed paraffin-embedded (FFPE) RNA), we sought to evaluate its applicability for the analysis of sEV miRNAs. Importantly, taking into account the recent technical clustering improvements of sequencing chips, we sought to adapt our transcript barcoding approach within a paired-end, dual index-compatible cDNA library preparation workflow to enhance our sequencing and multiplexing capabilities. Using RNA extracted from 8.4 &#215; 109 sEVs in 16 replicates, and from decreasing amounts of sEVs from 1010 sEVs to as low as 2.5 &#215; 107 sEVs, we evaluated the reproducibility and sensitivity of this methodology. The data demonstrate that the 16 3&#39; adenylated DNA barcodes allow for highly reproducible and sensitive detection of sEV-miRNA profiles across repeats using as low as 3.15 pg of total small non-coding RNAs or 1.35 pg of miRNAs.

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Ultrasensitive cDNA Library Preparation for Next-generation Sequencing of MicroRNAs from Small Extracellular Vesicles

Recent studies demonstrate that small extracellular vesicles (sEVs), which are found in all biofluids, play critical roles in intercellular communication ...

Organoids have emerged as a pivotal tool for advancing the understanding of tumorigenesis and cancer therapy. By generating human organoid models representing different tumor stages and performing histological analyses, it is possible to obtain a deeper understanding of the alterations in cellular morphology, spatial architecture, and the expression of key molecular markers as the tumor progresses. This study presents a comprehensive protocol for the establishment and culture of esophageal squamous cell organoids. Additionally, the protocol outlines methods for assessing the expression patterns and spatial organization of critical molecules within the organoids, utilizing techniques such as fixation, embedding, and staining. Through this protocol, significant changes were identified in the spatial structure of esophageal squamous epithelial cells and in the expression of various tumor biomarkers during tumorigenesis. The protocol facilitates the construction and histological analysis of organoids, enabling researchers to investigate the spatial architecture and molecular alterations of epithelial cells across different stages of tumorigenesis and therapeutic intervention.

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Establishment and Histological Analysis of Esophageal Organoids Modeling the Progression from Normal to Cancerous Tissues

Organoids have emerged as a pivotal tool for advancing the understanding of tumorigenesis and cancer therapy. By generating human organoid models ...

The CRISPR-Cas9 system has been harnessed and repurposed into a powerful genome editing tool. By leveraging this technology, researchers can precisely cut, paste, and even rewrite DNA sequences within living cells. Nevertheless, the application of CRISPR screen technology goes far beyond mere experimentation. It serves as a pivotal tool in the fight against genetic diseases, systematically dissecting complex genetic landscapes, empowering researchers to unravel the molecular mechanisms underlying biological phenomena, and enabling scientists to identify and target the root causes of illnesses such as cancer, cystic fibrosis, and sickle cell anemia. Among all, cancer poses a formidable challenge for medicine, spurring eradication efforts. Radiotherapy, as a traditional treatment, yields results but has limitations. It eradicates cancer cells but also damages healthy tissues, causing adverse effects that reduce quality of life. Additionally, not all cancer cells respond to radiotherapy, and some may develop resistance, worsening the condition. To address this, a comprehensive whole-genome CRISPR screen technology is introduced, as it enables the efficient identification of radiosensitive and radioresistant genes, thereby advancing the field of cancer research and treatment. A genome-wide CRISPR screen was conducted in lung adenocarcinoma cells exposed to irradiation following the described protocol, through which both radioresistance- and radiosensitivity-associated genes were identified.

genome-wide-crispr-screen-for-unveiling-radiosensitive-radioresistant

Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes

The CRISPR-Cas9 system has been harnessed and repurposed into a powerful genome editing tool. By leveraging this technology, researchers can precisely ...

Leptomeningeal metastasis (LM), the spread of cancer cells into the cerebrospinal fluid (CSF)-filled leptomeninges, is a rare yet devastating complication of advanced solid tumors. Patients with LM often have a poor prognosis, with survival measured in weeks to months. Developing in vivo models that accurately replicate the complexities of LM is essential for understanding its cellular and pathological mechanisms and evaluating potential therapies. Murine LM models are typically created through intra-cardiac, carotid artery, or cisterna magna injection of tumor cells. However, intra-cardiac or carotid injections often result in substantial extracranial and brain tumor burden, complicating bioluminescent imaging and leading to mortality unrelated to LM. Meanwhile, conventional cisterna magna injection requires invasive procedures, such as skin incision and muscle dissection, making it both traumatic and resource intensive. Here, we describe a minimally invasive procedure for tumor cell injection into the leptomeningeal space through the cisterna magna without the need for a skin incision. This approach reduces extracranial tumor formation, minimizes surgical trauma, and shortens the time and postoperative care required compared to other surgical methods. Importantly, it consistently induces LM with minimal brain parenchyma infiltration, as confirmed by two-photon microscopy and histological analysis. This streamlined approach offers an efficient and reliable model for studying LM in preclinical research.

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Minimally Invasive Cisterna Magna Injection Model for Leptomeningeal Metastasis Studies in Mice

Leptomeningeal metastasis (LM), the spread of cancer cells into the cerebrospinal fluid (CSF)-filled leptomeninges, is a rare yet devastating complication ...

Prostate cancer remains one of the most prevalent malignancies affecting men worldwide, making early detection and advancements in precision medicine crucial for effective intervention and treatment. A standardized protocol is presented for utilizing stimulated Raman histology (SRH) with integrated artificial intelligence (AI) in prostate cancer detection, offering significant advancements over conventional histopathological methods. SRH provides these advancements by enhancing efficiency through near-real-time, label-free imaging of fresh, unstained tissues, thereby eliminating the delays associated with traditional biopsy analysis.
By using stimulated Raman scattering (SRS) microscopy to detect the specific vibrational frequencies of CH2 bonds associated with lipids and CH3 bonds linked to proteins and DNA, cancerous and benign tissues in prostate biopsies can be differentiated. The AI model further enhances diagnostic precision, achieving 98.6% accuracy in identifying prostate cancer. The protocol outlines essential steps for sample preparation, imaging, and data analysis, facilitating improved biobanking processes and enabling downstream applications, such as transcriptomics and xenograft studies. This approach accelerates the diagnostic workflow and shows promise for intraoperative applications, potentially aiding surgeons in identifying positive margins intraoperatively. Additionally, the ability to re-scan and adjust cancer-to-tissue ratios allows for a more tailored analysis of biopsy samples, enhancing tumor detection in unprocessed tissues. Further research and validation are necessary for the widespread adoption of SRH in clinical practice.

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Detection and Isolation of Cancer in Prostate Biopsies Using Stimulated Raman Histology and Artificial Intelligence

Prostate cancer remains one of the most prevalent malignancies affecting men worldwide, making early detection and advancements in precision medicine ...

Laparoscopic anatomical liver resection is a standard treatment for liver cancer. Segmental resection of S4/5/7/8 is complex and lacks standardized procedures, leading to common complications. Innovative techniques are essential for enhancing safety and outcomes. A 45-year-old male with a history of hepatitis B, Child-Pugh Class A liver function, performance status (PS) score 0, and alpha-fetoprotein (AFP) level of 198.3 ng/mL was diagnosed with a 4 cm &#215; 5 cm &#215; 5 cm mass in S4/7/8, indicating primary hepatocellular carcinoma (HCC), closely associated with the middle and right hepatic veins (BCLC A). The 15-min retention rate of indocyanine green (ICG) was 7.8%. The standard liver volume (SLV) was 1073 mL, and the actual liver volume was 1345 mL. We performed laparoscopic resection of segments S4/5/8 and partial S7, resecting the middle hepatic vein (MHV) while preserving the right hepatic vein (RHV) because MHV was so closed with the tumor. The future liver remnant (FLR) was 590 mL, with an FLR/SLV ratio of 55%. The surgical procedure utilized Takasaki's approach to block the right anterior hepatic pedicle and fluorescence staining to identify the transection line. The operation lasted 205 min with an estimated blood loss of 150 mL. The patient experienced no postoperative complications and was discharged on the sixth day. Histopathology confirmed hepatocellular carcinoma with clear resection margins. Takasaki's approach, combined with ICG fluorescence navigation, significantly improves laparoscopic anatomical hepatectomy. This technique enhances visualization, reduces complications, and offers a new standard for complex liver resections.

laparoscopic-anatomical-hepatectomy-using-takasaki-s-approach

Laparoscopic Anatomical Hepatectomy Using Takasaki's Approach and Indocyanine Green Fluorescence Navigation

Laparoscopic anatomical liver resection is a standard treatment for liver cancer. Segmental resection of S4/5/7/8 is complex and lacks standardized ...

Journal - cancer research