The Establishment of a Fast and Safe Orthotopic Colon Cancer Model Using a Tissue Adhesive Technique.

PURPOSE: We aimed to develop a novel method for orthotopic colon cancer model, using tissue adhesive in place of conventional surgical method. MATERIALS AND METHODS: RFP HCT 116 cell line were used to establish the colon cancer model. Fresh tumor tissue harvested from a subcutaneous injection was grafted into twenty nude mice, divided into group A (suture method) and group B (tissue adhesive method). For the group A, we fixed the tissue on the serosa layer of proximal colon by 8-0 surgical suture. For the group B, tissue adhesive (10 µL) was used to fix the tumor. The mortality, tumor implantation success, tumor metastasis, primary tumor size, and operation time were compared between the two groups. Dissected tumor tissue was analyzed for the histology and immunohistochemistry. Also, we performed tumor marker analysis. RESULTS: We observed 30% increase in graft success and 20% decrease in mortality, by using tissue adhesive method, respectively. The median colon tumor size was significantly increased by 4 mm and operation time was shortened by 6.5 minutes. The H&E showed similar tumor structure between the two groups. The immunohistochemistry staining for cancer antigen 19-9, carcinoembryonic antigen, cytokeratin 20, and Ki-67 showed comparable intensities in both groups. Real-time quantitative reverse transcription analysis showed eight out of nine tumor markers are unchanged in the tissue adhesive group. Western blot indicated the tissue adhesive group expressed less p-JNK (apototic marker) and more p-MEK/p-p38 (proliferation marker) levels. CONCLUSION: We concluded the tissue adhesive method is a quick and safe way to generate orthotopic, colon cancer model.

Models of Prostate Cancer Bone Metastasis.

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.

Animal Models of Breast Cancer Bone Metastasis.

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.

Murine Models of Bone Sarcomas.

This chapter describes the procedures for inducing bone sarcoma in mice. Two models based on inoculation of cancer cells in paraosseous and intraosseous site will be described. In addition to providing technical aspects of anesthesia and surgical options, key information of cell preparation and postoperative follow-up will be discussed.

Establishment of Melanoma Tumor Xenograft Using Single Cell Line Suspension and Co-injection of Patient-Derived T Cells in Immune-Deficient NSG Mice.

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.

Mouse Models to Study Natural Killer Cell-Mediated Immunosurveillance and Metastatic Latency.

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.

Gold Standard Assessment of Immunogenic Cell Death in Oncological Mouse Models.

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.

Measuring In Vivo Tissue Metabolism Using 13C Glucose Infusions in Mice.

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.

Development of Patient-Derived Tumor Xenograft Models.

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).

Imaging Glioma Progression by Intravital Microscopy.

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.

Cytokine Profiling and Orthotopic Xenografing of Pancreatic Stellate Cells.

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.

Detection and Quantification of Macropinosomes in Pancreatic Tumors.

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.

Orthotopic Pancreatic Tumor Mouse Models of Liver Metastasis.

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.

Molecular and Physiological Evaluation of Pancreatic Cancer-Induced Cachexia.

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.

Intravital Imaging of Human Melanoma Cells in the Mouse Ear Skin by Two-Photon Excitation Microscopy.

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.

The Colorectal Cancer Microenvironment: Strategies for Studying the Role of Cancer-Associated Fibroblasts.

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.

Testing Cell-Based Immunotherapy for Colorectal Cancer.

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.

Patient-Derived Xenograft Models of Colorectal Cancer: Procedures for Engraftment and Propagation.

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.

Animal Model: Xenograft Mouse Models in Esophageal Adenocarcinoma.

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.

Identification of Cancer Stem Cells in Esophageal Adenocarcinoma.

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.