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Cell biology assays Drug discovery and analysis Tumor formation Tumor microenvironment Microenvironment Oncogenesis Proliferative signaling Replicative immortality Tumor immunologyQualitative in vivo Bioluminescence Imaging
Bioluminescence imaging (BLI) technology is an advanced method of carrying out molecular imaging on live laboratory animals in vivo. This powerful technique is widely-used in studying a variety of biological processes, and it has been an ideal tool in exploring tumor growth and metastatic spread in real-time. This technique ensures the optimal use of laboratory animal resources, particularly the ethical principle of reduction in animal use, given its non-invasive nature, ensuring that ongoing biological processes can be studied over time in the same animal, without the need to euthanize groups of mice at specific time points. In this protocol, the luciferase imaging technique was developed to study the effect of co-inoculating pericytes (contractile, αSMA+ mesenchymal stem cell-like cells, located abluminally in microvessels) on the growth and metastatic spread of ovarian cancers using an aggressive ovarian cancer cell line–OVCAR-5–as an example.
Zebrafish Embryo Xenograft and Metastasis Assay
Xenograft models, and in particular the mouse xenograft model, where human cancer cells are transplanted into immunocompromised mice, have been used extensively in cancer studies. Although these models have contributed enormously to our understanding of cancer biology, the zebrafish xenograft model offers several advantages over the mouse model. Zebrafish embryos can be easily cultured in large quantities, are small and easy to handle, making it possible to use a high number of embryos for each experimental condition. Young embryos lack an efficient immune system. Therefore the injected cancer cells are not rejected, and the formation of primary tumors and micrometastases is rapid. Transparency of the embryos enables imaging of primary tumors and metastases in an intact and living embryo. Here we describe a method where GFP expressing tumor cells are injected into pericardial space of zebrafish embryos. At four days post-injection, the embryos are imaged and the formation of primary tumor and distant micrometastases are analyzed.
Whole Mammary Gland Transplantation in Mice Protocol
Whole Mammary Gland Transplantation involves transplanting an excised mammary gland into another, more suitable host. This method can be used to extend the life of a mammary gland past the mouse’s life span by transplanting the mammary gland of an older mouse into a young healthy mouse. As you can see in the video below (Video 1), by attaching it to the abdomen of the mouse, the gland will receive a steady blood supply and both epithelial and stromal cells will remain viable for up to one year. Although this method is not used often, it has been part of several experiments including determining whether the stroma or epithelium is the primary target in chemically induced mouse mammary tumorigenesis (Medina and Kittrell, 2005). To monitor transplants, palpate every week for tumor formation. The transplanted mammary gland may also be passaged serially every 8-10 weeks. Keep transplanted gland in the same mouse for no longer than one year.
Video 1. Whole mammary gland transplantation
A Murine Orthotopic Allograft to Model Prostate Cancer Growth and Metastasis
Prostate cancer is one of the most common cancers in men in the United States. Comprehensive understanding of the biology contributing to prostate cancer will have important clinical implications. Animal models have greatly impacted our knowledge of disease and will continue to be a valuable resource for future studies. Herein, we describe a detailed protocol for the orthotopic engraftment of a murine prostate cancer cell line (Myc-CaP) into the anterior prostate of an immune competent mouse.
Establishment of Patient-Derived Xenografts in Mice
Patient-derived xenograft (PDX) models for cancer research have recently attracted considerable attention in both the academy and industry (Hidalgo et al., 2014; Wilding and Bodmer, 2014). PDX models have been developed from different tumor types including lung cancer to improve the drug development process. These models are used for pre-clinical drug evaluation and can be used for the predictive results of clinical outcomes because they conserve original tumor characteristics such as heterogeneity, complexity and molecular diversity (Kopetz et al., 2012). Additionally, PDX model provides the potential tool for the personalized drug therapy. In this protocol, we present methods for the establishment of PDX in mice using primary tumor tissues from patients with small cell lung cancer (SCLC).
Mouse Mammary Intraductal (MIND) Method for Transplantation of Patient Derived Primary DCIS Cells and Cell Lines
The MIND method involves intraductal injection of patient derived ductal carcinoma in situ (DCIS) cells and DCIS cell lines (MCF10DCIS.COM and SUM225) inside the mouse mammary ducts [Video 1 and Figure 1 in Behbod et al. (2009)]. This method mimics the normal environment of DCIS and facilitates study of the natural progression of human DCIS, i.e., their initial growth as carcinoma in situ within the ducts, followed by invasion into the stroma through the myoepithelial cell layer and basement membrane (Behbod et al., 2009; Valdez et al., 2011). In order to demonstrate that transplantation procedure is successful, the transplanted mammary glands may be excised as early as two weeks following intraductal injection of cells followed by Hematoxylin and Eosin (H&E) staining and/or immunofluorescence staining using human specific cytokeratin 5 and/or 19 [please see Figures 2-4 in Behbod et al. (2009)]. Additionally, the presence of trypan blue inside the mouse mammary ducts immediately following intraductal injection is the best indicator that the injection was successful (Video 1 starting at 4:33 sec).
In vivo Extravasation Assay
Tumor metastases develop when disseminated intravascular cancer cells acquire the ability to arrest by adhering to the capillary walls of distant organs, actively extravasate into their parenchyma, proliferate and establish secondary colonies. The extravasation assay described here is an in vivo technique aimed to analyze the ability of tumor cells to achieve early colonization of the lungs following tail vein injection in mice. Importantly, tumor cells need to be easily visible, therefore either they are fluorescent (e.g. expressing RFP or GFP) or they have to be pre-labelled with a fluorescent tracker prior to injection. Lungs are analyzed at different time points, experimentally determined by the researcher, depending on cell features and malignancy. Generally, an early time point is required to check equal lodging in the pulmonary vasculature for the various cells injected. At one or more later time points (from 6 to 48 h) extravasated cells dispersed in the lung parenchyma are quantitated. With our protocol extravasation is directly evaluated in the whole lungs ex vivo considering cell fluorescence. However, immunofluorescence stainings for endothelial markers and microscopic analyses of lung sections are recommended to evaluate positioning and status of tumor cells (i.e. inside, outside the vessels or associated to them; single cells or clusters). Since extravasation is not only influenced by tumor cell motility but also by their survival ability, the results obtained with this technique should be complemented with proliferation and apoptosis analyses.