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Although the current murine model of bone metastasis using intracardiac (IC) injection successfully recapitulates the process of bone metastasis, further progress in the study of bone metastasis requires a new model to circumvent some limitations of this model. Here, we present a new murine model of bone metastasis achieved by injecting cancer cells through the intra-caudal arterial (CA). This model does not require high technical proficiency, predominantly delivers cancer cells to bone marrow of hind limbs with much higher efficiency than IC injection, and greatly shortens the period of overt bone metastasis development. Moreover, CA injection barely causes acute death of mice, enabling us to inject a larger number of cancer cells to further accelerate the development of bone metastasis with a wide variety of cell lines. Our model may open a new avenue for understanding the bone metastatic processes and development of drugs preventing bone metastasis and recurrence. Reliably modelling bone metastasis in mice is essential for research. Here, the authors present a new murine model for cancer metastasis based on intra-caudal artery injection that reliably recapitulates bone metastasis.

By implementing the conditions for orthotopic implantation of different types of human breast tumors, the authors have created a publicly available bank of new mouse models that more faithfully recreate individual tumor properties and provide individualized information about tumor behavior and prognosis. Development and preclinical testing of new cancer therapies is limited by the scarcity of in vivo models that authentically reproduce tumor growth and metastatic progression. We report new models for breast tumor growth and metastasis in the form of transplantable tumors derived directly from individuals undergoing treatment for breast cancer. These tumor grafts illustrate the diversity of human breast cancer and maintain essential features of the original tumors, including metastasis to specific sites. Co-engraftment of primary human mesenchymal stem cells maintains phenotypic stability of the grafts and increases tumor growth by promoting angiogenesis. We also report that tumor engraftment is a prognostic indicator of disease outcome for women with newly diagnosed breast cancer; orthotopic breast tumor grafting is a step toward individualized models for tumor growth, metastasis and prognosis. This bank of tumor grafts also serves as a publicly available resource for new models in which to study the biology of breast cancer.

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Desmoplasia is a fibro-inflammatory process and a well-established feature of pancreatic cancer. A key contributor to pancreatic cancer desmoplasia is the pancreatic stellate cell. Various in vitro and in vivo methods have emerged for the isolation, characterization, and use of pancreatic stellate cells in models of cancer-associated fibrosis. In addition to cell culture models, genetically engineered animal models have been established that spontaneously develop pancreatic cancer with desmoplasia. These animal models are currently being used for the study of pancreatic cancer pathogenesis and for evaluating therapeutics against pancreatic cancer. Here, we review various in vitro and in vivo models that are being used or have the potential to be used to study desmoplasia in pancreatic cancer.

Cancer is a heterogeneous disease manifest in many forms. Tumor histopathology can differ significantly among patients and cellular heterogeneity within tumors is common. A primary goal of cancer biologists is to better understand tumorigenesis and cancer progression; however, the complex nature of tumors has posed a substantial challenge to unlocking cancer’s secrets. The cancer stem cell (CSC) paradigm for the pathobiology of solid tumors appropriately acknowledges phenotypic and functional tumor cell heterogeneity observed in solid tumors and accounts for the disconnect between drug approval based on response and the general inability of approved therapies to meaningfully impact survival due to their failure to eradicate these most important of cellular targets. First proposed to exist decades ago, CSC have only recently begun to be precisely identified due to technical advancements that facilitate identification, isolation, and interrogation of distinct tumor cell subpopulations with differing ability to form and perpetuate tumors. Precise identification of CSC populations and the complete hierarchy of cells within solid tumors will facilitate more accurate characterization of patient subtypes and ultimately contribute to more personalized and effective therapies. Rapid advancement in the understanding of tumor biology as it exists in patients requires cooperation among institutions, surgeons, pathologists, cancer biologists and patients alike, primarily because this translational research is best done with patient-derived tissue grown in the xenograft setting as patient-derived xenografts. This review calls for a broader change in the approaches taken to study cancer pathobiology, highlights what implications the CSC paradigm has for pathologists and cancer biologists alike, and calls for greater collaboration between institutions, physicians and scientists in order to more rapidly advance our collective understanding of cancer.

As an important regulator of intracellular protein degradation, the mechanism of the deubiquitinating enzyme family in tumour metastasis has received increasing attention. Our previous study revealed that USP3 promotes tumour progression and is highly expressed in gastric cancer (GC). Herein, we report two critical targets, COL9A3 and COL6A5, downstream of USP3, via the isobaric tags for relative and absolute quantification technique. Mechanistically, we observed that USP3 interacted with and stabilised COL9A3 and COL6A5 via deubiquitination in GC. Importantly, we found that COL9A3 and COL6A5 were essential mediators of USP3-modulated oncogenic activity in vitro and in vivo. Examination of clinical samples confirmed that elevated expression of USP3, concomitant with increased COL9A3 and COL6A5 abundance, correlates with human GC progression. These data suggest that USP3 promotes GC progression and metastasis by deubiquitinating COL9A3 and COL6A5. These findings identify a mechanism of GC metastasis regarding USP3-mediated deubiquitinating enzyme activity and suggest potential therapeutic targets for GC management.

For decades, xenografts using well-established human tumor cell lines have been the most commonly used models to study human cancers in mice. More recently, human tumors implanted directly into immunodeficient mice have become increasingly popular as evidence accrues that they more accurately recapitulate features of patient tumors. Here we describe our protocols for the orthotopic and heterotopic implantation of pancreatic cancer cell lines and freshly isolated patient tumors into immunodeficient mice. We also describe procedures for the digestion of tumors into single-cell suspensions for the isolation of subpopulations of tumor cells. Orthotopic or heterotopic implantation of established cell lines requires 1–2 h, with 1-cm tumors arising after 2–5 weeks. Engraftment of patient tumor samples takes ∼2 h and growth of palpable tumor requires ∼14 weeks. Once established, direct xenograft tumors require 2 and 5 h for heterotopic and orthotopic implantation, respectively, and 5–6 weeks for palpable tumor growth.

Surgical orthotopic implantation of human colon cancer tissue to the ceca of mice has been used to mimic behavior of cancer in human patients for the development of precision cancer medicine. However, with the current method of serosal surface implantation (SSI) of pieces of human colon cancer tissue, cancer cells are exposed to the peritoneum, which can artificially increase the rate of peritoneal carcinomatosis (PC) during the disease course. The objective of the present study was to introduce a tumor-sealing method (TSM) and compare it with SSI for the ability to produce clinically-relevant metastases without artificial PC. HCT116 colon cancer cells transfected with green fluorescence protein (GFP) were cultured and then injected into the subcutaneous layer of athymic nude mice. Subcutaneous tumors were allowed to grow sufficiently to supply adequate tumor for orthotopic implantation. For SSI, a 1 mm -sized tumor fragment was sutured to partially torn serosa of the cecum. For TSM, the blind end of the cecum was folded over the tumor fragment and sealed with sutures. At 20 days after implantation, all mice were opened to visualize PC by intravital fluorescence imaging. At necropsy, distant metastasis was investigated using frozen section of whole blocks of organs. At 20 days after implantation, PC rates in the SSI group and the TSM group were 80% (12/15) and 20% (3/15), respectively (p<0.001). The liver metastasis rate was 41.7% (5/12) in the SSI group and 50% (5/10) in the TSM group (p=0.696). The lung metastasis rate was 0% (0/12) in the SSI group and 10% (1/10) in the TSM group (p=0.201). The mean survival of mice without PC on the 20th day was significantly longer than that of mice with PC on the 20th day (69.1±14.7 vs. 44.5±12.4 days, p=0.001). These results suggest that TSM might be a more patient-like and useful method as a model of metastatic colon cancer than SSI.

Endometrial cancer (EC) is the most common malignancy of the genital tract among women in developed countries. Recently, a molecular classification of EC has been performed providing a system that, in conjunction with histological observations, reliably improves EC classification and enhances patient management. Patient-derived xenograft models (PDX) represent nowadays a promising tool for translational research, since they closely resemble patient tumour features and retain molecular and histological features. In EC, PDX models have already been used, mainly as an individualized approach to evaluate the efficacy of novel therapies and to identify treatment-response biomarkers; however, their uses in more global or holistic approaches are still missing. As a collaborative effort within the ENITEC network, here we describe one of the most extensive EC PDX cohorts developed from primary tumour and metastasis covering all EC subtypes. Our models are histologically and molecularly characterized and represent an excellent reservoir of EC tumour samples for translational research. This review compiles the information on current methods of EC PDX generation and their utility and provides new perspectives for the exploitation of these valuable tools in order to increase the success ratio for translating results to clinical practice.

In this protocol, the authors present an experimental metastasis assay in which cancer cells are injected into the tail vein of a mouse, and the resulting secondary organ colonization is assessed, primarily in the lung, 10 d later. We describe a sensitive, robust, high-throughput method for quantifying the ability of metastatic tumor cells to colonize a secondary organ. Metastasis is the leading cause of death in cancer patients, and successful colonization of the secondary organ is the rate-limiting step in the metastatic process; thus, experimental methods that can be used to interrogate the key factors required for this critical step are of great importance. The experimental metastasis assay we detail here includes tail-vein injection of cancer cells into the mouse and determination of the resulting secondary organ colonization, primarily in the lung, 10 d post dosing. This assay can be used to investigate factors that regulate metastatic colonization both at the tumor-cell-intrinsic level (via manipulation of the tumor cells before injection) and at the tumor-cell-extrinsic level (such as the tissue microenvironment, via the use of genetically modified (GM) mice or agents such as antibodies or drugs). Using this method, we have robustly screened more than 950 GM mouse lines to identify novel microenvironmental regulators of metastatic colonization. The experimental details discussed here include choosing of appropriate cell numbers, handling of the cells, selection of recipient animals and injection techniques. Furthermore, we discuss key experimental design considerations, including the choice of the method used to determine metastatic burden and statistical analysis of the results, as well as provide troubleshooting tips and identification of factors that contribute to experimental variability.