T cell activation and its maintenance have been a focus of research within groups that study immunotherapy approaches for cancer treatment. Therefore, agents that regulate T cell activity are often tested in vitro and in...T cell activation and its maintenance have been a focus of research within groups that study immunotherapy approaches for cancer treatment. Therefore, agents that regulate T cell activity are often tested in vitro and in vivo. In this chapter, we describe a technique to directly answer the question of whether an external agent employed at the time of T cell activation can impact the anti-tumor efficacy of activated T cells. We have used this technique to understand the timing of administering immune checkpoint inhibitors, to understand the effects of activation agents for professional antigen presenting cells, and similarly to understand the effects of vaccine adjuvants.
The field of molecular biology has undergone tremendous advancements in recent years, with the development of powerful techniques that allow for in-depth exploration of cellular processes at the molecular level. This cha...The field of molecular biology has undergone tremendous advancements in recent years, with the development of powerful techniques that allow for in-depth exploration of cellular processes at the molecular level. This chapter, "Advanced Molecular Biology Techniques," provides a detailed protocol of the molecular techniques. We begin with CRISPR-Cas9 genome editing, a transformative tool for precise and efficient gene manipulation, enabling targeted mutations and gene knockouts in various organisms. Gene amplification via Real-Time PCR is then discussed, highlighting its ability to quantify gene expression and detect rare genetic variants with high sensitivity. Flowcytometry follows, offering a robust platform for analyzing cellular populations based on specific markers, enabling the study of immune cells, cancer diagnostics, and cell cycle analysis. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) is explored as a method for mapping protein-DNA interactions, providing insights into gene regulation and epigenetic modifications. The chapter also covers Single-cell RNA sequencing (scRNA-Seq), a groundbreaking technique for profiling gene expression at the single-cell level, allowing for the discovery of cell heterogeneity and complex biological processes. Next, we explore into proteomics through Mass Spectrometry-Based Analysis, which offers detailed proteome characterization and biomarker discovery by identifying and quantifying proteins in complex samples. Finally, Fluorescence In Situ Hybridization (FISH) is discussed as a method for visualizing the spatial localization of specific nucleic acid sequences within intact cells or tissues. Together, these advanced molecular biology techniques offer unparalleled precision and insight into the molecular mechanisms underlying health, disease, and cellular function.
Cancer is a major global health concern marked by uncontrolled cellular proliferation and genetic modifications leading to malignancy. The disease's complexity encompasses various forms of cancer, increased rates of diag...Cancer is a major global health concern marked by uncontrolled cellular proliferation and genetic modifications leading to malignancy. The disease's complexity encompasses various forms of cancer, increased rates of diagnosis and prognosis and numerous treatment modalities, including surgery, chemotherapy, and radiation, each confronting problems such as medication resistance and side effects. Solid tumors, comprising approximately 85 % of malignancies, provide significant treatment challenges due to their uneven vascular supply and interstitial pressure, resulting in inadequate medication distribution and therapeutic failure. The tumor microenvironment (TME) comprises cancer cells and diverse supportive cells such as immune cells, endothelial cells and fibroblasts, which interact to facilitate tumor growth and progression. T lymphocytes, B lymphocytes, natural killer cells, and macrophages are only a few types of immune cells that can aid or impede cancer progression, which makes treatment more complicated. In this chapter we will explore the TME in solid cancers, focusing on its role in cancer biology and therapeutics strategies. In the future, advancing therapies that more precisely target TME components will minimize treatment resistance and improve patient outcomes.
This chapter introduces to the indications, biofluids used and laboratory methods of liquid biopsy. A detailed description of preanalytical factors, extraction methods, enrichment methods, quality control, storage and an...This chapter introduces to the indications, biofluids used and laboratory methods of liquid biopsy. A detailed description of preanalytical factors, extraction methods, enrichment methods, quality control, storage and analysis of various targets of liquid biopsy such as Circulating Tumor Nucleic Acids, Circulating Tumor Cells, Extracellular Vesicles, and Tumor-educated platelets has been included.
The process of editing genes has emerged as a game-changing instrument in the field of cancer research. It has the potential to provide a whole new understanding of the biology of tumors and to facilitate the creation of...The process of editing genes has emerged as a game-changing instrument in the field of cancer research. It has the potential to provide a whole new understanding of the biology of tumors and to facilitate the creation of tailored medicines. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system are the three basic methods of gene editing techniques that are discussed in this chapter. We investigate the protocol modifications that are specific to each approach, focusing on high-prevalence tumors, and we investigate the utility, efficiency, and application issues that are associated with each technique in oncology. In addition, we describe current developments in improving these methods to successfully target oncogenes and tumor suppressor genes, with the goal of driving forward advances in precision cancer therapy.
Identification of immune checkpoint proteins, PD-1 and CTLA-4 on T cells and the FDA approval of antibodies targeting them to boost anti-cancer immunity has ushered in the era of cancer immunotherapy and motivated the id...Identification of immune checkpoint proteins, PD-1 and CTLA-4 on T cells and the FDA approval of antibodies targeting them to boost anti-cancer immunity has ushered in the era of cancer immunotherapy and motivated the identification and establishment of additional key players of the immune system as viable therapeutic targets. Three distinct approaches have been developed as means to enhance the immune response against cancer: 1) Immune checkpoint blockade therapy utilizes antagonistic or agonistic antibodies against inhibitory and stimulatory proteins respectively on key cells or cell types of the immune system, 2) Cancer vaccine therapy uses abnormal proteins/antigens expressed on cancer cells as targets for cancer vaccine development and 3) CAR-T cell therapy involves the generation of recombinant T cells that express specific receptors enabling the recognition and elimination of cancer cells in an antigen-dependent manner.
Cancer is perceived as difficult to treat due to its capacity to manifest in different forms and lack of knowledge of mechanical details. Advances in the analytical techniques relevant to boosting cancer research require...Cancer is perceived as difficult to treat due to its capacity to manifest in different forms and lack of knowledge of mechanical details. Advances in the analytical techniques relevant to boosting cancer research require an hour. With the help of cell culture techniques, several significant advances in cancer research have been made in the recent past. The main difficulty associated with cell culture techniques of cancer research is to create an in vivo tumor microenvironment cost-effectively. Here, in this chapter, we have discussed the different protocols for utilizing cell culture techniques in cancer research. The 2D, 3D, scaffold and organoid based cell culture techniques have been covered in detail. In addition, we have presented a comparative analysis, including advantages and disadvantages of each type of cell culture technique. Moreover, the assays, which can be used for assessing the quality of cancer cell lines, have been listed in detail.
Tumor cells generate a type of cell called Cancer stem cells (CSCs) having self-renewal and differentiation properties, tumor initiation, metastasis and progression, as well as therapeutic resistance. CSCs analysis and t...Tumor cells generate a type of cell called Cancer stem cells (CSCs) having self-renewal and differentiation properties, tumor initiation, metastasis and progression, as well as therapeutic resistance. CSCs analysis and targeting are efficient ways in developing more effective therapeutics. In this book we will elaborate on the recent advancement in CSCs analysis like surface marker analysis, functional analysis, molecular techniques, as well as different cancer cell analysis. And elaborate the different therapeutic targeting strategies of CSCs like targeting surface cellular markers, ATP-driven efflux transporters, key signaling cascades, and tumor microenvironments. The chapter also addresses the challenges of CSC research, including cellular heterogeneity and resistance to conventional therapies, and discusses future directions to enhance CSC targeting efficacy. Real-world case studies and clinical trials provide practical insights, underscoring the potential of CSC-focused therapies to transform cancer treatment and advance towards more personalized and effective therapeutic strategies.
Mouse models have contributed to a better understanding of cancer biology and the development of new treatments. This chapter elaborates on the various types of mouse models applied in cancer research, such as xenograft,...Mouse models have contributed to a better understanding of cancer biology and the development of new treatments. This chapter elaborates on the various types of mouse models applied in cancer research, such as xenograft, syngeneic, and humanized models, together with the state-of-the-art techniques of genetic engineering involved in their generation. We described the methodologies of tumor induction and engraftment procedures and these model applications in drug development, efficacy testing, and studies on immuno-oncology. Further, the chapter covers ethical considerations and regulatory requirements on the use of animals in research, essentially aligned with international guidelines and those in India. The chapter illustrates that mouse models will not become outdated in preclinical testing any time soon but continue to be relevant for the study of tumor biology and the tumor microenvironment besides their use for investigating genetic and molecular pathways in cancer. Emerging technologies, such as CRISPR/Cas9 and organoid integration, are also highlighted for their work in improving the accuracy and translational potential of models. These developments combined with initiatives on collaborative and open science that enable the sharing of data and resources, hold great promise for the future of in vivo cancer modeling. The mouse models will continue to be one of the prime movers in advancing cancer research and formulating individual medication strategies that lead to improved patient outcomes through their integration of classical approaches with modern technologies.
Cancer biomarker discovery is crucial for early diagnosis, prognosis, and therapeutic monitoring, and proteomics and metabolomics have emerged as powerful tools in biomarker research. Proteomics, the large-scale study of...Cancer biomarker discovery is crucial for early diagnosis, prognosis, and therapeutic monitoring, and proteomics and metabolomics have emerged as powerful tools in biomarker research. Proteomics, the large-scale study of proteins, provides insights into the complex molecular changes that occur in cancer cells, offering potential biomarkers for tumor identification and monitoring treatment responses. Through techniques such as mass spectrometry and protein microarrays, proteomic analysis can identify differential protein expression, post-translational modifications, and protein-protein interactions that characterize different cancer stages. Similarly, metabolomics, the comprehensive analysis of small molecule metabolites, enables the identification of metabolic alterations associated with cancer. Tumor cells often exhibit reprogrammed metabolic pathways to sustain growth, making metabolites valuable as biomarkers for early cancer detection and treatment stratification. Both omics approaches allow for the identification of cancer-specific signatures, uncovering potential biomarkers with clinical relevance. This chapter describes the different proteomics and metabolomics techniques which are used in cancer biomarker discovery.
The understanding of cancer mechanisms has advanced, revealing the crucial roles of oncogenes, tumor suppressor genes, and epigenetics in cancer progression, making it the second deadliest disease. Genetic changes activa...The understanding of cancer mechanisms has advanced, revealing the crucial roles of oncogenes, tumor suppressor genes, and epigenetics in cancer progression, making it the second deadliest disease. Genetic changes activating oncogenes and causing uncontrolled cell growth include the Philadelphia chromosome translocation and Ras mutations. Epigenetic alterations like histone modifications and DNA methylation can also disrupt gene regulation in cancer cells. The combination of genetic and epigenetic changes speeds up cancer spread and provides new targets for treatment. Abnormalities in chromatin structure affect gene activity, impacting cellular functions. DNA methylation patterns affect tumor suppressor genes and proto-oncogenes, closely linked to cancer development and spread. DNMT inhibitors, such as Decitabine and Azacytidine, target DNA methylation and show promise in treating certain blood disorders. Non-nucleoside inhibitors are being developed to reduce the toxicity of nucleoside analogs. Cancer treatments focus on histone modifications like acetylation and methylation, crucial for gene control. In has been demonstrated that inhibitors that targets the demethylases and histone methyltransferases stop the proliferation of cancer cells. The FDA has approved HDAC inhibitors such as Panobistat and Vorinostat to trat some types of blood cancer. Novel substances targeting HATs and HDACs, such as PU141 and C646, exhibit inhibitory effects on these enzymes, limiting cancer cell growth. Research is ongoing on natural substances with HDAC inhibitory action, such as apicidin and amamistatin. The effectiveness and safety of the epigenetic cancer treatment are being assessed in the clinical trials. Overall, the potential of epigenetic changes in cancer therapy offers hope for improved outcomes in challenging cancers.
In investigations about transcriptomics, epigenomics, and genomics, high-throughput sequencing technologies have become indispensable. Several hundred million of DNA molecules may be sequenced at once thanks to high thro...In investigations about transcriptomics, epigenomics, and genomics, high-throughput sequencing technologies have become indispensable. Several hundred million of DNA molecules may be sequenced at once thanks to high throughput sequencing (HTS) technologies, which can simultaneously analyze many DNA molecules. Traditionally, sequencing information has been clarified utilizing a low throughput technique known as Sanger sequencing. This added value makes it feasible to employ HTS to generate tremendous amounts of data, which enhances the comprehension of the transcriptome and genetic fingerprints of cells during various stages of evolution and pathology. By identifying somatic changes, morphological deviations, and repetitive changes across the human genome, techniques such as whole exome sequencing (WES) and whole genome sequencing (WGS) provide information about cancer formation as well as prospective therapies. Identifying tumor biology and discovering biomarkers rely on the examination of aberrant networks and variations in gene expression that RNA DNA sequencing, or RNA-Seq, offers. By identifying rare cell kinds and their function in carcinogenesis, the sequencing of one cell offers illumination on the wide range of cells observed across tumors. Metagenomics and chromatin immunoprecipitation sequencing (ChIP-Seq) delivers essential knowledge by discovering alterations that affect the epigenetic configuration and the microenvironment that accompanies tumors. Integrating these recent developments will allow the development of personalized treatments that use unique genetic traits to determine every cancer patient, offering more individualized treatments. The revolutionary implications of high-throughput genome sequencing for cancer research and treatment are addressed in this book chapter, particularly concerning cancer precision as well as effective treatment outcomes.
Cancer is a serious illness caused by changes in the genome that lead to many mutations. These mutations can cause harmful tumor growth that impacts individuals physically and mentally. Cancer cells grow uncontrollably,...Cancer is a serious illness caused by changes in the genome that lead to many mutations. These mutations can cause harmful tumor growth that impacts individuals physically and mentally. Cancer cells grow uncontrollably, spread to other body parts, change genetically, form new blood vessels, and evade detection by the immune system. They develop ways to avoid destruction by the immune system, making it important for researchers to understand these mechanisms to enhance immune responses against cancer. Treatments like immune checkpoint inhibitors help the immune system recognize and attack cancer cells. Cancer affects cell growth and division, with any body cell being vulnerable, and many cancer cells can invade different areas. Certain factors, including diet, exposure to microbes, and environmental influences like radiation and tobacco smoke, can increase cancer risk. About 20 % of cancers are linked to microorganisms. A poor diet and lack of exercise are also associated with cancer, and a strong immune system can help fight it. Genetic factors account for 3-10 % of cases, making family history significant. Eating antioxidant-rich foods is important for prevention. While mutations contribute to tumors, they are not the only cause of cancer. Recent advancements in therapies have greatly improved cancer survival rates. Cytotoxic drugs target cancer cells that divide quickly, and new agents have improved treatment plans. A patient's overall health and disease stage influence their treatment journey. Surgery and chemotherapy have shown success, but they come with risks. Radiation therapy aims to kill cancer cells but can also harm healthy tissue. Research on nanoparticles in radiation therapy is promising. Other treatments like bone marrow transplants and cryoablation have been explored. Immunotherapy boosts the immune system's ability to combat cancer cells. The evolutionary view of cancer considers it a group of cells that mutate and adapt. This view emphasizes the role of tumor composition and environmental factors in how cancer develops. Recognizing tumor diversity is vital for creating personalized treatment plans, as it significantly affects cancer progression and treatment resistance. The way cancer cells interact with their environment, including their relationships with microorganisms, also affects their survival and growth. Understanding ecological principles may lead to new therapies and prevention methods, improving patient outcomes and survival rates.
Parkinson disease (PD) is the second most prevalent neurodegenerative disorder globally, trailing only Alzheimer´s disease. It currently affects nearly 3 % of individuals aged 65 and above. The disease is characterized b...Parkinson disease (PD) is the second most prevalent neurodegenerative disorder globally, trailing only Alzheimer´s disease. It currently affects nearly 3 % of individuals aged 65 and above. The disease is characterized by the progressive loss of dopaminergic neurons accompanied by a chronic neuroinflammatory process, which is responsible for both motor symptoms (tremor, rigidity, bradykinesia) and non-motor symptoms (depression, dysphagia, anxiety, constipation, and anosmia). To gain a deeper comprehension of the fundamental mechanisms underlying PD and to facilitate the development of efficacious therapeutic interventions, it is imperative to utilize animal models that accurately reflect the pathological characteristics observed in humans. This chapter provides a comprehensive overview of the methodologies employed in the generation of animal models of Parkinson's disease in laboratory settings. These models, which encompass a range of approaches, serve as invaluable tools for reproducing key aspects of neurodegeneration and neuroinflammation associated with PD. By establishing reliable animal models, we can investigate the cellular and molecular pathways driving disease progression, thereby gaining insights into potential therapeutic targets. Furthermore, the chapter discusses the limitations and advantages of different model systems, emphasizing their relevance in translational research aimed at finding effective treatments for PD patients.
Human Dental Pulp Stem Cells (hDPSCs) represent a remarkable cell source for tissue engineering and regenerative medicine, offering significant potential for use in personalized medicine and autologous therapies. Decellu...Human Dental Pulp Stem Cells (hDPSCs) represent a remarkable cell source for tissue engineering and regenerative medicine, offering significant potential for use in personalized medicine and autologous therapies. Decellularized extracellular matrix (ECM)-derived biological scaffolds show excellent properties for supporting cell delivery and growth in both in vitro and in vivo applications. These scaffolds provide essential biochemical cues that regulate cellular functions and offer a more accurate representation of the in vivo environment. Porcine decellularized adipose tissue (pDAT) is a very abundant source of ECM, constituting an ideal material for biologic scaffold preparation. The integration of hDPSCs with pDAT-derived ECM enables the patient-specific generation of diverse humanized tissues and their application in personalized drug screening platforms. This chapter details a three dimensional (3D) culture methodology utilizing hDPSCs and pDAT-derived scaffolds to engineer humanized bone tissue.
Breast cancer (BC) represents a major socio-economic challenge worldwide due to its high morbidity and mortality rates. Despite various therapeutic strategies, the heterogeneity of breast cancer and the resistance of tum...Breast cancer (BC) represents a major socio-economic challenge worldwide due to its high morbidity and mortality rates. Despite various therapeutic strategies, the heterogeneity of breast cancer and the resistance of tumour cells often lead to treatment failure. Consequently, the use of animal models of BC is crucial for understanding the cellular and molecular mechanisms involved in the different stages of carcinogenesis and for screening new drugs to assess their efficacy, potential safety and side effects. The accuracy, advantages and limitations of these models are the subject of ongoing debate among researchers, as no single model is standardised for all types of BC due to their unique genetic and phenotypic characteristics and the diversity of responses to treatment. This chapter focuses on the use of xenograft animal models by directly transplanting tumour cells into the BC microenvironment. It encompasses cell and animal preparation procedures, as well as transplantation methods. This technique provides a model of stage IV triple-negative BC in immune-competent animals, characterised by a short, non-surgical induction period. This model is particularly valuable for the study of carcinoma in situ and metastasis.
Mitochondrial dysfunction is a shared hallmark of neurodegenerative disorders, including Alzheimer's disease (AD) and tauopathies among others. Pathological alterations of the microtubule-associated protein Tau can disru...Mitochondrial dysfunction is a shared hallmark of neurodegenerative disorders, including Alzheimer's disease (AD) and tauopathies among others. Pathological alterations of the microtubule-associated protein Tau can disrupt mitochondrial dynamics, transport, and function, ultimately leading to neuronal toxicity and synaptic deficits. Understanding these processes is crucial for developing therapeutic interventions. The nematode Caenorhabditis elegans serves as a powerful model to study mitochondrial morphology and Tau-induced neurotoxicity due to its well-characterized nervous system and genetic tractability. Here, we describe a robust methodology for assessing mitochondrial morphology, Tau aggregation, and neuronal integrity in a nematode model of tauopathy. By combining confocal laser scanning microscopy and motility assays, we provide a comprehensive framework for investigating mitochondrial deficits. This approach offers valuable insights into the interplay between Tau pathology and mitochondrial dysfunction, thereby advancing our understanding of neurodegenerative mechanisms and potential therapeutic targets.
Chronic restraint stress (CRS) is a widely recognized model to study stress-induced anorexia and metabolic dysregulation in mice. Acyl-coenzyme A-binding protein (ACBP) has emerged as a critical player in metabolic regul...Chronic restraint stress (CRS) is a widely recognized model to study stress-induced anorexia and metabolic dysregulation in mice. Acyl-coenzyme A-binding protein (ACBP) has emerged as a critical player in metabolic regulation, with potential implications for stress-related disorders. This study presents two complementary methodologies to artificially elevate circulating Acyl-CoA-binding protein (ACBP) levels in mice under CRS. The first approach involves the continuous delivery of recombinant ACBP via subcutaneously implanted osmotic pumps. The second approach utilizes the retention using selective hooks (RUSH) system, a chemical-genetic platform enabling controlled secretion of ACBP through a biotin-activated mechanism. These methodologies aim to counteract the metabolic and behavioral impacts of CRS, offering a framework for investigating ACBP's therapeutic potential in mitigating anorexia and restoring metabolic homeostasis. The integration of these delivery systems provides a robust tool for advancing research on stress-related disorders.
Cystic fibrosis (CF) is a genetic disorder primarily known for its severe impact on lung function, but it also significantly affects the digestive system, leading to complications such as intestinal blockages, malabsorpt...Cystic fibrosis (CF) is a genetic disorder primarily known for its severe impact on lung function, but it also significantly affects the digestive system, leading to complications such as intestinal blockages, malabsorption, inflammation, and microbial dysbiosis. The study of CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) effects on intestinal physiology is critical for developing new effective treatments. This work highlights the use of the mouse intestine as a valuable model for analyzing cellular electrophysiology and CFTR function. The method reviews the molecular mechanisms underlying intestinal absorption in CF and the role of CFTR in maintaining gut homeostasis. The transgenic mouse models mimicking CF features allow researchers to explore drug responses, and potential therapeutic interventions by studying the molecular mechanisms involved, such as inflammation, autophagy or immunity. The Ussing chamber assay is a versatile tool for evaluating ion transport, barrier function, and the effects of drugs on intestinal epithelial integrity. This assay provides insights into CFTR functionality and the influence of various compounds on intestinal absorption and permeability. This protocol underscores the importance of understanding CF's gastrointestinal implications emphasizing the potential for mouse models to advance therapeutic strategies not only for CF but also for other gastrointestinal disorders.