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1.
Methods Mol Biol ; 1914: 295-308, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30729472

RESUMO

More than 80% of patients with advanced prostate cancer (PCa) experience bone metastasis, which negatively impacts overall survival and patient quality of life. Various mouse models have been used to study the mechanisms of bone metastasis over the years; however, there is currently no model that fully recapitulates what happens in humans because bone metastasis rarely occurs in spontaneous PCa mouse models. Nevertheless, animal models of bone metastasis using several different tumor inoculation routes have been developed to help study bone metastatic progression, which occurs particularly in late-stage PCa patients. This chapter describes the protocols commonly used to develop models of bone metastatic cancer in mice using different percutaneous injection methods (Intracardiac and Intraosseous). These models are useful for understanding the molecular mechanisms of bone metastatic progression, including tumor tissue tropism and tumor growth within the bone marrow microenvironment. Better understanding of the mechanisms involved in these processes will clearly lead to the development of new therapeutic strategies for PCa patients with bone metastases.


Assuntos
Neoplasias Ósseas/secundário , Osso e Ossos/patologia , Modelos Animais de Doenças , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Humanos , Luciferases/química , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Neoplasias da Próstata/diagnóstico por imagem , Microtomografia por Raio-X/instrumentação , Microtomografia por Raio-X/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
2.
Methods Mol Biol ; 1914: 309-330, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30729473

RESUMO

This chapter is designed to provide a comprehensive overview outlining the different in vivo models available for research into breast cancer bone metastasis. The main focus is to guide the researcher through the methodological processes required to establish and utilize these models within their own laboratory. These detailed methods are designed to enable the acquisition of accurate and meaningful results that can be used for publication and future translation into clinical benefit for women with breast cancer-induced bone metastasis.


Assuntos
Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Neoplasias da Mama/diagnóstico por imagem , Linhagem Celular Tumoral , Embrião não Mamífero , Feminino , Humanos , Luciferases/química , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Camundongos , Camundongos Nus , Camundongos SCID , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação , Peixe-Zebra
4.
Methods Mol Biol ; 1913: 207-215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30666609

RESUMO

When primary tumor cells are grown in vitro, they are exposed to an environment that is vastly different from the tumor environment they originate from. The in vitro environment can lack the three-dimensional structure of the tumor, other cell types present within the tumor microenvironment, and important growth factors. Humanized mouse models allow researchers to study primary tumor cells in a more natural environment. With further development of several strains of immune-deficient mice, the mouse model allows for observation of the patient-derived tumor xenograft (PDTX) growth alone as well as in the presence of a human immune system. We describe how this can be accomplished with injection of single cell suspension of melanoma tumor cells into immune-deficient NOD-scid IL2Rγnull (NSG) mice. We also describe how tumor cells and immune cells can be co-injected, using Winn assay, and the possibility to use that method to study immune therapies for cancer.


Assuntos
Melanoma/patologia , Cultura Primária de Células/métodos , Linfócitos T/transplante , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Cultura Primária de Células/instrumentação , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
5.
Methods Mol Biol ; 1862: 67-82, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30315460

RESUMO

Metabolic alterations are a hallmark of cancer. While determining metabolic changes in vitro has delivered valuable insight into the metabolism of cancer cells, it emerges that determining the in vivo metabolism adds an additional layer of information. Here, we therefore describe how to measure the in vivo metabolism of cancer tissue using 13C glucose infusions in mice.


Assuntos
Isótopos de Carbono/química , Glucose/administração & dosagem , Metabolômica/métodos , Animais , Cromatografia Gasosa-Espectrometria de Massas/instrumentação , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glucose/química , Glucose/metabolismo , Metabolômica/instrumentação , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
6.
Methods Mol Biol ; 1862: 217-225, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30315470

RESUMO

In spite of the latest advancements in understanding cancer development and progression, drugs successful in preclinical testing often fail upon reaching phase III clinical trials. A reason for this is the use of inappropriate preclinical models that do not preserve tumor heterogeneity. Although used for decades, cell cultures derived from patients substantially deviate from their original biopsy upon culturing; moreover, they cannot predict the response of an organism as a whole.Patient-derived xenograft (PDX) models are emerging as powerful tools since they have a predictive therapeutic value and preserve the heterogeneity of the original tumors. PDX are established by implanting freshly isolated tumors from patients into immunocompromised mice, allowing for the progressive growth and amplification of cancer tissue for in vivo testing. Here, we describe the detailed methods we developed to establish PDX from both surgically removed endometrial cancer fragments (endometrial cancer) and fine-needle aspiration biopsies (pancreatic cancer).


Assuntos
Neoplasias do Endométrio/patologia , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Criopreservação/instrumentação , Criopreservação/métodos , Neoplasias do Endométrio/cirurgia , Endométrio/patologia , Endométrio/cirurgia , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Pâncreas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
7.
Methods Mol Biol ; 1862: 227-243, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30315471

RESUMO

We describe here a method for generating mouse orthotopic gliomas in order to follow their progression over time by multi-photon laser scanning microscopy. After craniotomy of the parietal bone, glioma cells are implanted in the brain cortex and a glass window is cemented atop, allowing chronical imaging of the tumor. The expression of different fluorescent proteins in tumor cells and in specific cell types of a number of currently available transgenic mouse strains allows obtaining multicolor 3D images of the tumor over time. This technique is suitable both to evaluate the effect of pharmacological treatments and to unravel basic mechanisms of tumor-host interactions.


Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Glioma/diagnóstico por imagem , Microscopia Intravital/métodos , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral/transplante , Craniotomia , Modelos Animais de Doenças , Progressão da Doença , Glioma/patologia , Humanos , Imagem Tridimensional/instrumentação , Imagem Tridimensional/métodos , Microscopia Intravital/instrumentação , Proteínas Luminescentes/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
8.
Methods Mol Biol ; 1884: 141-150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465200

RESUMO

Metastatic latency is a major concern in the clinic, yet how these disseminated cancer cells survive and initiate metastases is unknown (Massagué and Obenauf, Nature 529:298-306, 2016). Here, we describe an approach to isolate latency competent cancer (LCC) cells from early stage human lung and breast carcinoma cell lines using mouse xenograft models (Malladi, Cell 165:45-60, 2016). Cancer cell lines labeled with GFP-luciferase and antibiotic selection markers were injected intracardially into athymic mice. Three months, post-injection, LCC cells were identified in situ and isolated. Upon reinjection, LCC cells retain their tumorigenic potential, enter a slow-cycling or quiescent state, and evade NK cell-mediated innate immune surveillance.


Assuntos
Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Genes Reporter/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Vigilância Imunológica/imunologia , Luciferases/química , Luciferases/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Metástase Neoplásica/patologia , Neoplasias/patologia , Transdução Genética/instrumentação , Transdução Genética/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
9.
Methods Mol Biol ; 1884: 297-315, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465212

RESUMO

The efficacy of cancer therapies strongly relies on their ability to reinstate cancer immunosurveillance. Numerous biomedical approaches with immunotherapeutic activity have been developed to reeducate the host immune system to detect and clear tumor cells. Cytotoxicants have been primarily designed to slow down malignant cell proliferation and to induce programmed cell death. Some cytotoxic stimuli are able to activate a particular type of apoptosis, which is referred to as immunogenic cell death (ICD), that de facto convert cancer cells into their own vaccine. This effect ultimately facilitates the establishment of an antitumor immune response that potentially annihilates spared malignant cells, as well as an immune memory that prevents cancer recurrence. Based on the characteristic hallmarks of ICD, protocols have been developed to validate ICD induction in vitro, ex vivo, and in vivo. These methods may contribute to identify novel ICD inducers and to design multimodal regimens with superior therapeutic efficacy. Moreover, their translation into clinical research could have prognostic or predictive value. This chapter will introduce the "gold standard" protocol for the in vivo assessment of ICD in mice. The procedure relies on vaccination with treated cancer cells, followed by rechallenge with living entities of the same type, in syngeneic immunocompetent animals.


Assuntos
Apoptose/imunologia , Vigilância Imunológica , Imunoterapia/métodos , Neoplasias/terapia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias/imunologia , Neoplasias/patologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto/normas
10.
Methods Mol Biol ; 1882: 157-160, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30378052

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the most lethal human malignancies with a poor prognosis due to systemic metastasis and a high recurrence rate. Interactions between tumor and stromal cells play a critical role in tumor progression. However, the interaction between PSCs and pancreatic cancer cells (PCCs) and the underlying mechanisms are poorly understood. Coculture system with PSCs and PCCs is very useful technique platform for the in vitro and in vivo study of the interaction between these two cellular components. In this protocol, we aim to describe the cytokine profiling technique for in vitro study of PSC-PCC intercellular communication, and orthotopic xenografting animal model with coinjection of primary PSCs and PCC cell line.


Assuntos
Carcinoma Ductal Pancreático/patologia , Comunicação Celular , Citocinas/metabolismo , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Camundongos Nus , Pâncreas/citologia , Pâncreas/patologia , Células Estreladas do Pâncreas/patologia , Cultura Primária de Células/instrumentação , Cultura Primária de Células/métodos , Células Estromais/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
11.
Methods Mol Biol ; 1882: 171-181, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30378054

RESUMO

Macropinocytosis is a mechanism of fluid-phase endocytosis that functions in the nonspecific internalization of extracellular fluid. This uptake pathway has specialized roles in different cell types and organisms, and its importance has recently been established in several diseases, including cancer. In cancer, macropinocytosis is stimulated by oncogenes, such as Ras, and macropinocytic cargo is targeted to lysosomes for degradation, providing a catabolic route for tumor cells to obtain amino acids from the tumor microenvironment. Here, we describe a protocol to employ fluorescently labeled dextran molecules in order to visualize and quantify the extent of macropinocytosis in pancreatic tumors. Multiple samples can be processed in parallel by this method, and the protocol can be easily customized for pancreatic tumor tissue isolated from subcutaneous, orthotopic and genetically engineered mouse models (GEMM), or human patients.


Assuntos
Neoplasias Pancreáticas/patologia , Pinocitose , Animais , Linhagem Celular Tumoral/transplante , Dextranos/química , Endossomos/patologia , Corantes Fluorescentes/química , Humanos , Lisossomos/patologia , Camundongos , Camundongos Nus , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
12.
Methods Mol Biol ; 1882: 309-320, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30378065

RESUMO

The survival from pancreatic cancer is poor because most patients are diagnosed after the cancer has metastasized. Liver is the most common site of pancreatic cancer metastasis. Orthotopic mouse models of liver metastasis by intrasplenically injecting the pancreatic tumor cells are useful in studying the molecular mechanisms of metastasis and evaluating therapeutic regimens.


Assuntos
Neoplasias Hepáticas/secundário , Fígado/patologia , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
13.
Methods Mol Biol ; 1882: 321-333, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30378066

RESUMO

Cachexia, a complex metabolic syndrome, is characterized by involuntary weight loss along with muscle wasting and fat depletion leading to poor quality of life of patients. About 80% of pancreatic cancer patients exhibit cachectic phenotype at the time of diagnosis. Here, we present the several molecular and physiological parameters, which we utilize to study the pancreatic cancer-induced cachexia in in vitro models and preclinical mice models of pancreatic cancer. We have described myotube and adipocyte-based in vitro models of muscle and fat wasting, including methods of cell culture, differentiation, and treatment with cancer cell-conditioned medium. Furthermore, we have explained the methods of evaluation of key cachectic markers for muscles. Next, we have detailed the orthotopic implantation mouse models of pancreatic cancer and evaluation of different physiological parameters, including body weight, food intake, body composition analysis, glucose tolerance test, insulin resistance test, grip strength measurement, and rotarod performance test. We have also explained morphological parameters and molecular markers to evaluate the muscle wasting in pancreatic cancer-induced cachexia.


Assuntos
Caquexia/patologia , Técnicas de Cultura de Células/métodos , Neoplasias Pancreáticas/complicações , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Células 3T3-L1 , Absorciometria de Fóton , Adipócitos/fisiologia , Animais , Caquexia/diagnóstico , Caquexia/etiologia , Caquexia/fisiopatologia , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Nus , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Neoplasias Pancreáticas/patologia , Teste de Desempenho do Rota-Rod/instrumentação , Teste de Desempenho do Rota-Rod/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
14.
Methods Mol Biol ; 1755: 223-232, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29671273

RESUMO

Noninvasive imaging of reporter gene expression by two-photon excitation (2PE) laser scanning microscopy is uniquely suited to perform dynamic and multidimensional imaging down to single-cell detection sensitivity in vivo in deep tissues. Here we used 2PE microscopy to visualize green fluorescent protein (GFP) as a reporter gene in human melanoma cells implanted into the dermis of the mouse ear skin. We first provide a step-by-step methodology to set up a 2PE imaging model of the mouse ear's skin and then apply it for the observation of the primary tumor and its associated vasculature in vivo. This approach is minimally invasive and allows repeated imaging over time and continuous visual monitoring of malignant growth within intact animals. Imaging fluorescence reporter gene expression in small living animals by 2PE provides a unique tool to investigate critical pathways and molecular events in cancer biology such as tumorigenesis and metastasis in vivo with high-spatial and temporal resolutions.


Assuntos
Genes Reporter/genética , Microscopia Intravital/métodos , Melanoma/patologia , Neoplasias Cutâneas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Derme/citologia , Derme/diagnóstico por imagem , Orelha Externa , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Injeções Intradérmicas , Microscopia Intravital/instrumentação , Melanoma/diagnóstico por imagem , Camundongos , Camundongos Nus , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neoplasias Cutâneas/diagnóstico por imagem , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
15.
Methods Mol Biol ; 1765: 87-98, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29589303

RESUMO

Colorectal cancer (CRC) is a key public health concern and the second highest cause of cancer related death in Western society. A dynamic interaction exists between CRC cells and the surrounding tumor microenvironment, which can stimulate not only the development of CRC, but its progression and metastasis, as well as the development of resistance to therapy. In this chapter, we focus on the role of fibroblasts within the CRC tumor microenvironment and describe some of the key methods for their study, as well as the evaluation of dynamic interactions within this biological ecosystem.


Assuntos
Fibroblastos Associados a Câncer/patologia , Neoplasias Colorretais/patologia , Cultura Primária de Células/métodos , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Neoplasias Colorretais/cirurgia , Progressão da Doença , Humanos , Microdissecção e Captura a Laser/instrumentação , Microdissecção e Captura a Laser/métodos , Camundongos , Camundongos Nus , Camundongos SCID , Cultura Primária de Células/instrumentação , Células Estromais/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
16.
Methods Mol Biol ; 1765: 299-305, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29589316

RESUMO

Cell-based immunotherapy for cancer is emerging as an attractive alternative to conventional small-molecule or antibody-based treatment. Due to the characteristics of cell-based therapy, validation of test materials before in vivo administration is required. Here we describe general validation steps for preclinical evaluation of cell-based immunotherapy. We also describe a xenograft model of human colorectal cancer. This model can be used for applied to preclinical evaluation of various cell-based therapy regimens for colorectal cancer treatment.


Assuntos
Transplante de Células/métodos , Neoplasias Colorretais/terapia , Imunoterapia/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Transplante de Células/instrumentação , Neoplasias Colorretais/imunologia , Células Dendríticas/imunologia , Células Dendríticas/transplante , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Humanos , Imunoterapia/instrumentação , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , Camundongos , Camundongos Nus , Camundongos SCID , Linfócitos T/imunologia , Linfócitos T/transplante , Estudos de Validação como Assunto , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
17.
Methods Mol Biol ; 1765: 307-314, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29589317

RESUMO

Preclinical compounds tested in animal models often demonstrate limited efficacy when transitioned into patients. As a result, individuals are assigned to treatment regimens that may be ineffective at treating their disease. The development of more clinically relevant models, such as patient-derived xenografts (PDXs), will (1) more completely mimic the human condition and (2) more accurately predict tumor responses to previously untested therapeutics.PDX models are clinically relevant as tumor tissue is implanted directly from human donor to the mouse recipient. Therefore, these models prevent cell population selection, intentional or unintentional, as the human tissue adapts to an in vitro, two-dimensional environment prior to implantation into a three-dimensional in vivo murine host. Often, cell heterogeneity and tumor architecture can be maintained from human to the PDX model in the mouse. This protocol describes the engraftment and propagation processes for establishing colorectal (CRC) PDX models in mice, using tumor tissue from human subjects.


Assuntos
Colo/patologia , Neoplasias Colorretais/patologia , Reto/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Cultura Primária de Células/instrumentação , Cultura Primária de Células/métodos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
18.
Methods Mol Biol ; 1756: 151-164, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29600368

RESUMO

Researchers often use murine models of esophageal cancer to evaluate novel therapies prior to clinical protocol treatment. Subcutaneous xenograft models are often used for testing the efficacy of anticancer agents in many cancers including esophageal adenocarcinoma. However, mice subcutaneous esophageal adenocarcinoma models only represent local tumor growth and do not provide any information about a survival benefit for a particular anticancer regimen, which is very crucial for experimental treatment efficacy. In addition, anticancer agents may well inhibit subcutaneous tumor growth without effecting overall animal survival. Herein, we describe a peritoneal dissemination mouse xenograft model for survival outcome analysis with intraperitoneal injection of human esophageal adenocarcinoma cell lines.


Assuntos
Adenocarcinoma/mortalidade , Antineoplásicos/uso terapêutico , Neoplasias Esofágicas/mortalidade , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Carboplatina/uso terapêutico , Linhagem Celular Tumoral/transplante , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/patologia , Feminino , Humanos , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Paclitaxel/uso terapêutico , Análise de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
19.
Methods Mol Biol ; 1756: 165-176, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29600369

RESUMO

Cancer stem cells (CSCs) are a subpopulation of cancer cells that have the ability to self-renew and to generate differentiated cells of various lineages. Due to their specific morphological and biological features, they are often resistant to therapy and in turn lead to metastasis and cancer recurrence. Because of their crucial roles in carcinogenesis and patient prognosis, identification and isolation of CSCs have become an important part of improved cancer management regime. Isolation, characterization, and development of targeted therapy against CSCs have potential efficacy in treating esophageal cancer. In addition, CSCs can act as a predictive tool for chemoradiotherapy response in esophageal adenocarcinoma. Different methods including functional assays, cell sorting using various intracellular, and cell surface markers and xenotransplantation techniques are used for the identification and separation of CSCs in different cancers. None of these methods solely can guarantee complete isolation of CSC population, thus a combination of methods could be used for reliable detection and isolation of CSCs. Here, we describe the identification and isolation of CSCs from esophageal adenocarcinoma cells by cell sorting after Hoechst 33342 staining followed by in vitro functional assays, and in vivo xenograft techniques.


Assuntos
Adenocarcinoma/patologia , Separação Celular/métodos , Neoplasias Esofágicas/patologia , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Células-Tronco Neoplásicas/patologia , Animais , Benzimidazóis/química , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Separação Celular/instrumentação , Esôfago/citologia , Esôfago/patologia , Citometria de Fluxo/instrumentação , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Esferoides Celulares , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
20.
Methods Mol Biol ; 1731: 247-260, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29318559

RESUMO

Proteases are "protein-cleaving" enzymes, which, in addition to their non-specific degrading function, also catalyze the highly specific and regulated process of proteolytic processing, thus regulating multiple biological functions. Alterations in proteolytic activity occur during pathological conditions such as cancer. One of the major deregulated classes of proteases in cancer is caspases, the proteolytic initiators and mediators of the apoptotic machinery. The ability to image apoptosis noninvasively in living cells and animal models of cancer can not only provide new insight into the biological basis of the disease but can also be used as a quantitative tool to screen and evaluate novel therapeutic strategies. Optical molecular imaging such as bioluminescence-based genetically engineered biosensors has been developed in our laboratory and exploited to study protease activity in animal models with a high signal to noise. Using the circularly permuted form of firefly luciferase, we have developed a reporter for Caspase 3/7, referred to as Caspase 3/7 GloSensor. Here, we discuss the use of the Caspase 3/7 GloSensor for imaging apoptotic activity in mouse xenografts and genetically engineered mouse models of cancer and present the potential of this powerful platform technology to image the proteolytic activity of numerous other proteases.


Assuntos
Substâncias Luminescentes/química , Imagem Molecular/métodos , Proteólise , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Apoptose , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular Tumoral , Feminino , Genes Reporter/genética , Engenharia Genética/instrumentação , Engenharia Genética/métodos , Humanos , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Camundongos , Camundongos Nus , Camundongos Transgênicos , Imagem Molecular/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
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