
==== Front
Chin Med J (Engl)
Chin Med J (Engl)
CM9
Chinese Medical Journal
0366-6999
2542-5641
Lippincott Williams & Wilkins Hagerstown, MD

38185826
CMJ-2023-747
10.1097/CM9.0000000000002889
00003
3
Review Article
Progress and perspective of organoid technology in breast cancer research
Huang Changsheng
Jin Hongyan
Li Jinjiao
Ji Yuanyuan
Department of Obstetrics and Gynecology, Peking University First Hospital, Beijing 100034, China.
Correspondence to: Associated Prof. Hongyan Jin, Department of Obstetrics and Gynecology, Peking University First Hospital, Beijing 100034, China E-Mail: maggijhy@163.com
08 1 2024
20 9 2024
137 18 21572168
27 3 2023
Copyright © 2024 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0

Abstract

Breast cancer, a malignant tumor with a high incidence in women, lacks in vitro research models that can represent the biological functions of breast tumors in vivo. As a new biological tool, the organoid model has unique advantages over traditional methods, such as cell culture and patient-derived xenografts. Combining organoids with other emerging technologies, such as gene engineering and microfluidic chip technology, provides an effective method to compensate for the deficiencies in organoid models of breast cancer in vivo. The emergence of breast cancer organoids has provided new tools and research directions in precision medicine, and drug research. In this review, we summarized the merits and demerits of organoids compared to traditional biological models, explored the latest developments in the combination of new technologies and organoid models, and discussed the construction methods and application prospects of different breast cancer organoid models.

Keywords:

Organoids
Breast cancer
Preclinical model
Bioengineering
OPEN-ACCESSTRUE
==== Body
pmcIntroduction

Since 2020, breast cancer has surpassed lung cancer as the most common malignancy worldwide, and is one of the most common causes of death among women.[1] In recent years, the incidence of breast cancer in our country has increased annually and the age of affected women has tended to decrease. In China, breast cancer is most common among people in their 40s and 50s, with an average age of 48–49 years, more than 10 years younger than that reported in Western countries.[2] Although the progesterone receptor (PR), estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER2) are commonly used to type and to guide the treatment of the breast cancer, there are hundreds of genes related to breast cancer.[3] The diversity of mutated genes and individual differences among patients are the reasons for the failure of breast cancer treatment. The pathogenesis of breast cancer has not been fully elucidated and the risk factors affecting the occurrence of breast cancer are varied.[4] Therefore, an accurate treatment for patients with breast cancer has become a popular research topic.

The selection of an appropriate biological model is the basis for studying the pathogenesis of breast cancer. Common biological models include two-dimensional (2D) cell culture models, patient-derived xenograft (PDX) models, and organotypic tissue slice cultures (OTSCs). The 2D model is simple, but cannot fully reflect the reactions of the human body. Although PDX models preserve the biological and molecular characteristics of patient tumors well, their shortcomings, such as high experimental costs and long experimental periods, limit their large-scale application. OTSCs have both advantages; however, they cannot be preserved for a long time and do not reflect the regenerative potential of the tumor. Therefore, there is an urgent need for a model that can simulate the biological functions of tumors in the human body.

Recently, emerging organoids have become a focus of research. Organoids are three-dimensional (3D) structures with multicellular complexity and some degrees of tissue structure and function. They are characterized by the stable proliferation and fast construction speed, can be frozen to form biobanks, and can be derived from patient tissues. Research on breast cancer organoids is rapidly developing. Embryonic and induced pluripotent stem cells (iPSCs) can be used to culture breast cancer organoids to study the growth and development of breast cancer and disease occurrence.[5]

In contrast, breast cancer organoids cultured directly with tumor cells maintain the original breast cancer genes and histological characteristics.[6] This interdisciplinary combination also greatly expands the application of organoids, and organoids modified by genetic engineering can be used for the experimental research and drug development; to a certain extent, microfluidic chip technology can compensate the lack of vascular systems in organoids. Breast cancer organoids lack a unified construction method, which is an important reason for the low reproducibility of organoid experiments. The lack of participation of the immune system is also a key factor that makes it difficult to accurately reflect the human responses. In this study, the characteristics of organoids and traditional biological models were compared, the advantages and disadvantages of the current organoid models were expounded, and the construction methods and application prospects of different breast organoid models were discussed.

Merits and Demerits of Different Biological Models

The most basic and crucial step in disease research is the construction of biological models. Common biological models for breast cancer include 2D cell culture, PDX, OTSC, and organoid models. A schematic diagram of the four methods for building biological models is shown in Figure 1.

Figure 1 Construction of the four biological models. The schemes show four ways to construct biological models. (A) 2D cell culture model uses scissors or other tools to separate tissues, add tissues into digestive juices to get single cells, and then transplant the single cells into the appropriate culture medium to get a monolayer cell structure. (B) PDX model isolates and amplifies tumor cells, and then the tumor cells are transplanted into immunodeficient mice, and the mice are cultured to produce tumor mice. (C) Organotypic tissue slice culture is to obtain the appropriate size of organ tissues, freeze or paraffin-wrap tissues, use the microtome to get tissue sections, and put them into a culture medium. (D) Organoid model uses the self-organizing function of somatic stem cells or human-induced stem cells, adding specific growth factors to induce and differentiate into organoids. 2D: Two-dimensional; PDX: Patient-derived xenograft.

2D cell culture model

2D cell culture model involves dipping stem cells into a culture medium and allowing them to grow along a 2D surface on the surface of the dish. 2D cell culture models have been a major tool in biological research for decades, ranging from basic research to stem cell and cancer research, and regenerative medicine. Almost all of our knowledge about basic biological processes comes from primary cells and established cell lines grown on 2D surfaces.[7] The same is true in breast cancer. 2D cell cultures have provided simple methods to construct biological models, convenient experimental operation for control variables, easier observation of changes in cells and the extracellular environment, lower experimental costs, and shorter construction time compared with other biological models.[8] However, the shortcomings of the 2D cell culture model are obvious, and the results of the 2D cell culture model are often inconsistent with the results of in vivo and clinical studies, or even directly contradictory. Peng et al[9] found that tumor cells cultured in a 2D cell culture model grew faster and had stronger variability than human tumors in the body. The result may be different owing to the lack of immunosuppression in the 2D cell culture models.[10] In the early stages of clinical trials, approximately 90% of the drugs were effective in the 2D cell culture model for tumor cell therapy, failed or did not reach the expected efficacy in the pre-clinical test, and 5% of the drugs failed because of various factors such as costs or experimental time. Less than 5% of candidate cancer drugs have been confirmed in clinical trials.[11] Some researchers believe that this may be related to the lack of cell-matrix and cell-cell contacts in 2D cell culture models. It is also believed that there are problems, such as poor biological stability and easy gene mutations, in the 2D cell culture model.[7]

PDX model

The PDX model is developed by transplanting cancer cells or tissues from a patient's primary tumor into immunodeficient mice, causing them to grow into transplanted tumors. PDX models have been widely used as in vivo tumor models for studying cancer metastasis and drug screening.[12] Compared with human cancer cell lines transplanted into mice, the PDX model can more accurately show the characteristics of human tumors.[13] Compared to traditional models, such as cell culture, mouse, and human tumor xenograft models, the PDX model better preserves the biological and molecular characteristics and heterogeneity of patient tumors.[10] Therefore, these models have been used in cancer research and drug testing.[14] However, this model has certain shortcomings. First, compared with other models, the PDX model is expensive and the experimental period is long. Second, the current PDX model lacks the most important immune function in the human body and cannot completely simulate the tumor immune response.[11,15] These are the critical problems to be solved by applying the PDX model in cancer research.[14]

OTSC model

The OTSC model refers to tissue slices prepared from solid tissue using a tissue microtome that preserves tissue viability and includes all cell types in the original tissue, allowing the preservation of intercellular and cell–matrix interactions. Tissue slices were first used to study the tumor tissue in culture early in the last century. However, the original model had significant limitations, as the slices were cut by hand with a razor blade and were therefore irregular in size, which, coupled with poor incubation conditions, led to a rapid loss of cell viability. With the development of special tissue microtomes and remarkable improvements in sectioning and hatching techniques, more attention has been paid to tissue section cultures.[16] Precision-cut tissue slices are 3D tissue explants that are usually derived from various human or animal organs and can be cultured outside the body for a long time. Unlike monolayer cell cultures, precision-cut tissue slices preserve the anatomical structure of the organ, the cells in their original tissue matrix structure, and other organ-specific features, such as metabolic activity, tissue homeostasis, and immunological functions.[17] However, a significant weakness of OTSC is that it is extremely difficult to culture biopsies for a long time because of the diversity of cell types in tissue slices, each requiring a specific medium, making it difficult to find a suitable medium component for all cells.[18]

Organoid model

Organoids are 3D structures with multicellular complexity and some degrees of organizational structure and function. The general meaning of organoids is "similar to organs". Organoids have been defined as 3D structures with specific functions and structures of organs developed from stem cells or organ progenitor cells and formed by self-organization.[19] Much research has been conducted on the obtention of human tumors based on organoids, including those of the colon, pancreas, prostate, breast, stomach, lung, esophagus, bladder, ovary, kidney, and liver. [20,21] As a disease model, organoids represent the parental tumors' in vivo structure, function, and genetic characteristics.[22,23] Compared with 2D cultured cancer cell lines, organoid models are more similar to original tumors and reflect intra-tumor heterogeneity.[24] The tumor microenvironment is indispensable for studying breast cancer development. The tumor microenvironment possessed by organoids maintains the contact between cells and the stroma, which cannot be achieved by 2D culture. Compared to xenograft models, organoid models can be expanded, cryopreserved, and genetically modified relatively quickly. These functions allow the generation of a living tumor organoid biobank and provide a platform for high-throughput drug screening.[6] To some extent, organoid modeling combines the advantages of the 2D cell culture and xenograft models, and thus is a promising technology.

Despite the many advantages of organoid models, they also have some limitations. One of the main drawbacks of organoids is limited maturity. Organoids often resemble fetal tissue rather than adult tissue;[25] therefore, these organoid models do not perfectly represent real organs. The key to organoids is their self-organization into organ-like tissues. Organoid models should possess the function and morphological structure of organs. However, the overall structure formed by self-organization is still significantly different from that of the real organs. To overcome these shortcomings, scientists have tried to influence the morphology of organoids after self-organization through external interventions (such as 3D printing technology and chemical induction)[26] to obtain organoids consistent with the morphology and structure of real organs. In addition, for tumors in the human body, both the vasculature and immune system are necessary for tumor tissue. However, the currently constructed organoid models do not have vasculature or immune functions. Therefore, some studies have been conducted to compensate the deficiencies in organoid construction by implanting artificial blood vessels into organoids or adopting organoid microarrays and other technologies.[27] For example, Wang et al[28] designed a microchip to generate an intact and perfusable microvascular network that connected microfluidic channels without significant leakage. This means that microfluidic technology can be used to mimic vasculature systems in organoids. In 2018, Dijkstra et al[29] tried to co-culture tumor organoids and immune cells to simulate parts of the immune characteristics of the tumor microenvironment. Furthermore, tumor organoid models can properly recapitulate the tumor immune microenvironment by culturing tumor spheroids in microfluidic devices to preserve autologous myeloid and lymphoid cell populations or by adding immune components, such as cancer-associated fibroblasts (CAFs), manipulating organoid vascularization, and perfusion to preserve endogenous stromal components.[30] Therefore, complex tumor organoid culture platforms can be used to model immunotherapy responses and facilitate immunotherapy preclinical testing.

Table 1 summarizes the advantages and disadvantages of these models, each with its applicable research scope.

Table 1 Advantages and disadvantages of the four biological models.[31]

Items	2D cell culture model	PDX models	OTSC	Organoid model	
Culture time	Several days	1–2 months	about 2 days	1–2 weeks	
Relative cost	Cheap	Expensive	Cheap	Medium	
Maintenance cost	+++	–	++	+	
Success rate	High	Medium	High	Low	
Genetic character retention	+	++	+++	++	
Genetic stability	+	++	–	++	
Gene manipulation	+++	–	+	++	
Tumor microenvironment	–	++	++	–	
Stem cell potential	++	+++	–	+++	
High throughput screening	+++	–	++	+++	
Low-flux screening	+++	+	++	+++	
3D structure	–	+++	++	++	
Note: (–) Not suitable, (+) general, (++) suitable, (+++) best (Adapted from Sachs and Clevers[31]). 2D: Two-dimension; 3D: Three-dimension; OTSC: Organotypic tissue slice culture; PDX: Patient-derived xenograft.

Development of Organoid Models

Although organoids emerged in the last decade, their origin can be traced back a hundred years ago. In 1907, Wilson[32] used isolated sponge cells to regenerate complete individuals with normal functioning through self-reproduction. This experiment caused a huge sensation and was the earliest experiment on the in vitro regeneration of individuals. Subsequently, Holtfreter dissociated and generated amphibian kidneys in 1944, and Weiss and Taylor[33] reconstructed several complete organs from single-cell suspensions of chicken embryos in 1960. These were the first organ regeneration experiments, which led to a boom in organ reconstruction research. Although the principles of organ reconstruction were not resolved at that time, researchers were eager to recreate human organs for experimental research and disease treatments.

In 1981, Evans[34] successfully discovered pluripotent stem cells (PSCs) in mouse embryos, and Martin[35] also successfully isolated PSCs in mouse embryos in the same year and named them "embryonic stem cells." As stem cell research became a new research hotspot, it has also become a key direction for organ research. In 1998, the first human embryonic stem cell line was successfully isolated from human blastocysts.

In 1987, Li et al[36] found that when breast tissue was cultured on extracellular matrix extracts from Engelbreth-Holm-Swarm tumors, the breast epithelial tissue formed a lumen with a 3D structure and appeared to have a secreting capacity. The findings spurred research on 3D organs. In 2009, Sato et al[37] successfully generated intestinal organoids using adult intestinal stem cells in a Matrigel medium and provided a well-defined and stable culture system. The establishment of this experimental system laid the foundation for the construction of other organoids from tissues. In 2011, Eiraku et al[38] used mouse embryonic stem cells to grow retinal organoids. In 2012, Nakano et al[39] developed retinal organoids using hPSCs. In 2013, Lancaster developed brain organoids from hPSCs, followed by the liver, kidney, pancreas, prostate, lung, mammary gland, fallopian tube, hippocampus, and other organoids.[20]

In 2014, Lancaster and Knoblich[19] defined organoids as a collection of organ-specific cell types that develop from stem cells or organ progenitor cells, and self-organize through cell classification and spatial restriction similar to that in vivo. Three basic organoid characteristics were proposed: (1) It is composed of various organ-specific cells. (2) It can summarize specific functions of organs (such as excretion, filtration, nerve activity, contraction, etc.). (3) It has a spatial structure similar to that of the organs.[19] The emergence of an organoid definition also indicates that organoid research has become more standardized, recognized, and focused.

The timeline of the emergence and development of organoids is illustrated in Figure 2.

Figure 2 Evolution of organoids. The timeline of organoid culture development. Key milestones and breakthroughs of organoid technologies. Organoid technology began in 2009. Cleve's team of Hubrecht Institute in the Netherlands successfully cultured adult stem cells into crypts and villi structures of the small intestine, which is a milestone in the development of organoids. ESCs: Embryonic stem cells; iPSCs: Induced pluripotent stem cells; PSCs: Pluripotent stem cells.

Breast Organoids with Different Cell Sources

There is an increasing number of organoid models of the breast; however, generally, breast organoids can be divided into three categories according to the cell source: breast organoid models derived from stem cells, normal breast cell lines, and breast tumor cells.

Breast organoids from stem cells

Stem cells are divided into three main classes: embryonic, human-induced pluripotent, and adult stem cells. iPSCs exhibit similar gene expression, epigenetic spectrum, and differentiation potential as embryonic stem cells, and can generate somatic cells of any type, which has become a hot topic in organoid model research. In 2015, Cravero et al[40] generated bovine-iPSCs and determined that they could generate mammary epithelial tissue, raising the question of whether human-iPSCs could generate mammary epithelial tissue. In 2017, Qu et al[41] successfully generated human mammalian organs by using human-iPSCs. They cultured human-iPSCs using the MammoCult culture medium and differentiated and enriched them into spheres of non-neuroectodermal cells, thus generating mamma-like organs. These breast organoids express common breast tissue, lumen, and basal markers, including ERs, and can be induced to produce milk proteins. This experiment showed that human-iPSCs could be directed to mammary lineage differentiation in vitro and provided an idea for generating mammary organoid models based on iPSCs. This study laid the foundation for studying the normal growth and development of mammary glands and constructing disease models for various mammary diseases, including breast cancer.[5]

Organoids from normal breast cells

As early as 1987, researchers found that normal breast epithelial tissue could form a lumen with a 3D structure and seemed to have secretory capacity when cultured on matrix extracts from Engelbreth-Holm-Swarm tumors.[36] This model may be the earliest mammary gland-like 3D in vitro culture. In 2011, Pasic et al[42] used tissues obtained from normal margins removed during breast surgery as raw materials to develop an organoid model with a normal breast duct network. This model represents the structure of a normally developed mammary gland with high fidelity in terms of the quantity and quality of the mammary gland acinus and mammary duct. In this model, real-time hormone stimulation and observation of the mammary glands after stimulation can be performed. Using this model, Pasic et al[42] found that mouse and human mammary glands have different demands for growth factors. Low concentrations of epidermal growth factors can promote the production of human breast ducts with normal morphology, whereas higher concentrations of epidermal growth factors are required for the stimulation of mouse breast organoids. Later, more studies have been performed on organoid models derived from breast cell lines. Some organoids were constructed with normal mouse breast tissue as raw materials,[43] while others were constructed with normal breast lines from other people as raw materials. Known cell lines that can be used to construct breast organoids include the mammary epithelial progenitor cell line D492 and the non-cancerous mammary cell lines HMT-3522S1 and MCF10A.[44,45] In general, great progress has been made in the culture of mouse, rat, and human mammary gland cell organoid models. Scientists are trying to develop organoid models of other species, such as dogs and goats.[46] The biggest feature of organoid models derived from breast cell lines is that the cell sources are varied, the materials are convenient, and the differences between individuals are fully preserved. This technology is expected to be developed to construct individual breast models, which is conducive to the realization of precision medicine for breast diseases.

Organoids from breast tumor cells

Breast cancer is the most important type of breast disease and has long been the focus of research by many scientists. In 2011, scientists established the first organoid tumor model (intestinal epithelial adenocarcinoma model).[47] Organoid models can effectively recapitulate the intra- and intertumor heterogeneity observed in human cancers.[24] Researchers have found that human breast cancer organoid cultures best represent the tissue origin in primary cultures.[48] In 2012, Kim-Vy Nguyen-Ngoc attempted to cultivate breast cancer organoids to study the importance of extracellular matrix in tumor cell invasion and metastasis.[49] In 2018, an cancer organoid[50] was constructed using mouse tumor cells to study the drug resistance of breast tumor organoids in BRCA-deficient mice. In 2019, Mazzucchelli et al[51] developed a new method to model breast cancer organoids from surgical and biopsy samples, and proposed a new enzymatic digestion process to improve the success rate of organoid construction. Recently, scientists have attempted to build organoid biobanks from normal and tumor-derived human breast tissues. They created 95 breast cancer organoids from 155 tumors that were prone to proliferation. Importantly, compared to the histological subtypes, grades, and receptors of the original breast cancer,[6] organoid models maintain the genetic and histological characteristics of the original breast cancer after long-term passage. These organoids serve as valuable preclinical models of breast cancer and can be widely used in academic, clinical, and pharmaceutical research. The different cell origins of organoids are summarized in Table 2.

Table 2 Organoids of different stem cell sources.[38,39,52–65]

Species	Cell origin	Organoid	References	
Mouse	Mouse embryonic stem cell	Mouse embryonic stem cells were arranged along the proximal-distal axis to form a hemispherical epithelial optic cup structure.	Eirakuet al[38]	
Mouse embryonic stem cells self-organize to form 3D adenohypophysial tissue that secretes adrenocorticotropic and growth-stimulating hormones.	Suga et al[53]	
Mouse embryonic stem cells differentiated into the sensory epithelium of the inner ear in 3D culture and showed the functional characteristics of natural mechanical sensitive hair cells.	Koehleret al[55]	
Mouse adult stem cell	Mouse Lgr5(+) stem cells form microstructures resembling the small intestine (crypt-villus complex).	Sato et al[37]	
Human	Human embryonic stem cell	Human embryonic stem cells were differentiated into cardiomyocytes and human heart tissue was prepared.	Stevenset al[52]	
Human embryonic stem cell culture can be self-organized to form a multi-layer tissue containing rod and cone cells.	Nakanoet al, 2012	
Human embryonic stem cells self-organize into a three-layer cell structure similar to the embryonic cerebellum and differentiate into Purkinje cells with electrophysiological functions.	Muguruma et al[54]	
Human pluripotent stem cells	hPSCs form a 3D intestinal epithelial structure containing functional intestinal cells, goblet cells, Pan's cells, and intestinal endocrine cells.	Spenceet al[25]	
hPSCS were cultured to form various discrete yet interdependent brain regions and reproduce features of human cerebral cortex development.	Lancasteret al[39]	
Vascularized and functional livers were generated from human-iPSCs using liver buds created in vitro (iPSC-LBs).	Takebeet al[57]	
Human pluripotent stem cells (HSCS) are differentiated into 3D human stomach tissues with gastric glands, antral mucous cells, and various gastric endocrine cells.	McCracken et al[58]	
Constructed an alveolar-like structure with an upper airway-like epithelium, basal cells, and immature ciliated cells surrounded by smooth muscle and myofibroblasts.	Dye et al[59]	
This protocol describes an in vitro 3D differentiation method in which hair cell-containing inner ear sensory epithelium is derived from hPSC.	Nie and Hashino[56]	
The use of iPSCs to grow heart organoids mimics how gut and heart tissue work together, starting with stem cells, and helps to study how different tissues interact during development.	Silva et al[63]	
The use of brain organoids reproduces many important cellular and molecular events in the developing cerebral cortex and contributes to the study of human cortical development.	Uzquianoet al[64]	
In vitro, human cortical organoids were transplanted into the developing thymus-free rat brain to observe integration and function.	Revahet al[62]	
Human adult stem cells	Isolated pancreatic ductal cells can be cultured into pancreatic organoids, and pancreatic organoids can be induced to differentiate into ductal and endocrine cells after transplantation.	Huch et al[60]	
The researchers used the distal lung parenchymal tissue to establish a lung organoid model to study the infection dynamics of SARS-CoV-2 and observe the changes in the cytodynamics of infected organoids, which is similar to the clinical characteristics of COVID-19 patients.	Wang et al[65]	
2D: Two-dimension; 3D: Three-dimension; COVID-19: Corona virus disease 2019; hPSCs: Human pluripotent stem cells; iPSCs: Induced pluripotent stem cells; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2.

Although these three organoid source models have unique characteristics, one of the main drawbacks is their limited cell maturity. Organoids often resemble fetal rather than adult tissues,[37] indicating that breast organoids cannot perfectly replace human organs. This might be one of the reasons why breast cancer organoids are different from actual human breast tumors, regarding not only the tissue types, but also the drug sensitivity and side effects.

Currently, the vast majority of in vitro organoid models neither simulate the tumor microenvironment, vasculature, and immune response, nor can they reflect nervous system regulation, which is why organoid technology cannot completely replicate human organs.

The current deficiencies of organoids can be partly remedied by integrating new technologies into the construction process of organoid models, such as genetic engineering and microfluidic chips, which may compensate for the limitation of 3D-cell culture organoids.

New Techniques in Breast Organoid Construction

Although organoid models are still in the exploratory research stage, the combination of organoids with new technologies has greatly expanded their applications. Next, we introduce the combinations and applications of breast organoids and emerging technologies [Figure 3].

Figure 3 Application of the breast organoid model. The diagram summarizes the combination and application of breast organoids and emerging technologies, including the combination of genetic engineering and microfluidic chip technology and their applications in the biological sample library, drug testing, and precision medicine.

New technologies in breast organoids

Breast organoids modified by genetic engineering

Organoid genetics is a new era of research that employs multiple genetic engineering methods.[66] Duarte[50] created a mouse breast cancer organoid model with BRCA1 and BRCA2 defects utilizing genetic engineering, and proposed that organoids constructed based on the genetically engineered mouse model (GEMM) with BRCA1 and BRCA2 defects could be easily modified.Organoid genetics provides a new direction for organoid research. Mice with the required genes can be easily obtained using genetic engineering, and organoids can be cultivated based on the mammary tissues of mice. Because organoid cultures can be effectively derived and rapidly amplified in vitro, the cost and time of breeding a large number of genetically engineered mice are reduced, and biological experiments requiring a large number of genetically engineered mice can be carried out. In addition to the above possible approaches, studies have used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to facilitate a more detailed study of tumor behavior in models resembling physiological conditions. [67,68] Drost used the CRISPR/Cas9 system to target and knock out four types of breast cancer-related tumor suppressor genes (P53, PTEN, RB1, and NF1),[68] and then cultivated breast cancer organoids showing ER positivity. Zhang et al[69] used CRISPR/Cas9 gene editing to create breast cancer organoids from breast stem cells. Gene mutations play a crucial role in the development of breast cancer; however, gene expression may vary among patients, pathological subtypes, and even within the same disease type. Editing organoids through genetic engineering can contribute to a better understanding of the molecular mechanisms underlying specific molecular subtypes of breast cancer and can also be used to develop drugs targeting specific genetic subtypes.

Microfluidic chips for breast organoids

The combination of organoid and chip technologies is not new. In 2010, scientists attempted to use chips to maintain the physiological functions of organs, such as the liver, brain, cortex, and bone marrow.[70] Organoids developed using microfluidic chip technology include the lungs, liver, kidneys, intestines, skin, and heart. In 2021, Hu et al[71] built a lung cancer organ-chip model for drug experiments and found that this model had clinical results similar to those of PDX models, gene mutations, and actual clinical efficacy, proving the reliability of this lung cancer organ-on-a-chip model in drug experiments. In organoids constructed with microfluidic chips, the flow of hormones can be easily controlled, a precise nutrient supply can be provided, and their geometric shape can be controlled. For example, combining the construction of breast cancer organoids with microfluidic chip technology can simulate the influence of hormones produced by other organs in the human body on mammary glands.[72] The microfluidic chip controls the concentration gradient of cytokines, such as morphogens, in different parts of the organoid to manage the change in the shape of the mammary organoid and finally form a shape similar to that of the mammary gland.

In addition, the microfluidic chip technology can accurately simulate the properties of blood vessels and blood flow, compensating the lack of vascular systems in organoid models.[29] Finally, with the development of microfluidic chips and organoid technologies, different organoid chips can be combined and assembled into a complete "human," which can mimic the interaction between different organs.[73] Although research on the combination of breast organoid and microfluidic chip technology is not perfect, the integration of the two technologies in the future is believed to have great potential for research on breast diseases, especially breast cancer.

A schematic diagram of an organoid chip is shown in Figure 4.

Figure 4 Construction of an organoid chip. The schematic diagram shows an idea of organoid-chip construction. Human iPSCs are cultured into corresponding breast organoids and other organoids (such as immune organs) through organoid-chip technology, and the combination of them through organoid-chip technology can not only help researchers to observe the changes of organoids by controlling the organ microenvironment and external stimuli, but also help them to observe the interactions between different organs. iPSC: Induced pluripotent stem cells.

Applications of breast organoids

Construction of breast organoid biobanks

The main function of a biobank is to preserve, organize, and process various types of biological samples, and maintain the biological information contained in the biological samples for the benefit of clinical research. The breast cancer organoid biobank established by Sachs et al[6] was a significant breakthrough in the study of breast cancer. The widespread adoption of biobanks, which are the cryopreservation of living tumor cells, is likely to accelerate the development of rational combination therapies, guide future clinical trials, and facilitate research on new antitumor drugs. Compared to other traditional breast cancer models, organoids have good genetic stability, low cost, short construction time, and long preservation time, all of which support the large-scale establishment of organoid biological specimen banks.

Application of breast organoids in drug testing

One of the main uses of organoids in breast cancer research is in drug testing. Organoids can be used to determine the safety and effectiveness of drugs or their metabolism in organs. Breast cancer organoids show similar histological characteristics, proliferation rates, and genetic heterogeneity to the original tumor. Walsh et al[74] demonstrated the drug sensitivity of breast cancer organoid models. They xenotransplanted the original tumor tissue and organoid tissue into mice for drug sensitivity experiments and found that the drug sensitivity of the organoids was similar to that of the original tumor tissue to some extent. Campaner et al[75] found that breast cancer organoids could represent the original tumor tissue, which could be used to test the efficacy of standard treatments and identify drug-resistant groups within the tumor. Compared to the 2D models used in traditional drug experiments, breast cancer models have higher drug resistance and are more similar to tumors in vivo, which may be caused by the larger contact surface between single-layer tissues and drugs and slower drug metabolism compared to 3D structures.[76] Generally speaking, in breast cancer drug experiments, the xenotransplantation model is closer to the tumor in vivo, limited by its high cost and slow time cycle. The 2D model, although low-cost and less time-consuming, differs from the tumor structure, function, and tumor microenvironment in vivo, which makes 2D model drug experiments less accurate. However, the effect was not good; therefore, the organoid model is now the first choice for drug experiments.[72]

Application of breast organoids in precision medicine

Precision medicine and personalized therapy are the goals of current medicine, and organoid technology is of great importance for the individualized treatment of breast diseases.[77] Organoids are highly consistent with primary tumors in terms of their morphology, genotype, specific function, mutation characteristics, and physiological and pathological changes, providing an important support for establishing an individualized medical system. Breast organoids can now be used to study the molecular events during breast development and degeneration, which could lead to the discovery of targets for predicting breast cancer risk and the development of new breast cancer therapies.[78] In addition, breast cancer organoids can be constructed using tissues obtained from patients with breast cancer, and drug experiments can be conducted on breast cancer organoid models to show patients' reactions to drugs to predict therapeutic effects and modify therapeutic plans,[77] which can be optimized to achieve precise treatment. Hofmann et al[77]used a new strategy to grow in vitro spheroids successfully from primary breast cancer tissue and used the spheroids to test drug sensitivity and drug side effects. Microfluidic chip technology has been added to the clinical applications of breast cancer organoid models. If this organ tandem technology is realized, it can not only study the efficacy of breast cancer treatment but also further evaluate the effect of cancer treatment on other organs, select the most beneficial treatment plan for individuals, and reduce the harm caused by the side effects of treatment and drugs. Owing to the short construction time of organoids, less equipment and reagents required, and good genetic stability and flexibility, the potential of organoids in clinical applications has not yet been estimated.[79]

To sum up, the current traditional models for breast cancer research have some shortcomings. As the earliest in vitro model to be constructed, the 2D cell culture model has been commonly used owing to its low cost and ease of operation. However, the 2D cell culture model has been unable to overcome the difficulty of being very different from the tumor in vivo; therefore, the experimental results are inconsistent with clinical situations. Although PDX models can well simulate the in vivo environment and have advantages such as tumor microenvironment and vasculature, their high research cost, long culture time in mice, and lack of an immune system become research difficulties. The OTSC models were consistent with the original tumor tissue to a certain extent, fully reflecting the tumor situation in vivo. However, for technical reasons, the organ tissue slice model cannot be cultured and preserved for a long time, which does not reflect the potential of tumor stem cells. As a new technology, organoids greatly compensate for defects in constructing breast cancer models. Breast cancer models constructed using organoids have the characteristics of low cost, short culture time, and proximity to the tumor in vivo. To some extent, it can be said that it combines the advantages of other models. Breast cancer organoid models play important roles in breast cancer research.

Although the breast cancer organoid model has many advantages, organoid construction as a novel technology faces many challenges. Organ-like technology is still in the exploratory stage compared with traditional models, and its fidelity, stability, reproducibility, and scalability should be improved in the future.

Owing to the birth of organoid technology more than 10 years ago, although there are many organoid model construction methods, a unified and recognized standard construction process is lacking. The reproducibility of many organoid experiments is not high.[76] Therefore, it is necessary to constantly adjust and optimize the construction of breast organoids, to identify the best program for breast organoid culture, and to establish a standard method of breast organoid culture. Second, conventionally cultured breast organoids lack vasculature or immune cells. Owing to the lack of corresponding vasculature, with an increase in the volume of organoids, tissue necrosis may occur owing to the lack of oxygen supply and obstruction of metabolic waste discharge, which limits the application of organoids.

The introduction of new subjects into routine organoid cultures has become an important means of solving organoid defects. For example, genetic engineering has been used to construct in vitro organoid models to study the mechanisms of gene regulation in specific breast cancer tumors to reduce the impact of tumor heterogeneity. Microfluidic chips can provide a vascular and immune microenvironment for organoids and compensate for defects in conventional organoid cultures. In 2018, Dijkstra et al[29] attempted to co-culture tumor organoids and immune cells to simulate some of the immune characteristics of the tumor microenvironment.[30] The combination of organoids and microfluidic chips can effectively simulate the properties of blood vessels and the fluid flow in blood vessels. Pham et al[80] co-cultured vascular endothelial cells derived from human iPSCs from the same source as brain organoids to generate cerebral vascular structures to solve the problem of the lack of organoid vascularization. In addition, attempts to organically combine multiple tissue-derived organ chips to simulate the interaction between different organs in vitro are also a future developmental direction in the field of organoids.

Basic and clinical research are the two major directions of medical research. Organoid technology can not only be used in basic research, but also plays an important role in clinical research. One of the main uses of organoids in breast cancer research is in drug testing. The safety, efficacy, and metabolic capacity of drugs can be evaluated by conducting drug experiments in organoid models. Moreover, drug experiments conducted on organoid models are more reliable because of the similarity between organoid models and the original tumor tissues in vivo. In addition, organoid technology can also be applied in precision medicine to try treatment strategies and drug use based on the organoid model of individual breast cancer patients and propose the most favorable treatment plan.

Funding

This work was supported by the National Natural Science Foundation of China (Nos. 81971348, 61673024), and the Fundamental Research Funds for the Central Universities (No. PKU2022XGK002)

Conflicts of interest

None.

How to cite this article: Huang CS, Jin HY. Progress and perspective of organoid technology in breast cancer research. Chin Med J 2024; 137: 2157–2168. doi: 10.1097/CM9.0000000000002889
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