Cancer Biology


Protocols in Current Issue
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0 Q&A 2842 Views Nov 20, 2021

Bone metastasis is a frequent and lethal complication of many cancer types (i.e., prostate cancer, breast cancer, and multiple myeloma), and a cure for bone metastasis remains elusive. To recapitulate the process of bone metastasis and understand how cancer cells metastasize to bone, intracardiac injection and intracaudal arterial animal models were developed. The intratibial injection animal model was established to investigate the communication between cancer cells and the bone microenvironment and to mimic the setting of prostate cancer patients with bone metastasis. Given that detailed protocols of intratibial injection and its quantitative analysis are still insufficient, in this protocol, we provide hands-on procedures for how to prepare cells, perform the tibial injection, monitor tibial tumor growth, and quantitatively evaluate the tibial tumors in pathological samples. This manuscript provides a ready-to-use experimental protocol for investigating cancer cell behaviors in bone and developing novel therapeutic strategies for bone metastatic cancer patients.

0 Q&A 6360 Views Apr 5, 2020
Metastasis accounts for the majority of cancer related deaths. The genetically engineered mouse (GEM) models and cell line-based subcutaneous and orthotopic mouse xenografts have been developed to study the metastatic process. By using lung cancer cell line A549 as an example, we present a modified protocol to establish the cell line-based xenograft. Our protocol ensures sufficient establishment of the mouse xenografts and allows us to monitor tumor growth and spontaneous metastasis. This protocol could be adapted to other types of established cancer cell lines or primary cancer cells to study the mechanism of metastatic process as well as to test the effect of the potential anti-cancer agents on tumor growth and metastatic capacity.
0 Q&A 3826 Views Nov 20, 2019
Tissues are comprised of different cell types whose interactions elicit distinct gene expression patterns that regulate tissue formation, regeneration, homeostasis and repair. Analysis of these gene expression patterns require methods that can capture as closely as possible the transcriptomes of cells of interest in their tissue microenvironment. Current technologies designed to study in situ transcriptomics are limited by their low sensitivity that require cell types to represent more than 1% of the total tissue, making it challenging to transcriptionally profile rare cell populations rapidly isolated from their native microenvironment. To address this problem, we developed fluorouracil-tagged RNA sequencing (Flura-seq) that utilizes cytosine deaminase (CD) to convert the non-natural pyrimidine fluorocytosine to fluorouracil. Expression of S. cerevisiae CD and exposure to fluorocytosine generates fluorouracil and metabolically labels newly synthesized RNAs specifically in cells of interest. Fluorouracil-tagged RNAs can then be immunopurified and used for downstream analysis. Here, we describe the detailed protocol to perform Flura-seq both in vitro and in vivo. The robustness, simplicity and lack of toxicity of Flura-seq make this tool broadly applicable to many studies in developmental, regenerative, and cancer biology.
0 Q&A 6502 Views Feb 5, 2019
Bone is one of common metastasis sites for many types of cancer. In bone metastatic microenvironment, tumor-bone interactions play a significant role in the regulation of osteolytic or osteoblastic bone metastasis. In order to investigate the direct interaction between tumor cells and bone tissue, it is essential to generate appropriate animal models that mimic the behavior of tumor cells in bone metastatic lesions. Calvarial implantation model (bone invasion model) is a newly-established animal model that accurately recapitulates the behavior of tumor cells in the tumor-bone microenvironment. The surgical technique for tumor cell implantation is simpler than intracardiac, intra-arterial, or intraosseous injection techniques. This model can be useful for the identification of key factors driving tumor-induced osteolytic or osteoblastic changes.
1 Q&A 8612 Views Sep 20, 2018
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.
1 Q&A 12308 Views Sep 20, 2018
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.
0 Q&A 7879 Views Jun 5, 2017
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
1 Q&A 18561 Views Feb 20, 2017
Platelets and coagulation have long been known to be essential for metastasis in experimental models. In order to study the interactions between tumor cells, platelets and endothelium, we have adapted methods used in coagulation research for the isolation of platelets and their reintroduction into mice. Anti-coagulated murine blood served as the source for platelets. Platelets were separated from other elements of the whole blood by centrifugation. Here the critical elements are first inhibition of coagulation and second isolation and maintenance of the platelets in the presence of inhibitors of platelet activation. We then used the vital dye PKH26 to fluorescently label the platelets. Infusion of these labelled platelets allows microscopic observation of the introduced platelets. After reintroduction, these platelets appear to function normally and comprise approximately 50% of the total platelets. Because they are fluorescently labelled, they can easily be identified. Finally it would be possible to use these methods for the determination of specific effects of altered gene expression in platelets by using platelets from genetically engineered mice. These methods have facilitated study of the interactions between platelets and tumor cells in tissue culture and in murine models. They would also be applicable to video microscopy. Here we provide details of the methods we have used for platelet isolation from mice and their staining for further microscopy and re-introduction into mice.
0 Q&A 10516 Views Feb 20, 2017
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.
0 Q&A 13980 Views Nov 20, 2016
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).

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