Exploring the interaction between extracellular matrix components in a 3D organoid disease model to replicate the pathophysiology of breast cancer

An organoid is a small, three-dimensional (3D) cell assembly that is heterocellular in nature. Primary organoids are composed of cells that come from donor tissues and can self-organize and change into different cell types to mimic the complex cellular organization and makeup of an organ. Organoids provide a more accurate picture of human health than traditional 2D cell culture and animal models. This makes them an important tool for biomedical research [1]. Organoids derived from human breast tissue are a unique way to study the biology and pathology of breast cancer in a manner that is controlled and specific to each patient. Personalized treatment and the study of how different tumors are from each other are both possible because of the primary breast cancer biopsies [2]. Organoids provide a more accurate picture of how a patient’s tumor works than standard 2D cell cultures or animal models, because they maintain the cell complexity and characteristics of the original tumor. Researchers can use organoids to look for drugs and measure how each patient responds to a certain course of treatment as a personalized therapy [3]. A 3D multicellular heterogenic scaffold created without the help of a scaffold material (synthetic, polymeric, or extracellular matrix) is referred to as a scaffold-free organoid. The traditional organoid production approach involves embedding cells within a gel-like matrix such as Matrigel or other cross-linkable/thermoresponsive hydrogels, which offer structural support for their development and growth. However, scaffold-free organoids allow cells to self-assemble and organize into complex structures without the use of an external scaffolding substance [4]. High consistency and reproducibility are the two significant benefits of scaffold-free organoid models. They enable the accurate measurement of cellular responses, such as extracellular matrix synthesis, without being hampered by exogenous collagen.

A scaffold or support structure is used to create a three-dimensional (3D) structure that resembles the native tissue. This structure is called a scaffold-based organoid, and it is stable and resembles the organ being studied or made [5].. To create scaffold-based organoids, cells are placed inside a material called a scaffold, which provides structural support, helps cells stick together, and makes it easier for tissue structures to form in an organized manner. Scaffolds can be fabricated from biomaterials such as hydrogels, polymers, and parts of the extracellular matrix (ECM). These supports provide cells with a three-dimensional structure for interacting with their surroundings and arranging themselves into structures that resemble tissues in the target organ. Organoids built on scaffolds have several advantages. First, it provides a good microenvironment that helps cells grow into different types of cells and form new tissues. It is possible to recreate the position of cells, presence of ECM components, and other important parts of the structure of the original tissue. Second, scaffold-based organoids can be prepared to include specific cell types or cells obtained from a patient. This makes it possible to use personalized medical methods and model diseases. Third, they can be used to study organ growth, physiology, and pathology in controlled laboratory settings. Depending on where and how they are used, scaffold-based organoids have different limitations. In general, the following are the problems with scaffold-based organoid systems: To help organoids grow and keep cells alive, the support must be biocompatible and have a certain structure. However, some scaffolds might need to work better with certain cell types or regions of interest, which could lead to growth or differentiation that is not as good as possible. Its biocompatibility should be carefully examined to ensure that its structure does not affect the cell lineage in organoids [6, 7]. Scaffolds are often made and prepared using complicated steps, and differences in the way scaffolds are made can affect the organoids, which in turn affects their reproducibility [8]. Scaffolds can make it harder for nutrients to penetrate through the organoid structure, which can affect the health of the cells and make the core areas hypoxic, nutrient deficient, and growth factor deficient. The thickness and porosity of the scaffold can affect how nutrients and release factors are distributed, which could affect how organoids grow and work as a whole. The phenotype and behavior of cells can be changed in response to different materials used. Sometimes, the scaffold can change the cellular lineage and affect differentiation, maturation, and signaling pathways. It is important to ensure that the scaffold has the same capabilities as those of natural tissues [9]. Although scaffold-based organoids try to mimic the structure of native tissues, they may not be able to fully replicate the complex structure and biological organization found in vivo. The scaffold could be used as a general structural support, but it might not be able to replicate the complex relationships between cells and their arrangement in natural organs [10].

In this review we have dive into the critical discussion of the following question.

The extracellular matrix (ECM) is an arrangement or network of extracellular macromolecules that structurally and biochemically supports neighboring cells. Cell adhesion, cell-to-cell communication, and differentiation are standard ECM functions. It is essential for many biological functions, including maintaining tissue integrity and controlling cell behavior. It is a scaffold for cells that enables their adhesion, migration, and interaction with their surroundings [11]. Native tissues and organs are sources of natural ECMs components. They comprise a complex network of proteins (e.g., collagen, fibronectin, laminin, and elastin) and polysaccharides (e.g., glycosaminoglycans). They serve as structural scaffolds and offer biochemical cues to support cellular activities. Researchers have attempted to make artificial or synthetic ECMs biocompatible and can communicate with cells via ligand-receptor interactions. However, their limited in vivo stability and susceptibility to enzymatic degradation restrict their long-term applications in tissue engineering [12].

The Co-Culture System approach involves pre-culturing various cell types separately and then combining them to allow their self-assembly into spheroids [119, 120]. Mammary epithelial cells can be broadly divided into luminal and basal cells based on their location within the bilayer breast epithelium. To develop organoids that closely resemble the in vivo breast microenvironment, these cells were isolated from breast tissue samples and grown in a three-dimensional culture system. The efficiency of this procedure has recently increased, allowing for the long-term culture of breast cancer (BC) organoids and preservation of several lineages within the breast epithelium, including progenitor cells [121].

Researchers have used genetic manipulation methods in breast organoids to study the biology of breast cancer and drug responses. Oncogenic transformation in various breast cancer subtypes and specific genetic alterations or mutations have been introduced into organoids to mimic tumor characteristics. The progesterone receptor (PR) regulates the expression of various genes involved in cell adhesion, immune response, and survival, such as receptor activators of the NFκB ligand and calcitonin [122]. Specific genetic alterations have also been associated with different histological tumor types, such as inactivation of E-cadherin in lobular breast cancer and HER2 gene amplification in poorly differentiated ductal cancer [123]. Furthermore, germline mutations in BRCA1 and BRCA2 have been linked to genetic predisposition to breast cancer [124]. However, there are concerns and limitations associated with xenotransplantation, including the risk of contamination, and the need for further research and validation before considering clinical applications [125].

Humanized cancer models in rodents involve a combination of mouse models with xenografted or spontaneous human cancer cells, along with the human immune system (HIS) mice. These models have become more sophisticated and robust, allowing for in vivo exploration of human cancer immunology and immunotherapy [126]. The laboratory mouse is the most common animal model used in cancer research because of its genetic variability, physiological similarities with humans, and ability to generate humanized mouse models by incorporating the human immune system with human tumor xenografts [127]. Breast cancer organoids have been xenotransplanted into immunocompromised mice to examine therapeutic interventions, such as non-obese diabetic severe combined immunodeficiency or NOD-scid Mice, which are highly immunodeficient and suitable for the transplantation of human tissues because they lack functional B and T cells. A more sophisticated immunodeficiency model is provided by NOD-SCID IL2Rγnull (NSG) mice, which are deficient in B and T cells and functional NK and IL2R signaling. NSG mice have gained popularity as a popular choice for xenotransplantation studies because of their improved engraftment efficiency. Another strain of mice with multiple immunodeficiencies, including a problem with IL2R signaling, are NOG (NOD/Shi-scid/IL2Rγnull) mice, which are suitable for engrafting human tissues. Similar to NSG mice, NSI (NOD/Shi-scid IL2Rγnull) mice lack the IL2R chain, which enhances their capacity to engraft human tissue [128, 129]. Breast xenotransplantation models have advantages and disadvantages. The breast xenotransplantation model allows for the study of human breast cancer in an animal model and can provide insights into the self-renewal capacity and differentiation potential of distinct cell populations or individual cells in the mammary gland. The disadvantages of xenotransplantation models may not fully replicate the complexity of the human tumor microenvironment. The theoretical hazard of causing new human infections through the intermingling of tissues from different species has been a concern in the field of xenotransplantation [130].

Heterotypic organoids are created by cultivating pluripotent or multipotent stem cells in a three-dimensional (3D) matrix under conditions that encourage self-organization and the presence of various cell types. Organoids are ex vivo multicellular fragments produced by cultivating pluripotent or multipotent stem cells in a 3D matrix under conditions that promote self-organization. These conditions are established experimentally, and frequently use information regarding the signals involved in organ development or regeneration [131‐133].

Normal fibroblasts are mesenchymal cells responsible for maintaining tissue homeostasis, whereas cancer-associated fibroblasts (CAFs) are fibroblasts that have been chronically misregulated in epithelial cancers. CAFs are a dominant and heterogeneous cell type within the tumor microenvironment (TME) and play a pivotal role in controlling cancer cell invasion, metastasis, immune evasion, angiogenesis, and chemotherapy resistance [134]. Unlike normal fibroblasts, CAFs have tumor-promoting functions and can influence tumor progression, invasion, and response to therapy. CAFs communicate with cancer cells and other cells in the TME through various mechanisms, including metabolite exchange, paracrine signaling, desmoplasia, and acidosis [134, 135]. CAFs have been found to promote cell survival during detachment, block anoikis, and facilitate luminal filling in three-dimensional cell culture [136]. CAFs also secrete insulin-like growth factor-binding proteins (IGFBPs) that stabilize the anti-apoptotic protein Mcl-1, contributing to anoikis inhibition [137]. Additionally, CAFs promote epithelial-mesenchymal transition (EMT) by secreting collagen triple helix repeat containing-1 (CTHRC1), which activates the Wnt/β-catenin signaling pathway [138, 139]. Human adipose tissue-derived stem cells (hASCs) have been identified as a potential source of CAFs because they can differentiate into a CAF-like myofibroblastic phenotype when exposed to conditioned medium from breast cancer cell lines [140]. Fibroblasts were added to the 3D tissue models to replicate the stromal environment found in the breast tissue and produce a more accurate representation of the breast cancer model. Fibroblasts play a crucial role in maintaining scaffold-free conditions by promoting cell migration and proliferation within a collagen matrix [141]. Fibroblasts also shed microvesicles from their plasma membranes, which then spread throughout the matrix. The presence of fibroblasts provides favorable conditions for simulating collagen processing in vitro and for understanding the mechanisms controlling cell uptake and intracellular degradation [142]. Additionally, fibroblasts encapsulated in a collagen gel show enhanced extracellular matrix (ECM) production, including collagen type I and elastin expression. This suggests that fibroblasts contribute to the maintenance of scaffold-free conditions by actively participating in ECM synthesis and remodeling. Fibroblasts influence the behavior of immune and endothelial cells within the tumor microenvironment. They secrete fibroblast growth factor (FGF), which attracts immune cells to the tumor site and promotes their activation and differentiation. By releasing pro-angiogenic factors that encourage endothelial cell migration and proliferation, fibroblasts also help in the development of new blood vessels that supply the tumor with nutrients.

Endothelial cells play a crucial role in tumor angiogenesis, which involves the development of new blood vessels that supply nutrients and oxygen to the developing tumor. These endothelial cells interact with cancer cells and other stromal cells in the tumor microenvironment to promote vascularization and tumor development. Endothelial cells are essential players in tumor angiogenesis, and their interactions with fibroblasts and immune cells can affect their behavior. The formation of blood vessels and development of tumors can be aided by activated fibroblasts, which can increase the production of proangiogenic factors (PAF) in endothelial cells. Vascular endothelial growth factor (VEGF), which affects endothelial cell behavior by regulating permeability and functionality within the tumor microenvironment, can also be secreted by immune cells [143]. Sustained stress-activated myofibroblasts have an altered secretory phenotype, producing factors, such as TGF-β and VEGF, to promote proliferation and recruit other cells. Macrophages and fibroblasts have physiological functions in tissue homeostasis, immune response, angiogenesis, and wound healing.

Cancer stem cells (CSCs) have been shown to play a critical role in breast cancer initiation, progression, metastasis, and drug resistance. These cells possess long-term proliferative potential and the ability to regenerate phenotypically heterogeneous cell types. CSCs in breast cancer often exhibit attributes of cells that have undergone an epithelial-mesenchymal transition (EMT) [144]. Breast cancer stem cells (BCSCs) are driven by the persistent activation of developmental pathways such as Notch, Wnt, Hippo, and Hedgehog [145]. This trilayer breast organoid serves as a reliable model for studying breast cancer and provides valuable insights into this disease. These organoids enable researchers to better understand the molecular characteristics of breast cancer, which can help assess the therapeutic response. Additionally, trilayer breast organoids have the potential to identify new druggable targets for targeted therapy [146]. Their inclusion in 3D breast cancer models is crucial for a better understanding of tumor angiogenesis and vascular interactions [147].

Various cell types within the organoid, reflecting the cellular variety in the associated organ, are heterotypic aspects of multicellular organoid culture, essential for simulating the interactions and crosstalk between various cell populations in the organ, which supports physiological processes, can be disturbed in diseases such as cancer, and contributes to their maintenance [148].

Scaffold-free breast organoids display characteristics resembling those of normal human breast acini, including a hollow lumen and secondary acini, and express mammary gland-specific progenitor markers [149]. Scaffold-free organoids also have high consistency and reproducibility, as well as the ability to measure cellular collagen I production without noise from exogenous collagen, and can be subjected to various stimuli from the microenvironment and exogenous treatments with precise timing without concern for matrix binding [150]. Additionally, scaffold-free breast organoids can be generated from primary mammary carcinomas, retaining the high-grade spindle cell morphology of the primary tumors.

Breast tumor-derived fibroblasts secrete extracellular matrix (ECM) components that induce morphogenesis and growth of breast epithelial cells. Adipose progenitor cells have been shown to assemble the fibronectin (Fn) matrix in response to soluble factors secreted by breast cancer cells, leading to increased stiffness of the tumor stroma [151]. ECM proteins upregulated in breast tumor tissue were found to have cell line-specific effects on cell migration and invasion, with cell adhesion, elongation, and irregularity being key determinants [152]. Multiple cell types, such as mammary epithelial, tumor, and stromal cells, all of which contribute to the synthesis and remodeling of ECM, are involved in the development of breast organoids. In turn, the ECM maintains organoid structure and functionality by creating a microenvironment that resembles that of both normal breast tissue and tumors. The primary cell types involved in developing breast organoids are mammary epithelial cells, which help produce ECM constituents, including collagen, laminins, fibronectin, and proteoglycans. The ductal and lobular structures found in the mammary gland are maintained by mammary epithelial cells, which also develop epithelial compartments in the organoids.

One study focused on spheroid cell culture methodological factors to improve reproducibility and physiological significance when investigating the metabolic effects of drug treatment in breast epithelial cells. Spheroids were formed by co-culturing MCF10A breast epithelial cells and MDA-MB-231 breast cancer cells in standardized and enriched media (DMEM or RPMI). Spheroid analysis was used to assess metabolic behavior and integrity using Spheroid-Sizer software, confocal microscopy, and western blotting [153]. Extracellular matrix-stromal cell interactions contribute to the neoplastic phenotype of breast epithelial cells. A previous review examined the role of the extracellular matrix and stromal cells in influencing the neoplastic phenotype of epithelial cells during the development of breast cancer. In breast cancer, epithelial-mesenchymal interactions involve stromal microenvironmental factors that influence epithelial growth, hormonal responses, morphogenesis, and plasticity [154]. 3D Cell Structures created a vascular endothelial-breast epithelial cell coculture model. In a study, a 3D model of vascular endothelial-breast epithelial cell interactions was developed, focusing on cell-cell interactions between endothelial and breast epithelial cells. Breast epithelial cells migrated out of their spheroids and along HUVEC networks, which appeared to be partly mediated by secreted EGF and cell-cell contact [155]. Another study comprehensively investigated altered lipid metabolism in breast cancer, examined changes in lipid composition, identified critical regulators, and analyzed their impact on cancer progression. This study revealed novel lipidomic changes in EMT-induced breast cancer and emphasized the importance of ELOVL2 in cancer progression. Cancer-associated fibroblast CAFs, a type of mesenchymal cell found in the tumor stroma, have been shown to require proline synthesis by PYCR1 to deposit a pro-tumorigenic ECM. CAF subpopulations that produce collagen-rich ECM, such as myofibroblast-like CAFs (myCAFs), contribute to tumor progression and metastasis [156]. Tumor cells from patient samples are also a part of the organoid culture in the case of breast cancer organoids. As they still possess the capacity to produce ECM elements resembling those found in the tumor microenvironment, tumor cells contribute to ECM synthesis and remodeling within organoids [157].

Tumor cell secretion of vascular endothelial growth factor (VEGF) in response to hypoxia stimulates endothelial cell proliferation and angiogenesis within the tumor microenvironment [158]. The formation of capillary-like structures during the assembly and growth of tumor cell-endothelial cell (TC:EC) spheroids suggests the formation of a network of blood vessels within these models. This formation is critical for the nutrient supply to growing tumor cells and indicates spatial invasiveness within the ECM. These spheroid shapes and surface textures can provide information regarding the invasive potential of cells within the ECM. These findings emphasize the importance of understanding the dynamic interactions between tumors and endothelial cells in the context of 3D models [159].

Breast cancer is a multifactorial disease that includes many separate entities with markedly different biological characteristics and clinical manifestations. Based on the expression of human epidermal growth factor receptor 2 (HER2) and hormone receptors (HRs) (progesterone and estrogen) breast cancer is categorized into four subtypes: Luminal A, Luminal B, HER2 enriched, and Triple-negative breast cancer (TNBC) [160]. Out of all breast cancers, 50 to 60% are known to be luminal A (LABC; ER/PR+, HER2-, and low expression of Ki-67). This subtype has a great prognosis with limited invasiveness, with a relapse rate that is 27.8% lower than other subtypes [161]. Luminal B is further classified into two types i.e. Luminal B like HER2- (ER+ but ER and PR expression lower than in luminal A-like; HER2-; high Ki67 index) and Luminal B like HER 2+ (ER+ but lower ER and PR expression than luminal A-like; HER2+; high Ki67 index) [162]. About 15–20% of breast cancers are HER2+, which is defined as having evidence of HER2 protein overexpression and determined by immunohistochemistry status (IHC3+), fluorescence in-situ hybridization (FISH) measurement of a copy number of six or more for the HER2 gene, or a HER2/CEP17 ratio of 2·0 or higher. In 2013, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) revised their criteria, reintroducing a cutoff value of 2·0 or above for the HER2/CEP17 ratio and full staining of more than 10% of the cells [163]. TNBC is characterized by the absence of progesterone receptor (PR) and estrogen receptor (ER) expression, as well as the lack of HER2 overexpression and/or gene amplification. According to the 2010 ASCO/CAP recommendation, invasive breast tumors should be classified as ER-positive if their immunohistochemical ER expression is ≥1% [164]. While the therapeutic relevance of several genetic subtypes of breast cancer has been extensively acknowledged, the importance of tumor extracellular matrix heterogeneity has been mainly overlooked [165]. The significance of tissue-specific ECM and tissue-mimicking biomaterials in tissue/organ regeneration has been emphasized by advances in tissue engineering. Tumor-derived extracellular matrix (ECM) may be more effective than tissue engineering at simulating the intricate physiology of the natural microenvironment [166]. Tan et al. 2023 in their research compared the composition, organization, and intended application of ECM obtained from two genetic subtypes of breast cancer: TNBC (very aggressive, ERα-)-derived ECM (TN-ECM) and luminal-A breast cancer (less aggressive, ERα+)-derived ECM (LA-ECM). Through comparison, they discovered that Tumor-derived ECM displayed altered architecture and increased levels of pro-collagen I, fibronectin, and laminin compared to normal breast tissue-derived ECMs (B-ECM). They also explored that HER2+ tumor subtypes have been related to higher collagen deposition levels. In TNBC and HER2+ breast cancers, fibronectin was substantially expressed in both the primary and metastatic tumors [167]. These results highlight the significance of tissue-mimicking microenvironments in drug testing by potentially clarifying the distinct microenvironments linked to native tumor matrices. Rafaeva, Maria et al. Fibroblast-derived matrix (FDM) model. By employing this model, they demonstrate that, in contrast to FDMs originating from non-malignant tissue (normal) fibroblasts, cancer cells exhibit enhanced proliferation on cancer-associated FDMs. At the primary tumor site, they evaluate changes in ECM characteristics from normal to cancer-associated stroma [168]. There are presently very few temporally resolved proteomic studies available, that more accurately reflect deposited extracellular matrix during the course of disease progression. Their FDM proteomics approach can be used to bridge this gap by investigating the ECM deposition by the CAF subtypes.

Campaner et al. developed patient-derived organoids (PDOs) derived from various subtypes of breast cancer (luminal A, luminal B, HER2-enriched, and TNBC). Through the application of Masson’s trichrome histochemical staining, which enables the assessment of extracellular matrix deposition, they observed that thesss tumor tissue was characterized by desmoplastic stroma that was enhanced by an excessively fibrous collagen matrix [61]. PDOs can serve as in vitro platforms for testing combination treatments meant to overcome drug resistance as well as for evaluating the sensitivity of cancer cells to conventional therapy. Charles et al. in their research revealed that, regardless of patient age or race, collagen 1 (COL 1) expression elevated considerably in most ER+/PR+ breast cancer subtypes. To objectively prove a correlation between fibrillar COL expression and receptor status. RNA sequencing from the SCANB and TCGA data sets was utilized to assess the expression of COL1A1 and COL1A2 in HER2+ and ER+/PR+ cancers. ER−/PR-cancers exhibited significantly (p < 0.0001) lower expression of COL1A1 and COL1A2 than ER+ tumors. No correlation was found between the expression of COL1A1 and COL1A2 and HER2 status [169]. The disadvantage of this study is that they used a single 3-day time point for endpoint analysis. Cells had time to adjust to the new culture conditions at this point. Although induced protein changes were not identified, more time points could be needed to observe the translational effects of matrix adherence. Additionally, the use of monoculture, which is not representative of the heterogeneous cell population noticed in vivo, was a drawback of this work. (Figs. 4, 5, 6) (Table 2)

Microvasculature integration with parenchyma and breast tissue organoid stroma is required to develop vascularized breast organoids. This is necessary for accurately simulating the native tissue environment, enabling physiologically relevant perfusion of the organoids, and supporting cellular dynamics within the tissue model via perivascular niche cells [175, 176]. Vascularization of breast organoids can be performed in various ways. One method to develop a vascular network inside organoids is to coculture organoids with microvessels or endothelial cells. This method enables the integration of the microvasculature with the breast tissue model, allowing for perfusion and nutrient supply to the cells. Microinjection methods or exposing organoids to endothelial cells in a two-dimensional (2D) layer can be used to incorporate microvessels.

Providing structural support and regulating cellular behavior are essential functions of the extracellular matrix. Vascularized breast organoids may use ECM components to promote vascularization and tissue development [177]. Collagen-alginate hydrogels with filamentous architectures have been used to mimic the ECM of breast tumor microenvironments in the context of breast cancer spheroids and organoids. These hydrogels vary in stiffness by varying the crosslinking of alginate molecules, which influences the mechanical properties of the ECM. The filamentous architecture of collagen–alginate hydrogels mimics the ECM structure in a breast tumor environment. It has been used to study the growth of breast tumor spheroids and their response to chemotherapy [178]. Collagen-rich ECM environments have been shown to promote cell growth and behavior, including those of vascular endothelial cells. In tissue cultures, collagen within the ECM can provide cues for angiogenesis and vascularization, which are critical for the development of functional blood vessels within spheroids and organoids.

Once vascularized breast organoids have been developed, it is crucial to maintain a perfusion system that enables constant circulation of nutrients and oxygen within the tissue model. Long-term tissue viability and functionality were preserved by perfusion.

Microfabricated and microfluidic platforms such as microfluidics and microprinting offer promising tools for addressing organoid and spheroid production limitations. These platforms can improve the nutrient delivery and culture conditions, thereby producing more uniform and reproducible organoids and spheroids. This makes it possible to create size-controlled culture areas that enhance vascularization. However, these techniques may be challenging to implement and require specialized equipment [179]. The co-culture of pluripotent stem cells and endothelial cells on 3D substrate matrices has been proposed to produce vascularized organoids. This method enables the creation of functional organoids with a more accurate representation of corresponding tissues. It allows for the study of physiological processes and disease manifestations in a controlled in vitro environment. However, it is difficult to optimize culture conditions and precisely integrate vascular networks [180]. Researchers have utilized spheroid-based engineering to generate the human vasculature in mice. This approach involves creating 3D spheroids of cells that mimic the tissue architecture and subsequently implanting them into a living host. The advantage of this method is its ability to generate a functional vasculature in vivo. However, controlling the precise formation of vascular networks may be challenging, and host factors can influence outcomes.

Vascularized breast cancer organoids can be used as living biobanks to evaluate drugs and to develop individualized treatments. They are helpful tools for researching drug responses and developing specialized therapies because of their capacity to mimic the tumor microenvironment and heterogeneity of individual patients [181]..

Organoid vascularization is necessary to improve biological relevance and to ensure sufficient oxygen and food supply [182]. Organoid vascularization approaches can be classified into two types: in vitro and in vivo approaches. In the in vivo method, nonvascularized organoids are inserted and left to be vascularized by the host’s peripheral vascular system. To ensure that organoid cells have access to sufficient nutrients for survival, this approach depends on timely invasion of the host vasculature into the non-vascularized organoid through angiogenic sprouting. Naturally, organoids would require more time for vascularization when this method is used. The time required for in vivo vascularization may be too long, resulting in necrosis before the development of a functional vascular network. This limitation has led to the use of in vitro vascularization procedures to develop organoids that are pre-vascularized before implantation, which has definite advantages over nonvascularized organoids [183]. In vitro vascularization can be achieved by co-cultivation of vascular cells or tissue engineering. In vitro vascularization techniques can be divided into templating and self-organizing approaches [184, 185]. Templating methods include 3D bioprinting, DMD patterning, and sacrificial molding. On the other hand, self-organizing methods include co-culture of organoids with endothelial cells in a compartmentalized chamber and neo-angiogenesis in a microfluidic device using control fluid dynamics [186].

The stroma is an essential part of the breast tissue because it offers a favorable microenvironment that affects mammary cell fate, differentiation, and tissue homeostasis. It is becoming increasingly apparent that stromal interactions are essential for the development of healthy mammary tissue and breast cancer. Mammary epithelial cell behavior, including proliferation, migration, and differentiation, is influenced by cues and signals provided by stromal cells. In organoid culture systems, stromal cells play a significant role in promoting the growth and maintenance of mammary epithelial cells in vitro, allowing for long-term culture and study of breast organoids [222].

Breast organoids contain stromal cells that secrete cytokines, chemokines, and growth factors that control the behavior of mammary epithelial cells. For instance, tumor necrosis factor (TNF-α) and transforming growth factor (TGF-β) produced by stromal cells can affect the invasiveness of breast cancer cells, as can stromal-derived factors (SDF-1), which stimulate the growth of mammary progenitor cells. Furthermore, stromal cells have the potential to produce matrix metalloproteinases (MMPs), such as MMP-2, MMP-7, MMP-9, and MMP-11, which are essential for remodeling the extracellular matrix and facilitating tumor invasion and metastasis.

Within the breast organoids, stromal and mammary epithelial cells interact dynamically and reciprocally. Although stromal cells play a crucial role in the growth and function of mammary epithelial cells, mammary epithelial cells, particularly tumor cells, can also influence the phenotype and behavior of stromal cells in the microenvironment. Reciprocal crosstalk between stromal and epithelial cells greatly aids tumor development, invasion, and metastasis [223].

The immune system is another crucial element in the breast tissue microenvironment. Interactions among immune, stromal, and epithelial cells significantly influence breast organoid growth and cancer development. Involvement of the immune system in breast organoids is essential for research on immune responses, inflammation, and potential immunotherapies for breast cancer [224].

Macrophages are essential elements of the tumor microenvironment (TME) and are important for tumor development. The well-established M1/M2 macrophage paradigm emphasizes the distinct functional polarization states of macrophages in response to TME. Previous studies have shown that M1 macrophages are anti-tumor, whereas tumor-associated macrophages (TAMs), also known as M2-polarized macrophages, are linked to pro-tumorigenic outcomes in cancer [225].

M1 macrophages are classically activated macrophages with proinflammatory and tumoricidal phenotypes. They are induced by inflammatory cytokines such as lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). M1 macrophages are characterized by the production of pro-inflammatory cytokines (like interleukin- 1β, interleukin-6, and tumor necrosis factor-alpha) and reactive oxygen species, which support the immune response against tumors and the phagocytosis of tumor cells. In addition, they participate in antigen presentation, promoting the activation of cytotoxic T cells and adaptive immune responses against cancer cells. In contrast, M2 macrophages exhibit pro-tumorigenic and anti-inflammatory phenotypes. They are brought on by anti-inflammatory cytokines frequently found in the TME, like interleukin-4 (IL-4) and interleukin-13 (IL-13). Growth factors secreted by M2 macrophages, such as transforming growth factor-beta (TGF-β) and vascular endothelial growth factor (VEGF), promote angiogenesis, tumor cell proliferation, and metastasis. Additionally, M2 macrophages participate in immune suppression, impairing the anti-tumor immune response by promoting an immunosuppressive environment [226].

M1 and M2 macrophages significantly affected the rapid development of breast cancer in the context of the breast organoid microenvironment. The protumorigenic properties of M2 macrophages, which are TAMs, are thought to aid tumor growth, invasion, and metastasis. In addition to promoting angiogenesis, which aids in vascularization and nutrient supply to the tumor, they also alter the extracellular matrix to promote tumor cell invasion. The ratio of M1 to M2 macrophages within the TME is dynamic and is influenced by several factors, including cytokines, chemokines, and signals from tumor and stromal cells. The interaction of these factors determines whether macrophages are pro- or anti-tumorigenic (M1 or M2) and affects breast cancer development. To develop targeted therapeutic strategies that can modify macrophage polarization and promote anti-tumor immune responses while limiting tumor-promoting effects, it is essential to understand the role of M1 and M2 macrophages in the breast organoid microenvironment.

Immune cells can be incorporated into 3D structures in organoid culture systems, enabling the study of immune cell behavior and interactions with mammary epithelial cells. For example, immune organoids have been used to study immune conditions and to understand the structures and functions of immune tissues in a setting that closely resembles the in vivo microenvironment [227]. Immune breast organoids are three-dimensional culture models created in vitro from patient-derived tumor cells or cell lines using three-dimensional culture technology and cytokines that encourage breast cancer cell proliferation while preventing their apoptosis. Compared with xenografts, which are made from different species and might not accurately reflect the patient’s condition, these organoids have a structure similar to that of breast tumors in the body and provide a more accurate representation of the disease [228]. Cytokines, tumor necrosis factors, interleukins, and transforming growth factor-β play significant roles in breast cancer development and progression. The increased production of cytokines in the tumor microenvironment influences tumor initiation, angiogenesis, and metastasis. Cytokines, such as IL-1, IL-6, IL-11, and TGF-β, promote cancer cell proliferation and invasion. TGF-β has a dual role, acting as a tumor suppressor in the early stages of BC but later promoting tumor progression. TGF-β also mediates the epithelial-to-mesenchymal transition (EMT), which is linked to BC progression and metastasis [229, 230]. Immune breast organoids help research how the immune system and the tumor microenvironment interact. These interactions are essential for cancer growth and response to therapy. Organoids help researchers to better understand the tumor-immune microenvironment and model the immune response against breast cancer cells. This information can direct the development of innovative immunotherapies and enhance the effectiveness of breast cancer treatments [231]. Although immune breast organoids have shown great promise in the study and treatment of cancer, it is important to remember that they still have limitations. For example, they may not accurately replicate the complexity of the human immune system. The accuracy and usefulness of organoids for precision immuno-oncology applications are continuously improving through ongoing research and technological advancements [232].

Additionally, immune cells in the microenvironment of the breast tissue contribute to both the growth of breast organoids and cancer development. On the one hand, immune cells can encourage the development and spread of tumors by secreting cytokines, chemokines, and growth factors that foster a microenvironment favorable to tumors. In contrast, the activation of cytotoxic T cells and natural killer cells enables immune cells to mount anti-tumor responses and inhibit tumor growth.

Recent studies have highlighted the importance of lymphocyte-stromal cell interactions in autoimmune and inflammatory diseases. These interactions most likely affect the immune response within the organoid and its microenvironment, thereby affecting the development of the breast organoids [233]. Researchers can produce more accurate models of breast tissue that replicate crucial elements of the tumor microenvironment by adding stromal components and immune cells to organoid culture. Owing to this improved representation, the investigation of tumor-stroma crosstalk, immune responses, and drug responses can now be performed in a physiologically relevant setting. Breast organoids can be used to study the interactions between mammary epithelial, stromal, and immune cells, which may provide important information regarding the earliest stages of oncogenic transformation and the particular cell types that give rise to various breast cancer subtypes. Additionally, this study may help identify potential therapeutic targets in the immune and stromal compartments to create new breast cancer treatment plans.