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1.
Methods Mol Biol ; 2174: 3-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813240

RESUMO

The inherent or developed resistance of many cancer cells to chemotherapy and irradiation is actually the main challenge to overcome in cancer treatment. It is well known that cancer cells are characterized by several hallmarks, and it seems that the ability to evolve ways to evade stressful conditions and killing therapies must be consider another typical characteristic displayed by all malignant cells. This overview aims to provide a concise description of the main mechanisms involved in the promotion of resistance to anticancer therapy and to describe the most frequent challenges faced in the war against cancer therapy resistance.

2.
Methods Mol Biol ; 2174: 13-17, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813241

RESUMO

Prostate cancer is one of the main causes of cancer and the sixth cause of death among men worldwide. One of the major challenges in prostate cancer research is cell heterogeneity defined as the different genomic and phenotypic characteristics in each individual cell making more difficult to assess the proper prostate cancer diagnosis and therapy. Tumor 3D spatial arrangement allow a strong interaction between the different cellular lineages and components which modulate cell proliferation, differentiation, and morphology. Prostate cancer spheroids are a cellular model which is capable to mimic the mechanical tensions of tumor tissue, providing a more representative pathophysiological model than the use of conventional 2D culture. Here, we describe a protocol to develop a 3D model of spheroids using prostate cancer cell lines (LNCaP, PC3, VCaP) which can be used to improve research considering tumoral heterogeneity role in cancer development, prognosis, and therapy.

3.
Methods Mol Biol ; 2174: 19-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813242

RESUMO

Glioblastomas (GBM) are the most frequent and aggressive brain tumors due to their recurrence and resistance to current therapies. These characteristics are associated with the presence of glioma stem cells (GSCs), mainly identified by the detection of the membrane antigens CD133 and CD15. The main source of GSCs has been biopsies of tumors. However, alternatives are sought from cell lines because more homogeneous populations can be obtained with high yields. This chapter describes a method for the enrichment and characterization of GSCs from cell lines derived from human GBM by selective culture with serum-free neural stem cell medium and growth factors. The technique offers alternatives for the enrichment and characterization of GSCs, that could contribute to a better understanding of the biology of GBMs.

4.
Methods Mol Biol ; 2174: 31-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813243

RESUMO

Molecular docking is a useful and powerful computational method for the identification of potential interactions between small molecules and pharmacological targets. In reverse docking, the ability of one or a few compounds to bind a large dataset of proteins is evaluated in silico. This strategy is useful for identifying molecular targets of orphan bioactive compounds, proposing new molecular mechanisms, finding alternative indications of drugs, or predicting drug toxicity. Herein, we describe a detailed reverse docking protocol for the identification of potential targets for 4-hydroxycoumarin (4-HC). Our results showed that RAC1 is a target of 4-HC, which partially explains the biological activities of 4-HC on cancer cells. The strategy reported here can be easily applied to other compounds and protein datasets.

5.
Methods Mol Biol ; 2174: 45-69, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813244

RESUMO

Colon cancer is a highly anabolic entity with upregulation of glycolysis, glutaminolysis, and de novo synthesis of fatty acids, which also induces a hypercatabolic state in the patient. The blockade of either cancer anabolism or host catabolism has been previously proven to be a successful anticancer experimental treatment. However, it is still unclear whether the simultaneous blockade of both metabolic counterparts can limit malignant survival and the energetic consequences of such an approach. In this chapter, by using the CT26.WT murine colon adenocarcinoma cell line as a model of study, we provide a method to simultaneously perform a pharmacological blockade of tumor anabolism and host catabolism, as a feasible therapeutic approach to treat cancer, and to limit its energetic supply.

6.
Methods Mol Biol ; 2174: 73-88, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813245

RESUMO

In the present work we describe a novel system for the identification of microRNAs (miRNAs) in fluids. The method is based on combined novel 3D microarray technology using silk as scaffold and total internal reflection fluorescence microscopy (TIRFM), which allows for the rapid identification of miRNAs using a portable device.

7.
Methods Mol Biol ; 2174: 89-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813246

RESUMO

With the advances in sequencing technology and transcriptome analysis, it is estimated that up to 75% of the human genome is transcribed into RNAs. This finding prompted intensive investigations on the biological functions of noncoding RNAs and led to very exciting discoveries of microRNAs as important players in disease pathogenesis and therapeutic applications. Research on long noncoding RNAs (lncRNAs) is in its infancy, yet a broad spectrum of biological regulations has been attributed to lncRNAs. Here, we provide a collection of detailed experimental protocols for lncRNA studies, including lncRNA immunoprecipitation, lncRNA pull-down, lncRNA northern blot analysis, lncRNA in situ hybridization, and lncRNA knockdown. We hope that the information included in this chapter can speed up research on lncRNAs biology and eventually lead to the development of clinical applications with lncRNA as novel prognostic markers and therapeutic targets.

8.
Methods Mol Biol ; 2174: 119-131, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813247

RESUMO

Sequencing analysis finds many applications in various fields of biology from comparative genomics to clinical research. Recent studies, using high-throughput sequencing method, has generated terabytes of data. It is challenging to interpret and draw a meaningful conclusion without the proper understanding of various steps involved in the analysis of such data. This chapter deals with the pipeline to be followed to process the raw RNA sequencing (RNA-Seq) reads, align, assemble, and quantify them in order to draw significant clinical conclusions from them.

9.
Methods Mol Biol ; 2174: 135-141, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813248

RESUMO

Head and neck squamous cell carcinoma (HNSCC) is a deadly disease which in many cases is managed with a combination of radiation and chemotherapy. Unfortunately, the ability to monitor treatment response in real time is limited. Thus, truly individualized therapy remains an unrealized goal. We have previously investigated the possibility of combining advanced imaging using magnetic resonance imaging (MRI) combined with the analysis of circulating tumor cells to classify response in HNSCC as part of a prospective trial (PREDICT-HN). An adaption of the methodology from that trial is described herein in hopes of allowing for recapitulation and further development of this exciting methodology.

10.
Methods Mol Biol ; 2174: 143-170, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813249

RESUMO

Extracellular vesicles (EVs) produced by cancer cells function as a unique form of intercellular communication that can promote cell growth and survival, help shape the tumor microenvironment, and increase invasive and metastatic activity. There are two major classes of EVs, microvesicles (MVs) and exosomes, and they differ in how they are formed. MVs are generated by the outward budding and fission of the plasma membrane. On the other hand, exosomes are derived as multivesicular bodies (MVBs) fuse with the plasma membrane and release their contents. What makes EVs especially interesting is how they mediate their effects. Both MVs and exosomes have been shown to contain a wide-variety of bioactive cargo, including cell surface, cytosolic, and nuclear proteins, as well as RNA transcripts, micro-RNAs (miRNAs), and even fragments of DNA. EVs, and their associated cargo, can be transferred to other cancer cells, as well as to normal cell types, causing the recipient cells to undergo phenotypic changes that promote different aspects of cancer progression. These findings, combined with those demonstrating that the amounts and contents of EVs produced by cancer cells can vary depending on their cell of origin, stage of development, or response to therapies, have raised the exciting possibility that EVs can be used for diagnostic purposes. Moreover, the pharmaceutical community is aggressively pursuing the use of EVs as a potential drug delivery platform. Here, in this chapter, we will highlight what is currently known about how EVs are generated, how they impact cancer progression, and the different ways they are being exploited for clinical applications.

11.
Methods Mol Biol ; 2174: 171-191, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813250

RESUMO

The study of tumor exosomes has gained relevance in the last decades due to their potential use for therapeutic and diagnostic application. Although there is extensive knowledge of exosome biology, some biological samples like tumor-derived exosomes have been difficult to characterize due to their complexity and heterogeneity. This distinctive feature makes difficult the identification of specific exosome subpopulations with a shared molecular signature that could allow for targeting of exosomes with therapeutic and diagnostic potential use in cancer patients. Nanoscale flow cytometry has lately emerged as an alternative tool that can be adapted to the study of nanoparticles, such as exosomes. However, the physicochemical properties of these particles are an important issue to consider as nanoparticles need the application of specific settings which differ from those used in conventional flow cytometry of cells. Therefore, in the last few years, one of the main aims has been the optimization of technical and experimental protocols to improve exosome analysis. In this chapter, we discuss several aspects of cytometric systems with a special emphasis in technical considerations of samples and equipment.

12.
Methods Mol Biol ; 2174: 193-206, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813251

RESUMO

Beyond cell proliferation, one of the most outstanding characteristics of the cancerous cells that promotes the tumoral progression is their high capacity to migrate and invade the surrounding healthy tissue. These cellular processes (migration and invasion) are critical steps to metastasis. Metastatic progression of the tumors is often the leading cause of morbidity and mortality in cancer patients. Critical genes and cell signaling pathways involved in cell migration and invasion of tumor cells have been identified, and several clinical efforts to alleviate cancer are focused on them; however, once the tumor has metastasized, it is extremely difficult to stop the progression of very aggressive forms of cancer such as glioblastomas. Therefore, it is crucial to elucidate the specific molecular mechanisms underlying tumor progression. In this chapter, we describe some methods to study tumor progression by assessing migration and cell invasion in 2D and 3D cell culture conditions.

13.
Methods Mol Biol ; 2174: 207-216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813252

RESUMO

From the knowledge that hematopoiesis does not occur randomly in the bone marrow but is regulated by the different components of the microenvironment, the use of in vitro coculture systems has been used as a powerful tool in the analysis of different processes that are involved in the maintenance of blood cells. In this chapter, we describe a methodological strategy to perform a coculture between primitive hematopoietic cells and endothelial cells to evaluate cell cycle, an aspect of relevant importance in the permanence of primitive leukemic cells.

14.
Methods Mol Biol ; 2174: 219-244, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813253

RESUMO

The use of immunotherapy as an alternative treatment for cancer patients has become of great interest in the scientific community as it is required to overcome many of the currently unsolved problems such as tumor escape, immunosuppression and unwanted unspecific toxicity. The use of chimeric antigen receptor T cells has been a very successful strategy in some hematologic malignancies. However, the application of CAR T cells has been limited to solid tumors, and this has aimed the development of new generation of CARs with enhanced effectivity and specificity. Here, we review the state of the art of CAR T cell therapy with special emphasis on the current challenges and opportunities.

15.
Methods Mol Biol ; 2174: 245-254, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813254

RESUMO

In the treatment of cancer, over the last decade different drugs delivery systems have been developed to increase therapeutic specificity to improve drug's efficacy, and safety by increasing bioavailability. Among these systems, small nucleic acid molecules with a three-dimensional structure, known as aptamers, have shown several advantages. Several approaches to design aptamers require modifications from starting libraries of DNA sequences. Here, we describe cell-internalization SELEX (Systematic Evolution of Ligands by Exponential Enrichment), a sophisticated technique based on RNA aptamers as a starting point, that enables design functional aptamers as drug-delivery tools. This variation of the original SELEX technique using RNA aptamers instead DNA aptamers allows to obtain aptamers that are internalized in prostate cancer cells using as a starting point an RNA aptamer library with 76 nucleotides. The major advantage of this technique is that modifications are not required in the initial library, as initial T7 transcription promoter or 2'F nucleotides before sequencing.

16.
Methods Mol Biol ; 2174: 255-262, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813255

RESUMO

CRISPR-Cas9 is a method for genome editing that can be used efficiently for in vivo applications; the basic implementation of this method is used to generate genome site-directed sequence eliminations. Here we describe a protocol for genome editing using CRISPR-Cas9 in zebrafish (Danio rerio) one-cell embryos.

17.
Methods Mol Biol ; 2174: 263-275, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813256

RESUMO

In recent decades, zebrafish (Danio rerio) has become a major in vivo model for the evaluation of drug efficacies and toxicities. In the field of drug delivery research, zebrafish larvae are a suitable model for the use of fluorescent-labeled chemicals, nanoparticle, liposome, or micelle-mediated delivery systems because of their transparent body wall. In the current chapter, we describe the method to perform micelle-based siRNA delivery using cancer cells implanted into the circulation of zebrafish.

18.
Methods Mol Biol ; 2174: 277-297, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813257

RESUMO

Stiffness control of cell culture platforms provides researchers in cell biology with the ability to study different experimental models in conditions of mimicking physiological or pathological microenvironments. Nevertheless, the signal transduction pathways and drug sensibility of cancer cells have been poorly characterized widely using biomimetic platforms because the limited experience of cancer cell biology groups about handling substrates with specific mechanical properties. The protein cross-linking and stiffening control are crucial checkpoints that could strongly affect cell adhesion and spreading, misrepresenting the data acquired, and also generating inaccurate cellular models. Here, we introduce a simple method to adhere to polyacrylamide (PAA) hydrogels on glass coverslips without any special treatment for mechanics studies in cancer cell biology. By using a commercial photosensitive glue, Loctite 3525, it is possible to polymerize PAA hydrogels directly on glass surfaces. Furthermore, we describe a cross-linking reaction method to attach proteins to PAA as an alternative method to Sulfo-SANPAH cross-linking, which is sometimes difficult to implement and reproduce. In this chapter, we describe a reliable procedure to fabricate ECM protein-cross-linked PAA hydrogels for mechanotransduction studies on cancer cells.

19.
Methods Mol Biol ; 2170: 1-18, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797447

RESUMO

Application of the CRISPR-Cas prokaryotic immune system for single-stranded RNA targeting will have significant impacts on RNA analysis and engineering. The class 2 Type VI CRISPR-Cas13 system is an RNA-guided RNA-nuclease system capable of binding and cleaving target single-stranded RNA substrates in a sequence-specific manner. In addition to RNA interference, the Cas13a system has application from manipulating RNA modifications, to editing RNA sequence, to use as a nucleic acid detection tool. This protocol uses the Cas13a ortholog from Leptotrichia buccalis for transient expression in plant cells providing antiviral defense. We cover all the necessary information for cloning the Cas13 protein, crRNA guide cassette, performing transient Agrobacterium-mediated expression of the necessary Cas13a components and target RNA-virus, visualization of virus infection, and molecular quantification of viral accumulation using quantitative PCR.

20.
Methods Mol Biol ; 2170: 35-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797449

RESUMO

Laser capture microdissection (LCM) has become a powerful technique that allows analyzing gene expression in specific target cells from complex tissues. Widely used in animal research, still few studies on plants have been carried out. We have applied this technique to the plant-nematode interaction by isolating feeding cells (giant cells; GCs) immersed inside complex swelled root structures (galls) induced by root-knot nematodes. For this purpose, a protocol that combines good morphology preservation with RNA integrity maintenance was developed, and successfully applied to Arabidopsis and tomato galls. Specifically, early developing GCs at 3 and 7 days post-infection (dpi) were analyzed; RNA from LCM GCs was amplified and used successfully for microarray assays.

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