Breast cancer refers to the most common malignancy in women. Its incidence rate ranks first among female malignancies, and it has been elevating (1). With the rise in the new patients and the potential variations in the molecular phenotype and genotype of breast cancer, a more efficient culture system maintaining the biological characteristics of breast cancer in vivo is critical to clinical research and transformation. Traditional breast disease models consist of cells and PDX. The former is simple and easy to adjust culture conditions, however, there are various cross-contamination problems, and the genetic information and tumor heterogeneity of patients are difficult to retain in the passage process; the latter can act as a gold standard for revelant tumor studies. To a certain extent, it can indicate the occurance and evolution of patients’ tumors, whereas its time-consuming, low success rate, high cost and ethical issues. Thus, novel breast cancer research models should be developed essentially. Organoids exploit cells exhibiting different differentiation potentials (e.g., embryonic stem cells, pluripotent stem cells, and tumor cells, and other sources of cells; they are combined with in vitro three-dimensional culture technology and introduce extracellular matrix analogs and cytokines to maintain stem cell proliferation and differentiation, as well as other components. Thus, the cells grow in a culture system close to the microenvironment of the body, and a cell tissue structure is formed similar to the structure of the source organ in the body. As compared with in vitro culture models (e.g., conventional cell line culture, human xenograft, and simple biological materials), tissues formed by organoid culture systems can achieve relatively complex structures and functions, currently the ideal in vitro culture in vitro. system. Moreover, organoid culture requires less tissue and exhibits a high success rate and avoids ethical disputes and species differences.

Tumor-like organ culture refers to a cross-concept accompanying the development of organoids and oncology, namely, tumor cells form microstructures similar to the source tumor tissue structure and gene expression lineage under appropriate culturing conditions, which can reveal the biology of the source tumor tissue. It acts as an effective model to study tumors and high-throughput screening drugs in vitro. At present, tumors (e.g., colorectal cancer, pancreatic cancer, liver cancer, prostate cancer and breast cancer) have achieved relatively mature organoid culture systems (2-6). To be specific, the breast cancer culture system usually introduces various cell growth factors to the matrigel to simulate the in vivo microenvironment of breast cancer cells; thus, breast cancer stem cells can maintain their original differentiation ability and long-term subculture in vitro. It has become a feasible biological model for in vitro studies on the occurrence and development of breast cancer, and it has been extensively employed in individualized treatment of breast cancer patients.

Cells exhibit a three-dimensional growth pattern in the body. Under conventional two-dimensional planar culture conditions, the cells are gradually flattened, losing some of their biological characteristics in the body; Thus, a low degree of reduction of the results of in vitro experiments is caused. Three-dimensional cultivation technology has been progressively developed. In 1907, Wilson (7) demonstrated that mechanically dissociated sponge cells can be recombined to form a complete organism. In 1977, Emerman and Pitelka (8) initially inoculated cells obtained from mouse breast tissue on a three-dimensional matrix surface; Besides, they added insulin, hydrocortisone and prolactin to differentiate isolated breast cells into acinar structures and the source. The tissues were compared and reported to exhibit a similar structure to that of mammary acinus, which was a precedent for “breast organoid” culture. In 1992, Petersen et al. (9) initially termed the use of breast cells to form microstructures as “organoids”. With the advancement of stem cell biology, people have come to realize that breast cancer stem cells are the key to form organoids. Therefore, some researchers have attempted to add transforming growth factor α (TGF-α) and fibroblast growth factor 7 (FGF7) to the medium to activate the MAPK signaling pathway on the breast, promote the proliferation and differentiation of breast cancer stem cells, sa well as forming a breast branch phenotype (10). Subsequently, Sato et al. (11) added Wnt and R-Sponding1, which induced the proliferation of Lgr5+ stem cells and stimulated β-catenin and Tcf4 to be up-regulated, as a result, the stem cell activity of the tumor cells was activated, as well as in vitro intestinal organoid culture. In 2012, de Visser et al. (12) reported that breast Lgr5+ cells as breast stem cells can long exist during the passage; under the action of cytokines, breast stem cells were found to be arranged to form a normal duct structure similar to breast. According to this theory, Sachs et al. (13) added the Neuregulin1 nuclear factor that facilitates self-proliferation and differentiation of breast cancer stem cells, as well as small molecule inhibitors A83-01 and SB202190 that inhibit apoptosis of breast cancer stem cells based on the original organ culture program. Therefore, the synthetic medium for breast cancer organoids was used to develop the system. These researchers cultivated 95 breast cancer organoids from 155 breast cancer surgical tissues and successfully established the first breast cancer organoid bank. The breast cancer-like organoids in the library cover a range of pathological types, molecular phenotypes and genotypes of breast cancer, which were adopted for high-throughput screening of drugs and editing of mutated genes for precision medical purposes. Over the past few years, with the optimization of digestion steps and the continuous improvement of experimental techniques (e.g., screening of breast cancer stem cells), the success rate of breast cancer organoid culture has been elevated to 87.5%, arousing a wave of study and application of breast cancer organoids (Fig. 1) (14).

Breast organoids act as vital branches of tumor organoids, whereas the construction of the culture system has long been explored and optimized to form an organoid culture system that exhibits common characteristics with other tumor organoids as well as targets breast cancer cell characteristics. The construction of breast cancer organoids primarily consists of three elements (i.e., breast cancer cells, stroma and cytokines).

Organoid functions effectively reflect tumor heterogeneity, and gene sequencing of organoids formed in culture reveals that most organoid functions can display genes of source tissues well, even after prolonged passage, organoids and primary tumors are mutated. The similarity of characteristics and gene profile expression can reach 60% or more (28). Moreover, breast cancer organoids present all subtypes of solid tumors, which can be used as an effective in vitro model for this study’s use of breast cancer organoids for high-throughput screening and personalized treatment, in combination with gene editing technology and the use of normal tissue-derived organoids for drug toxicity and side effects detection (Fig. 3).

The in vitro culture of breast cancer organoids creates the optimal model for tumor biology at the current stage, and its application in the screening of novel tumor drugs has significantly elevated the rate of clinical transformation. At present, the organoids generally take 4∼6 weeks from cultivation to testing, which are used to evaluate clinical efficacy. For patients who are urgent need of treatment, acquiring to the relating consequences of high-throughput screening of drugs, doctors are usually followed to guidelines and empirical plans, which leads to excessive thera-pies. Though breast cancer organoids can better reflect the patient’s disease situation, we also note that when the culture system is being built, complete technology is still lacked to simultaneously connect both organoids and tumor microenvironment (37). Heterogeneity remains different from solid tumors. Moreover, More critical aspects affecting yield include whether the source tissue is healthy or tumor tissue, as well as the number of stem cells obtained after enzyme digestion. Healthy tissue organoids achieve the success rate of nearly 20.83%, while that of cancer tissue reaches 87.5% (5). at present, the breast cancer-like organs that we cultivate from patients are primarily surgical tissue or puncture, whereas the tissues in different locations cover different numbers of breast cancer cells, and some tissues are liquefied layer and over-differentiated from the outside to the inside layer, tumor cell layer, and necrotic layer in the center of the tumor, in the sampling process, it is difficult to ensure that all are tumor tissues, and the number of cells obtained after enzyme digestion is different, which affects the survival rate of organoid culture. Moreover, matrigel and cytokines have different concentrations in different laboratories, which is not conducive to a unified evaluation of the formation rate of breast cancer organ cultures (38-40). To be specific, it lacks mesenchymal cells, so it has no nerve and vascular system, and it has great differences with solid tumors in constituting the tumor microenvironment.

With the advent of advanced imaging technologies (e.g., optical metabolic imaging (OMI), the time of using organoids to determine the effectiveness of treatment options can be reduced to 72 h, and the invalid scheme of organoids can be more accurately identified, so as to avoid patients having received excessive treatment (41).

In response to the problem of low cell content in tumor tissue, we can use multi-point biopsy. For experimental differences caused by different product batches and inconsistent schemes in the culture system, we can adjust matrigel by adding collagen protein composition, so the same substrate is applied in independent experiments to ensure the repeatability of the experiment (14). Moreover, Besides, recent studies have continuously pointed out that we can solve the shortcomings of single organoid system components by adding other cells to form a “co-culture” system. Dijkstra et al. (36) employed the patient’s tumor-like organs to activate T lymphocytes to amplify the patient’s immune function. The results of their experiment provide novel insights into the study on breast cancer immuno-therapy. Moreover, the continuous advancement of microfluidic technology and new equipment will further expand the application scenarios and enhance the efficiency of tumor organoids.

In the conventional cell models, oxygen, nutrients, metabolities and signaling molecules can be used infinitely, however, the cancer cells in the body have limited utilization of various substances due to the three-dimensional structure of the tumor mass, consequently, causing various degrees of influence on the expression of cancer cell molecules and biological behaviors. Besides, patient-derived xenografts cannot be screened for high-throughput of drugs. Compared with the above models, organoids are easy to be cultured, and involve the advantages of cell models and PDX. Therefore, it has become the first choice for emerging breast cancer in a short time of research models in vitro.

Although breast cancer organoids are widely used, there are still apparent deficiencies, such as: lack of mesenchymal cells, nerves, blood vessels, and immune system. The model that is constituted by single breast cancer is still quite different from the solid tumor in terms of tumor heterogeneity. The relating studies have continuously pointed out that we can form a “co-culture” system by adding corresponding cell components to restore the “tumor microenvironment” to a certain extent, for instance: lymphocytes andfibroblasts, to achieve the purpose of highly reproducing solid tumors in vitro.

Breast cancer organoids can not only explore the role of tumor stem cells, so that we can study the occurrence, development and driving factors of tumors from normal cells, invasive cancers to metastatic cancers; but used to the chemotherapy, combined with gene editing technology to interfere with the progress of breast cancer, assessment of drug toxicity and immunotherapy autologous transplan-tation. Organoids can be generated from surgical samples, as well as from endoscopic fine needle aspiration samples. Thus, the model is capable of summarizing various disease stages and clinical conditions. Moreover, organoids can be generated from a small amount of tissue and completed efficiently, which can be employed for drug testing and assessment of potential diagnostic biomarkers; they can also verify multiple drivers of tumor progression in vitro. Overall, it is considered that as fueled by the advancement of technology and research in relevant fields, the application of breast cancer organs will be increasingly widespread, bringing invaluable value to more clinical treatment and academic study.