Gold Nanoparticles Inhibit Extravasation of Canine Osteosarcoma Cells in the Ex Ovo Chicken Embryo Chorioallantoic Membrane Model.

Canine osteosarcoma (OS) is an aggressive bone tumor with high metastatic potential and poor prognosis, mainly due to metastatic disease. Nanomedicine-based agents can be used to improve both primary and metastatic tumor treatment. Recently, gold nanoparticles were shown to inhibit different stages of the metastatic cascade in various human cancers. Here, we assessed the potential inhibitory effect of the glutathione-stabilized gold nanoparticles (Au-GSH NPs) on canine OS cells extravasation, utilizing the ex ovo chick embryo chorioallantoic membrane (CAM) model. The calculation of cells extravasation rates was performed using wide-field fluorescent microscopy. Transmission electron microscopy and Microwave Plasma Atomic Emission Spectroscopy revealed Au-GSH NPs absorption by OS cells. We demonstrated that Au-GSH NPs are non-toxic and significantly inhibit canine OS cells extravasation rates, regardless of their aggressiveness phenotype. The results indicate that Au-GSH NPs can act as a possible anti metastatic agent for OS treatment. Furthermore, the implemented CAM model may be used as a valuable preclinical platform in veterinary medicine, such as testing anti-metastatic agents.

Toxicological Analysis by Assessment of Vascularization and Cell Viability Using the Chicken's Chorioallantoic Membrane (CAM Assay).

The chorioallantoic membrane (CAM) assay is an increasingly popular method using a hen's egg as an experimental organism. Animal models have been established in scientific research for centuries. Yet, awareness of animal welfare in society rises, and the transferability of findings obtained in rodent models to human physiology is challenged. Thus, using fertilized eggs as an alternative platform for animal experimentation might be a promising alternative. The CAM assay is utilized for toxicological analysis by determination of CAM irritation as well as analysis of organ damage and ultimately death of the embryo. Furthermore the CAM provides a micromilieu suited for the implantation of xenografts. Xenogene tissues and tumors grow on the CAM due to a lack of rejection by the immune system and a dense vascular network providing oxygen and nutrients. Multiple analytical methods including in vivo microscopy and various imaging techniques are applicable to this model. Additionally, ethical aspects, a comparatively low financial burden, and low bureaucratic hurdles legitimize the CAM assay.We here describe an in ovo model utilized for xenotransplantation of a human tumor. The model can be used to evaluate the efficacy as well as the toxicity of different therapeutic agents after intravascular injection. Additionally, we present the evaluation of vascularization and viability by intravital microscopy, ultrasonography, and immunohistochemistry.

Use of Chicken Embryos as an Angiogenesis Model for Central Nervous System Malignant Tumor Research.

AIM: To demonstrate the usability of chicken chorioallantoic membrane (CAM) as an angiogenesis model for the development and treatment of malignant tumors of the central nervous system. MATERIAL AND METHODS: A fresh tumor tissue piece taken from Glioblastoma patients, a malignant tumor of the central nervous system, was transferred to the CAM of chicken embryos and left to incubate in the incubator and their development was monitored. After examining the results of the study macroscopically, CAM tissue samples were evaluated both histochemically and immunohistochemically in terms of angiogenic factors VEGF (Vascular Endothelial Growth Factor), bFGF (basic Fibroblast Growth Factor) and PDGF (Platelet Derived Growth Factor). RESULTS: According to histochemical findings obtained from our study when compared with control embryos, blood vessels, fibroblast count and inflammatory infiltration were observed more in the tumor transplanted groups, especially in the tumordeveloping CAM region. There was also intense pleomorphism and marked hypercellularity in the cells. In our immunohistochemical findings, it was determined that bFGF, PDGF, VEGF staining intensities were higher in tumor transplanted groups compared to control groups, and this elevation was more pronounced in the tumor-developing region. CONCLUSION: As a result, it has been shown that the chicken embryo CAM model may be a suitable in vivo model for cancer angiogenesis studies. The protocol we created in this study will be a source for projects related to the use of therapeutic agents in cancer angiogenesis.

In ovo model in cancer research and tumor immunology.

Considering cancer not only as malignant cells on their own but as a complex disease in which tumor cells interact and communicate with their microenvironment has motivated the establishment of clinically relevant 3D models in past years. Technological advances gave rise to novel bioengineered models, improved organoid systems, and microfabrication approaches, increasing scientific importance in preclinical research. Notwithstanding, mammalian in vivo models remain closest to mimic the patient's situation but are limited by cost, time, and ethical constraints. Herein, the in ovo model bridges the gap as an advanced model for basic and translational cancer research without the need for ethical approval. With the avian embryo being a naturally immunodeficient host, tumor cells and primary tissues can be engrafted on the vascularized chorioallantoic membrane (CAM) with high efficiencies regardless of species-specific restrictions. The extraembryonic membranes are connected to the embryo through a continuous circulatory system, readily accessible for manipulation or longitudinal monitoring of tumor growth, metastasis, angiogenesis, and matrix remodeling. However, its applicability in immunoncological research is largely underexplored. Dual engrafting of malignant and immune cells could provide a platform to study tumor-immune cell interactions in a complex, heterogenic and dynamic microenvironment with high reproducibility. With some caveats to keep in mind, versatile methods for in and ex ovo monitoring of cellular and molecular dynamics already established in ovo are applicable alike. In this view, the present review aims to emphasize and discuss opportunities and limitations of the chicken embryo model for pre-clinical research in cancer and cancer immunology.

Tumors derived from lung cancer cells respond differently to treatment with sodium valproate (a HDAC inhibitor) in a chicken embryo chorioallantoic membrane model.

Lung cancer is the most frequent cause of cancer death. Some human lung malignant tumors have a combined small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) histology, with tumor cell phenotype changing during tumor progression. Valproic acid is used as an anti-seizure medication to treat migraine, and bipolar mood disorders. Recently, its efficacy as an adjuvant therapy was shown in cancer due to its histone deacetylase (HDAC) inhibitory property. HDACs are upregulated in lung tumors, and HDAC inhibitors, including valproic acid, inhibit endothelial cell proliferation in vitro and in vivo and have antiproliferative and antimigratory properties. We tested valproic acid for possible antiangiogenic and antimigratory effects on experimental lung tumors grafted onto the chicken embryo chorioallantoic membrane (CAM). Tumors were formed from two NSCLC cell lines and a single SCLC cell line. To investigate tumor and CAM interactions, in vivo biomicroscopy, visualization of blood vessels with injected fluorescent dextran, histological, immunohistochemical and histomorphometric methods were applied. Our results showed that a sodium valproate (NaVP) treatment-induced a dose-dependent decrease of experimental tumor invasion into the CAM mesenchyme and a reduction in angiogenesis. Both the invasion and the angiogenic response were dependent on the type of cell line used: invasion and angiogenesis of tumors derived from A549 and NCI-H146 cell lines responded to increasing doses of NaVP from 4 to 8 mM, whereas Sk_Lu_1 cells response were antimigratory and antiangiogenic when NaVP was used up to 6 mM. When 8mM NaVP was used, stimulated invasion and angiogenesis in tumors from Sk_Lu_1 cells were observed.

CAM-Delam Assay to Score Metastatic Properties by Quantifying Delamination and Invasion Capacity of Cancer Cells.

The major cause of cancer-related deaths is metastasis formation (i.e., when cancer cells spread from the primary tumor to distant organs and form secondary tumors). Delamination, defined as the degradation of the basal lamina and basement membrane, is the initial process that facilitates the transmigration and spread of cancer cells to other tissues and organs. Scoring the delamination capacity of cancer cells would indicate the metastatic potential of these cells. We have developed a standardized method, the ex ovo CAM-Delam assay, to visualize and quantify the ability of cancer cells to delaminate and invade, thereby being able to assess metastatic aggressiveness. Briefly, the CAM-Delam method includes seeding cancer cells in silicone rings on the chick chorioallantoic membrane (CAM) at embryonic day 10, followed by incubation from hours to a few days. The CAM-Delam assay includes the use of an internal humidified chamber during chick embryo incubation. This novel approach increased embryo survival from 10%-50% to 80%-90%, which resolved previous technical problems with low embryo survival rates in different CAM assays. Next, the CAM samples with associated cancer cell clusters were isolated, fixed, and frozen. Finally, cryostat-sectioned samples were visualized and analyzed for basement membrane damage and cancer cell invasion using immunohistochemistry. By evaluating various known metastatic and non-metastatic cancer cell lines designed to express green fluorescent protein (GFP), the CAM-Delam quantitative results showed that the delamination capacity patterns reflect metastatic aggressiveness and can be scored into four categories. Future use of this assay, apart from quantifying delamination capacity as an indication of metastatic aggressiveness, is to unravel the molecular mechanisms that control delamination, invasion, the formation of micrometastases, and changes in the tumor microenvironment.

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy.

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The 3D in vivo chorioallantoic membrane model and its role in breast cancer research.

PURPOSE: We aimed to evaluate the role of the chorioallantoic membrane model (CAM) in breast cancer research. METHODS: The following is an overview of the use of the CAM in the field of breast cancer research based on a PubMed literature query. RESULTS: The CAM is a 3D in vivo model that can be used for the analysis of tumor growth, biology and angiogenesis of primary tumor tissue or tumor cell lines. The CAM model has been used in breast cancer research for drug testing, migration assays and the evaluation of vascularization, amongst others. The CAM model is a valuable method that offers a better imitation of the physiological phenomena compared to 2D or 3D in vitro models. CONCLUSION: The CAM model has primarily and successfully been utilized for the assessment of the tumor biology of established breast cancer cell lines. Further, the CAM model is a promising method to analyze patient derived primary tumor material and could be used as a "patient-specific 3D-tumor-therapy-model" for the cost-efficient evaluation of anti-cancer drugs to find the optimal treatment for breast cancer patients.

Chorioallantoic membrane (CAM) assay to study treatment effects in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor. While there are a number of in vivo rodent models for evaluating tumor biology and response to therapy, these models require significant time and resources. Here, we established the chick-embryo chorioallantoic (CAM) assay as an affordable and time efficient xenograft model for testing a variety of treatment approaches for DIPG. We found that patient-derived DIPG tumors develop in the CAM and maintain the same genetic and epigenetic characteristics of native DIPG tumors. We monitored tumor response to pharmaco- and radiation therapy by 3-D ultrasound volumetric and vasculature analysis. In this study, we established and validated the CAM model as a potential intermediate xenograft model for DIPG and its use for testing novel treatment approaches that include pharmacotherapy or radiation.

Patterns of Tumor Vasculogenesis on a Chick Embryo Chorioallantoic Membrane In Vivo Model of Breast Cancer.

BACKGROUND/AIM: Understanding tumor vasculogenesis is a cornerstone for the inhibition of tumor progression. This study aimed to generate an in vivo breast cancer environment to analyze the patterns of tumor vasculogenesis. MATERIALS AND METHODS: Human mesenchymal stem cells (hMSC) and breast cancer MCF-7 cells (MCF-7) were seeded onto a chorioallantoic membrane (CAM) and, after a 7-day incubation, we performed a morphological and immunohistochemical analysis of CAM. RESULTS: hMSC and MCF-7 activated vasculogenesis and hematopoiesis on CAM. They stimulated the development of cord/capillary-like structures (CLS), formed by endothelial-like cells and hematopoietic cells. CLS presented a polygonal pattern, evolving towards a clearly visible plexus. Immunohistochemically, CLS were CD105+/AC133+/Oct3/4+, and the intensity was weak-moderate in the endothelial-like cells (inconstant) and weak in the hematopoietic cells. CONCLUSION: Tumor and embryonic vasculogenesis share a common paradigm, while CD105, AC133, and Oct3/4 were found to play a role in establishing the vasculogenic and hematopoietic stage.

Tumor perivascular cell-derived extracellular vesicles promote angiogenesis via the Gas6/Axl pathway.

Aberrant angiogenesis is a hallmark of cancer and is critically associated with tumor progression. Perivascular cells are essential components of blood vessels, and the role of tumor perivascular cell-derived extracellular vesicles (TPC-EVs) in angiogenesis remains elusive. In the present study, using genetic mouse models and pharmacological inhibitors, we found that ablation of perivascular cells inhibited angiogenesis in allografted colorectal cancer tumors. Further studies demonstrated that TPC-EVs promoted the proliferation, migration, invasion, viability, and tube formation of HUVECs. They also facilitated vessel spouting in rat aortic rings and induced neovascularization in chick chorioallantoic membranes (CAMs). Silencing of Gas6 or blockade of the Axl pathway suppressed TPC-EV-induced angiogenesis in vitro and ex vivo. Moreover, inhibition of the Gas6/Axl signaling pathway impaired TPC-EV-mediated angiogenesis in vivo. Our findings present a deeper insight into the biological functions of TPCs and TPC-EVs in tumor angiogenesis and demonstrate that TPC-EV-derived Gas6 could be an attractive and innovative regulator of tumor angiogenesis.

Applications of the Chick Chorioallantoic Membrane as an Alternative Model for Cancer Studies.

A variety of in vivo experimental models have been established for the studies of human cancer using both cancer cell lines and patient-derived xenografts (PDXs). In order to meet the aspiration of precision medicine, the in vivomurine models have been widely adopted. However, common constraints such as high cost, long duration of experiments, and low engraftment efficiency remained to be resolved. The chick embryo chorioallantoic membrane (CAM) is an alternative model to overcome some of these limitations. Here, we provide an overview of the applications of the chick CAM model in the study of oncology. The CAM model has shown significant retention of tumor heterogeneity alongside increased xenograft take rates in several PDX studies. Various imaging techniques and data analysis have been applied to study tumor metastasis, angiogenesis, and therapeutic response to novel agents. Lastly, to practically illustrate the feasibility of utilizing the CAM model, we summarize the general protocol used in a case study utilizing an ovarian cancer PDX.

The CAM Model for CIC-DUX4 Sarcoma and Its Potential Use for Precision Medicine.

(1) Background: CIC-DUX4 sarcoma is a rare mesenchymal small round cell tumor which belongs to rare cancers that occupy a significant percentage of cancer cases as a whole, despite each being rare. Importantly, each rare cancer type has different features, and thus there is a need to develop a model that mimics the features of each of these cancers. We evaluated the idea that the chicken chorioallantoic membrane assay (CAM), a convenient and versatile animal model, can be established for the CIC-DUX4 sarcoma. (2) Methods: Patient-derived cell lines of CIC-DUX4 were applied. These cells were transplanted onto the CAM membrane and tumor formation was examined by H&E staining, immunohistochemistry and Western blotting. The CAM tumor was transferred onto a fresh CAM and was also used to form organoids. Retention of the fusion gene was examined. (3) Results: H&E staining as well as molecular characterization demonstrated the formation of the CIC-DUX4 tumor on the CAM membrane. Expression of cyclin D2 and ETV4 was identified. The CAM tumor was transferred to a fresh CAM to form the second-generation CAM tumor. In addition, we were successful in forming tumor organoids using the CAM tumor. Retention of the fusion gene CIC-DUX4 in the CAM, second-generation CAM, and in the CAM-derived organoids was confirmed by RT-PCR. (4) Conclusions: The CAM assay provides a promising model for CIC-DUX4 sarcoma.

A novel method of screening combinations of angiostatics identifies bevacizumab and temsirolimus as synergistic inhibitors of glioma-induced angiogenesis.

Tumor angiogenesis is critical for the growth and progression of cancer. As such, angiostasis is a treatment modality for cancer with potential utility for multiple types of cancer and fewer side effects. However, clinical success of angiostatic monotherapies has been moderate, at best, causing angiostatic treatments to lose their early luster. Previous studies demonstrated compensatory mechanisms that drive tumor vascularization despite the use of angiostatic monotherapies, as well as the potential for combination angiostatic therapies to overcome these compensatory mechanisms. We screened clinically approved angiostatics to identify specific combinations that confer potent inhibition of tumor-induced angiogenesis. We used a novel modification of the ex ovo chick chorioallantoic membrane (CAM) model that combined confocal and automated analyses to quantify tumor angiogenesis induced by glioblastoma tumor onplants. This model is advantageous due to its low cost and moderate throughput capabilities, while maintaining complex in vivo cellular interactions that are difficult to replicate in vitro. After screening multiple combinations, we determined that glioblastoma-induced angiogenesis was significantly reduced using a combination of bevacizumab (Avastin®) and temsirolimus (Torisel®) at doses below those where neither monotherapy demonstrated activity. These preliminary results were verified extensively, with this combination therapy effective even at concentrations further reduced 10-fold with a CI value of 2.42E-5, demonstrating high levels of synergy. Thus, combining bevacizumab and temsirolimus has great potential to increase the efficacy of angiostatic therapy and lower required dosing for improved clinical success and reduced side effects in glioblastoma patients.

Targeting neovascularization and respiration of tumor grafts grown on chick embryo chorioallantoic membranes.

Since growing tumors stimulate angiogenesis, via vascular endothelial growth factor (VEGF), angiogenesis inhibitors (AIs, blockers of the VEGF signaling pathway) have been introduced to cancer therapy. However, AIs often yielded only modest and short-lived gains in cancer patients and more invasive tumor phenotypes in animal models. Combining anti-VEGF strategies with lactate uptake blockers may boost both efficacy and safety of AIs. We assessed this hypothesis by using the ex ovo chorioallantoic membrane (CAM) assay. We show that AI-based monotherapy (Avastin®, AVA) increases tumor hypoxia in human CAM cancer cell xenografts and cell spread in human as well as canine CAM cancer cell xenografts. In contrast, combining AVA treatment with lactate importer MCT1 inhibitors (α-cyano-4-hydroxycinnamic acid (CHC) or AZD3965 (AZD)) reduced both tumor growth and cell dissemination of human and canine explants. Moreover, combining AVA+AZD diminished blood perfusion and tumor hypoxia in human explants. Thus, the ex ovo CAM assay as an easy, fast and cheap experimental setup is useful for pre-clinical cancer research. Moreover, as an animal-free experimental setup the CAM assay can reduce the high number of laboratory animals used in pre-clinical cancer research.

The Chicken Egg Chorioallantoic Membrane (CAM) Model as an In Vivo Method for the Investigation of the Invasion and Metastasis Cascade of Malignant Tumor Cells.

The CAM model enables an in vivo analysis of the individual sub-steps of the metastatic cascade like local invasion, intravasation, or the establishment of metastasis in particular organs. Incubated fertilized chicken eggs are inoculated with human tumor cells and further processed for up to 9-10 days. The invasion and metastasis of these cells is then detected quantitatively with high specificity and sensitivity by means of a PCR for human ALU sequences, using the genomic DNA isolated from distant portions of the CAM, as well as from diverse internal organs of the developing embryo.

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets. The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

The CAM assay in the study of the metastatic process.

Among the in vivo experimental models, the chick embryo chorioallantoic membrane (CAM) has been routinely used to implant several malignant cell lines or tumor tissues to study their angiogenic and metastatic capability. Since the chick embryo is naturally immunodeficient, the CAM can support the engraftment of tumor cells, and their growth therein can faithfully recapitulate most of the characteristics of the carcinogenic process including: growth, invasion, angiogenesis and colonization of distant tissues. This review article is focused on the discussion of the more recent literature data concerning the use of the CAM to investigate the metastatic process.

Hyaluronic acid engrafted metformin loaded graphene oxide nanoparticle as CD44 targeted anti-cancer therapy for triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with limited treatment modalities. It is associated with high propensity of cancer recurrence. METHODS: UV Spectroscopy, FTIR, DLS, Zeta potential, TEM and SEM were employed to characterize nanoparticles. MTT assay, Wound healing assay, SEM, Immunocytochemistry analysis, Western blot, RT-PCR, mammosphere formation assay were employed to study apoptosis, cell migration and stemness. Tumor regression was studied in chick embryo xenograft and BALB/c mice model. RESULTS: Hylaluronic acid engrafted metformin loaded graphene oxide (HA-GO-Met) nanoparticles exhibited an anti-cancer efficacy at much lower dosage as compared to metformin alone. HA-GO-Met nanoparticles induced apoptosis and inhibited cell migration of TNBC cells by targeting miR-10b/PTEN axis via NFkB-p65. Upregulation of PTEN affected pAKT(473) expression that induced apoptosis. Cell migration was inhibited by reduction of pFAK/integrinß1 expressions. Treatment inhibited epithelial mesenchymal transition (EMT) and reduced stemness as evident from the increase in E-cadherin expression, inhibition of mammosphere formation and low expression levels of stemness markers including nanog, oct4 and sox2 as compared to control. Moreover, tumor regression was studied in chick embryo xenograft and BALB/c mice model. HA-GO-Met nanoparticle treatment reduced tumor load and nullified toxicity in peripheral organs imparted by tumor. CONCLUSIONS: HA-GO-Met nanoparticles exhibited an enormous anti-cancer efficacy in TNBC in vitro and in vivo. GENERAL SIGNIFICANCE: HA-GO-Met nanoparticles induced apoptosis and attenuated cell migration in TNBC. It nullified overall toxicity imparted by tumor load. It inhibited EMT and reduced stemness and thereby addressed the issue of cancer recurrence.

Non-rodent animal models of osteosarcoma: A review.

Osteosarcoma is extremely malignant, and the most common cancer that affects bone. Current treatments involve surgical resection of the affected area and multi-agent chemotherapy, though survival rate is generally poor for those affected by metastases. As treatment for osteosarcoma has remained unchanged for the past few decades, there is a need for further advancements in the understanding of osteosarcoma biology and therapeutics. Thus, reliable animal models that can accurately recapitulate the disease are required. Though rodents represent the most popular animal model of osteosarcoma, they may not model the disease best. This review analyzes emerging alternative non-rodent animal models of osteosarcoma, such as the chick chorioallantoic membrane (CAM) assay, pigs, and canines. Each of these alternatives offer advantages over classic rodent models for pre-clinical research. Research of these cross-species platforms imparts knowledge of metastases biology and potential new treatments for osteosarcoma.