Background

Breast cancer remains a significant global health concern. It is the most commonly diagnosed cancer in women and a leading cause of cancer-related deaths worldwide [1,2]. Epidemiological data indicate a continuous rise in incidence, largely associated with lifestyle factors, aging populations, and increased screening [3]. Despite substantial progress in clinical management driven by molecular subtyping, targeted therapies, immunotherapy, and improvements in early detection in recent decades, the clinical outcome for many patients continues to be negatively influenced by high tumor heterogeneity and frequent therapeutic resistance acquisition [4]. These characteristics limit the long-term success of current treatments and highlight the necessity of experimental approaches that allow a more precise understanding of breast cancer biology [2]. To achieve this, research models must not only replicate tumor-intrinsic molecular features but also key components of the tumor microenvironment, including stromal interactions, extracellular matrix composition, angiogenic processes, and the dynamic exchange of soluble factors [5]. Conventional two-dimensional (2D) cell culture systems have played an essential role in elucidating molecular pathways associated with proliferation, migration, and survival; however, they fail to reproduce the architectural and biochemical complexity of tumors in vivo [6]. Recently, three-dimensional (3D) culture platforms, including spheroids, scaffolds, organoids, and bioprinted constructs, have improved the modeling of cell–cell and cell–matrix interactions. However, limitations still exist when using these platforms to study angiogenesis, invasion, immune interactions, and metastatic potential [7]. For this reason, animal models are often necessary to validate in vitro observations and evaluate therapeutic responses in a context that is physiologically relevant [8].

Among animal systems, murine xenograft models have historically been the gold standard in preclinical breast cancer research. They permit long-term tumor development and allow evaluation of metastasis, immune modulation, and therapeutic efficacy. Nonetheless, their routine use poses significant challenges [9]. Rodent studies require specialized facilities, rigorous ethical reviews, significant financial resources, and extended timeframes for experimentation. These challenges, coupled with the growing emphasis on reducing, refining, and replacing animal experimentation (the 3Rs), have driven the development of alternative models that minimize animal suffering while maintaining biological relevance [10,11].

In this context, the chorioallantoic membrane (CAM) of the chick embryo has emerged as a powerful, versatile, and ethically advantageous platform for in vivo cancer research [12]. The CAM is a highly vascularized extraembryonic membrane that supports rapid tumor engraftment and neovascularization [13,14]. Importantly, the chicken embryo lacks a fully developed immune system during early incubation, which allows implantation of human cancer cells without the need for immunosuppression [15,16]. This cost-effective model requires minimal infrastructure and enables experimental timelines that are significantly shorter than those observed in rodents. Additionally, the CAM is not innervated during the early stages of development, which minimizes potential pain and reduces ethical concerns [17–21].

The present protocol was developed to provide a detailed, standardized, and reproducible methodology for inducing tumors on the CAM using human breast cancer cell lines. While the model has been successfully applied to melanoma, glioblastoma, colorectal, and head and neck cancers, breast cancer remains one of the most studied tumor types in CAM systems due to its strong angiogenic profile and rapid growth [22]. Here, we outline the entire workflow from cell preparation and embryo windowing to tumor implantation, monitoring, histological processing, and downstream analysis. Using MDA-MB-231 and MCF-7 cells, this protocol enables the formation of measurable tumors within 72 h, allowing researchers to quickly assess tumor morphology and angiogenic characteristics. The model is also compatible with drug testing, gene silencing, overexpression, extracellular vesicle delivery, and co-culture with stromal or immune components [23,24].

Despite its advantages, the CAM system has limitations. Because embryonic development progresses rapidly, experiments must be completed within a limited timeframe, which prevents long-term studies of metastasis or tumor dormancy. Additionally, the immature immune system of the embryo hinders the evaluation of intricate adaptive immune responses. The relatively small size of the embryo and tumors may also restrict surgical manipulation and imaging resolution [25–27]. However, ongoing technical innovations (including intravital microscopy, micro-CT imaging, transcriptomic profiling, and nanoparticle tracking) are expanding the versatility of CAM-based approaches [23,28].

Overall, the CAM model provides an ethical, economical, and biologically relevant alternative for preclinical breast cancer research. Studies have demonstrated that CAM-derived tumors preserve histological architecture, proliferative markers, and angiogenic profiles closely resembling those observed in murine models and human patient specimens [23,29]. Consequently, this platform facilitates rapid, high-throughput in vivo experimentation while significantly reducing the use of mammalian animals [30]. Although the CAM model offers several experimental advantages, it also has intrinsic biological limitations that need to be considered. The experimental timeframe is limited, typically concluding around day 14 of embryonic development, which restricts long-term investigations of tumor growth, metastasis, and delayed therapeutic responses [31]. Furthermore, the chick embryo’s immunologically immature environment does not fully replicate the complex tumor–immune interactions observed in adult organisms, limiting its use for immunotherapy studies [32]. Therefore, the CAM assay should be viewed as a complementary preclinical platform rather than a substitute for established murine models. This protocol provides a practical, highly adaptable tool for studying tumor biology, evaluating therapeutic interventions, and connecting in vitro systems with mammalian models. While the primary focus of this study is breast cancer, the CAM model has also been successfully used to generate tumors from other cancer cell lines in our laboratory. These include neuroblastoma [SH-SY5Y, Be(2)-C], cervical cancer (HeLa, SiHa), lung cancer (H1299), and melanoma (A-375). This demonstrates the versatility of the CAM system and its potential applicability to a variety of cancer types.