Cancer Biology

Breast cancer remains one of the most prevalent and deadly malignancies affecting women worldwide. Its progression and metastatic behavior are driven by complex mechanisms. To develop more effective therapeutic strategies, it is crucial to understand tumor growth, angiogenesis, and microenvironmental interactions. Although traditional in vivo models such as murine xenografts have long been used to study tumor biology, these approaches are often time-consuming, costly, and ethically constrained. In contrast, the chick embryo chorioallantoic membrane (CAM) assay offers a rapid, cost-effective, and ethically flexible alternative for evaluating tumor development and angiogenesis. This protocol describes an in ovo CAM-based xenograft model in which human breast cancer cells are implanted onto the vascularized CAM of chick embryos. This method enables real-time evaluation of tumor growth. Furthermore, the model allows for manipulation of experimental conditions, including pharmacological treatments or genetic modifications, to study specific molecular mechanisms involved in breast cancer progression. The major advantages of this protocol lie in its simplicity, reduced cost, and capacity for high-throughput screening, making it a valuable tool for translational cancer research.

Glioblastoma (GBM) is the most aggressive brain tumor, and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. A label-free, mass spectrometry–based quantitative proteomics has been developed to identify and characterize proteins that are differentially expressed in GBM to gain a better understanding of the interactions and functions that lead to the pathological state focusing on the extracellular matrix (ECM). The main challenge in GBM research has been to identify novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. To better investigate the GBM secretome upon in vitro treatment with histone deacetylase inhibitor (iHDAC), we employed a high-throughput label-free methodology of protein identification and quantification based on mass spectrometry followed by in silico studies. Our analysis revealed significant changes in the ECM protein profile, particularly those associated with the angiogenic matrisome. Proteins such as decorin, ADAM10, ADAM12, and ADAM15 were differentially regulated upon in silico analysis. In contrast, key angiogenesis markers such as VEGF and ECM proteins like fibronectin and integrins did not display significant changes. These results suggest that iHDAC inhibitors may modulate or suppress tumor behavior growth by targeting ECM proteins’ secretion rather than directly inhibiting angiogenesis.

Vascular smooth muscle cells (VSMCs) have been cultured for decades to study the role of these cells in cardiovascular disorders. The most common source of VSMCs is the rat aorta. Here we show the adaptation of this method to isolate and culture mouse aortic VSMCs. The advantage of this method is that there are many more transgenic mouse lines available compared to rats. The protocol consists of the isolation of the aorta, the liberation of vascular cells by the action of collagenase, culturing of VSCMs, and analyzing filamentous actin and alpha smooth muscle actin by fluorescence microscopy. VSCMs can be further used to study mechanisms underlying cardiovascular diseases.

Graphic abstract

Figure 1. Working steps

Nuclear blood pool imaging using radiolabeled red blood cells has been used in the clinical setting for the evaluation of a number of medical conditions including gastrointestinal hemorrhage, impaired cardiac contractility, and altered cerebrovascular blood flow. Nuclear blood pool imaging is typically performed using Technetium-99m-labeled (^99mTc) human erythrocytes (i.e., the “tagged RBC” scan) and gamma camera-based planar scintigraphic imaging. When compared to typical clinical planar scintigraphy and single-photon emission computed tomographic (SPECT) imaging platforms, positron emission tomography (PET) provides superior image quality and sensitivity. A number of PET-based radionuclide agents have been proposed for blood pool imaging, but none have yet to be used widely in the clinical setting. In this protocol, we described a simple and fast procedure for imaging the vasculature of immunodeficient mice through a combination of a small animal positron emission tomography/computed tomography (PET/CT) scanner and human erythrocytes labeled with the PET tracer 2-deoxy-2-(¹⁸F)fluoro-D-glucose (¹⁸F-FDG). This technique is expected to have significant advantages over traditional ^99mTc -labeled erythrocyte scintigraphic nuclear imaging for these reasons.

Angiogenesis, the formation of new blood vessels from pre-existing ones plays an important role during organ development, regeneration and tumor progression. The spheroid-based sprouting assay is a well-established and robust method to study the influence of genetic alterations or pharmacological compounds on capillary-like tube formation of primary cultured endothelial cells. A major advantage of this assay is the possibility to study angiogenesis in a 3D environment. Endothelial cells are cultured as hanging drops to form spheroids. Those spheroids are embedded into a collagen matrix and tube formation is analyzed 24 h later. By analyzing sprout number and sprout length the effects of genetic manipulation or drug treatment on angiogenesis can be investigated.

Angiogenesis, the growth of new blood vessels from pre-existing vessels, is a critical process that occurs during normal development and tumor formation. Targeting tumor angiogenesis by blocking the activity of vascular endothelial growth factor (VEGF) has demonstrated some clinical benefit; nevertheless there is a great need to target additional angiogenic pathways. We have found that the human umbilical vein endothelial cell (HUVEC) fibrin bead sprouting assay (FBA) is a robust and predictive in vitro assay to evaluate the activity of angiogenesis inhibitors. Here, we describe an optimized FBA protocol for the assessment of biological inhibitors of angiogenesis and the automated quantification of key endpoints.

Angiogenesis is a multifactorial event which requires the migration, proliferation, differentiation and structure rearrangement of endothelial cells. This angiogenic process has been commonly studied using in vitro assays such as Boyden chamber assay, wound healing assay and tube formation assay. These assays mainly use monolayers of endothelial cells which are modified by repeated passages and are fully proliferative, a situation far away from physiology. In addition, not only endothelial cells are involved in this process but surrounding cells (such as pericytes, smooth muscle cells, fibroblasts) and the supporting matrix are also major players.

The three-dimensional ex vivo aortic ring model recapitulates the complexities of angiogenesis and combines the advantages of in vitro and in vivo models. The aortic ring is cultivated in a chemically defined culture environment. Microvessels which grow in this system are lumenized vessels with surrounding supporting cells and are essentially indistinguishable from microvessels formed during angiogenesis in vivo. The efficacy of pro-or anti-angiogenic factors can be determined in the absence of serum molecules which may otherwise interfere with the substances being tested (Nicosia and Ottinetti, 1990). However, this system requires access to fresh rat tissue but several samples can be prepared from one aorta.

Angiogenesis is the process of formation of new blood vessels from pre-existing vessels or endothelial cell progenitors. It plays a crucial role in tumor growth and metastasis. Tumor angiogenesis have been widely studied as an important target for suppressing tumor growth and metastasis. Here, we describe an in vivo chick embryo chorioallantoic membrane (CAM) model. The chick embryo chorioallantoic membrane is an extraembryonic and is rich of blood vessels. After exposing the vascular zone of the CAM, a sterilized filter-paper disk is employed, which is used as a carrier for being loaded with various chemicals, drugs or virus. Finally, the CAM was fixed and spread on glass slide, and the blood vessels were quantified by counting the number of blood vessel branch points. Compared with the matrigel plug angiogenesis assay, in which tumor cells are mixed with the matrigel gel (expensive) and injected into the mice, subsequently using immunohistochemistry (IHC) staining (time consuming) with the endothelial marker to indicate the presence of the newly formed capillaries, the main advantages of CAM model are its low cost, simplicity, reproducibility, and reliability. Thus, the CAM can be widely used in vivo to study both angiogenesis and anti-angiogenesis.

Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. During angiogenesis, endothelial cells (ECs) undergo activation after binding of angiogenic factors to their receptors, release of proteases to dissolve the basement membrane, migration towards an angiogenic signal, proliferation, and an increase in cell number for new blood vessel formation. Finally, reorganization of ECs forms the three-dimensional vasculature. HUVEC tube-formation assay is one of the simple, but well-established in vitro angiogenesis assays based on the ability of ECs to form three-dimensional capillary-like tubular structures, when cultured on a gel of growth factor-reduced basement membrane extracts. During the assay, ECs differentiate, directionally migrate to align, branch, and form the tubular polygonal networks of blood vessels.

The matrigel plug angiogenesis assay is a simple in vivo technique to detect the newly formed blood vessels in the transplanted gel plugs in nude mice. The matrigel matrix is derived from the engelbroth-holm-swarm (EHS) mouse sarcoma, and its composition is comparable to the basement membrane proteins. The matrigel can induce differentiation of a variety of cell types such as hepatocytes, mammary epithelial cells, and endothelial cells. In our case, tumor cells are mixed with the matrigel gel and are injected into the mice. The later immunohistochemistry (IHC) staining with the endothelial marker indicates the presence of the newly formed capillaries in the sectioned gel plugs.