Breast cancer (BC) has replaced lung cancer as the most frequently diagnosed cancer in women globally. The International Agency for Research on Cancer (IARC) estimates that approximately 19.3 million new cancer cases and 9.7 million cancer-related deaths occurred globally in 2022 (1). Metastatic progression remains a primary contributor to mortality in breast cancer patients, frequently demonstrating resistance to conventional therapies. The American Cancer Society and the National Cancer Institute reported 313,510 new breast cancer in 2024, with approximately 42,780 resulting in mortality (2). Triple-negative breast cancer (TNBC), characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression, represents the most aggressive breast cancer subtype. Accounting for 10–20% of all breast cancer cases (3), TNBC exhibits aggressive features including tumor heterogeneity, rapid metastasis to distant organs (particularly the brain, lungs, and bone) (4), and a high recurrence rate. Unlike hormone receptor-positive breast cancer (HR+ BC)—the most common subtype (5)—TNBC does not respond to endocrine or HER2-targeted therapies, and its treatment primarily relies on chemotherapy (6). In contrast, HR+ BC typically progresses more slowly, driven by hormonal signaling pathways (5), and benefits from well-established targeted treatment options such as endocrine therapies (7). This biological divergence extends to preclinical modeling: The scarcity of clear therapeutic targets in TNBC necessitates high-throughput drug screening using zebrafish models (8), whereas HR+ BC often fails to develop a typical phenotype in zebrafish. Furthermore, modeling HR+ BC resistance mechanisms (e.g., endocrine therapy resistance)—which involve significantly more complex host-tumor interactions (9)—is inherently limited in zebrafish systems. These characteristics pose significant therapeutic challenges. Zebrafish models have emerged as valuable tools for BC (especially TNBC) research, enabling the identification of genes underlying invasive metastasis and tumor angiogenesis, while facilitating the development of targeted therapies.

Over the past two decades, zebrafish (Danio rerio) has developed into an indispensable model organism for cancer research. Through the construction of various transgenic lines, its application value has been significantly increased, especially in the real-time monitoring of tumor angiogenesis and the study of tumor immune microenvironment, which demonstrates its unique advantages. The zebrafish embryonic vascular system is highly conserved evolutionarily, and its functional circulatory system can be constructed within 24 hours after fertilization (10). The optically transparent nature of embryonic tissues provides an ideal window for dynamic observation of developmental biological processes and pathological changes. By fluorescently labeling vascular endothelial cells, researchers can observe the early stages of tumorigenesis with high spatial and temporal resolution, tracking the behavior of neovascularization (10). Conversely, a dual-fluorescent-labeling system for tumor cells and host cells enables precise dynamic analysis at the single-cell level during metastasis (11). In addition, the high fecundity of zebrafish, the in vitro developmental characteristics of the embryo (10), and the tiny size of the zebrafish provide significant convenience for experimental manipulation. Together, these features enable the model to maintain a highly developmentally synchronized population of embryos in a limited space. Based on its cost-effectiveness, developmental homogeneity and miniaturization, the zebrafish system has become an ideal platform for high-throughput screening for potency assessment and toxicity testing of anticancer drugs. In this review, the multidimensional application of the zebrafish model in BC research will be systematically elaborated, focusing on its research progress in the areas of metastasis and invasion mechanism, tumor angiogenesis regulation and preclinical drug screening.

Conventionally, genetically engineered murine (GEM) models and human cancer cell-derived xenograft models in immunodeficient strains (e.g., NOD-SCID) (12) have served as gold-standard platforms for preclinical evaluation of anticancer therapy safety and efficacy. Nevertheless, murine models present limitations (12) including prolonged experimental timelines and technical constraints that hinder high-throughput drug screening and comprehensive toxicity profiling. The zebrafish model has consequently emerged as a transformative platform in cancer research, demonstrating unique capabilities in revolutionizing precision oncology and advancing personalized medicine approaches.

Zebrafish cancer models are constructed using three main strategies: genetic engineering modification, chemically induced carcinogenesis and xenograft models. In the genetic engineering strategy, researchers have induced tumorigenesis by targeting and integrating oncogenic gene expression vectors or using targeted knockdown of tumor suppressor genes mediated by the CRISPR/Cas9 system (13). However, this model system has translational medicine limitations, mainly reflected in the lack of BRCA1 direct homologous gene in the zebrafish genome and only 22% amino acid sequence identity between its brac2 gene and human BRCA2 (14). To break through this limitation, the research team developed a morpholino oligonucleotide-mediated BRCA2 gene silencing model and a CRISPR knockout line, and these model systems provide key experimental evidence for resolving the DNA damage repair mechanism during breast cancer development (15). Chemically induced carcinogenesis allows for the establishment of mechanistically well-defined tumor models in multiple organ systems such as the liver, pancreas, gastrointestinal tract, epidermis, musculoskeletal system, vasculature, and testis through the exposure of specific carcinogens (16–20). The lack of mammary organs to induce breast cancer in zebrafish can be compensated for by xenotransplantation.

Xenotransplantation serves as the primary methodology for developing zebrafish breast cancer models ( Figure 1 ), involving engraftment of foreign tissues across species barriers. Embryonic xenotransplantation exploits the immunologically privileged window prior to 28 days post-fertilization, when adaptive immunity remains undeveloped (21), effectively preventing graft rejection. The miniature scale of zebrafish embryos permits tumor engraftment with as few as hundreds of cells, enabling single-cell resolution tracking of xenograft dynamics via intravital imaging. This low cellular inoculum closely recapitulates early tumorigenic events (22), mirroring initial stages of human cancer progression. This unique attribute makes zebrafish models particularly adept at modeling rare cell populations, including cancer stem cells (23–25) and circulating tumor cells (26, 27), with unprecedented spatiotemporal resolution. Cell dosage represents a critical experimental parameter requiring optimization in xenotransplantation protocols. Current protocols use cancer cell injections of 25-2,000 cells, with most research teams favoring injections of 50-200 (glioblastoma) (28) or 100-200 (colorectal cancer) (29) cells. We summarized the number of cells commonly used for different injection sites for most of the studies in this review as 200–300 for the yolk sac, 400 for the PVS, and 300–500 for the Duct of Cuvier. Vanda Póvoa et al. (30) used a zebrafish xenograft model to study the implantation efficiency of multiple human mammary glands. Breast cancer cells Hs578T showed high implantation rates (~95% implantation rate) whereas the implantation rate of MDA-MB-468 was less proliferative and less apoptotic. In actual studies, the number of cells implanted in successful xenografts varies by cell line, and optimization of each cell line is usually required (28).

Zebrafish xenograft models provide versatile implantation sites including: yolk sac (23–25), perivitelline space (PVS), common cardinal vein (CCV) (31, 32), and hindbrain ventricle (HBV). The yolk sac provides a lipid-rich microenvironment (cholesterol, phosphatidylcholine, triglycerides) (33) that enables high-contrast visualization of fluorescently labeled cells through standardized protocols. The yolk sac is the site of choice for the study of survival, cell division, proliferation and migration (34). This lipid matrix not only sustains xenograft viability but also potentiates tumor proliferation through nutrient enrichment. The perivitelline space (PVS), located between the periderm and the yolk syncytial layer, serves as a key site for xenografting cancer cells in zebrafish embryos. This model enables real-time study of neoangiogenesis dynamics and metastatic cascades (35, 36), as well as quantitative assessment of primary tumor regression in response to therapies (37, 38). Despite its experimental advantages, PVS microinjection requires advanced technical proficiency compared to yolk sac procedures (39). Common cardinal vein (CCV) or duct of Cuvier injection enables direct intravascular delivery (40) of tumor cells, facilitating real-time monitoring of metastatic processes (41). This technique demands exceptional microsurgical expertise, particularly in adult zebrafish models with reduced vascular lumen diameters (42). The hindbrain ventricle (HBV), characterized by dense vascularization, provides an optimal platform for investigating hematogenous metastasis mechanisms (43). However, the procedure demands submicron-level spatial precision during microinjection, as technical inaccuracies may induce structural damage to adjacent neural architectures. Optimal site selection requires careful consideration of microenvironmental relevance versus technical feasibility, depending on specific research objectives (e.g., metastatic mechanisms vs. high-throughput drug screening).

Xenografts originate from two primary sources: established cancer cell lines or patient-derived specimens (PDX, Patient-Derived Xenografts) obtained through surgical resection or biopsy. Standard preclinical workflows predominantly employ commercially available immortalized cell lines for tumor model establishment. The zebrafish xenograft literature documents multiple BC cell line options, necessitating careful selection based on specific research objectives. TNBC studies demonstrate preferential use of MDA-MB-231 (58% prevalence) (24, 25), followed by MDA-MB-468 (22%) (44), with BT549, CAL-148, HCC38, HCC70, MDA-MB-436, and MDA-MB-453 collectively accounting for <20% utilization. However, chronic 2D culture on plastic substrates coupled with repeated freeze-thaw cycles drives genetic drift and phenotypic convergence, compromising clinical translatability of findings (41). These limitations underscore the necessity of PDX models that preserve original tumor heterogeneity and drug response profiles. Contemporary precision oncology prioritizes individualized therapeutic strategies over conventional subtype classification, driven by recognition of intra-/inter-tumoral heterogeneity. Emerging PDX-zebrafish platforms now enable in vivo assessment of tumor progression dynamics and drug sensitivity profiles, directly informing personalized treatment regimens. Innovative approaches incorporating three-dimensional (3D) spheroid models (45) further enhance physiological relevance. Ambrosio et al. (45) demonstrated that high-glucose (HG) conditions potentiate mammary adipose tissue-derived mesenchymal stromal/stem cell (MAT-MSC)-mediated BC cell invasion through zebrafish xenograft studies. Zebrafish xenografts of HG-primed MCF7/MAT-MSC spheroids exhibited significantly increased metastatic dissemination, confirming microenvironment-mediated pro-invasive crosstalk. These advances position zebrafish models as transformative tools in precision oncology research.

The standard workflow for utilizing zebrafish models in breast cancer (BC) research involves dissociating tumor cells into single-cell suspensions, labeling them with CM-Dil (red fluorescence) (11), and microinjecting the labeled cells into target sites (e.g., yolk sac, perivitelline space) of anesthetized Tg(fli1a:EGFP) embryos (46). Tumor burden is quantified pre- and post-treatment via semi-automated fluorescence intensity segmentation (i.e., FIJI/ImageJ) of confocal microscopy images (47). Metastatic progression is assessed through 3D reconstruction of Z-stacked image series and quantification of the number of disseminated tumor cells to the caudal hematopoietic plexus (48–50). Key quantitative endpoints include: (1) Tumor volume (mm³), calculated from XYZ-axis measurements; (2) Metastatic index (scored 0–4); (3) Angiogenic sprouting density (vessels/mm²); (4) Kaplan-Meier survival analysis with median survival duration. The zebrafish model’s high-throughput compatibility and optical transparency establish it as a premier platform for in vivo investigation of metastatic cascades, neovascular dynamics, and preclinical drug efficacy.

There are also some limitations to the use of the zebrafish model in breast cancer research. Although zebrafish is homologous to many mammalian pathways and organ systems, it does lack several mammalian tissues (lung, breast, and prostate) (105), but may be able to “add” desired cells or growth signals to allow normal growth cues associated with in situ injections (34). For example, transgenic fish (15) expressing human growth factors, receptors and/or cytokines could be generated to enhance implantation and tumor growth.

There are inherent temperature differences between zebrafish and human systems that may influence tumor cell proliferation rates and zebrafish physiology. Zebrafish embryos develop optimally at 27°C-28°C (39, 106), whereas human tumor cells thrive at 37°C (107), a temperature required for mammalian cell viability. To address this discrepancy, xenograft experiments methodologically implement a compromise temperature that balances optimal growth conditions in the host with the metabolic requirements of the tumor cells. This temperature must be sufficiently high to sustain proliferation of xenografted cells while maintaining embryo viability and preventing developmental defects. In recent years, the standard temperature for performing xenograft assays in the literature has been increased to 34°C at the expense of shortening the incubation time to 3 to 6 days post-injection (dpi) (39). Incubation temperatures and incubation times remain unbalanced. In an experimental design comparing incubation temperatures (28, 34 and 36°C), Pablo et al. (108) systematically evaluated morphological abnormalities and developmental effects in injected and control embryos at multiple time points. Their results showed that incubation at 36°C for 48 h was most favorable for xenograft survival, and no significant morphological or metabolic disturbances were observed in either host embryos or tumor cells, allowing them to proliferate near their respective optimal temperatures. Protocols are adapted to tumor cell requirements: while 34°C (39) remains widely used for viability balance, temperature escalation (35-36°C) (109, 110) may be implemented for short-term drug efficacy assays with mortality controls.

Zebrafish xenograft models faithfully replicate conserved metastatic mechanisms documented in murine models, including evolutionarily maintained signaling pathways (e.g., HTR2A/2B-mediated EMT induction (111)) and prototypical metastatic cascades such as hematogenous dissemination. However, phylogenetic divergences in immunoinhibitory checkpoint regulation and organotropic metastasis predilection compel the implementation of complementary methodology combining both model organisms to optimize experimental validity and translational relevance (12, 51). Paul et al. (112) demonstrated that cell line pairs that preferentially target bone marrow and brain niches in mice show similar targeting in the larval fish by 5 dpi following injection to the circulatory system. The authors (112) validated the conservation of organotropism across species by injecting zebrafish with mouse-derived 4T1Br4 (brain-homing) and 4T1BM2 (bone marrow-tropic) cells, and observed metastatic targeting patterns that aligned with murine experimental data. The optical transparency of zebrafish embryos enables real-time visualization of tumor cell-microenvironment interactions, a technical challenge in opaque mammalian models. Fluorescent lineage tracing in zebrafish has enabled precise tracking of metastatic processes, including vascular migration and micrometastatic colonization (51). Whereas zebrafish larval models enable rapid metastasis assays (days-scale), murine models require extended observation periods (weeks to months) (113). Crucially, murine models maintain indispensable translational relevance by reconstituting humanized adaptive immune microenvironments and supporting evolutionarily conserved myeloid differentiation pathways that generate metastasis-associated macrophages (MAMs) (114), a process incompletely modeled in zebrafish. While zebrafish excel in modeling early metastatic dissemination, murine models remain superior for studying organ-specific metastases [e.g., bone metastasis (115)] and immune microenvironment dynamics, as evidenced by studies of breast cancer bone metastasis employing transgenic mouse models [e.g., MMTV-PyMT (116)]. Recent methodological advances now enable implantation of patient-derived tumor samples in zebrafish, significantly improving human TME complexity recapitulation and demonstrating high treatment response predictability (117). As demonstrated by the results of Mendes et al. (118), the zebrafish TNBC xenograft model effectively differentiates between sensitivity to anthracyclines and paclitaxel, and the efficacy of combination chemotherapy is superior to that of single-agent treatment. The BC zAvatars model can be efficiently established (including early and late stage tumors), retains key biomarkers (e.g., ER), and its therapeutic response is highly concordant with the patients’ clinical outcomes (18/18 perfect match). The model also accurately reflects tumor staging and micrometastatic characteristics, providing a basis for personalized decisions on adjuvant treatment intensity and monitoring regimens. Nevertheless, key limitations persist (51).

Furthermore, while implanting a small number of transplanted cells (around 100–200 per embryo) can ensure successful engraftment, it may not necessarily include cancer-driving stem cells, which are indicative of the genetic diversity present in human tumors. Another disadvantage of embryo xenotransplantation is that the developing embryo provides a different biological microenvironment than the adult human body, and therefore the transplanted cancer cells are surrounded by new signals, which may have implications for cancer biology. The delayed maturation of zebrafish adaptive immunity imposes temporal limitations on longitudinal studies of tumor immunobiology or therapeutic response kinetics. While the immunodeficient larval stage facilitates xenotransplantation, it precludes investigation of adaptive immunity’s dual roles in tumor suppression and therapy potentiation (34). Although zebrafish are useful for screening TME-centered chemicals, mammalian models remain necessary to develop these findings into drugs with favorable pharmacokinetic/pharmacodynamic properties (119). From a technical point of view, most studies have been developed by using membrane and cytoplasmic dyes to color tumor cells. These dyes do not correctly distinguish between dead and living cells, leading to biased overestimation of tumor mass and growth. As the zebrafish model becomes more widely used, the limitations will be dissolved.

Zebrafish have emerged as a valuable in vivo model for human disease research, particularly in oncology, due to their unique biological advantages. Zebrafish xenotransplantation represents a powerful experimental strategy that enables cost-efficient, high-throughput investigation of human malignancies, overcoming the limitations of small sample sizes encountered in mammalian models. Nevertheless, critical knowledge gaps persist, including the development of immunocompromised zebrafish strains resistant to tumor rejection and the long-term viability of xenografts. These gaps underscore the urgent need for further research, especially given the clinical challenges faced by patients with TNBC. Patients with TNBC—who derive limited benefit from anti-HER2 or endocrine therapies, exhibit poor five-year survival rates and elevated early recurrence risk. Elucidating the molecular mechanisms underlying breast cancer progression through systematic investigation is essential for identifying novel therapeutic targets to improve clinical outcomes in this high-risk patient population. Zebrafish have emerged as an indispensable tool in this endeavor, with accumulating evidence reviewed herein supporting the transformative potential of zebrafish xenografts in both fundamental breast cancer research and therapeutic development. The advent of 3D multicellular co-culture platforms enables precise dissection of tumor-stroma crosstalk. Integrating these systems with zebrafish xenografts could map spatial-temporal interactions between malignant cells and specific TME components. Future efforts should primarily concentrate on standardizing zebrafish model protocols and validating their clinical translatability. This can be achieved through strategies like integrating organoid co-culture systems, ultimately aiming to enhance predictive accuracy and ensure a smoother transition from bench to bedside. Consequently, leveraging zebrafish models in high-throughput drug screening pipelines may accelerate the translation of preclinical discoveries into targeted therapies for breast cancer patients. Taken together, these advancements position zebrafish as a cornerstone model in precision oncology and personalized medicine paradigms.